J. Nat. Prod. 2009, 72, 1331–1334
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Isolation, Biomimetic Synthesis, and Cytotoxic Activity of Bis(pseudopterane) Amines Abhijeet S. Kate,†,‡ Jason K. Pearson,† Balaji Ramanathan,‡ Kelly Richard,‡ and Russell G. Kerr*,†,‡ Department of Chemistry, UniVersity of Prince Edward Island, Charlottetown, Prince Edward Island, Canada C1A 4P3, and Department of Biomedical Sciences, Atlantic Veterinary College, UniVersity of Prince Edward Island, Charlottetown, Prince Edward Island, Canada C1A 4P3 ReceiVed December 23, 2008
LC-MS/MS-based screening of the dichloromethane extract of the gorgonian coral Pseudopterogorgia acerosa led to the isolation of a novel bis(pseudopterane) amine (1). The structural assignment of 1 was achieved by 1D and 2D NMR and mass spectrometry analysis. A biomimetic synthesis of 1 and the known symmetrical diterpene 2 from pseudopterolide (3) is described in this report. Bis(pseudopterane) amine showed selective growth inhibition activity against cancer cell lines with IC50 values of 4.2 µM (HCT116) and 42 µM (HeLa). The gorgonian coral Pseudopterogorgia acerosa has received considerable attention from the natural products community due to a diversity of terpene chemistry.1-6 Fenical and Clardy reported2 the isolation of pseudopterolide, a structurally intriguing diterpene with an apparently ring-contracted cembranoid skeleton from P. acerosa collected in the Florida Keys. Later studies of this gorgonian collected in Puerto Rico, Tobago, and Martinique indicated the presence of an extensive array of diterpenes with the pseudopterane skeleton.1-6 We recently reported7 the isolation of a series of lipidyl pseudopteranes from a Bahamian collection of P. acerosa, which exhibit selective inhibitory activity against protein tyrosine phosphatase 1B. From a subsequent expedition to the Bahamas, a collection of P. acerosa has led to the isolation of a novel amine bridging bis(pseudopterane) amine (1) along with several known diterpenes including pseudopterolide (3), gorgiacerodiol (4), and bis(gorgiacerol) amine (2). The mass spectrometric fragmentation pattern of the pseudopterane diterpenoids were useful for the development of a data-dependent screening method called the doubleplay method.8,9 The advantage of this method is that minor components can be identified in the presence of major metabolites of extract fractions. After development of the doubleplay method with an ion-trap mass spectrometer, a range of fractions from a C18 flash column of the dichloromethane extract of P. acerosa was screened. The resulting data indicated the presence of several peaks within the m/z range 740 to 790 with fragmentation similar to that of gorgiacerodiol (4), and we subsequently isolated two of the more abundant of these compounds. We report here the isolation, characterization, biomimetic synthesis, and cytotoxic activity of one new and one previously reported4 bis-diterpenoid (1 and 2).
* To whom correspondence should be addressed. Tel: (902) 566-0565. Fax: (902) 566-7445. E-mail:
[email protected]. † Department of Chemistry. ‡ Department of Biomedical Sciences.
10.1021/np8008144 CCC: $40.75
The sample of P. acerosa investigated in this study was collected at Sweetings Cay, Bahamas, and the dried coral was exhaustively extracted with CH2Cl2/CH3OH (1:1). The crude extract was partitioned between CH3OH/H2O and hexanes and then with CH2Cl2. The latter organic extract was subjected to C18 flash chromatography to afford six fractions. UPLC-MS/MS analysis of the CH3CN fraction from the C18 column showed the presence of two peaks (1 and 2) both with pseudomolecular ions of m/z 758. The MS2 fragmentation pattern for those two peaks indicated that these metabolites were related. Both compounds were purified by preparative TLC followed by semipreparative HPLC using a phenylhexyl column. The 1H NMR spectrum of both 1 and 2 confirmed that these are two closely related compounds, and 1H and 13C NMR, MS, and database searches confirmed the identification of 2 as the previously isolated bis(gorgiacerol) amine (2).4 HRMS analysis of 1 indicated a pseudomolecular ion [M + H]+ of m/z 758.3127, which corresponds to a molecular formula of C42H47NO12 and indicates 20 degrees of unsaturation. The IR spectrum showed absorptions characteristic for an unsaturated γ-lactone (1739 cm-1), conjugated ester (1721 cm-1), hydroxyl group (3464 cm-1), and alkene (3078 cm-1). The 13C NMR spectrum of 1 indicated the presence of 42 carbons including six methyl, six methylene, 14 methine, and 16 quaternary carbons on the basis of HMQC and DEPTQ experiments (Table 1). Proton resonances at δ 6.67 (1H) and 5.42 (1H) with corresponding carbon signals at δ 149.2 and 80.6, and quaternary carbons at δ 132.61 and 173.6 with long-range HMBC correlations to the signal at δ 6.67, suggested the presence of an R,γ-disubstituted R,β-unsaturated γ-lactone, illustrated in Figure 1 (partial structure A). Quaternary carbon signals at δ 131.9 and 170.7 showed HMBC correlations with a proton signal at δ 4.87, which was attached to a carbon at δ 86.0 (HMQC). The proton at δ 4.87 also exhibited an HMBC correlation with a signal at δ 62.9 (CH), while a proton at δ 5.70 showed HMBC correlations with signals at δ 170.7 and 62.9. This, together with a broad IR absorption at 1739 cm-1, suggests the presence of partial structure B. Proton signals at δH 6.34 (1H), 6.31 (1H), 3.82 (3H), and 3.85 (3H) along with carbon signals at δC 163.8 (C), 163.7 (C), 160.3 (C), 160.4 (C), 150.4 (C), 150.6 (C), 116.2 (C), 116.1 (C), 112.0 (CH), 110.1 (CH), 51.56 (CH3), and 51.64 (CH3) suggested the presence of two R,R′disubstituted β-furan moieties, as shown in substructure C. This was supported by IR and long-range HMBC correlations. A methyl singlet at δ 1.82 showed HMBC correlations with carbons at δ 114.0, 145.1, and 49.9, suggesting an isopropylene group. A similar pattern was observed with three other methyl signals at δ 1.95, 1.94, and 1.91, indicating the presence of four isopropylene groups (substructure D). These substructures were connected using 1H-1H COSY and HMBC correlations (Figure 2). The presence of three exchangeable protons was confirmed by a D2O exchange experiment. The amine
2009 American Chemical Society and American Society of Pharmacognosy Published on Web 07/02/2009
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Journal of Natural Products, 2009, Vol. 72, No. 7
Notes
Table 1. NMR Data for Bis(pseudopterane) Amine (1) in CDCl3 (300 MHz for 1H and 75 MHz for 13C) position
Ca
13
1 2
43.9, CH 31.7, CH2
3 4 5 6 7 8 9 10 11 12 13 14
160.4, qC 116.2, qC 110.1, CH 150.4, qC 48.1, CH 80.6, CH 149.2, CH 132.6, qC 61.3, CH 75.8,CH 144.3, qC 116.8, CH2
15 16 17 18
22.3, CH3 163.8, qC 139.2, qC 115.3, CH2
19 20 21 NH 12(OH) 1′ 2′
21.5, CH3 173.6, qC 51.56, CH3
3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′
160.3, qC 116.1, qC 112.0, CH 150.6, qC 48.8, CH 86.0, CH 62.9, CH 131.9, qC 140.2, CH 69.7, CH 145.1, qC 114.0, CH2
15′ 16′ 17′ 18′
18.9, CH3 163.7, qC 140.8, qC 116.0, CH2
19′ 20′ 21′ 12′(OH)
22.1, CH3 170.7, qC 51.64, CH3
49.9, CH 29.2, CH2
1
H (J in Hz)
3.13, dd (13.1, 3.6) 3.65, dd (15.5, 13.1) 2.67, dd (15.5, 3.6)
COSY 2a, 2b 2b, 1 2a, 1
6.34, s
NOESY
HMBC 13C
2b 2b 2a, 1 7
4, 6, 3 9, 6 8, 10, 20
3.78, d (4.3) 5.42, dd (4.38,
