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New potent cytotoxic lamellarin alkaloids from Indian ascidian Didemnum obscurum
Tetrahedron 61 (2005) 9242–9247
New potent cytotoxic lamellarin alkaloids from Indian ascidian Didemnum obscurum* S. Malla Reddy,a M. Srinivasulu,a N. Satyanarayana,b Anand K. Kondapib and Y. Venkateswarlua,* a
Organic Chemistry Division-I, Natural Products Laboratory, Indian Institute of Chemical Technology, Hyderabad 500 007, India b Department of Biochemistry, University of Hyderabad, Hyderabad 500 046, India Received 30 May 2005; revised 7 July 2005; accepted 21 July 2005 Available online 8 August 2005
Abstract—Chemical investigations of the ascidian Didemnum obscurum has resulted in the isolation of four new lamellarin alkaloids, lamellarin-z (1), lamellarin-h (2), lamellarin-f (3) and lamellarin-c (4) along with seven known lamellarins, lamellarin-K (5), lamellarin-I (6), lamellarin-J (7), lamellarin-K triacetate (8), lamellarin-L triacetate (9), lamellarin-F (10) and lamellarin-T diacetate (11). The structures of the compounds 1–11 were established by detailed analysis of NMR spectral data. Cytotoxic activity of the isolates has been done against coloractal cancer cells. q 2005 Elsevier Ltd. All rights reserved.
1. Introduction Ascidians (tunicates) are known to be a rich source of unique and biologically active secondary metabolites1 viz. a cyclic depsipeptide didemnin-B,2 eudistomin-C,3 the lissoclinamides,4 ascididemnin,5 eilatin and the segolins.6 The biomedical potential for ascidian secondary metabolites has resulted focused interest on these primitive chordates by several groups. The major secondary metabolites of ascidians are amino acid derived components,7 and from the titled Didemnum species tyrosine and tryptophan amino acid derived compounds were isolated.8 Lamellarins are a group of DOPA (2-amino, 3-(3 0 ,4 0 dihydroxy phenyl)propionic acid) derived pyrrole hexacyclic alkaloids, which were first isolated from a prosobranch mollusc Lamellaria species by Faulkner and his co-workers in 1985.9 Since that time a total of 38 lamellarins was isolated from different ascidisans.10 Lamellarins not only have an interesting structural feature, but also exhibit a wide array of significant biological activities; including cell division inhibition, cytotoxicity, HIV-I integrase inhibition, and immuno modulatory *
IICT Communication # 050515.
Keywords: Lamellarin alkaloids; Ascidians; Cytotoxic activity; Coloractal cancer cells. * Corresponding author. Tel.: C91 40 27193167; fax: C91 40 27160512; e-mail: [email protected] 0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tet.2005.07.067
activity.11 Lamellarin-I showed sensitizing effects to doxorubicin in multidrug resistant P388/schabelcells at concentrations as low as 0.2 mm and showed full potentiation at a concentration 10 times lower than that of the prototype MDR inhibitor verapamil.12
2. Results and discussions Previously from our group, we reported several lamellarins and their activities.10b,11b,13 Our continuous interest in isolating the bioactive secondary metabolites from ascidians (tunicates),10b,11b,13 we have collected a red colonial ascidian Didemnum obscurum, from Tiruchandur coast, Tamilnadu, India, during August-2002. The DCM–MeOH (1/1) extract of the ascidian was subjected to Sephadex LH-20 gel filtration chromatography and grouped into two fractions, Fraction-I and Fraction-II. Fraction-I was further subjected to silica-gel column chromatography, followed by reversed phase (C-18) HPLC column chromatography, using MeOH–H2O (80/20) as eluent to afford two new lamellarins, lamellarin-z (1), and lamellarin-h (2) along with four known lamellarins, lamellarin-K (5),14 lamellarin-I (6),14 lamellarin-J (7),15 and lamellarin-F (10).14 Fraction-II was acetylated (Ac2O/NaOAc) and purified sequentially on silica-gel column chromatography, and reversed phase (C-18) HPLC using MeOH–H2O (60/40) as eluent, to afford two new lamellarins as acetates, lamellarin-f triacetate (3), and lamellarin-c triacetate (4) along with three known lamellarins as acetates, lamellarin-K triacetate (8),10b lamellarin-L
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triacetate (9),14 and lamellarin-T diacetate (11).13
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signals on benzenoid rings were assigned by conventional HMQC, HMBC (Table 1) and NOESY correlations. In the 1H–1H NOESY spectrum of lamellarin-z (1), the methoxyl signals at d 3.47 (3H, s, H-29), 3.88 (3H, s, H-28), 4.10 (3H, s, H-27), and 3.42 (3H, s, H-26) showed correlations with the protons appearing at d 6.64 (1H, s, H-22), 7.14 (1H, d, JZ8.0 Hz, H-15), 7.12 (1H, d, JZ 1.5 Hz, H-12), and 6.95 (1H, s, H-10), respectively. The methoxyls at d 3.92 (3H, s, H-25), and 3.98 (3H, s, H-24) did not show any cross peaks in the NOESY spectrum. In the HMBC spectrum of lamellarin-z, the H-22 proton signal at d 6.64 (s) showed correlations with the carbons at d 129.30 (C-2), 146.33 (C-18), 143.33 (C-20), and 147.01 (C-21); the H-19 aromatic signal at d 6.98 (s) showed correlations with the carbons C-17 (d 109.75), C-21 (d 147.01), and C-20 (d 143.33). Similarly, in the trisubstituted aromatic ring the proton at d 7.14 (1H, d, JZ8.0 Hz, H-15) showed correlations with the carbons C-13 (d 149.89), and C-11 (d 128.30); the proton at d 7.18 (1H, dd, JZ8.0, 1.5 Hz, H-16) showed correlation with the carbons C-2 (d 129.30), C-1 (d 111.64), C-12 (d 114.24), and C-14 (d 149.04) and the proton at d 7.12 (1H, d, JZ1.5 Hz, H-12) showed correlation peaks with the carbons C-1 (d 111.64), C-16 (d 124.02), and C-14 (d 149.04). The proton at d 6.95 (1H, s, H-10) showed correlation peaks with the carbons C-10b (d Table 1. Spectral data for lamellarin-z (1)
Lamellarin-z (1) was isolated as an optically inactive white solid and its molecular formula was deduced to be C31H27NO9 from its HRFAB mass m/z 558.1760 and was supported by 13C NMR, indicating the presence of 31 carbons. Its UV spectrum in methanol showed absorption maxima similar to those of lamellarins possessing a D5,6 double bond,10b,11b,13 and its IR spectrum indicated the presence of phenolic and aromatic ester functionalities. Its 1 H NMR spectrum showed eight aromatic protons, of which three protons appeared as singlets at d 6.64 (1H, s, H-22), 6.95 (1H, s, H-10), and 6.98 (1H, s, H-19), three protons were ascribed to an ABX pattern of 1,3,5-trisubstituted benzene ring, at d 7.12, (1H, d, JZ1.5 Hz, H-12), 7.18, (1H, dd, JZ1.5, 8.0 Hz, H-16), and 7.14, (1H, d, JZ8.0 Hz, H15) and two ortho-coupled protons at d 7.36 (1H, d, JZ 7.6 Hz, H-6), and d 9.20 (1H, d, JZ7.6 Hz, H-5) were assigned to the isoquinoline system. Further, its 1H NMR spectrum displayed six methoxyl signals at d 3.42 (3H, s, H26), 3.47 (3H, s, H-29), 3.88 (3H, s, H-28), 3.92 (3H, s, H25), 3.98 (3H, s, H-24), and 4.10 (3H, s, H-27); one D2O exchangeable signal appeared at d 5.80 as a broad singlet. Using the foregoing spectral data and a literature survey revealed that compound 1 belongs to lamellarin type of alkaloids, viz. lamellarin-B1 and lamellarin-3,10b which possess a D5,6double bond. The positions of the methoxyl
Position
1
H NMR multiplicity (J in Hz)a
13
C-1 C-2 C-3 C-5 C-6 C-6a C-7 C-8 C-9 C-10
— — — 9.2, d, (7.6) 7.36, d, (7.6) — — — — 6.95, s
111.64 129.30 108.15 122.89 106.91 119.43 148.44 153.22 142.22 101.55
C-10a C-10b C-11 C-12 C-13 C-14 C-15 C-16
121.26 133.77 128.30 114.24 149.89 149.04 111.91 124.02
C-17 C-18 C-19 C-20 C-21 C-22
— — — 7.12, d, (1.5) — — 7.14, d, (8.0) 7.18, dd, (8.0, 1.5) — — 6.98, s — — 6.64, s
C-23 C-24 C-25 C-26 C-27 C-28 C-29
— 3.98, s 3.92, s 3.42, s 4.10, s 3.88, s 3.47, s
155.48 56.29 61.12 55.18 61.66 56.18 55.54
a
Measured in CDCl3, 600 MHz. Measured in CDCl3, 150 MHz. c Measured in CDCl3, 600 MHz. b
C NMRb
109.75 146.33 103.58 143.33 147.01 104.57
HMBC (J in Hz)c
— — — C-6a, C-10b, C-6 C-7, C-10a, C-5 — — — — C-6a, C-8, C-10b, C-9 — — — C-1, C-14, C-16 — — C-11, C-13 C-1, C-12, C-14, C-2 — — C-17, C-21, C-20 — — C-2, C-18, C-20, C-21 — C-7 C-9 C-8 C-14 C-13 C-20
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133.77), C-6a (d 119.43), C-8 (d 153.22), and C-9 (d 142.22). The isoquinoline H-6 proton at d 7.36 showed correlations with the carbons C-5 (d 122.89), C-10a (d 121.26), and C-7 (d 148.44); the other isoquinoline H-5 at d 9.2 showed correlation peaks with the carbons C-10b (d 133.77), C-6 (d 106.91), and C-6a (d 119.43). The foregoing spectral data established the structure of lamellarin-z as 1. Lamellarin-h (2) was obtained as an optically inactive white solid, and its molecular formula was deduced to be C30H25NO8 by HRFABMS m/z 528.1654 and was supported by 13C NMR indicating the presence of 30 carbons. Its UV spectrum in methanol showed absorption maxima similar to those of lamellarin-z (1). Its IR spectrum indicated the presence of phenolic and ester functionalities. The 1H NMR spectrum showed nine aromatic protons of which four appeared as singlets at d 6.68 (1H, s, H-22), 6.96 (1H, s, H-19), 7.14 (1H, s, H-10), and 7.05 (1H, s, H-7); three signals were ascribed to a 1,3,4 tri substituted benzene at d 7.13 (1H, d, JZ1.8 Hz, H-12), 7.138 (1H, d, JZ8.1 Hz, H-15), and 7.20 (1H, dd, JZ8.1, 1.8 Hz, H-16) and the two isoquinoline moiety protons at d 9.18 (1H, d, JZ7.6 Hz, H-5), and 7.00 (1H, d, JZ7.6 Hz, H-6). Further, the 1H NMR spectrum displayed five methoxyl groups at d 3.48 Table 2. Spectral data of lamellarin-h (2) Position
1 H NMR multiplicity (J in Hz)a
13
C-1 C-2 C-3 C-5 C-6 C-6a C-7
— — — 9.18, d, (7.6) 7.00, d, (7.6) — 7.05, s
110.71 129.41 107.75 123.28 112.24 124.79 107.36
C-8 C-9 C-10
— — 7.14, s
149.13 150.07 111.90
C-10a C-10b C-11 C-12
— — — 7.13, d, (1.8)
119.02 134.29 128.31 105.22
C-13 C-14 C-15 C-16
149.01 149.87 114.38 124.11
C-17 C-18 C-19
— — 7.13, d, (8.0) 7.20, dd, (8.0, 1.8) — — 6.96, s
C-20 C-21 C-22
— — 6.68, s
143.31 146.99 104.61
C-23 C-24 C-25 C-26 C-27 C-28
— 3.96, s 3.44, s 3.87, s 3.98, s 3.48, s
155.45 55.93 55.17 56.17 56.27 55.53
a
Measured inCDCl3 600 MHz. Measured in CDCl3, 150 MHz. c Measured in CDCl3, 600 MHz. b
C NMRb
109.78 146.31 103.53
HMBC (J in Hz)c
— — — C-6, C-6a, C-10b C-7, C-10a, C-5 — C-6, C-10a, C-9, C-8 — — C-8, C-6a, C-10b, C-9 — — — C-14, C-16, C-1, C-13 — — C-13, C-11, C-16 C-1, C-15 — — C-17, C-21, C-18, C-20 — — C-2, C-20, C-21, C-18 — C-9 C-8 C-14 C-13 C-20
(3H, s, H-28), 3.98 (3H, s, H-27), 3.87 (3H, s, H-26), 3.44 (3H, s, H-25), and 3.96 (3H, s, H-24). A literature survey revealed that compound 2 is closely related to lamellarina.10b The positions of methoxyl groups were deduced by its HMQC and HMBC (Table 2) and NOESY correlations. In its NOESY spectrum, the methoxyls at d 3.48 (3H, s, H-28), 3.98 (3H, s, H-27), 3.87 (H-26), and 3.44 (H-25) showed correlations with the protons at d 6.68 (1H, s, H-22), 7.138 (1H, d, JZ8.1 Hz, H-15), 7.13 (1H, d, JZ1.8 Hz, H-12), and 7.14 (1H, s, H-10), respectively. The methoxyl at d 3.96 (3H, s, H-24) showed correlation with the proton at d 7.05 (1H, s, H-7), which in turn showed correlation with the proton at d 7.00 (1H, d, JZ7.6 Hz, H-6). The HMBC spectrum of lamellarin-h, revealed that the H-22 aromatic proton at d 6.68 (1H, s) showed correlations with carbons C-2 (d 129.41), C-17 (d 109.78), and C-20 (d 143.31). The H-19 aromatic signal at d 6.96 (1H, s) showed correlations with carbons C-17 (d 109.78), C-21 (d 146.99), C-18 (d 146.31), and C-20 (d 143.31). The H-12 proton of trisubstituted benzene ring at d 7.13 (1H, d, JZ1.8 Hz) showed correlations with the carbons C-1 (d 110.71), C-16 (d 124.11), C-14 (d 149.87), and C-13 (d 149.01); the H-15 aromatic proton at d 7.138 (1H, d, JZ8.1 Hz, H-15) showed correlations with the carbons C-13 (d 149.01), C-11 (d 128.31), and C-16 (d 124.11); and the proton H-16 at d 7.20 (1H, dd, JZ8.1, 1.8 Hz, H-16) showed correlations with the carbons C-1 (d 110.71), and C-15 (d 114.38). The H-10 aromatic signal at d 7.14 showed correlations with the carbons C-10b (d 134.29), C-6a (d 124.79), C-8 (d 149.13), and C-9 (d 150.07); the aromatic H-7 proton appeared at d 7.05 (1H, s) showed correlation with the carbons C-9 (d 150.07), C-8 (d 149.13), C-10a (d 119.02), and C-6 (d 112.24). One of the isoquinoline protons appeared at d 7.00 (1H, d, JZ7.6 Hz, H-6) and showed correlation with the carbons C-7 (d 107.36), C-10a (d 119.02), and C-5 (d 123.28); while the other proton at d 9.18 (1H, d, JZ7.6 Hz) showed correlation with the carbons C-6 (d 112.24), C-6a (d 124.79), and C-10b (d 134.29). Thus, the structure of lamellarin-h was established as 2. Lamellarin-f (3) was obtained as an optically inactive white solid, and the molecular formula was established as C35H29NO12, by HRFABMS, m/z 656.1762. Its UV spectrum is similar to those of lamellarin-z and lamellarin-h. Its 1H NMR spectrum displayed eight aromatic protons, of which three appeared at d 6.80 (1H, s, H-22), 7.15 (1H, s, H-10) and 7.1 (1H, s, H-19); three protons were attributed to the pendant 1,3,4-trisubstituted benzene at d 7.18 (1H, d, JZ1.9 Hz, H-12), 7.23 (1H, dd, JZ8.0, 1.9 Hz, H-16), d 7.28 (1H, d, JZ8.0 Hz, H-15) and the remaining two were attributed to isoquinoline system at d 9.25 (1H, d, JZ7.5 Hz, H-5) and d 7.08 (1H, d, JZ7.5 Hz, H-6). Further, its 1H NMR spectrum displayed signals for three acetyl groups at d 2.32 (3H, s), 2.38 (3H, s), and 2.48 (3H, s) and four methoxyl groups at d 3.45 (3H, s, H-31), 3.89 (3H, s, H-25), 3.488 (3H, s, H-24) and 3.82 (3H, s, H-28). A literature survey and the foregoing spectral data revealed that compound 3 is closely related to lamellarin-M14 where 7-hydroxy and 9-methoxy of lamellarin-M were interchanged in compound 3.
S. Malla Reddy et al. / Tetrahedron 61 (2005) 9242–9247
The positions of the methoxyl groups and thereby structure of lamellarin-f was determined from the study of its NOESY spectrum. In its NOESY spectrum, the methoxyl at d 3.45 (3H, s, 27-H), showed correlation with the proton at d 6.80 (1H, s, H-22), which infers presence of a methoxyl at C-21. In the lamellarin group of alkaloids the H-10 and H-22 protons will appear upfield compared with the rest of the aromatic protons due to the shielding effect of the pendant benzene ring attached at C-1.9 The methoxyl protons at d 3.48 (3H, s, H-24) showed a correlation with the proton at d 7.08 (1H, d, JZ7.5 Hz, H-6), which in turn showed linear correlation (1H–1H COSY) with the proton at d 9.24 (1H, d, JZ7.5 Hz, H-5). Further, the methoxyl protons at d 3.82 (3H, s, H-28) showed correlation with the proton appeared at d 7.18 (1H, d, JZ1.9 Hz, H-12). From the foregoing spectral data the structure of lamellarin-f was established as 3. Lamellarin-c (4) was obtained as an optically inactive white solid, and its molecular formula was deduced as C34H29NO11 by HRFABMS. The 1H NMR spectrum of lamellarin-c showed seven aromatic signals out of which four appeared as singlets at d 6.70 (1H, s, H-22), 6.80 (1H, s, H-10), 6.95 (1H, s, H-19), and 7.09 (1H, s, H-7); and the remaining three aromatic protons were ascribed to the pendant 1,3,4 tri substituted benzene ring at d 7.10 (1H, d, JZ2.0 Hz, H-12), 7.14 (1H, dd, JZ8.0, 2.0 Hz, H-16), and 7.21 (1H, d, JZ8.0 Hz, H-15). Further, its 1H NMR spectrum displayed three methoxyl groups at d 3.42 (3H, s, H-26), 3.81 (3H, s, H-27), and 3.33 (3H, s, H-30); three acetyl groups at d 2.34 (3H, s), 2.30 (3H, s), and 2.28 (3H, s); and two linearly coupled methylene groups at d 3.12 (2H, t, JZ6.3 Hz, H-6), d 4.77 (1H, quintet, JZ13.2, 6.3 Hz, H-5a), and d 4.88 (1H, quintet, JZ13.2, 6.3 Hz, H-5b). The foregoing spectral data and a literature survey revealed that compound 4 is closely related to lamellarin-L14 except for the hydroxyl and methoxy substitution being interchanged on the pendant benzene ring attached at C-1. The disposition of the methoxyl groups was deduced from the study of its NOESY spectral analysis. In its NOESY spectrum the methoxyl group at d 3.33 (3H, s, H-30) showed correlation with the proton at d 6.70 (1H, s, H-22), the methoxyl group at d 3.42 (3H, s, H-26) showed correlation with the proton at d 6.80 (1H, s, H-10) and the remaining methoxyl group at d 3.81 (3H, s, H-27) showed correlation with the aromatic proton at d 7.10 (1H, d, JZ2 Hz, H-12), which belongs to the C-1 substituted aromatic ring. Hence, the structure of the lamellarin-c was established as 4. As it was well demonstrated that lamellarin alkaloids exhibit cytotoxic activity, the isolates (1–4, 6–11) were tested in vitro for their cytotoxic activity against coloractal cancer cells (COLO-205) and the IC50 values are given in Table 3. Compounds 1, 4, 9 and 10 have shown excellent activity against test cancer cell lines. Further work is in progress.
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3. Experimental 3.1. General experimental procedures Melting points were obtained on a Mel-Temp apparatus and are uncorrected. The optical rotations were measured on a JASCO DIP-370 polarimeter. UV and IR spectra were recorded on Shimadzu-240 and Perkin-Elmer 240-C instruments, respectively. The 1H and 13C NMR was recorded on 600 MHz (Inova), 500 MHz (Bruker) instruments using TMS as internal standard. Chemical shifts are reported in d (ppm) and coupling constants (J) are expressed in hertz. The MS were recorded on a VG Auto Spec-M instrument. Preparative scale HPLC was performed using a Supelcosil C18 column (60A8, 12 mm, 25 cm!21.2 mm). Animal material. The ascidian D. obscurum F. Monnit, 1969 (Didemnidae) was collected from the Tiruchandur coast in the Gulf of Mannar, Tamilnadu, India, during August-2002. A voucher specimen (IIC-484) was deposited at the National Institute of Oceanography, Goa, India. Extraction and isolation. The freshly collected ascidian specimens (2.5 kg wet weight) were soaked in methanol at the site of collection until workup. The initial methanol extract was decanted and the ascidian material was extracted with 1:1 dichloromethane/methanol (3!3 L) at room temperature. The combined extract including initial methanol extract was filtered, and the solvent was removed under reduced pressure to give predominantly an aqueous suspension, and it was extracted into ethyl acetate (3! 0.5 L), and was concentrated under reduced pressure to give a dark brown gummy mass (6.5 g). This crude extract was subjected to gel filtration chromatography (Sephadex LH20, 1:1 CH2Cl2/MeOH, 35 mm!950 mm) by collecting a total of 25 continuous fractions (30 ml each). Following the TLC pattern, the 25 fractions were pooled into two fractions, Fraction-I and Fraction-II. Fraction-I was then subjected to silica-gel column chromatography, followed by reversed phase (C-18) HPLC column (Methanol/H2O, 80: 20) at a flow rate of 5 ml per min to afford two new lamellarins, lamellarin-z (1), and Lamellarin-h (2) along with four known lamellarins, lamellarin-K (5),14 lamellarinI (6),14 lamellarin-J (7),15 and lamellarin-F (10).14 FractionII was then acetylated (Ac2O/NaOAc) and then subjected to silica-gel column chromatography, followed by reversed phase (C-18) HPLC using MeOH–H2O (60/40) as eluent, to afford two new lamellarins as its acetates, lamellarin-f triacetate (3), and lamellarin-c triacetate (4) along with three known lamellarins as acetates, lamellarin-K triacetate (8),10b lamellarin-L triacetate (9),14 and lamellarin-T diacetate (11).13 3.2. Biological assay 0.2 million cells in complete medium were seeded into a micro well plate. The cells were incubated in presence of
Table 3. IC50 values of lamellarins against COLO-205 cell lines Compound
1
2
3
4
6
7
8
9
10
11
IC50 (mM)
0.0056
0.178
0.056
0.0002
0.025
0.05
0.7
0.00025
0.009
0.08
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increasing concentrations of test compounds at 37 8C in a CO2 incubator for 16 h. After 16 h the cells were centrifuged at 800!g for 10 min and the supernatant was discarded. The cells were re-suspended in 200 ml complete medium with 20 ml of 5 mg/ml of 3-(4,5-dimethylthiozol-3yl)-2,5-diphenyl tetrazolium bromide (MTT) and incubated for 4 h at 37 8C in a CO2 incubator. The cells were centrifuged at 800!g for 10 min, 100 ml of supernatant was discarded. The insoluble crystals formed due to the reduction of MTT by viable cells were dissolved in 0.1 M acidic isopropanol and quantified in a micro plate reader at 570 nm.16 The percentage of inhibition of cell viability was computed with reference to the MTT reduction in control cells without test compound. The experimental measurements were made in triplicate and the average value was taken as percentage inhibition. The concentration of test compound required for 50% inhibition of cell viability (IC50) was determined. 3.2.1. Lamellarin-z (1). White solid (4 mg), mp 268– 272 8C, IR (KBr): nmax: 3444.8, 1637, 1425, 1219, 772 cmK1. UV (MeOH): l max (log 3): 230.0 (0.3626), 404.5 (0.0226) nm. 1H NMR (600 MHz, CDCl3): d 9.20 (1H, d, JZ7.6 Hz, H-5), 7.36 (1H, d, JZ7.6 Hz, H-6), 7.18 (1H, dd, JZ8.0, 1.5 Hz, H-16), 7.14 (1H, d, JZ8.0 Hz, H-15), 7.12 (1H, JZ1.5 Hz, H-12), 6.98 (1H, s, H-19), 6.95 (1H, s, H-10), 6.64 (1H, s, H-22), 4.10 (3H, s, H-27), 3.98 (3H, s, H-24), 3.92 (3H, s, H-25), 3.88 (3H, s, H-28), 3.47 (3H, s, H-29), 3.42 (3H, s, H-26). 13C NMR: (150 MHz, CDCl3): see Table 1. FABMS obsd m/z (%) 558 (MCC1, 25), 557 (MC, 25), 464 (9), 391 (40), 329 (12), 307 (21), 279 (8), 228 (6), 176 (33), 154 (100), 136 (94), 107 (53), 69 (83), 57 (94). HRFABMS m/z: 558.1760; (calcd for C 31H 28NO9 , 558.1764; D 0.7 ppm). 3.2.2. Lamellarin-h (2). White solid (9 mg), mp 265– 269 8C, IR (KBr): nmax: 3441, 2928.3, 1669.7, 1424.1, 1266.3, 1223.9, 1049.6, 857.2, 760.2 cmK1. UV (MeOH): lmax (log 3): 236.5 (0.7618); 308 (0.5499); 392.5 (0.163), 412.0 (0.1945) nm. 1H NMR (600 MHz, CDCl3): d 9.18 (1H, d, JZ7.6 Hz, H-5), 7.20 (1H, dd, JZ8.0, 1.8 Hz, H-16), 7.14 (1H, s, H-10), 7.13 (1H, d, JZ8.0 Hz, H-15), 7.13 (1H, d, JZ1.8 Hz, H-12), 7.05 (1H, s, H-7), 7.00 (1H, d, JZ7.6 Hz, H-6), 6.96 (1H, s, H-19), 6.68 (1H, s, H-22), 5.79 (1H, br s, OH), 3.98 (3H, s, H-27), 3.96 (3H, s, H-24), 3.87 (3H, s, H-26), 3.48 (3H, s, H-28), 3.44 (3H, s, H-25). 13 C NMR: (150 MHz, CDCl3): see Table 2. FABMS obsd m/z (%) 528 (MCC1, 22), 527 (MC, 21), 467 (12), 439 (8), 391 (18), 367 (10), 176 (18), 154 (32), 137 (33), 109 (22), 81 (44), 69 (72), 55 (100). HRFABMS m/z: 528.1654; (calcd for C30H26NO8, 528.1658; D 0.7 ppm). 3.2.3. Lamellarin-f (3). White solid (4 mg), mp 276– 279 8C, IR (KBr): nmax: 1738, 1665, 1637, 1552, 772.1 cmK1. UV (MeOH) lmax (log 3): 224.5 (0.444), 298.0 (0.3169), 380 (0.0792), 399.5 (0.0983) nm. 1H NMR (500 MHz, CDCl3): d 9.25 (1H, d, JZ7.5 Hz, H-5), 7.28 (1H, d, JZ 8.0 Hz, H-15), 7.23 (1H, dd, JZ8.0, 1.9 Hz, H-16), 7.18 (1H, d, JZ1.9 Hz, H-12), 7.15 (1H, s, H-10), 7.10 (1H, s, H19), 7.08 (1H, d, JZ7.5 Hz, H-6), 6.80 (1H, s, H-22), 3.89 (3H, s, H-25), 3.82 (3H, s, H-28), 3.48 (3H, s, H-24), 3.45 (3H, s, H-31), 2.48 (3H, s), 2.38 (3H, s), 2.32 (3H, s); FABMS obsd m/z (%) 656 (MCC1, 32), 614 (8), 307 (23),
289 (16), 154 (92), 137 (100), 135 (92), 120 (19), 89 (26), 77 (32), 65 (12). HRFABMS m/z: 656.1762; (calcd for C35H30NO12, 656.1768; D 0.9 ppm.). 3.2.4. Lamellarin-c (4). White solid (5 mg), mp 164– 166 8C, IR (KBr): nmax: 1735, 1695, 1656, 1550, 1427, 1265, 1041, 858, and 758 cmK1. UV (MeOH) lmax (log 3): 208.0 (0.5547), 274.0 (0.3184), 313.0 (0.2745) nm. 1H NMR (500 MHz, CDCl3): d 7.21 (1H, d, JZ8.0 Hz, H-15), 7.14 (1H, dd, JZ8.0, 2.0 Hz, H-16), 7.10 (1H, d, JZ2.0 Hz, H-12), 7.09 (1H, s, H-7), 6.95 (1H, s, H-19), 6.80 (1H, s, H-10), 6.70 (1H, s, H-22), 4.88 (1H, quintet, JZ13.2, 6.3 Hz, H-5b), 4.77 (1H, quintet, JZ13.2, 6.3 Hz, H-5a), 3.81 (3H, s, H-27), 3.42 (3H, s, H-26), 3.33 (3H, s, H-30), 3.12 (2H, t, JZ6.3 Hz, H-6), 2.34 (3H, s), 2.30 (3H, s), 2.28 (3H, s); FABMS obsd m/z (%) 628 (MCC1, 6), 586 (5), 544 (4), 340 (10), 308 (12), 290 (8), 155 (100), 138 (72), 122 (22), 108 (28), 91 (22), 81 (28), 69 (42), 55 (38). HRFABMS m/z: 628.192; (calcd for C 34 H30NO11, 628.1818; D 16 ppm).
Acknowledgements We are thankful to Dr. V. K. Meenakshi, Department of Zoology, APC Mahalaxmi College for Women, Tuticorin628 002, Tamilnadu, India, for identifying the ascidian, the Department of Ocean Development, New Delhi, India, for financial assistance, Dr. J. S. Yadav, Director IICT for his constant encouragement, and CSIR, New Delhi, India, for providing fellowships to S.M.R. and M.S. Y.V. is thankful to CSIR, India and DAAD, Germany for financial assistance.
References and notes 1. (a) Bloor, S. J.; Schmitz, F. J. J. Am. Chem. Soc. 1987, 109, 6134–6136. (b) Kobayashi, J.; Tsuda, M.; Tanabe, A.; Ishibashi, M. J. Nat. Prod. 1991, 54, 1634–1638. (c) Copp, B. R.; Ireland, C. M.; Barrows, L. R. J. Org. Chem. 1991, 56, 4596–4597. 2. Rinehat, K. L., Jr.; Gloer, J. B.; Renis, H. E.; McGovern, J. P.; Swynenberg, E. G.; Stringfellow, D. A.; Kuentzel, S. L.; Li, L. H. Science 1981, 212, 933–935. 3. Rinehart, K. L., Jr.; Kobayashi, J.; Harbour, G. C.; Hughes, R. G., Jr.; Mizsak, S. A.; Scahill, T. A. J. Am. Chem. Soc. 1984, 106, 1524–1526. 4. Schmitz, F. J.; Ksebati, M. B.; Chang, J. S.; Wang, J. H.; Hossain, M. B.; Vander Helm, D. J. Org. Chem. 1989, 54, 3463–3472. 5. Kobayashi, J.; Chang, J.; Walchli, M. R.; Nakamura, H.; Yoshimasa, H.; Takuma, S.; Ohizumi, T. J. Org. Chem. 1988, 53, 1800–1804. 6. Rudi, A.; Kashman, Y. J. Org. Chem. 1989, 54, 5331–5337. 7. Davidson, B. S. Chem. Rev. 1994, 93, 1771–1791. 8. Faulkner, D. J. Nat. Prod. Rep. 2001, 18, 1–49. 9. Andersen, R. J.; Faulkner, D. J.; He, C. H.; Van Duyne, G. D.; Clardy, J. J. Am. Chem. Soc. 1985, 107, 5492–5495. 10. (a) Ploypradith, P.; Mahidol, C.; Sahakitpichan, P.; Wongbundit, S.; Ruchirawat, S. Angew. Chem., Int. Ed. 2004, 43,
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866–868. (b) Krishnaiah, P.; Reddy, V. L. N.; Venkataramana, G.; Ravinder, K.; Srinivasulu, M.; Raju, T. V.; Ravikumar, K.; Chandrashekhar, D.; Ramakrishna, S.; Venkateswarlu, Y. J. Nat. Prod. 2004, 67, 1168–1171. 11. (a) Urban, S.; Hickford, S. J. H.; Blunt, J. W.; Murno, M. H. G. Curr. Org. Chem. 2000, 4, 765–807. (b) Venkata Rami Reddy, M.; Rama Rao, M.; Rhodes, D.; Hansen, M. S. T.; Rubins, K.; Bushman, F. D.; Venkateswarlu, Y.; Faulkner, D. J. J. Med. Chem. 1999, 42, 1901–1907. 12. Quesada, A. R.; Garcia Gravalos, M. D.; Fernandez Puentes, J. L. Br. J. Cancer 1996, 74, 677–682.
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13. Venkata Rami Reddy, M.; Faulkner, D. J.; Venkateswarlu, Y.; Rama Rao, M. Tetrahedron 1997, 53, 3457–3466. 14. Carrol, A. R.; Bowden, B. F.; Coll, J. C. Aust. J. Chem. 1993, 46, 489–501. 15. Davis, R. A.; Carrol, A. R.; Pierens, G. K.; Quinn, R. J. J. Nat. Prod. 1999, 62, 419–424. 16. (a) Mosmann, T. J. Immunol. Methods 1983, 65, 55–63. (b) Cole, S. P. C. Cancer Chemother. Pharmacol. 1986, 17, 259–263.
New potent cytotoxic lamellarin alkaloids from Indian ascidian Didemnum obscurum* S. Malla Reddy,a M. Srinivasulu,a N. Satyanarayana,b Anand K. Kondapib and Y. Venkateswarlua,* a
Organic Chemistry Division-I, Natural Products Laboratory, Indian Institute of Chemical Technology, Hyderabad 500 007, India b Department of Biochemistry, University of Hyderabad, Hyderabad 500 046, India Received 30 May 2005; revised 7 July 2005; accepted 21 July 2005 Available online 8 August 2005
Abstract—Chemical investigations of the ascidian Didemnum obscurum has resulted in the isolation of four new lamellarin alkaloids, lamellarin-z (1), lamellarin-h (2), lamellarin-f (3) and lamellarin-c (4) along with seven known lamellarins, lamellarin-K (5), lamellarin-I (6), lamellarin-J (7), lamellarin-K triacetate (8), lamellarin-L triacetate (9), lamellarin-F (10) and lamellarin-T diacetate (11). The structures of the compounds 1–11 were established by detailed analysis of NMR spectral data. Cytotoxic activity of the isolates has been done against coloractal cancer cells. q 2005 Elsevier Ltd. All rights reserved.
1. Introduction Ascidians (tunicates) are known to be a rich source of unique and biologically active secondary metabolites1 viz. a cyclic depsipeptide didemnin-B,2 eudistomin-C,3 the lissoclinamides,4 ascididemnin,5 eilatin and the segolins.6 The biomedical potential for ascidian secondary metabolites has resulted focused interest on these primitive chordates by several groups. The major secondary metabolites of ascidians are amino acid derived components,7 and from the titled Didemnum species tyrosine and tryptophan amino acid derived compounds were isolated.8 Lamellarins are a group of DOPA (2-amino, 3-(3 0 ,4 0 dihydroxy phenyl)propionic acid) derived pyrrole hexacyclic alkaloids, which were first isolated from a prosobranch mollusc Lamellaria species by Faulkner and his co-workers in 1985.9 Since that time a total of 38 lamellarins was isolated from different ascidisans.10 Lamellarins not only have an interesting structural feature, but also exhibit a wide array of significant biological activities; including cell division inhibition, cytotoxicity, HIV-I integrase inhibition, and immuno modulatory *
IICT Communication # 050515.
Keywords: Lamellarin alkaloids; Ascidians; Cytotoxic activity; Coloractal cancer cells. * Corresponding author. Tel.: C91 40 27193167; fax: C91 40 27160512; e-mail: [email protected] 0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tet.2005.07.067
activity.11 Lamellarin-I showed sensitizing effects to doxorubicin in multidrug resistant P388/schabelcells at concentrations as low as 0.2 mm and showed full potentiation at a concentration 10 times lower than that of the prototype MDR inhibitor verapamil.12
2. Results and discussions Previously from our group, we reported several lamellarins and their activities.10b,11b,13 Our continuous interest in isolating the bioactive secondary metabolites from ascidians (tunicates),10b,11b,13 we have collected a red colonial ascidian Didemnum obscurum, from Tiruchandur coast, Tamilnadu, India, during August-2002. The DCM–MeOH (1/1) extract of the ascidian was subjected to Sephadex LH-20 gel filtration chromatography and grouped into two fractions, Fraction-I and Fraction-II. Fraction-I was further subjected to silica-gel column chromatography, followed by reversed phase (C-18) HPLC column chromatography, using MeOH–H2O (80/20) as eluent to afford two new lamellarins, lamellarin-z (1), and lamellarin-h (2) along with four known lamellarins, lamellarin-K (5),14 lamellarin-I (6),14 lamellarin-J (7),15 and lamellarin-F (10).14 Fraction-II was acetylated (Ac2O/NaOAc) and purified sequentially on silica-gel column chromatography, and reversed phase (C-18) HPLC using MeOH–H2O (60/40) as eluent, to afford two new lamellarins as acetates, lamellarin-f triacetate (3), and lamellarin-c triacetate (4) along with three known lamellarins as acetates, lamellarin-K triacetate (8),10b lamellarin-L
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triacetate (9),14 and lamellarin-T diacetate (11).13
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signals on benzenoid rings were assigned by conventional HMQC, HMBC (Table 1) and NOESY correlations. In the 1H–1H NOESY spectrum of lamellarin-z (1), the methoxyl signals at d 3.47 (3H, s, H-29), 3.88 (3H, s, H-28), 4.10 (3H, s, H-27), and 3.42 (3H, s, H-26) showed correlations with the protons appearing at d 6.64 (1H, s, H-22), 7.14 (1H, d, JZ8.0 Hz, H-15), 7.12 (1H, d, JZ 1.5 Hz, H-12), and 6.95 (1H, s, H-10), respectively. The methoxyls at d 3.92 (3H, s, H-25), and 3.98 (3H, s, H-24) did not show any cross peaks in the NOESY spectrum. In the HMBC spectrum of lamellarin-z, the H-22 proton signal at d 6.64 (s) showed correlations with the carbons at d 129.30 (C-2), 146.33 (C-18), 143.33 (C-20), and 147.01 (C-21); the H-19 aromatic signal at d 6.98 (s) showed correlations with the carbons C-17 (d 109.75), C-21 (d 147.01), and C-20 (d 143.33). Similarly, in the trisubstituted aromatic ring the proton at d 7.14 (1H, d, JZ8.0 Hz, H-15) showed correlations with the carbons C-13 (d 149.89), and C-11 (d 128.30); the proton at d 7.18 (1H, dd, JZ8.0, 1.5 Hz, H-16) showed correlation with the carbons C-2 (d 129.30), C-1 (d 111.64), C-12 (d 114.24), and C-14 (d 149.04) and the proton at d 7.12 (1H, d, JZ1.5 Hz, H-12) showed correlation peaks with the carbons C-1 (d 111.64), C-16 (d 124.02), and C-14 (d 149.04). The proton at d 6.95 (1H, s, H-10) showed correlation peaks with the carbons C-10b (d Table 1. Spectral data for lamellarin-z (1)
Lamellarin-z (1) was isolated as an optically inactive white solid and its molecular formula was deduced to be C31H27NO9 from its HRFAB mass m/z 558.1760 and was supported by 13C NMR, indicating the presence of 31 carbons. Its UV spectrum in methanol showed absorption maxima similar to those of lamellarins possessing a D5,6 double bond,10b,11b,13 and its IR spectrum indicated the presence of phenolic and aromatic ester functionalities. Its 1 H NMR spectrum showed eight aromatic protons, of which three protons appeared as singlets at d 6.64 (1H, s, H-22), 6.95 (1H, s, H-10), and 6.98 (1H, s, H-19), three protons were ascribed to an ABX pattern of 1,3,5-trisubstituted benzene ring, at d 7.12, (1H, d, JZ1.5 Hz, H-12), 7.18, (1H, dd, JZ1.5, 8.0 Hz, H-16), and 7.14, (1H, d, JZ8.0 Hz, H15) and two ortho-coupled protons at d 7.36 (1H, d, JZ 7.6 Hz, H-6), and d 9.20 (1H, d, JZ7.6 Hz, H-5) were assigned to the isoquinoline system. Further, its 1H NMR spectrum displayed six methoxyl signals at d 3.42 (3H, s, H26), 3.47 (3H, s, H-29), 3.88 (3H, s, H-28), 3.92 (3H, s, H25), 3.98 (3H, s, H-24), and 4.10 (3H, s, H-27); one D2O exchangeable signal appeared at d 5.80 as a broad singlet. Using the foregoing spectral data and a literature survey revealed that compound 1 belongs to lamellarin type of alkaloids, viz. lamellarin-B1 and lamellarin-3,10b which possess a D5,6double bond. The positions of the methoxyl
Position
1
H NMR multiplicity (J in Hz)a
13
C-1 C-2 C-3 C-5 C-6 C-6a C-7 C-8 C-9 C-10
— — — 9.2, d, (7.6) 7.36, d, (7.6) — — — — 6.95, s
111.64 129.30 108.15 122.89 106.91 119.43 148.44 153.22 142.22 101.55
C-10a C-10b C-11 C-12 C-13 C-14 C-15 C-16
121.26 133.77 128.30 114.24 149.89 149.04 111.91 124.02
C-17 C-18 C-19 C-20 C-21 C-22
— — — 7.12, d, (1.5) — — 7.14, d, (8.0) 7.18, dd, (8.0, 1.5) — — 6.98, s — — 6.64, s
C-23 C-24 C-25 C-26 C-27 C-28 C-29
— 3.98, s 3.92, s 3.42, s 4.10, s 3.88, s 3.47, s
155.48 56.29 61.12 55.18 61.66 56.18 55.54
a
Measured in CDCl3, 600 MHz. Measured in CDCl3, 150 MHz. c Measured in CDCl3, 600 MHz. b
C NMRb
109.75 146.33 103.58 143.33 147.01 104.57
HMBC (J in Hz)c
— — — C-6a, C-10b, C-6 C-7, C-10a, C-5 — — — — C-6a, C-8, C-10b, C-9 — — — C-1, C-14, C-16 — — C-11, C-13 C-1, C-12, C-14, C-2 — — C-17, C-21, C-20 — — C-2, C-18, C-20, C-21 — C-7 C-9 C-8 C-14 C-13 C-20
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133.77), C-6a (d 119.43), C-8 (d 153.22), and C-9 (d 142.22). The isoquinoline H-6 proton at d 7.36 showed correlations with the carbons C-5 (d 122.89), C-10a (d 121.26), and C-7 (d 148.44); the other isoquinoline H-5 at d 9.2 showed correlation peaks with the carbons C-10b (d 133.77), C-6 (d 106.91), and C-6a (d 119.43). The foregoing spectral data established the structure of lamellarin-z as 1. Lamellarin-h (2) was obtained as an optically inactive white solid, and its molecular formula was deduced to be C30H25NO8 by HRFABMS m/z 528.1654 and was supported by 13C NMR indicating the presence of 30 carbons. Its UV spectrum in methanol showed absorption maxima similar to those of lamellarin-z (1). Its IR spectrum indicated the presence of phenolic and ester functionalities. The 1H NMR spectrum showed nine aromatic protons of which four appeared as singlets at d 6.68 (1H, s, H-22), 6.96 (1H, s, H-19), 7.14 (1H, s, H-10), and 7.05 (1H, s, H-7); three signals were ascribed to a 1,3,4 tri substituted benzene at d 7.13 (1H, d, JZ1.8 Hz, H-12), 7.138 (1H, d, JZ8.1 Hz, H-15), and 7.20 (1H, dd, JZ8.1, 1.8 Hz, H-16) and the two isoquinoline moiety protons at d 9.18 (1H, d, JZ7.6 Hz, H-5), and 7.00 (1H, d, JZ7.6 Hz, H-6). Further, the 1H NMR spectrum displayed five methoxyl groups at d 3.48 Table 2. Spectral data of lamellarin-h (2) Position
1 H NMR multiplicity (J in Hz)a
13
C-1 C-2 C-3 C-5 C-6 C-6a C-7
— — — 9.18, d, (7.6) 7.00, d, (7.6) — 7.05, s
110.71 129.41 107.75 123.28 112.24 124.79 107.36
C-8 C-9 C-10
— — 7.14, s
149.13 150.07 111.90
C-10a C-10b C-11 C-12
— — — 7.13, d, (1.8)
119.02 134.29 128.31 105.22
C-13 C-14 C-15 C-16
149.01 149.87 114.38 124.11
C-17 C-18 C-19
— — 7.13, d, (8.0) 7.20, dd, (8.0, 1.8) — — 6.96, s
C-20 C-21 C-22
— — 6.68, s
143.31 146.99 104.61
C-23 C-24 C-25 C-26 C-27 C-28
— 3.96, s 3.44, s 3.87, s 3.98, s 3.48, s
155.45 55.93 55.17 56.17 56.27 55.53
a
Measured inCDCl3 600 MHz. Measured in CDCl3, 150 MHz. c Measured in CDCl3, 600 MHz. b
C NMRb
109.78 146.31 103.53
HMBC (J in Hz)c
— — — C-6, C-6a, C-10b C-7, C-10a, C-5 — C-6, C-10a, C-9, C-8 — — C-8, C-6a, C-10b, C-9 — — — C-14, C-16, C-1, C-13 — — C-13, C-11, C-16 C-1, C-15 — — C-17, C-21, C-18, C-20 — — C-2, C-20, C-21, C-18 — C-9 C-8 C-14 C-13 C-20
(3H, s, H-28), 3.98 (3H, s, H-27), 3.87 (3H, s, H-26), 3.44 (3H, s, H-25), and 3.96 (3H, s, H-24). A literature survey revealed that compound 2 is closely related to lamellarina.10b The positions of methoxyl groups were deduced by its HMQC and HMBC (Table 2) and NOESY correlations. In its NOESY spectrum, the methoxyls at d 3.48 (3H, s, H-28), 3.98 (3H, s, H-27), 3.87 (H-26), and 3.44 (H-25) showed correlations with the protons at d 6.68 (1H, s, H-22), 7.138 (1H, d, JZ8.1 Hz, H-15), 7.13 (1H, d, JZ1.8 Hz, H-12), and 7.14 (1H, s, H-10), respectively. The methoxyl at d 3.96 (3H, s, H-24) showed correlation with the proton at d 7.05 (1H, s, H-7), which in turn showed correlation with the proton at d 7.00 (1H, d, JZ7.6 Hz, H-6). The HMBC spectrum of lamellarin-h, revealed that the H-22 aromatic proton at d 6.68 (1H, s) showed correlations with carbons C-2 (d 129.41), C-17 (d 109.78), and C-20 (d 143.31). The H-19 aromatic signal at d 6.96 (1H, s) showed correlations with carbons C-17 (d 109.78), C-21 (d 146.99), C-18 (d 146.31), and C-20 (d 143.31). The H-12 proton of trisubstituted benzene ring at d 7.13 (1H, d, JZ1.8 Hz) showed correlations with the carbons C-1 (d 110.71), C-16 (d 124.11), C-14 (d 149.87), and C-13 (d 149.01); the H-15 aromatic proton at d 7.138 (1H, d, JZ8.1 Hz, H-15) showed correlations with the carbons C-13 (d 149.01), C-11 (d 128.31), and C-16 (d 124.11); and the proton H-16 at d 7.20 (1H, dd, JZ8.1, 1.8 Hz, H-16) showed correlations with the carbons C-1 (d 110.71), and C-15 (d 114.38). The H-10 aromatic signal at d 7.14 showed correlations with the carbons C-10b (d 134.29), C-6a (d 124.79), C-8 (d 149.13), and C-9 (d 150.07); the aromatic H-7 proton appeared at d 7.05 (1H, s) showed correlation with the carbons C-9 (d 150.07), C-8 (d 149.13), C-10a (d 119.02), and C-6 (d 112.24). One of the isoquinoline protons appeared at d 7.00 (1H, d, JZ7.6 Hz, H-6) and showed correlation with the carbons C-7 (d 107.36), C-10a (d 119.02), and C-5 (d 123.28); while the other proton at d 9.18 (1H, d, JZ7.6 Hz) showed correlation with the carbons C-6 (d 112.24), C-6a (d 124.79), and C-10b (d 134.29). Thus, the structure of lamellarin-h was established as 2. Lamellarin-f (3) was obtained as an optically inactive white solid, and the molecular formula was established as C35H29NO12, by HRFABMS, m/z 656.1762. Its UV spectrum is similar to those of lamellarin-z and lamellarin-h. Its 1H NMR spectrum displayed eight aromatic protons, of which three appeared at d 6.80 (1H, s, H-22), 7.15 (1H, s, H-10) and 7.1 (1H, s, H-19); three protons were attributed to the pendant 1,3,4-trisubstituted benzene at d 7.18 (1H, d, JZ1.9 Hz, H-12), 7.23 (1H, dd, JZ8.0, 1.9 Hz, H-16), d 7.28 (1H, d, JZ8.0 Hz, H-15) and the remaining two were attributed to isoquinoline system at d 9.25 (1H, d, JZ7.5 Hz, H-5) and d 7.08 (1H, d, JZ7.5 Hz, H-6). Further, its 1H NMR spectrum displayed signals for three acetyl groups at d 2.32 (3H, s), 2.38 (3H, s), and 2.48 (3H, s) and four methoxyl groups at d 3.45 (3H, s, H-31), 3.89 (3H, s, H-25), 3.488 (3H, s, H-24) and 3.82 (3H, s, H-28). A literature survey and the foregoing spectral data revealed that compound 3 is closely related to lamellarin-M14 where 7-hydroxy and 9-methoxy of lamellarin-M were interchanged in compound 3.
S. Malla Reddy et al. / Tetrahedron 61 (2005) 9242–9247
The positions of the methoxyl groups and thereby structure of lamellarin-f was determined from the study of its NOESY spectrum. In its NOESY spectrum, the methoxyl at d 3.45 (3H, s, 27-H), showed correlation with the proton at d 6.80 (1H, s, H-22), which infers presence of a methoxyl at C-21. In the lamellarin group of alkaloids the H-10 and H-22 protons will appear upfield compared with the rest of the aromatic protons due to the shielding effect of the pendant benzene ring attached at C-1.9 The methoxyl protons at d 3.48 (3H, s, H-24) showed a correlation with the proton at d 7.08 (1H, d, JZ7.5 Hz, H-6), which in turn showed linear correlation (1H–1H COSY) with the proton at d 9.24 (1H, d, JZ7.5 Hz, H-5). Further, the methoxyl protons at d 3.82 (3H, s, H-28) showed correlation with the proton appeared at d 7.18 (1H, d, JZ1.9 Hz, H-12). From the foregoing spectral data the structure of lamellarin-f was established as 3. Lamellarin-c (4) was obtained as an optically inactive white solid, and its molecular formula was deduced as C34H29NO11 by HRFABMS. The 1H NMR spectrum of lamellarin-c showed seven aromatic signals out of which four appeared as singlets at d 6.70 (1H, s, H-22), 6.80 (1H, s, H-10), 6.95 (1H, s, H-19), and 7.09 (1H, s, H-7); and the remaining three aromatic protons were ascribed to the pendant 1,3,4 tri substituted benzene ring at d 7.10 (1H, d, JZ2.0 Hz, H-12), 7.14 (1H, dd, JZ8.0, 2.0 Hz, H-16), and 7.21 (1H, d, JZ8.0 Hz, H-15). Further, its 1H NMR spectrum displayed three methoxyl groups at d 3.42 (3H, s, H-26), 3.81 (3H, s, H-27), and 3.33 (3H, s, H-30); three acetyl groups at d 2.34 (3H, s), 2.30 (3H, s), and 2.28 (3H, s); and two linearly coupled methylene groups at d 3.12 (2H, t, JZ6.3 Hz, H-6), d 4.77 (1H, quintet, JZ13.2, 6.3 Hz, H-5a), and d 4.88 (1H, quintet, JZ13.2, 6.3 Hz, H-5b). The foregoing spectral data and a literature survey revealed that compound 4 is closely related to lamellarin-L14 except for the hydroxyl and methoxy substitution being interchanged on the pendant benzene ring attached at C-1. The disposition of the methoxyl groups was deduced from the study of its NOESY spectral analysis. In its NOESY spectrum the methoxyl group at d 3.33 (3H, s, H-30) showed correlation with the proton at d 6.70 (1H, s, H-22), the methoxyl group at d 3.42 (3H, s, H-26) showed correlation with the proton at d 6.80 (1H, s, H-10) and the remaining methoxyl group at d 3.81 (3H, s, H-27) showed correlation with the aromatic proton at d 7.10 (1H, d, JZ2 Hz, H-12), which belongs to the C-1 substituted aromatic ring. Hence, the structure of the lamellarin-c was established as 4. As it was well demonstrated that lamellarin alkaloids exhibit cytotoxic activity, the isolates (1–4, 6–11) were tested in vitro for their cytotoxic activity against coloractal cancer cells (COLO-205) and the IC50 values are given in Table 3. Compounds 1, 4, 9 and 10 have shown excellent activity against test cancer cell lines. Further work is in progress.
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3. Experimental 3.1. General experimental procedures Melting points were obtained on a Mel-Temp apparatus and are uncorrected. The optical rotations were measured on a JASCO DIP-370 polarimeter. UV and IR spectra were recorded on Shimadzu-240 and Perkin-Elmer 240-C instruments, respectively. The 1H and 13C NMR was recorded on 600 MHz (Inova), 500 MHz (Bruker) instruments using TMS as internal standard. Chemical shifts are reported in d (ppm) and coupling constants (J) are expressed in hertz. The MS were recorded on a VG Auto Spec-M instrument. Preparative scale HPLC was performed using a Supelcosil C18 column (60A8, 12 mm, 25 cm!21.2 mm). Animal material. The ascidian D. obscurum F. Monnit, 1969 (Didemnidae) was collected from the Tiruchandur coast in the Gulf of Mannar, Tamilnadu, India, during August-2002. A voucher specimen (IIC-484) was deposited at the National Institute of Oceanography, Goa, India. Extraction and isolation. The freshly collected ascidian specimens (2.5 kg wet weight) were soaked in methanol at the site of collection until workup. The initial methanol extract was decanted and the ascidian material was extracted with 1:1 dichloromethane/methanol (3!3 L) at room temperature. The combined extract including initial methanol extract was filtered, and the solvent was removed under reduced pressure to give predominantly an aqueous suspension, and it was extracted into ethyl acetate (3! 0.5 L), and was concentrated under reduced pressure to give a dark brown gummy mass (6.5 g). This crude extract was subjected to gel filtration chromatography (Sephadex LH20, 1:1 CH2Cl2/MeOH, 35 mm!950 mm) by collecting a total of 25 continuous fractions (30 ml each). Following the TLC pattern, the 25 fractions were pooled into two fractions, Fraction-I and Fraction-II. Fraction-I was then subjected to silica-gel column chromatography, followed by reversed phase (C-18) HPLC column (Methanol/H2O, 80: 20) at a flow rate of 5 ml per min to afford two new lamellarins, lamellarin-z (1), and Lamellarin-h (2) along with four known lamellarins, lamellarin-K (5),14 lamellarinI (6),14 lamellarin-J (7),15 and lamellarin-F (10).14 FractionII was then acetylated (Ac2O/NaOAc) and then subjected to silica-gel column chromatography, followed by reversed phase (C-18) HPLC using MeOH–H2O (60/40) as eluent, to afford two new lamellarins as its acetates, lamellarin-f triacetate (3), and lamellarin-c triacetate (4) along with three known lamellarins as acetates, lamellarin-K triacetate (8),10b lamellarin-L triacetate (9),14 and lamellarin-T diacetate (11).13 3.2. Biological assay 0.2 million cells in complete medium were seeded into a micro well plate. The cells were incubated in presence of
Table 3. IC50 values of lamellarins against COLO-205 cell lines Compound
1
2
3
4
6
7
8
9
10
11
IC50 (mM)
0.0056
0.178
0.056
0.0002
0.025
0.05
0.7
0.00025
0.009
0.08
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increasing concentrations of test compounds at 37 8C in a CO2 incubator for 16 h. After 16 h the cells were centrifuged at 800!g for 10 min and the supernatant was discarded. The cells were re-suspended in 200 ml complete medium with 20 ml of 5 mg/ml of 3-(4,5-dimethylthiozol-3yl)-2,5-diphenyl tetrazolium bromide (MTT) and incubated for 4 h at 37 8C in a CO2 incubator. The cells were centrifuged at 800!g for 10 min, 100 ml of supernatant was discarded. The insoluble crystals formed due to the reduction of MTT by viable cells were dissolved in 0.1 M acidic isopropanol and quantified in a micro plate reader at 570 nm.16 The percentage of inhibition of cell viability was computed with reference to the MTT reduction in control cells without test compound. The experimental measurements were made in triplicate and the average value was taken as percentage inhibition. The concentration of test compound required for 50% inhibition of cell viability (IC50) was determined. 3.2.1. Lamellarin-z (1). White solid (4 mg), mp 268– 272 8C, IR (KBr): nmax: 3444.8, 1637, 1425, 1219, 772 cmK1. UV (MeOH): l max (log 3): 230.0 (0.3626), 404.5 (0.0226) nm. 1H NMR (600 MHz, CDCl3): d 9.20 (1H, d, JZ7.6 Hz, H-5), 7.36 (1H, d, JZ7.6 Hz, H-6), 7.18 (1H, dd, JZ8.0, 1.5 Hz, H-16), 7.14 (1H, d, JZ8.0 Hz, H-15), 7.12 (1H, JZ1.5 Hz, H-12), 6.98 (1H, s, H-19), 6.95 (1H, s, H-10), 6.64 (1H, s, H-22), 4.10 (3H, s, H-27), 3.98 (3H, s, H-24), 3.92 (3H, s, H-25), 3.88 (3H, s, H-28), 3.47 (3H, s, H-29), 3.42 (3H, s, H-26). 13C NMR: (150 MHz, CDCl3): see Table 1. FABMS obsd m/z (%) 558 (MCC1, 25), 557 (MC, 25), 464 (9), 391 (40), 329 (12), 307 (21), 279 (8), 228 (6), 176 (33), 154 (100), 136 (94), 107 (53), 69 (83), 57 (94). HRFABMS m/z: 558.1760; (calcd for C 31H 28NO9 , 558.1764; D 0.7 ppm). 3.2.2. Lamellarin-h (2). White solid (9 mg), mp 265– 269 8C, IR (KBr): nmax: 3441, 2928.3, 1669.7, 1424.1, 1266.3, 1223.9, 1049.6, 857.2, 760.2 cmK1. UV (MeOH): lmax (log 3): 236.5 (0.7618); 308 (0.5499); 392.5 (0.163), 412.0 (0.1945) nm. 1H NMR (600 MHz, CDCl3): d 9.18 (1H, d, JZ7.6 Hz, H-5), 7.20 (1H, dd, JZ8.0, 1.8 Hz, H-16), 7.14 (1H, s, H-10), 7.13 (1H, d, JZ8.0 Hz, H-15), 7.13 (1H, d, JZ1.8 Hz, H-12), 7.05 (1H, s, H-7), 7.00 (1H, d, JZ7.6 Hz, H-6), 6.96 (1H, s, H-19), 6.68 (1H, s, H-22), 5.79 (1H, br s, OH), 3.98 (3H, s, H-27), 3.96 (3H, s, H-24), 3.87 (3H, s, H-26), 3.48 (3H, s, H-28), 3.44 (3H, s, H-25). 13 C NMR: (150 MHz, CDCl3): see Table 2. FABMS obsd m/z (%) 528 (MCC1, 22), 527 (MC, 21), 467 (12), 439 (8), 391 (18), 367 (10), 176 (18), 154 (32), 137 (33), 109 (22), 81 (44), 69 (72), 55 (100). HRFABMS m/z: 528.1654; (calcd for C30H26NO8, 528.1658; D 0.7 ppm). 3.2.3. Lamellarin-f (3). White solid (4 mg), mp 276– 279 8C, IR (KBr): nmax: 1738, 1665, 1637, 1552, 772.1 cmK1. UV (MeOH) lmax (log 3): 224.5 (0.444), 298.0 (0.3169), 380 (0.0792), 399.5 (0.0983) nm. 1H NMR (500 MHz, CDCl3): d 9.25 (1H, d, JZ7.5 Hz, H-5), 7.28 (1H, d, JZ 8.0 Hz, H-15), 7.23 (1H, dd, JZ8.0, 1.9 Hz, H-16), 7.18 (1H, d, JZ1.9 Hz, H-12), 7.15 (1H, s, H-10), 7.10 (1H, s, H19), 7.08 (1H, d, JZ7.5 Hz, H-6), 6.80 (1H, s, H-22), 3.89 (3H, s, H-25), 3.82 (3H, s, H-28), 3.48 (3H, s, H-24), 3.45 (3H, s, H-31), 2.48 (3H, s), 2.38 (3H, s), 2.32 (3H, s); FABMS obsd m/z (%) 656 (MCC1, 32), 614 (8), 307 (23),
289 (16), 154 (92), 137 (100), 135 (92), 120 (19), 89 (26), 77 (32), 65 (12). HRFABMS m/z: 656.1762; (calcd for C35H30NO12, 656.1768; D 0.9 ppm.). 3.2.4. Lamellarin-c (4). White solid (5 mg), mp 164– 166 8C, IR (KBr): nmax: 1735, 1695, 1656, 1550, 1427, 1265, 1041, 858, and 758 cmK1. UV (MeOH) lmax (log 3): 208.0 (0.5547), 274.0 (0.3184), 313.0 (0.2745) nm. 1H NMR (500 MHz, CDCl3): d 7.21 (1H, d, JZ8.0 Hz, H-15), 7.14 (1H, dd, JZ8.0, 2.0 Hz, H-16), 7.10 (1H, d, JZ2.0 Hz, H-12), 7.09 (1H, s, H-7), 6.95 (1H, s, H-19), 6.80 (1H, s, H-10), 6.70 (1H, s, H-22), 4.88 (1H, quintet, JZ13.2, 6.3 Hz, H-5b), 4.77 (1H, quintet, JZ13.2, 6.3 Hz, H-5a), 3.81 (3H, s, H-27), 3.42 (3H, s, H-26), 3.33 (3H, s, H-30), 3.12 (2H, t, JZ6.3 Hz, H-6), 2.34 (3H, s), 2.30 (3H, s), 2.28 (3H, s); FABMS obsd m/z (%) 628 (MCC1, 6), 586 (5), 544 (4), 340 (10), 308 (12), 290 (8), 155 (100), 138 (72), 122 (22), 108 (28), 91 (22), 81 (28), 69 (42), 55 (38). HRFABMS m/z: 628.192; (calcd for C 34 H30NO11, 628.1818; D 16 ppm).
Acknowledgements We are thankful to Dr. V. K. Meenakshi, Department of Zoology, APC Mahalaxmi College for Women, Tuticorin628 002, Tamilnadu, India, for identifying the ascidian, the Department of Ocean Development, New Delhi, India, for financial assistance, Dr. J. S. Yadav, Director IICT for his constant encouragement, and CSIR, New Delhi, India, for providing fellowships to S.M.R. and M.S. Y.V. is thankful to CSIR, India and DAAD, Germany for financial assistance.
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