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Pambanolides A–C from the South Indian soft coral Sinularia inelegans
Accepted Manuscript Pambanolides A-C from the South Indian Soft Coral Sinularia inelegans Dr. Bhujanga Rao Chitturi, Vinay Bharadwaj Tatipamula, Chanti Babu Dokuburra, Usha Kiranmayi Mangamuri, Venugopal Raju Tuniki, Shasi Vardhan Kalivendi, Richard A. Bunce, Venkateswarlu Yenamandra PII:
S0040-4020(16)30118-1
DOI:
10.1016/j.tet.2016.02.056
Reference:
TET 27526
To appear in:
Tetrahedron
Received Date: 6 September 2015 Revised Date:
22 February 2016
Accepted Date: 25 February 2016
Please cite this article as: Chitturi BR, Tatipamula VB, Dokuburra CB, Mangamuri UK, Tuniki VR, Kalivendi SV, Bunce RA, Yenamandra V, Pambanolides A-C from the South Indian Soft Coral Sinularia inelegans, Tetrahedron (2016), doi: 10.1016/j.tet.2016.02.056. 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 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.
ACCEPTED MANUSCRIPT Graphical Abstract Pambanolides A-C from the South Indian Soft Coral Sinularia inelegans
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Bhujanga Rao Chitturi,*Vinay Bharadwaj Tatipamula, Chanti Babu Dokuburra, Usha Kiranmayi Mangamuri, Venugopal Raju Tuniki, Shasi Vardhan Kalivendi, Richard A. Bunce, Venkateswarlu Yenamandra* E-Mail: [email protected].
Please include as a footer on p. 1: †
This paper is dedicated to the memory of our late mentor, Dr. Venkateswarlu Yenamandra, who passed away suddenly on 17 July 2013.
Tetrahedron
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Tetrahedron journal homepage: www.elsevier.com
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Pambanolides A-C from the South Indian Soft Coral Sinularia inelegans Bhujanga Rao Chitturi,a* Vinay Bharadwaj Tatipamula,a Chanti Babu Dokuburra,a Usha Kiranmayi Mangamuri,a Venugopal Raju Tuniki,b Shasi Vardhan Kalivendi,c Richard A. Bunce,d Venkateswarlu Yenamandraa†* a
Natural Products Chemistry Division, Indian Institute of Chemical Technology, Hyderabad, Telangana-500007, India. Nuclear Magnetic Resonance Spectroscopy Division, Indian Institute of Chemical Technology, Hyderabad, Telangana-500007, India. c Centre for Chemical Biology, Indian Institute of Chemical Technology, Hyderabad, Telangana-500007, India. d Department of Chemistry, Oklahoma State University, 107 Physical Sciences, Stillwater, OK 74078-3071
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ABSTRACT
Article history: Received Received in revised form Accepted Available online
Five new diterpenoids, pambanolides A-C (1, 2a, 2b and 3) and 4,5-secosinulochmodin C (4) were isolated from the soft coral Sinularia inelegans along with five known compounds (5-9). The structures of all metabolites were elucidated by comprehensive analysis of spectroscopic data. A biosynthetic pathway has been proposed for the formation of pambanolides A (1) and rameswaralide (6). Seven of the isolated compounds were evaluated for cytotoxic activity against a panel of four human cancer cell lines A549, DU145, HeLa and MCF-7 and were found to have weak cytotoxicity, but promising antiproliferative properties.
1. Introduction
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Keywords: Soft coral Sinularia inelegans Pambanolides Structure elucidation Biosynthesis
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ARTICLE INFO
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The aquatic environment is one of the leading sources for producing chemical entities with diverse structural and biological properties.1 Out of 27 diverse phyla of life on planet Earth, only 17 occur on land, while all 27 are available in the ocean. Thus, there are intact treasures of biodiversity present in the sea, and there has been an immense interest in finding biologically active compounds from marine flora and fauna.2 Despite the large number of biologically active molecules being isolated from marine sources yearly, only a few of them advance to preclinical trials.3 Nevertheless, great challenges and possibilities remain in this area of research. Marine soft corals, sponges, tunicates and other microorganisms produce different chemical compounds for metabolism as well as for defense mechanisms against predators during their life cycles.4 Several of these secondary metabolites and their synthetic derivatives with potent biological activities are being used as remedies for the treatment of diseases affecting human health.2,5 Marine diterpenoids are gaining much attention as they are the major constituents of marine segregates and show vast diversity in structure and biological activity. In continuation of our work to identify bioactive secondary metabolites from marine sources,6-8 we have isolated five new compounds, named pambanolides A-C (1, 2a, 2b and 3) and 4,5-secosinulochmodin C (4), along with five known diterpenoids (5-9) from the soft coral Sinularia inelegans collected from the Gulf of Mannar on
the South Indian coast. These compounds were evaluated for their cytotoxic effects against four human cancer cell lines. Herein, we report the isolation, structural elucidation and in vitro antiproliferative activities of these compounds. 18 19 8
5
10
19
4
11
OH 2
14
O
H
O
12 13 15
2
O
13
H 4
O
12
3
17 1
9 11
H
2
18
13
17
COOMe 20
3
H
15
12
O
OH
16
1
14
8 10
11
COOMe
O
H
22
COOMe OH
OMe O
HO H
15
H
MeOOC 16
4
O
9 17
OH H
20
14
O H
19
1 15
O
H
2
O 3 OH
7
16
5 6
6
23
3
14
4
2a (5-H ); 2b (5-H )
O O H 10
H
COOMe 5
MeO
4 13
O
1
7
12
OMe 18 21 COOMe 5 O
16
O
8
H 20
17
20
19
6
H
10 1
H
7 11
9
3
O
H
21
18
22
O 8
6
7
9
21
COOMe
O H
5
O
H
H 6
O
COOMe
COOMe O
OMe
OMe O O OH
O OH
O
COOMe
O 7
COOMe
O 8
OH O 9
O
Figure 1. Structures of compounds (1 – 9) isolated from marine soft coral Sinularia inelegans
2
Tetrahedron
13-H showed strong cross peaks with C12 and proton 14-H also ACCEPTED MANUSCRIPT
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The collected specimens (ca 3 kg wet) were exhaustively extracted with methanol and dichloromethane and the combined extracts were concentrated to give a residue (94 g). This material was suspended in 125 mL water and extracted four times with 250 mL of ethyl acetate. The combined organic layers were concentrated to a crude extract (37 g), which was subjected to column chromatography on silica gel for a preliminary fractionation. Bioactive fractions were identified using an in vitro bioassay and subjected to further chromatographic processing. Final purification was achieved by reversed phase HPLC, yielding five new compounds: 1 (8 mg), 2a and 2b (11.2 mg), 3 (16 mg) and 4 (3 mg) along with five known compounds 5-9 (total ca 2 g). Pambanolide A (1) was obtained as a white solid. Its molecular formula was determined to be C21H24O7 by high resolution ESI-MS, showing a pseudomolecular ion peak at m/z 411.14036 [M+Na]+ (calcd for C21H24O7Na is 411.14197) which requires ten degrees of unsaturation. The IR spectrum showed absorption bands for the presence of hydroxyl and carbonyl groups at 3403, 1745 and 1710 cm-1. The 13C NMR and DEPT spectra resolved 21 carbon resonances, including eight sp2 carbons, two sp3 quaternary carbons and three sp3 methylene carbons. Among the eight sp2 carbons, two belonged to carbonyl groups and six belonged to three double bonds. In combination with the 1H and 13C NMRs and IR, the spectral data for compound 1 (Table 1) indicated the presence of an isopropenyl group (C15: δC 149.0; C16: δC 109.8; δH 4.80, 1H, s and 4.82, 1H, s; C17: δC 20.3, δH 1.83, 3H, s), a quaternary methyl (C19: δC 21.3, δH 1.49, 3H, s), an α,β-unsaturated methyl ester (C18: δC 165.1; C21: δC 51.6, δH 3.72, 3H, s) and a trisubstituted double bond (C13: δC 145.4, δH 6.68, 1H, dt). The molecular formula (C21H24O7) and spectral data of 1 account for the ten degrees of unsaturation, which include three double bonds and two carbonyl groups, suggesting that 1 is a pentacyclic structure (see Figure 2 for number scheme). The 1H-1H DQF-COSY spectral data revealed the presence of two individual spin systems. Among these two spin systems, one occurred as a result of the protons on linearly connected carbons from C13 to C14 to C1 to C2 and the other was due to the protons on linearly connected carbons from C9 to C10 to C11 to C7 (Figure 2). In the 1H NMR of 1, proton 6-H (δH 5.94, s) appeared as a singlet instead of a doublet and was not coupled with its neighboring methine proton 7-H. Conceivably, these two protons (6-H and 7-H) are oriented at a dihedral angle of 90o,8 and thus, do not show any correlations in the DQF-COSY spectrum.
showed a cross peak with C12. Therefore, these two spin systems (fragments a and b) were connected to each other via C12 establishing the C11-C12-C13 linkage. Carbons C12 and C13 are sp2 hybridized, and thus, constitute a double bond. Furthermore, the HMBC correlations of 1 revealed the presence of an α,βunsaturated γ-lactone ring (H/C: 10-H/20; 11-H/12, 20; 13-H/20) made up of C10 to C13 and C20 carbons in the skeleton. The total HMBC correlations of 1 restricted the pentacyclic system to be made up of an 11-membered furanobicyclic ring (H/C: 1H/14; 2-H/1, 3, 4, 14; 6-H/3, 4, 5, 7; 7-H/6, 11; 11-H/7, 12, 13; 13-H/1, 11, 12, 14) which was fused to a cyclopentane ring (H/C: 7-H/8, 10, 11; 9-H/7, 8, 10, 11; 10-H/7, 8, 9; 11-H/7, 8, 9) at the C7 and C11 carbons and with the γ-lactone at the C11 and C12 carbons. In fragment "b" (Figure 2), the only sp3 hybridized methine group proton 1-H showed an HMBC correlation with C15, which belonged to the isopropenyl group (H/C: 16-H/15, 17; 17-H/15, 16). Protons 14-H and 2-H also showed HMBC cross peaks with C15. Thus, the isopropenyl group is attached to C1 of fragment “b” (Figure 2). In the 13C NMR spectrum of 1, the chemical shift of quaternary carbon C8 (δC 92.4 ppm) was shifted downfield when compared with the 13C data of rameswaralide (6) (C8, δC 78.6 ppm),6b which suggested that the oxygen atom on the C8 quaternary carbon could be contained in a cyclic ether linkage. Additionally, the HMBC correlation cross-peaks between 6-H (δ 5.07)/C5, C7, C8 and 7-H (δ 2.64)/C6, C8 indicated the presence of a tetrahydrofuran ring with an oxygen bridge (ether linkage) between the C5 and C8 carbons. The 1H and 13C NMR chemical shift values for fragments C7 to C13, C20 and the isopropenyl group of compound 1 also seemed to correlate with similar groups present in rameswaralide (6).6b All these findings together with an exhaustive literature survey of similar NMR data9 permitted us to establish a structure for 1 (Figure 1), which incorporates an unprecedented pentacyclic (5/5/5/5/10) scaffold.
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2.1 Isolation and identification of compounds
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2. Results and Discussion
18
COOMe
COOMe
O H3C
19
OH
H3C
O
8
5
H
4
OH
6
H
O
7 H
9
H
a
b
O 14
11
10
3 2
H 1
H
H
21
12
13
O
O
15
20
O
Figure 2. Selected HMBC ( ), COSY ( correlations for pambanolide A (1)
O
) and NOESY (
17
16
)
In addition to the two spin systems (from COSY), the HMBC correlations (Table 1 and Figure 2) were consistent with a rigid structure for 1. In the HMBC spectrum, protons 11-H and
Figure 3. One predicted low energy conformation of pambanolide A (1)
In compound 1, the stereochemistry of fragments C7 to C13 and C1 closely resembled that of rameswaralide (6).6b The relative configuration of 1 was determined by NOE experimental data correlations with the support of conformational analysis (Figure 3). As shown in one of the low energy conformers of 1, clear NOE cross peaks between 7-H, 19-CH3, 9-H (δH 2.62 (Ηα)), 10-H and 11-H as well as between 7-H and 11-H and 6-H and 19-CH3 were observed in the NOEDIFF spectra. All of these NOEs were consistent with an α orientation for all of these protons, except for 1-H which did not exhibit enhancement with any of these protons in 1. The NOE cross peaks between C1H/C16-H in 1 (Figure 2) suggested that both protons were on the same face (β orientation) of the structure, as observed in rameswaralide (6). The proton attached to C6, which belongs to the pentasubstituted dihydrofuran ring, is geminal to the oxygen of this ring. Indeed, this dihydrofuran ring, with a double bond between C4 and C5, is a rigid scaffold within the molecule. Since
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Tetrahedron
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Table 1. 1D and 2D NMR data of compounds 1 and 2 (2a / 2b mixture) recorded in CDCl3
H site 1 2
Pambanolide A (1) δC mult COSY 44.5 CH 2, 14 33.3 CH2 1
HMBC 16, 17, 15, 14 1, 3, 4, 14, 15
Pambanolide B1 (2a) δH (J in Hz) δC mult 2.52 m 40.6 CH 3.29 t (13.2) 44.9 CH2 2.49 t (13.2) -205.4 qC -72.3 qC 3.90 s 86.6 CH -190.8 qC
Pambanolide B2 (2b) δH (J in Hz) δC mult 2.52 m 40.3 CH 3.29 t (13.2) 44.8 CH2 2.94 t (13.2) -205.2 qC -72.6 qC 3.86 s 87.9 CH -190.7 qC
131.9 qC 153.9 qC 45.0 CH2
--3.06 dd (8.8, 9.7) 2.72 dd (5.8, 9.7)
-----
---3, 4, 5, 7, 10
2.64 d (10.8) -2.62 d (6.5) 1.96 dd (6.5, 15.8)
59.4 CH 92.4 qC 43.0 CH2
11 -10
6, 8, 10, 11,19 -7, 8, 10, 11,19
--3.06 dd (8.8, 9.7) 2.72 dd (5.8, 9.7)
10 11
4.95 td (6.5, 9.1) 3.91 m (1.4, 9.1, 10.8)
83.0 CH 47.5 CH
9, 11 7, 10
7, 8, 20 7, 8, 12, 13,20
5.15 m 3.48 m
77.4 CH 51.7 CH
12 13
-6.68 dt (1.4, 12.0)
125.4 qC 145.4 CH
-14
-1, 11, 12,14,20
2.29 t (11.5) 3.11 m
14
3.21 ddd (7.4, 12.0, 13.6) 2.13 t (12.0) -4.82 s, 4.80 s 1.83 s -1.49 s -3.72 s --
32.3 CH2
13, 1
1, 2, 12, 13,20
149.0 qC 109.8 CH2 20.3 CH3 165.1 qC 21.3 CH3 168.5 qC 51.6 CH3 --
-17 16 -----
--1, 15, 17 1, 15, 16 -7, 8, 9 -18 --
3.10 m 1.52 m -4.79 s, 4.73 s 1.74 s -2.21 s -3.83 s 3.44 s
*The combined COSY and HMBC correlations for the mixture of 2a and 2b
(2a / 2b)* HMBC 3, 15 1, 3, 15 --3, 4, 13, 22 --
132.0 qC 154.1 qC 45.2 CH2
--10
--7, 8, 10, 11, 19
5.15 m 3.48 m
77.4 CH 51.5 CH
9, 11 12, 10
7, 9, 12, 20 6, 7, 8, 13, 20
44.1 CH 45.0 CH
2.29 t (11.5) 3.11 m
44.1 CH 45.0 CH
11, 13 12, 14
4, 7, 11, 13, 20 4
28.8 CH2
3.10 m 1.52 t (13.4) -4.79 s, 4.75 s 1.72 s -2.21 s -3.83 s 3.40 s
29.0 CH2
13, 1
2, 15
146.0 qC 110.2 CH2 20.3 CH3 168.8 qC 16.0 CH3 173.9 qC 52.8 CH3 60.7 CH3
-17 16 ------
-1, 15(2a), 17 1, 15, 16 -6, 7, 8, 9 -18 5
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7 8 9
COSY 2, 14 1 -----
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119.0 qC 103.3 qC 174.7 qC 88.5 CH
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3 4 5 6
δH (J in Hz) 3.53 m 2.93 d (12.7) 2.13 t (12.7) ---5.07 s
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146.2 qC 110.5 CH2 20.3 CH3 170.7 qC 16.0 CH3 173.9 qC 52.8 CH3 60.5 CH3
4
Tetrahedron
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α,β-unsaturated carbonyl group, along with a quaternary methyl the NOESY results suggested that C6-H hadACCEPTED an α orientation, MANUSCRIPT and one methyl ester. The 1H-1H DQF-COSY correlations of 2 the oxygen atom of the dihydrofuran could be positioned with a β revealed the presence of only one spin system consisting of eight orientation and subsequently the OH group on C3 should occupy a pseudoequatorial position in the envelope conformation of the different kinds of protons (out of 11 total) on the linearly dihydrofuran ring. The geometry of the trisubstituted double bond connected carbons C9 to C14 to C1 to C2 (see Figure 4 for number scheme). Furthermore, the HMBC correlations (Table 1) between C12 and C13 (δH 6.68) appeared to have the Econfiguration in the lowest energy conformer of 1. The energysuggested that compound 2 was a constrained structure, which minimized structure of 1 (Figure 3 and Table 1 in the SI) is contained one cyclohexanone ring, one cycloheptanone ring and virtually in agreement with the assigned structure based on NMR a cyclopentene ring together with one γ-lactone (H/C: 10-H/20; spectroscopic data. 11-H/20). The C1 methine proton 1-H (δH 2.52 m) showed an Pambanolide A (1) and rameswaralide (6) are constitutional HMBC correlation with C15 of the isopropenyl group (H/C: 16isomers. Both 1 and 6 exhibit the same molecular formula deduced H/15, 17; 17-H/15, 16). Protons 2-H and 14-H also showed from mass spectrometry (ESI, m/z = 411 [M+Na]+ ion), and the HMBC correlations with C15. The C17 methyl group (δH 1.72, s) 13 C NMR data show 21 carbon resonances. Compounds 1 and 6 also gave an HMBC cross peak with C1, hence the isopropenyl share some structural features, such as the isopropenyl group, the substituent was connected to the C1 methine center. The exocyclic α,β-unsaturated γ-lactone, the pentasubstituted molecular formula (C22H26O7) and spectral data of 2 accounted cyclopentane ring, and an α,β-unsaturated methyl ester function, for nine degrees of unsaturation including two double bonds and together with an oxygenated quaternary carbon bearing a methyl four carbonyl groups suggesting that 2 possesses a tetracyclic group. The structure of pambanolide A (1) has also been assigned (5/5/7/6) motif. A tentative structure for 2 was established by 1D on the basis of a postulated biosynthetic pathway (Scheme 1). and 2D NMR data correlations (Figure 4). According to Scheme 1, pambanolide A (1) and rameswaralide (6) could be obtained from the same precursor metabolite mandapamate (10) [Note: madapamate (10) and isomandapamate (5) are stereoisomers].
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Figure 4. Important HMBC ( ), COSY ( ) and NOESY ( correlations for pambanolide B1 (2a, 5-Hβ), pambanolide B2 (2b, 5-Hα)
Scheme 1. Proposed biosynthetic pathway for pambanolide A (1) and rameswaralide (6)
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Pambanolide B (2) was obtained as a white solid. The molecular formula C22H26O7 was deduced from the HRMS m/z 425.15685 [M+Na]+ (calcd for C22H26NaO7 is 425.15762), which required ten degrees of unsaturation. The IR spectrum showed multiple absorption bands for carbonyl groups at 1768, 1745, and 1710 cm-1. The structural determination of 2 was carried out by detailed interpretation of 1D and 2D NMR spectroscopic data. In the 13C NMR spectrum of 2, a total of 36 carbon resonances were observed. Among these 36 signals, eleven identified as pairs and twelve detected as individual peaks with increased (double) intensity. The remaining two carbons (δC 170.7 and 168.8 ppm) were found to be separate peaks with standard intensity. However, from the calculated molecular formula (C22H26O7) and 13 C NMR data assessments, it could be concluded that 2 is comprised of a mixture of two structurally similar compounds, pambanolide B1 (2a) and pambanolide B2 (2b). The 13C NMR and DEPT spectral data of 2 revealed the presence of eight sp2 carbons along with three sp3 methylenes, six sp3 methines and four methyl groups. Among the eight sp2 carbons, two belonged to ketone functions, two were part of ester carbonyl groups, and the remainder corresponded to two double bonds. Of the four methyl group signals, two appeared in the methoxy region (δC 60.5 and 52.8 ppm) of the 13C spectrum. Analysis of the 1H NMR, 13C NMR (Table 1) and IR data for 2 clearly indicated the presence of an isopropenyl group and an
)
Table 2. NOESY correlations for compounds 1, 2a and 2b recorded in CDCl3
1
H Site 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
1 NOESY 16-H ----19-CH3 11-H, 19-CH3 -10-H, 19-CH3 9-H (1.96), 11-H 10-H, 7-H -14-H (2.13) 13-H -1-H --6-H, 9-H (1.96), 19-CH3 -----
2a NOESY -------10-H 9-H (3.06), 11-H 10-H, 13-H -11-H ------
2b NOESY ----11-H, 13-H ---10-H 9-H (3.06), 5-H, 11-H 5-H, 10-H, 13-H -5-H, 11-H -----
--
--
----
---
--
--
Tetrahedron
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single crystal X-ray analysis. The ORTEP diagram for 2b is depicted in Figure 5.
Figure 5. Crystal structure of pambanolide B2 (2b). [CCDC No. 941403]
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Pambanolide C (3) was obtained as viscous, colorless liquid. The 13C NMR and HR ESI-MS spectral data of 3 afforded a molecular formula C23H30O8 consistent with a pseudomolecular ion peak at m/z 457.18179 [M+Na]+ (calcd for C23H30NaO8 is 457.18384). The IR spectrum showed the absorption bands of a hydroxy group at 3434 cm-1 and carbonyl groups at 1721 and 1714 cm-1. The 13C NMR and DEPT spectral data distinguished 23 carbon resonances, including signals for three carbonyl carbons, eight double bond carbons and four sp3 methylene carbons. The 1H and 13C NMR (Table 3) spectral data were isopropenyl group (C15: δC 145.9; C16: δC 111.4; δH 4.86, 1H, s and 4.79, 1H, s; C17: δC 20.8, δH 1.81, 3H, s), two α,βunsaturated methyl esters (C18: δC 163.9; C21: δC 51.3, δH 3.79, 3H, s; C20: δC 167.2; C22: δC 52.0, δH 3.72, 3H, s) and a quaternary methyl group (C19: δC 29.3, δH 1.35 s) (see Figure 6 for number scheme). As its molecular formula (C23H30O8) and
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The methoxy group (C22-OMe) protons [2a (δH 3.44, s) and 2b (δH 3.40, s)] on C5 appeared as two singlets in the 1H NMR, and together integrated to only three protons. However, they showed individual HSQC correlations with carbon pair signals C22 [2a (δC 60.5) and 2b (δC 60.7)], respectively. In combination with the 1H NMR, the HSQC spectrum of 2a/2b (see Figure S8, SI) clearly revealed the differences in matching the 5-H signals [2a (δH 3.90, s) and 2b (δH 3.86)] with the C5 carbon pair [2a (δC 86.6) and 2b (δC 87.9)]. A detailed examination of the 1D and 2D NMR data correlations of 2 suggested it was an inseparable mixture of two stereoisomers (2a/2b) present in a ~2:3 ratio (based on NMR integration). The relative configurations of 2 were determined by a NOESY experiment (Table 2 and Figure 4). In the NOESY spectrum, 10-H showed clear NOE correlations with 9-H (δH 3.06) and 11-H, which in turn, showed NOEs with 13-H. In addition, a clear NOE correlation was also observed between 12H and 1-H. In the NOEDIFF spectrum, proton 5-H (δH 3.86, s) in 2b showed strong NOEs with 11-H as well as 13-H, indicating an α orientation. Proton 5-H (δH 3.90) in 2a did not show NOE correlations with either 11-Hα or 13-Hα, and hence, 5-H of 2a should be in the β orientation. Indeed, the stereochemistry of 2a/2b appears to be analogous to rameswaralide (6)6b at the C1, C10 and C11 stereocenters. Consistent with this reasoning, the individual relative stereochemistries of 2a and 2b were assigned as shown in Figure 4. The structure of 2b was unambiguously established by X-ray crystal studies. Recrystallization of 2a/2b was accomplished in a methanol/water dual solvent system. Needle like crystals formed in the crystallization pot, and we were able to separate a tiny (monoclinic) crystal of 2b, which was subsequently submitted for
Table 3. 1D and 2D NMR data for compounds 3 and 4 recorded in CDCl3
H site 1 2
δH (J in Hz) 2.48 m 3.10 m --6.62 s -4.06 s --
9
2.48 d (18.0) 3.16 d (18.0) -3.58 d (8.0) --
13 14 15 16 17 18 19 20 21 22 23
-------
AC C
10 11 12
161.0 qC 114.6 qC 112.2CH 149.0 qC 83.7 CH 73.0 qC
HMBC 15, 13, 2 3, 1, 15, 4
--3, 4, 6, 7, 18 -19, 9, 23, 8,5 --
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3 4 5 6 7 8
Pambanolide C (3) COSY δC mult 44.0 CH 2, 14 30.5 CH2 1
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1
6.96 dd (3.0, 10.0) 2.26 d (8.0) 2.44 d (8.0) -4.79 s 4.86 s 1.81 s -1.35 s -3.79 s 3.72 s 3.25 s
45.9 CH2
--
19, 8, 7, 10
208.5 qC 42.0 CH2 125.2 qC
-12, 10 --
-12, 13,20 --
144.0 CH 31.4 CH2
14 13, 1
14, 11, 1, 12, 20 2, 1, 12, 13, 15
145.9 qC 111.4 CH2
-17
-17, 1, 15
20.8 CH3 163.9 qC 29.3 CH3 167.2 qC 51.3 CH3 52.0 CH3 57.7 qC
16 -------
1, 16, 15 -9, 8, 7, 6 -18 20 7
4,5-Secosinulochmodin C (4) COSY δH (J in Hz) δC mult 2.78 m 39.3 CH 2, 12 3.20 m, 30.7 CH2 1, 3 1.90 dm (12.5) 2.40 dt (3.0, 12.5) 36.2 CH 2, 11 5.20 s 79.5 CH --210.5 qC -2.70 d (10.0) 62.0 CH 10 -90.8 qC -2.55 d (15.5) 45.3 CH2 9 2.17 dd (8.5, 16.1) 5.10 t (8.2) 83.2 CH 8, 10
HMBC 2, 13, 15 1, 3, 15 5, 6, 8,11 3, 5, 6, 11 4, 5, 10 -6, 7, 9, 10 18
3.47 m 3.04 dd (4.8, 12.8) 2.66 ddd (1.5, 4.2, 15.2) 2.58 d (6.7) ---
43.6 CH 46.1 CH 42.9 CH2
6, 9, 11 3, 10 1
1, 11, 18 3, 6, 18 1, 13, 14, 15
201.2 qC 166.0 qC
---
---
-4.92 s 4.71 s 1.72 s -1.30 s 3.76 s ----
146.0 qC 113.0 CH2
-17
-1, 15, 17
22.3 CH3 175.5 qC 21.0 CH3 52.6 CH3 ----
16 -------
1, 15, 16 -6, 7, 8 14 ----
6
Tetrahedron
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The 1H-1H DQF-COSY spectral data revealed only a single linear correlation between 13-H, 14-H, 1-H and 2-H. The HMBC spectral data correlations (Table 3) established a complete structure for 3. The HMBC correlations suggested that compound 3 was a bicyclic structure, which contained a trisubstituted furan ring (H/C: 2-H/3, 4, 1; 5-H/3, 4, 6, 7; 7-H/5) and a 13-membered macrocyclic ring (H/C: 7-H/8, 9; 9-H/7, 8, 10; 11-H/12, 13; 13H/11, 12, 14, 1; 14-H/12, 13, 1, 2; 1-H/2, 13; 2-H/1, 3, 4) bridging the furan ring at C3 and C6. The C1 methine proton 1-H showed an HMBC correlation with C15, which belonged to an isopropenyl group (H/C: 16-H/15, 17; 17-H/15, 16). The protons 14-H and 2-H also showed HMBC cross peaks with C15. Thus, the isopropenyl group was located on the C1 methine carbon. In the HMBC spectrum, C23-OMe protons (δH 3.25, s) showed a cross peak with the C7 methine (δC 83.7), the 7-H showed correlations with C5, C8, C9, C19 and C23 on the 14-membered ring (Figure 6). Thus, from the foregoing data analysis and an exhaustive literature survey,8,9 the structure of 3 was assigned as shown in Figure 1. Both 3 and 8 exhibited the same molecular formula deduced from mass spectrometry (ESI, m/z 457 [M+Na]+) and each 13C NMR spectrum showed 23 carbon resonances with similar functionalities, including an isopropenyl group, two α,β-unsaturated methyl esters, one ketone, a quaternary methyl and a methoxy group.
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measurements and showed an m/z 377.15841 [M+H]+ (calcd for spectral data indicated nine degrees of unsaturation including MANUSCRIPT ACCEPTED four double bonds and three carbonyl groups, it was concluded C20H25O7 is 377.16003 [M+H]+). The NMR (1H, 13C and DEPT) spectral data (Table 4) indicated the presence of 20 carbons, that 3 had a bicyclic skeletal structure. including carbons for one isopropenyl group, one methyl ester, one quaternary methyl group, two ketones, three sp3 methylenes and seven sp3 methines. The molecular formula (C20H24O7) indicated nine degrees of unsaturation, and with one double bond and four carbonyl groups, confirmed that the structure consisted of a tetracyclic carbon framework. In the 1H-1H DQF-COSY spectrum, the presence of a single, wide spin system was noted with eight different kinds of protons (out of 11 total) on carbons C8 to C11 to C3 to C1 to C12 in a linear connection and one proton on C6 that branched off of C10 (see Figure 7 for number scheme). In the 1H-NMR spectrum, proton 4-H (δH 5.20) appeared as a singlet instead of the expected doublet from coupling with its neighboring proton 3-H (δH 2.40), suggesting that they showed a dihedral angle of 90o to each other.8 Figure 6. Selected HMBC ( ), COSY ( ) and NOESY ( ) Consequently protons 3-H and 4-H did not show any correlation correlations for pambanolide C (3) in the COSY spectral experiment.
Scheme 2. A proposed pathway for formation of pambanolide C (3) from sethukarailin (8)
The NOEDIFF spectrum for 3 showed clear NOEs from 1-H to 19-CH3, and from 19-CH3 to 7-H. Generally, 14-membered cembrane type diterpenoids9 exhibit an α isopropenyl group attached to the C1 stereocenter. In addition, 3 could represent an artifact derived from sethukarailin (8) (Scheme 2). According to the results of 2D NMR studies described here and information found in the literature, the relative stereochemistry of 3 is assigned as shown in Figure 6. 4,5-Secosinulochmodin C (4) was obtained as a white solid. It had a molecular formula of C20H24O7 according to HR ESI-MS
Figure 7. Selected HMBC ( ), COSY ( correlations for 4,5-secosinulochmodin C (4)
) and NOESY (
)
Table 4. NOESY correlations for compounds 3 and 4 recorded in CDCl3 1
H Site 1 2 3 6 7 8 9 10 11 17 18 19
Pambanolide C (3)
4,5-Secosinulochmodin C (4)
NOESY 19-CH3
NOESY 2-H (1.90) 1-H, 11-H 11-H, 10-H, 19-CH3 -9-H, 19-CH3 8-H (2.17), 10-H 9-H, 11-H, 6-H 1-H, 3-H,10-H 1-H -6-H, 8-H (2.17)
--19-CH3 -19-CH3 ----1-H, 7-H, 9-H (3.16)
The total HMBC correlations of 4 restricted the tetracyclic formulation to be made up of a cyclohexanone ring (H/C: 3-H/4, 5, 11; 4-H/3, 5, 6, 11; 6-H/4, 5, 10; 10-H/11; 11-H/3, 6), which was fused with a cyclopentane ring (H/C: 6-H/10; 8-H/6, 7, 8, 10;
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Scheme 3. A possible pathway for the formation of compound 4 from sinulochmodin C (12)
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The structures of known compounds 5-9 (Figure 1) were established by interpretation of the 1D and 2D NMR spectral data. Among these known compounds, isomandapamate (5), rameswaralide (6) and sethukarailin (8) have been previously reported from our group. 6,7 The revised and exact stereochemical structure of rameswaralide11 was confirmed for the first time by single crystal X-ray crystallographic studies (Figure 9). Water in methanol was the solvent system used for the crystallization of 6. Compounds 7 and 9 are known and have been described in an earlier report.12
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10-H/8) at the C6 and C10 carbon centers. A γ-lactone ring (H/C: 9-H/11, 18; 10-H/11, 18; 11-H/18), fused to the cyclohexanone at C10 and C11, was also observed from HMBC correlations of 4. From the HMBC correlations (H/C: H-4/C5, C6; H-6/C4, C5 and H-19/C6, C7) and the 13C chemical shift values for C4 and C7, it was obvious that, an oxygen atom had to be placed between these two carbons to form a tetrahydrofuran ring. The 1H and 13C NMR chemical shifts of the C4 methine group (δH 5.20; δC 79.5) and the downfield 13C chemical shift value for C7 (δC 90.8) provided strong evidence for a tetrahydrofuranone ring in the structure of 4. By extensive analysis of HMBC spectral data in combination with DQF-COSY spectral data correlations, it was determined that the structure of 4 appears to be the product of a redox bond cleavage at C4-C5 bond in sinulochmodin C,10 and consists of a tetracyclic (5/5/5/6) core with an eight-carbon side chain residue incorporating an α-keto ester functionality (H/C: 12-H/C13, C14) and an isopropenyl group. The 1-H showed an HMBC correlation with C15, which is part of the isopropenyl substituent (H/C: 16H/15, 17; 17-H/15, 16). Protons 2-H and 12-H also showed HMBC cross peaks with C15. Thus, C15 of the isopropenyl group is connected to the C1 methine center. From the above data analysis and information taken from the literature,9 it is clear that 4 is a tetracyclic norditerpenoid (19 carbon skeleton) bearing an additional methoxy group.
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Figure 9. The crystal structure of rameswaralide (6) [CCDC No. 933433]
2.2. Postulated Biogenetic Pathway to Compounds 1, 4 and 6
Figure 8. One energy minimized structure of compound 4
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The NOESY spectrum of 4 displayed a clear correlation (Figure 7) between H-6 and H-19. On the other hand, H-19 showed an NOE correlation with one of the methylene protons at C8 (δΗ 2.17 (Ηα)) and C8-Hα showed a clear correlation with H9, which in turn, showed the correlation with H-10. In the NOESY spectrum of 4, the NOE interaction between H-6, H-19, H-8α, H-9 and H-10 reflected the α orientation of H-6 and subsequently the β-orientation of the “C4-O-C7” ethereal bridge. Proton 1-H did not show any NOEs in the NOEDIFF spectrum with protons at the other stereocenters. Rather, the proton attached to C2 (δΗ 1.90, dm) showed NOE correlations with the 1-H and 11-H protons, indicating that they were on the same side of the structure. The stereochemistry of 4 appeared to be analogous to sinulochmodin C (12) except for the chiral center at C1. The energy-minimized structure as shown in Figure 8 (Table 2 in the SI) was also in agreement with the assigned structure of 4 from the NMR data. On the basis of the above observations and consideration of the proposed biosynthetic pathway (Scheme 3), the structure of 4 was fully established and found to possess the 1S, 5S, 7S, 8R, 10S, 11S, 12R, 13S absolute configuration at the respective stereocenters. Thus, 4,5-secosinulochmodin C (4) was assigned the norditerpenoid tetracyclic (5/5/5/6) core skeleton shown in Figure 8.
To the best of our knowledge, compound 1 is an unprecedented skeleton with an unusual pentacyclic (5/5/5/5/10) framework. A conceivable biogenetic pathway has been proposed for compounds 1 and 6 in Scheme 1. They could have a common precursor, probably mandapamate (10). Cleavage of the C6-C14 bond in mandapamate (10) or cleavage of the C5-C14 bond in rameswarlide (6) could lead to unstable intermediate (11), which could undergo a ring closing oxa-Michael reaction to afford 1. The new furanocembranoid 3 seems to be an artifact of the known compound sethukarailin (8) (Scheme 2). The previously unreported tetracyclic (5/5/5/6) norditerpenoid (4) with an eightcarbon side chain bearing an isopropenyl substituent and terminated by an α-keto ester functionality, appears to be a C4C5 redox bond cleavage product of sinulochmodin C (12). 2.3. Biological Activity In vitro cytotoxicity for seven selected compounds (1-3, 5-7 and 9) isolated from Sinularia inelegans was assayed. For these in vitro studies, we used four human cancer cell lines, which included A549 (human epithelial lung carcinoma), DU145 (human prostate cancer), HeLa (human epithelial cervical cancer) and MCF-7 (human breast adenocarcinoma).13 Each cell line was treated with four different concentrations of each compound ranging from 1 to 100 µM concentration for 48 h and the obtained IC50 values are summarized in Table 3 (SI). All these compounds showed weak cytotoxic activity towards the four
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compounds: 5 (38 mg), 6 (17 mg), 7 (130 mg), 8 (1.8 g), and 9 cancer cell lines tested. Among these compounds, 2 and 3 MANUSCRIPT ACCEPTED (12 mg). showed slightly higher activity against the DU145 and A549 cancer cell lines when compared to the other compounds tested. While compound 3 exhibited IC50 values of 41 µM and 69 µM, 3.2.1. Pambanolide A (1) was a white solid, mp 231-235 oC. compound 2 showed IC50 values of 63 µM and 55 µM against the [α]D25 –24.39 (c 0.82, CHCl3); for the 1H and 13C NMR, see Table DU145 and A549 cell lines, respectively. Among all of the 1 and Figures S1 and S2 (SI); EI-MS m/z = 411.2 [M+Na]+; HR compounds tested, cembranoid diterpene 3 showed the best ESI-MS, m/z = 411.14036 [M+Na]+ (calcd for C21H24NaO7 is cytotoxic activity across the entire panel of cell lines. Cell 411.14197); IR (neat): 3403, 1745 and 1710 cm-1; UV: 256 nm proliferation studies indicated that growth of these cell lines was (MeOH). The detailed molecular structure was deduced by slowed significantly by these same seven compounds. interpretation of the NMR spectroscopic data, which included 1H, 13 C, DEPT, HSQC, COSY, HMBC and NOESY spectra. 3. Experimental Section 3.2.2. Pambanolide B1 (2a) and Pambanolide B2 (2b), as a diastereomeric mixture, were obtained as a white solid, mp 189Commercially available chemicals were used for the isolation 196 oC. [α]D25 –9.17 (c 0.71, CHCl3); for the 1H and 13C NMR, protocols without further purification. The 1D and 2D 1H and 13C see Table 1 and Figures S6 and S7 in the SI; EI-MS m/z 425 NMR spectra of compounds were recorded on Bruker 300, [M+Na]+; HR ESI-MS m/z 425.15685 ([M+Na]+) C22H26O7 Avance 500 and Avance 600 instruments in CDCl3 solvent at 25 (calcd for C22H26NaO7 is 425.15762); IR (neat): 1768, 1745, 1710 °C; chemical shifts are reported in δ (ppm) and are referenced to cm-1; UV: 226 nm (MeOH). The detailed molecular structure was the CHCl3 solvent peak at δ 7.25 and the CDCl3 solvent peak at elucidated by interpretation of the NMR spectroscopic data, δ 77.00. FT-IR spectra for the compounds were measured as which included 1H, 13C, DEPT, HSQC, COSY, HMBC and solutions (CHCl3) or liquid films between KBr plates. Mass NOESY spectra. spectra were calculated from ESI and HR ESI-MS (Q-TOF, Agilent tech. (Mass Spectroscopy Division IICT-Hyderabad)) spectrometers. Optical rotations were measured in CHCl3 solvent. 3.2.3. Pambanolide C (2b) crystallography data: C22H26O7, M = Isolation and purification of compounds were done by silica gel 402.43, colorless block, 0.34 × 0.24 × 0.20 mm3, monoclinic, (100-200 and 230-400 mesh) column chromatography. Further space group P2 (1), a = 9.3368(13) Å, b = 10.7714(12) Å, c = purification, if necessary, was performed by reversed phase 0.5965(17) Å, V = 1017.0(16) Å3, Z = 2, Dc = 1.314 g/cm3, F000 = HPLC. Analytical TLC was carried out on pre-coated plates 428, CCD area detector, MoKα, radiation, λ = 0.71073 Å, T = (Merck, silica gel 60 PF254), and spots were visualized with UV 298(2) K, 2θmax = 50.0º, 4340 reflections collected, 1957 unique light, I2, or by charring after treatment with anisaldehyde (or (Rint = 0.0173). Final GoF = 1.036, R1 = 0.0446, wR2 = 0.0968, sulfuric acid). All chromatographic solvents were distilled prior final R indices (I > 2σ(I)), R1=0.0563, WR2 = 0.1063, R indices to use. (all data). Crystallographic data have been deposited for compound 2b with the Cambridge Crystallographic Data Centre 3.1. Collection of Soft Coral [CCDC No. 941403]. Copies of the data can be obtained free of charge at www.ccdc.cam.ac.uk/conts/retrieving.html [or from the Cambridge Crystallographic Data Centre (CCDC), 12 Union The soft coral Sinularia inelegans was collected from the sea at a depth of 20-30 m by skin diving along the Mandapam coast Road, Cambridge CB2 1EZ, UK; fax: +44(0) 1223 336 033; email: [email protected]]. (N 9° 18'; E 79° 08') in the Gulf of Mannar on the southern coast of India, during April 2011. The specimens (ca 3 kg wet) were preserved in methanol until worked up. Voucher material has 3.2.4. Pambanolide D (3) was obtained as viscous colorless oil. been deposited at the INO in Goa, India. [α]D25 –103.29 (c 0.82, CHCl3); for 1H and 13C NMR, see Table 3 and Figures S12 and S13 in the SI); HR ESI-MS m/z = 457.18179 [M+Na]+ (calcd for C23H30NaO8 is 457.18384). IR 3.2. Purification of compounds (neat): 3434, 1714 and 1666 cm-1; UV: 216 nm (MeOH). The detailed molecular structure was determined by interpretation of Immediately after collection, the wet samples were soaked in the NMR spectroscopic data, which included 1H, 13C, DEPT, methanol. After five days, the methanol was decanted and the HSQC, COSY, HMBC and NOESY spectra. sample was extracted with a 1:1 mixture of MeOH and DCM for three days. The later extraction was repeated three times. The 3.2.5. 4,5-Secosinulochmodin C (4) was a white solid, 208-214 resulting extracts were combined with the initial methanol o C. [α]D25 –27.39 (c 0.1, CHCl3); for 1H and 13C NMR, see Table preservative and concentrated under reduced pressure. 2 and Figures S17 and S18 in the SI); EI-MS m/z = 377 [M+Na]+; Subsequently, the residue (94 g) was suspended in 125 mL water HR ESI-MS (calcd for C20H25O7 is 377.16003; found 377.15841 and extracted with EtOAc (4 × 250 mL). The combined organic [M+H]+). The detailed molecular structure was deduced by layers were concentrated to afford 37 g of residue. The final interpretation of the NMR spectroscopic data, which included 1H, crude extract was directly separated by silica gel (100-200 mesh) 13 C, DEPT, HSQC, COSY, HMBC and NOESY spectra. column chromatography by using EtOAc in n-hexane (0%, 5%, 10%, 20%, 40%, 60%, 80% and 100%) and methanol in EtOAc 3.2.6. Rameswaralide (6) crystallography data: C21H24O7, M = (5%, 10% and 20%) as 24 fractions. On the basis of a preliminary bioassay and 1H-NMR spectral data, some of the 24 fractions (11, 388.40, colorless block, 0.22 × 0.17 × 0.11 mm3, orthorhombic, 12, 15, 16, 18, 19, 20, 21. 23 and 24) were purified by repeated space group P212121, a = 10.6679(10), b = 11.7688(11), c = silica gel (230-400 mesh) column chromatography and further 15.4535(15) Å, V = 1940.2(3) Å3, Z = 4, Dc = 1.330 g/cm3, F000 = purified (when necessary) by preparative HPLC using a reversed 824, CCD area detector, MoKα radiation λ = 0.71073 Å, T = phase C-18 column (5 µM particle size) eluted with 20% water in 294(2) K, 2θmax = 50.0º, 18622 reflections collected, 1957 unique acetonitrile to give five new compounds: 1 (8 mg), 2a and 2b (Rint = 0.0228). Final GoF = 1.036, R1 = 0.0285, wR2 = 0.0778, (mixture, 11.2 mg), 3 (16 mg), and 4 (3 mg); and five known R indices based on 1878 reflections with I > 2σ(I) (refinement on F2), 264 parameters, 0 restraints µ = 0.100 mm-1.
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6.
3.3. Bioassays Methods and procedures are outlined in the SI. 4. Conclusion
Corresponding Author: Dr. Chitturi Bhujanga Rao, c/o Dr. U. V. Mallavadhani, Principal Scientist, Natural Products Chemistry Division, Indian Institute of Chemical Technology, Taranaka, Hyderabad, A.P. India-500007. Corresponding author: E-Mail: [email protected].
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Acknowledgments
The authors are sincerely thankful to the CSIR, New Delhi and the Ministry of Earth Sciences, SMiLE project, CSIR, India for financial support and to the Director, IICT for his constant encouragement.
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(a) Harvey, A. L. Drug Discovery Today. 2008, 13, 894-901. b) Chin, Y. W.; Balunas, M. J.; Chai, H. B.; Kinghorn, A. D. The AAPS Journal. 2006, 8, 239-253. (a) Sarma, N. S.; Krishna, M. S.; Pasha, Sk. G.; Rao, T. S. P.; Venkateswarlu, Y.; Parameswaran, P. S. Chem. Rev. 2009, 109, 2803-2828. (b) Butler, M. S. Nat. Prod. Rep., 2005, 22, 162-195. (c) Mahido, C.; Ruchirawat, S.; Prawat, H.; Pisutjaroenpong, S.; Engprasert, S.; Chumsri, P.; Tengchaisri, T.; Siriinha, S.; Pichas, P. Pure Appl. Chem. 1998, 70, 20652072. (d) Newman, D. j.; Cragg, G. M. J. Nat. Prod. 2007, 70, 461-477. (e) Gerwick, W. H.; Simmons, T. L.; Andrianasolo, E.; McPhail, K.; Flatt, P. Mol. Cancer Ther. 2005, 4, 333-342. (f) Supriya, J.; Yogesh, C. IJRAP. 2010, 1, 55-62. (g) Boopathy, N. S.; Kathiresan, K. J. Oncology 2010, DOI:10.1155/2010/214186. (a) Sawadogo, W. R.; Schumacher, M.; Teiten, M. H.; Cerella, C.; Dicato, M.; Diederich, M. Molecules. 2013, 18, 36413673. (b) Ovenden, S. P. B.; Nielson, J. L.; Liptrot, C. H.; Willis, R. H.; Tapiolas, D. M.; Wright, A. D.; Motti, C. A. Molecules. 2012, 17, 2929-2938. (c) Kumar, S. V.; Parameswaran, P. S. Biotechnol. Adv. 2013, 31, 1826-1845. (a) Paul, V. J.; Arthur, K. E.; Williams, R. R.; Ross, C.; Sharp, K. Biol. Bull. 2007, 213, 226-251. (b) Mollica, A.; Locatelli, M.; Stefanucci, A.; Pinnen, F. Molecules. 2012, 17, 60836099.
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References and notes
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9. 10.
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We have isolated five new compounds (three pure and two as a mixture) and five known compounds from the marine soft coral Sinularia inelegans. The structures of the new compounds have been confirmed on the basis of 1D and 2D NMR spectroscopic data. Single crystal X-ray diffraction studies have also been carried out for compounds 2b and 6. The in vitro biological activity studies demonstrated that all compounds exhibited weak cytotoxic activity (IC50 values > 50 µM) against a panel of four human cancer cell lines. The observed antiproliferative effects of the compounds, however, suggest that several may serve as lead molecules for the subsequent development of new synthetic analogs with enhanced therapeutic values.
(a) Harvey, A. L. Drug Discovery Today. 2008, 13, 894-901. (b) Kinghorn, A. D.; Salim, A. A.; Chin, Y. W. “Bioactive Molecules and Medicinal Plants”, Chapter 1, Springer-Verlag Berlin Heidelberg, 2008, pp.1-24. (c) Kinghorn, A. D.; Chin, Y. W.; Balunas, M. J.; Chai, H. B. The AAPS Journal. 2006, 8, 239-253. (a) Sarma, N. S.; Krishna, M. S.; Pasha, Sk. G.; Rao, T. S. P.; Venkateswarlu, Y.; Parameswaran, P. S. Chem. Rev. 2009, 109, 2803-2828. (b) Ramesh, P.; Reddy, N. S.; Reddy, M. V. R.; Venkateswarlu, Y.; Faulkner, D. J. Tetrahedron Lett. 1998, 39, 8217-8220. (c) Venkateswarlu, Y.; Biabani, M. A. F.; Reddy, M. V. R.; Rao, T. P.; Kunwar, A. C. ; Faulkner, D. J. Tetrahedron Lett. 1994, 35, 2249-2252. (d) Reddy, M. V. R.; Lakshman, S.; Rama Rao, A. V.; Rao, J. V.; Venkateswarlu, Y. J. Nat. Prod. 1993, 56, 970-972. (e) Reddy, N. S.; Goud, T. V.; Venkateswarlu. Y. J. Nat. Prod. 2002, 65, 1059-1060. (a) Ravinder, K.; Reddy, A. V.; Krishnaiah, P.; Ramesh, P.; Ramakrishna, S.; Laatsch, H.; Venkateswarlu, Y. Tetrahedron Lett. 2005, 46, 5475-5478. (b) Krishnaiah, P.; Reddy, V. L. N.; Ravinder, K.; Venkateswarlu, Y. J. Nat. Prod., 2004, 67, 1168-1171. (a) Venkateswarlu, Y.; Sridevi, K. V.; Rama Rao. M. J. Nat. Prod. 1999, 62, 756-758. (b) Kamel, H. N.; Ferreira, D.; Fernandez, L. F. G.; Slattery, M. J. Nat. Prod. 2007, 70, 12231227. Li, Y.; Pattenden, G. Nat. Prod. Rep. 2011, 28, 1269-1310. Tseng, Y. J.; Ahmed, A. F.; Dai, C. F.; Chiang, M. Y.; Sheu, J. H. Org. Lett. 2005, 7, 3813-3816. (a) Earlier we reported rameswaralide (6)6b from the soft coral Sinularia dissecta with the relative stereochemistry C1-Hβ, C5-Hβ, C7-Hβ, C8-OHα, C10-β, C11-Hβ and C14-Hβ. In the present work, we have reported the absolute stereochemistry (C1-Hα, C5-Hα, C7-Hα, C8-OHβ, C10-α, C11-Hα, and C14Hα) of rameswaralide (6) which has been confirmed by the single crystal X-Ray crystallography (Figure 6). (a) Sato, A.; Fenical, W. Tetrahedron. 1985, 41, 4303-4308. (b) Yang, B.; Zhou, X.; Huang, H.; Yang, X. W.; Liu, J.; Lin, X.; Li, X.; Peng, Y.; Liu, Y. Mar. Drugs, 2012, 10, 20232032. (a) Reddy, M. A.; Jain, N.; Yada, D.; Kishore, C.; Reddy, V. J.; Reddy, P. S.; Addlagatta, A.; Kalivendi, S. V.; Sreedhar, B. J. Med. Chem. 2011, 54, 6751- 6760.
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Crystallographic data has been deposited for compound 6 with the Cambridge Crystallographic Data Centre [CCDC No. 933433]. Copies of the data can be obtained free of charge at www.ccdc.cam.ac.uk/conts/retrieving.html [or from the Cambridge Crystallographic Data Centre (CCDC), 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44(0) 1223 336 033; email: [email protected]].
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S0040-4020(16)30118-1
DOI:
10.1016/j.tet.2016.02.056
Reference:
TET 27526
To appear in:
Tetrahedron
Received Date: 6 September 2015 Revised Date:
22 February 2016
Accepted Date: 25 February 2016
Please cite this article as: Chitturi BR, Tatipamula VB, Dokuburra CB, Mangamuri UK, Tuniki VR, Kalivendi SV, Bunce RA, Yenamandra V, Pambanolides A-C from the South Indian Soft Coral Sinularia inelegans, Tetrahedron (2016), doi: 10.1016/j.tet.2016.02.056. 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 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.
ACCEPTED MANUSCRIPT Graphical Abstract Pambanolides A-C from the South Indian Soft Coral Sinularia inelegans
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Bhujanga Rao Chitturi,*Vinay Bharadwaj Tatipamula, Chanti Babu Dokuburra, Usha Kiranmayi Mangamuri, Venugopal Raju Tuniki, Shasi Vardhan Kalivendi, Richard A. Bunce, Venkateswarlu Yenamandra* E-Mail: [email protected].
Please include as a footer on p. 1: †
This paper is dedicated to the memory of our late mentor, Dr. Venkateswarlu Yenamandra, who passed away suddenly on 17 July 2013.
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Tetrahedron journal homepage: www.elsevier.com
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Pambanolides A-C from the South Indian Soft Coral Sinularia inelegans Bhujanga Rao Chitturi,a* Vinay Bharadwaj Tatipamula,a Chanti Babu Dokuburra,a Usha Kiranmayi Mangamuri,a Venugopal Raju Tuniki,b Shasi Vardhan Kalivendi,c Richard A. Bunce,d Venkateswarlu Yenamandraa†* a
Natural Products Chemistry Division, Indian Institute of Chemical Technology, Hyderabad, Telangana-500007, India. Nuclear Magnetic Resonance Spectroscopy Division, Indian Institute of Chemical Technology, Hyderabad, Telangana-500007, India. c Centre for Chemical Biology, Indian Institute of Chemical Technology, Hyderabad, Telangana-500007, India. d Department of Chemistry, Oklahoma State University, 107 Physical Sciences, Stillwater, OK 74078-3071
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b
ABSTRACT
Article history: Received Received in revised form Accepted Available online
Five new diterpenoids, pambanolides A-C (1, 2a, 2b and 3) and 4,5-secosinulochmodin C (4) were isolated from the soft coral Sinularia inelegans along with five known compounds (5-9). The structures of all metabolites were elucidated by comprehensive analysis of spectroscopic data. A biosynthetic pathway has been proposed for the formation of pambanolides A (1) and rameswaralide (6). Seven of the isolated compounds were evaluated for cytotoxic activity against a panel of four human cancer cell lines A549, DU145, HeLa and MCF-7 and were found to have weak cytotoxicity, but promising antiproliferative properties.
1. Introduction
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Keywords: Soft coral Sinularia inelegans Pambanolides Structure elucidation Biosynthesis
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ARTICLE INFO
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The aquatic environment is one of the leading sources for producing chemical entities with diverse structural and biological properties.1 Out of 27 diverse phyla of life on planet Earth, only 17 occur on land, while all 27 are available in the ocean. Thus, there are intact treasures of biodiversity present in the sea, and there has been an immense interest in finding biologically active compounds from marine flora and fauna.2 Despite the large number of biologically active molecules being isolated from marine sources yearly, only a few of them advance to preclinical trials.3 Nevertheless, great challenges and possibilities remain in this area of research. Marine soft corals, sponges, tunicates and other microorganisms produce different chemical compounds for metabolism as well as for defense mechanisms against predators during their life cycles.4 Several of these secondary metabolites and their synthetic derivatives with potent biological activities are being used as remedies for the treatment of diseases affecting human health.2,5 Marine diterpenoids are gaining much attention as they are the major constituents of marine segregates and show vast diversity in structure and biological activity. In continuation of our work to identify bioactive secondary metabolites from marine sources,6-8 we have isolated five new compounds, named pambanolides A-C (1, 2a, 2b and 3) and 4,5-secosinulochmodin C (4), along with five known diterpenoids (5-9) from the soft coral Sinularia inelegans collected from the Gulf of Mannar on
the South Indian coast. These compounds were evaluated for their cytotoxic effects against four human cancer cell lines. Herein, we report the isolation, structural elucidation and in vitro antiproliferative activities of these compounds. 18 19 8
5
10
19
4
11
OH 2
14
O
H
O
12 13 15
2
O
13
H 4
O
12
3
17 1
9 11
H
2
18
13
17
COOMe 20
3
H
15
12
O
OH
16
1
14
8 10
11
COOMe
O
H
22
COOMe OH
OMe O
HO H
15
H
MeOOC 16
4
O
9 17
OH H
20
14
O H
19
1 15
O
H
2
O 3 OH
7
16
5 6
6
23
3
14
4
2a (5-H ); 2b (5-H )
O O H 10
H
COOMe 5
MeO
4 13
O
1
7
12
OMe 18 21 COOMe 5 O
16
O
8
H 20
17
20
19
6
H
10 1
H
7 11
9
3
O
H
21
18
22
O 8
6
7
9
21
COOMe
O H
5
O
H
H 6
O
COOMe
COOMe O
OMe
OMe O O OH
O OH
O
COOMe
O 7
COOMe
O 8
OH O 9
O
Figure 1. Structures of compounds (1 – 9) isolated from marine soft coral Sinularia inelegans
2
Tetrahedron
13-H showed strong cross peaks with C12 and proton 14-H also ACCEPTED MANUSCRIPT
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The collected specimens (ca 3 kg wet) were exhaustively extracted with methanol and dichloromethane and the combined extracts were concentrated to give a residue (94 g). This material was suspended in 125 mL water and extracted four times with 250 mL of ethyl acetate. The combined organic layers were concentrated to a crude extract (37 g), which was subjected to column chromatography on silica gel for a preliminary fractionation. Bioactive fractions were identified using an in vitro bioassay and subjected to further chromatographic processing. Final purification was achieved by reversed phase HPLC, yielding five new compounds: 1 (8 mg), 2a and 2b (11.2 mg), 3 (16 mg) and 4 (3 mg) along with five known compounds 5-9 (total ca 2 g). Pambanolide A (1) was obtained as a white solid. Its molecular formula was determined to be C21H24O7 by high resolution ESI-MS, showing a pseudomolecular ion peak at m/z 411.14036 [M+Na]+ (calcd for C21H24O7Na is 411.14197) which requires ten degrees of unsaturation. The IR spectrum showed absorption bands for the presence of hydroxyl and carbonyl groups at 3403, 1745 and 1710 cm-1. The 13C NMR and DEPT spectra resolved 21 carbon resonances, including eight sp2 carbons, two sp3 quaternary carbons and three sp3 methylene carbons. Among the eight sp2 carbons, two belonged to carbonyl groups and six belonged to three double bonds. In combination with the 1H and 13C NMRs and IR, the spectral data for compound 1 (Table 1) indicated the presence of an isopropenyl group (C15: δC 149.0; C16: δC 109.8; δH 4.80, 1H, s and 4.82, 1H, s; C17: δC 20.3, δH 1.83, 3H, s), a quaternary methyl (C19: δC 21.3, δH 1.49, 3H, s), an α,β-unsaturated methyl ester (C18: δC 165.1; C21: δC 51.6, δH 3.72, 3H, s) and a trisubstituted double bond (C13: δC 145.4, δH 6.68, 1H, dt). The molecular formula (C21H24O7) and spectral data of 1 account for the ten degrees of unsaturation, which include three double bonds and two carbonyl groups, suggesting that 1 is a pentacyclic structure (see Figure 2 for number scheme). The 1H-1H DQF-COSY spectral data revealed the presence of two individual spin systems. Among these two spin systems, one occurred as a result of the protons on linearly connected carbons from C13 to C14 to C1 to C2 and the other was due to the protons on linearly connected carbons from C9 to C10 to C11 to C7 (Figure 2). In the 1H NMR of 1, proton 6-H (δH 5.94, s) appeared as a singlet instead of a doublet and was not coupled with its neighboring methine proton 7-H. Conceivably, these two protons (6-H and 7-H) are oriented at a dihedral angle of 90o,8 and thus, do not show any correlations in the DQF-COSY spectrum.
showed a cross peak with C12. Therefore, these two spin systems (fragments a and b) were connected to each other via C12 establishing the C11-C12-C13 linkage. Carbons C12 and C13 are sp2 hybridized, and thus, constitute a double bond. Furthermore, the HMBC correlations of 1 revealed the presence of an α,βunsaturated γ-lactone ring (H/C: 10-H/20; 11-H/12, 20; 13-H/20) made up of C10 to C13 and C20 carbons in the skeleton. The total HMBC correlations of 1 restricted the pentacyclic system to be made up of an 11-membered furanobicyclic ring (H/C: 1H/14; 2-H/1, 3, 4, 14; 6-H/3, 4, 5, 7; 7-H/6, 11; 11-H/7, 12, 13; 13-H/1, 11, 12, 14) which was fused to a cyclopentane ring (H/C: 7-H/8, 10, 11; 9-H/7, 8, 10, 11; 10-H/7, 8, 9; 11-H/7, 8, 9) at the C7 and C11 carbons and with the γ-lactone at the C11 and C12 carbons. In fragment "b" (Figure 2), the only sp3 hybridized methine group proton 1-H showed an HMBC correlation with C15, which belonged to the isopropenyl group (H/C: 16-H/15, 17; 17-H/15, 16). Protons 14-H and 2-H also showed HMBC cross peaks with C15. Thus, the isopropenyl group is attached to C1 of fragment “b” (Figure 2). In the 13C NMR spectrum of 1, the chemical shift of quaternary carbon C8 (δC 92.4 ppm) was shifted downfield when compared with the 13C data of rameswaralide (6) (C8, δC 78.6 ppm),6b which suggested that the oxygen atom on the C8 quaternary carbon could be contained in a cyclic ether linkage. Additionally, the HMBC correlation cross-peaks between 6-H (δ 5.07)/C5, C7, C8 and 7-H (δ 2.64)/C6, C8 indicated the presence of a tetrahydrofuran ring with an oxygen bridge (ether linkage) between the C5 and C8 carbons. The 1H and 13C NMR chemical shift values for fragments C7 to C13, C20 and the isopropenyl group of compound 1 also seemed to correlate with similar groups present in rameswaralide (6).6b All these findings together with an exhaustive literature survey of similar NMR data9 permitted us to establish a structure for 1 (Figure 1), which incorporates an unprecedented pentacyclic (5/5/5/5/10) scaffold.
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2.1 Isolation and identification of compounds
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2. Results and Discussion
18
COOMe
COOMe
O H3C
19
OH
H3C
O
8
5
H
4
OH
6
H
O
7 H
9
H
a
b
O 14
11
10
3 2
H 1
H
H
21
12
13
O
O
15
20
O
Figure 2. Selected HMBC ( ), COSY ( correlations for pambanolide A (1)
O
) and NOESY (
17
16
)
In addition to the two spin systems (from COSY), the HMBC correlations (Table 1 and Figure 2) were consistent with a rigid structure for 1. In the HMBC spectrum, protons 11-H and
Figure 3. One predicted low energy conformation of pambanolide A (1)
In compound 1, the stereochemistry of fragments C7 to C13 and C1 closely resembled that of rameswaralide (6).6b The relative configuration of 1 was determined by NOE experimental data correlations with the support of conformational analysis (Figure 3). As shown in one of the low energy conformers of 1, clear NOE cross peaks between 7-H, 19-CH3, 9-H (δH 2.62 (Ηα)), 10-H and 11-H as well as between 7-H and 11-H and 6-H and 19-CH3 were observed in the NOEDIFF spectra. All of these NOEs were consistent with an α orientation for all of these protons, except for 1-H which did not exhibit enhancement with any of these protons in 1. The NOE cross peaks between C1H/C16-H in 1 (Figure 2) suggested that both protons were on the same face (β orientation) of the structure, as observed in rameswaralide (6). The proton attached to C6, which belongs to the pentasubstituted dihydrofuran ring, is geminal to the oxygen of this ring. Indeed, this dihydrofuran ring, with a double bond between C4 and C5, is a rigid scaffold within the molecule. Since
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Tetrahedron
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Table 1. 1D and 2D NMR data of compounds 1 and 2 (2a / 2b mixture) recorded in CDCl3
H site 1 2
Pambanolide A (1) δC mult COSY 44.5 CH 2, 14 33.3 CH2 1
HMBC 16, 17, 15, 14 1, 3, 4, 14, 15
Pambanolide B1 (2a) δH (J in Hz) δC mult 2.52 m 40.6 CH 3.29 t (13.2) 44.9 CH2 2.49 t (13.2) -205.4 qC -72.3 qC 3.90 s 86.6 CH -190.8 qC
Pambanolide B2 (2b) δH (J in Hz) δC mult 2.52 m 40.3 CH 3.29 t (13.2) 44.8 CH2 2.94 t (13.2) -205.2 qC -72.6 qC 3.86 s 87.9 CH -190.7 qC
131.9 qC 153.9 qC 45.0 CH2
--3.06 dd (8.8, 9.7) 2.72 dd (5.8, 9.7)
-----
---3, 4, 5, 7, 10
2.64 d (10.8) -2.62 d (6.5) 1.96 dd (6.5, 15.8)
59.4 CH 92.4 qC 43.0 CH2
11 -10
6, 8, 10, 11,19 -7, 8, 10, 11,19
--3.06 dd (8.8, 9.7) 2.72 dd (5.8, 9.7)
10 11
4.95 td (6.5, 9.1) 3.91 m (1.4, 9.1, 10.8)
83.0 CH 47.5 CH
9, 11 7, 10
7, 8, 20 7, 8, 12, 13,20
5.15 m 3.48 m
77.4 CH 51.7 CH
12 13
-6.68 dt (1.4, 12.0)
125.4 qC 145.4 CH
-14
-1, 11, 12,14,20
2.29 t (11.5) 3.11 m
14
3.21 ddd (7.4, 12.0, 13.6) 2.13 t (12.0) -4.82 s, 4.80 s 1.83 s -1.49 s -3.72 s --
32.3 CH2
13, 1
1, 2, 12, 13,20
149.0 qC 109.8 CH2 20.3 CH3 165.1 qC 21.3 CH3 168.5 qC 51.6 CH3 --
-17 16 -----
--1, 15, 17 1, 15, 16 -7, 8, 9 -18 --
3.10 m 1.52 m -4.79 s, 4.73 s 1.74 s -2.21 s -3.83 s 3.44 s
*The combined COSY and HMBC correlations for the mixture of 2a and 2b
(2a / 2b)* HMBC 3, 15 1, 3, 15 --3, 4, 13, 22 --
132.0 qC 154.1 qC 45.2 CH2
--10
--7, 8, 10, 11, 19
5.15 m 3.48 m
77.4 CH 51.5 CH
9, 11 12, 10
7, 9, 12, 20 6, 7, 8, 13, 20
44.1 CH 45.0 CH
2.29 t (11.5) 3.11 m
44.1 CH 45.0 CH
11, 13 12, 14
4, 7, 11, 13, 20 4
28.8 CH2
3.10 m 1.52 t (13.4) -4.79 s, 4.75 s 1.72 s -2.21 s -3.83 s 3.40 s
29.0 CH2
13, 1
2, 15
146.0 qC 110.2 CH2 20.3 CH3 168.8 qC 16.0 CH3 173.9 qC 52.8 CH3 60.7 CH3
-17 16 ------
-1, 15(2a), 17 1, 15, 16 -6, 7, 8, 9 -18 5
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15 16 17 18 19 20 21 22
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7 8 9
COSY 2, 14 1 -----
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119.0 qC 103.3 qC 174.7 qC 88.5 CH
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3 4 5 6
δH (J in Hz) 3.53 m 2.93 d (12.7) 2.13 t (12.7) ---5.07 s
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1
146.2 qC 110.5 CH2 20.3 CH3 170.7 qC 16.0 CH3 173.9 qC 52.8 CH3 60.5 CH3
4
Tetrahedron
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α,β-unsaturated carbonyl group, along with a quaternary methyl the NOESY results suggested that C6-H hadACCEPTED an α orientation, MANUSCRIPT and one methyl ester. The 1H-1H DQF-COSY correlations of 2 the oxygen atom of the dihydrofuran could be positioned with a β revealed the presence of only one spin system consisting of eight orientation and subsequently the OH group on C3 should occupy a pseudoequatorial position in the envelope conformation of the different kinds of protons (out of 11 total) on the linearly dihydrofuran ring. The geometry of the trisubstituted double bond connected carbons C9 to C14 to C1 to C2 (see Figure 4 for number scheme). Furthermore, the HMBC correlations (Table 1) between C12 and C13 (δH 6.68) appeared to have the Econfiguration in the lowest energy conformer of 1. The energysuggested that compound 2 was a constrained structure, which minimized structure of 1 (Figure 3 and Table 1 in the SI) is contained one cyclohexanone ring, one cycloheptanone ring and virtually in agreement with the assigned structure based on NMR a cyclopentene ring together with one γ-lactone (H/C: 10-H/20; spectroscopic data. 11-H/20). The C1 methine proton 1-H (δH 2.52 m) showed an Pambanolide A (1) and rameswaralide (6) are constitutional HMBC correlation with C15 of the isopropenyl group (H/C: 16isomers. Both 1 and 6 exhibit the same molecular formula deduced H/15, 17; 17-H/15, 16). Protons 2-H and 14-H also showed from mass spectrometry (ESI, m/z = 411 [M+Na]+ ion), and the HMBC correlations with C15. The C17 methyl group (δH 1.72, s) 13 C NMR data show 21 carbon resonances. Compounds 1 and 6 also gave an HMBC cross peak with C1, hence the isopropenyl share some structural features, such as the isopropenyl group, the substituent was connected to the C1 methine center. The exocyclic α,β-unsaturated γ-lactone, the pentasubstituted molecular formula (C22H26O7) and spectral data of 2 accounted cyclopentane ring, and an α,β-unsaturated methyl ester function, for nine degrees of unsaturation including two double bonds and together with an oxygenated quaternary carbon bearing a methyl four carbonyl groups suggesting that 2 possesses a tetracyclic group. The structure of pambanolide A (1) has also been assigned (5/5/7/6) motif. A tentative structure for 2 was established by 1D on the basis of a postulated biosynthetic pathway (Scheme 1). and 2D NMR data correlations (Figure 4). According to Scheme 1, pambanolide A (1) and rameswaralide (6) could be obtained from the same precursor metabolite mandapamate (10) [Note: madapamate (10) and isomandapamate (5) are stereoisomers].
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Figure 4. Important HMBC ( ), COSY ( ) and NOESY ( correlations for pambanolide B1 (2a, 5-Hβ), pambanolide B2 (2b, 5-Hα)
Scheme 1. Proposed biosynthetic pathway for pambanolide A (1) and rameswaralide (6)
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Pambanolide B (2) was obtained as a white solid. The molecular formula C22H26O7 was deduced from the HRMS m/z 425.15685 [M+Na]+ (calcd for C22H26NaO7 is 425.15762), which required ten degrees of unsaturation. The IR spectrum showed multiple absorption bands for carbonyl groups at 1768, 1745, and 1710 cm-1. The structural determination of 2 was carried out by detailed interpretation of 1D and 2D NMR spectroscopic data. In the 13C NMR spectrum of 2, a total of 36 carbon resonances were observed. Among these 36 signals, eleven identified as pairs and twelve detected as individual peaks with increased (double) intensity. The remaining two carbons (δC 170.7 and 168.8 ppm) were found to be separate peaks with standard intensity. However, from the calculated molecular formula (C22H26O7) and 13 C NMR data assessments, it could be concluded that 2 is comprised of a mixture of two structurally similar compounds, pambanolide B1 (2a) and pambanolide B2 (2b). The 13C NMR and DEPT spectral data of 2 revealed the presence of eight sp2 carbons along with three sp3 methylenes, six sp3 methines and four methyl groups. Among the eight sp2 carbons, two belonged to ketone functions, two were part of ester carbonyl groups, and the remainder corresponded to two double bonds. Of the four methyl group signals, two appeared in the methoxy region (δC 60.5 and 52.8 ppm) of the 13C spectrum. Analysis of the 1H NMR, 13C NMR (Table 1) and IR data for 2 clearly indicated the presence of an isopropenyl group and an
)
Table 2. NOESY correlations for compounds 1, 2a and 2b recorded in CDCl3
1
H Site 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
1 NOESY 16-H ----19-CH3 11-H, 19-CH3 -10-H, 19-CH3 9-H (1.96), 11-H 10-H, 7-H -14-H (2.13) 13-H -1-H --6-H, 9-H (1.96), 19-CH3 -----
2a NOESY -------10-H 9-H (3.06), 11-H 10-H, 13-H -11-H ------
2b NOESY ----11-H, 13-H ---10-H 9-H (3.06), 5-H, 11-H 5-H, 10-H, 13-H -5-H, 11-H -----
--
--
----
---
--
--
Tetrahedron
5
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single crystal X-ray analysis. The ORTEP diagram for 2b is depicted in Figure 5.
Figure 5. Crystal structure of pambanolide B2 (2b). [CCDC No. 941403]
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Pambanolide C (3) was obtained as viscous, colorless liquid. The 13C NMR and HR ESI-MS spectral data of 3 afforded a molecular formula C23H30O8 consistent with a pseudomolecular ion peak at m/z 457.18179 [M+Na]+ (calcd for C23H30NaO8 is 457.18384). The IR spectrum showed the absorption bands of a hydroxy group at 3434 cm-1 and carbonyl groups at 1721 and 1714 cm-1. The 13C NMR and DEPT spectral data distinguished 23 carbon resonances, including signals for three carbonyl carbons, eight double bond carbons and four sp3 methylene carbons. The 1H and 13C NMR (Table 3) spectral data were isopropenyl group (C15: δC 145.9; C16: δC 111.4; δH 4.86, 1H, s and 4.79, 1H, s; C17: δC 20.8, δH 1.81, 3H, s), two α,βunsaturated methyl esters (C18: δC 163.9; C21: δC 51.3, δH 3.79, 3H, s; C20: δC 167.2; C22: δC 52.0, δH 3.72, 3H, s) and a quaternary methyl group (C19: δC 29.3, δH 1.35 s) (see Figure 6 for number scheme). As its molecular formula (C23H30O8) and
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The methoxy group (C22-OMe) protons [2a (δH 3.44, s) and 2b (δH 3.40, s)] on C5 appeared as two singlets in the 1H NMR, and together integrated to only three protons. However, they showed individual HSQC correlations with carbon pair signals C22 [2a (δC 60.5) and 2b (δC 60.7)], respectively. In combination with the 1H NMR, the HSQC spectrum of 2a/2b (see Figure S8, SI) clearly revealed the differences in matching the 5-H signals [2a (δH 3.90, s) and 2b (δH 3.86)] with the C5 carbon pair [2a (δC 86.6) and 2b (δC 87.9)]. A detailed examination of the 1D and 2D NMR data correlations of 2 suggested it was an inseparable mixture of two stereoisomers (2a/2b) present in a ~2:3 ratio (based on NMR integration). The relative configurations of 2 were determined by a NOESY experiment (Table 2 and Figure 4). In the NOESY spectrum, 10-H showed clear NOE correlations with 9-H (δH 3.06) and 11-H, which in turn, showed NOEs with 13-H. In addition, a clear NOE correlation was also observed between 12H and 1-H. In the NOEDIFF spectrum, proton 5-H (δH 3.86, s) in 2b showed strong NOEs with 11-H as well as 13-H, indicating an α orientation. Proton 5-H (δH 3.90) in 2a did not show NOE correlations with either 11-Hα or 13-Hα, and hence, 5-H of 2a should be in the β orientation. Indeed, the stereochemistry of 2a/2b appears to be analogous to rameswaralide (6)6b at the C1, C10 and C11 stereocenters. Consistent with this reasoning, the individual relative stereochemistries of 2a and 2b were assigned as shown in Figure 4. The structure of 2b was unambiguously established by X-ray crystal studies. Recrystallization of 2a/2b was accomplished in a methanol/water dual solvent system. Needle like crystals formed in the crystallization pot, and we were able to separate a tiny (monoclinic) crystal of 2b, which was subsequently submitted for
Table 3. 1D and 2D NMR data for compounds 3 and 4 recorded in CDCl3
H site 1 2
δH (J in Hz) 2.48 m 3.10 m --6.62 s -4.06 s --
9
2.48 d (18.0) 3.16 d (18.0) -3.58 d (8.0) --
13 14 15 16 17 18 19 20 21 22 23
-------
AC C
10 11 12
161.0 qC 114.6 qC 112.2CH 149.0 qC 83.7 CH 73.0 qC
HMBC 15, 13, 2 3, 1, 15, 4
--3, 4, 6, 7, 18 -19, 9, 23, 8,5 --
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3 4 5 6 7 8
Pambanolide C (3) COSY δC mult 44.0 CH 2, 14 30.5 CH2 1
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6.96 dd (3.0, 10.0) 2.26 d (8.0) 2.44 d (8.0) -4.79 s 4.86 s 1.81 s -1.35 s -3.79 s 3.72 s 3.25 s
45.9 CH2
--
19, 8, 7, 10
208.5 qC 42.0 CH2 125.2 qC
-12, 10 --
-12, 13,20 --
144.0 CH 31.4 CH2
14 13, 1
14, 11, 1, 12, 20 2, 1, 12, 13, 15
145.9 qC 111.4 CH2
-17
-17, 1, 15
20.8 CH3 163.9 qC 29.3 CH3 167.2 qC 51.3 CH3 52.0 CH3 57.7 qC
16 -------
1, 16, 15 -9, 8, 7, 6 -18 20 7
4,5-Secosinulochmodin C (4) COSY δH (J in Hz) δC mult 2.78 m 39.3 CH 2, 12 3.20 m, 30.7 CH2 1, 3 1.90 dm (12.5) 2.40 dt (3.0, 12.5) 36.2 CH 2, 11 5.20 s 79.5 CH --210.5 qC -2.70 d (10.0) 62.0 CH 10 -90.8 qC -2.55 d (15.5) 45.3 CH2 9 2.17 dd (8.5, 16.1) 5.10 t (8.2) 83.2 CH 8, 10
HMBC 2, 13, 15 1, 3, 15 5, 6, 8,11 3, 5, 6, 11 4, 5, 10 -6, 7, 9, 10 18
3.47 m 3.04 dd (4.8, 12.8) 2.66 ddd (1.5, 4.2, 15.2) 2.58 d (6.7) ---
43.6 CH 46.1 CH 42.9 CH2
6, 9, 11 3, 10 1
1, 11, 18 3, 6, 18 1, 13, 14, 15
201.2 qC 166.0 qC
---
---
-4.92 s 4.71 s 1.72 s -1.30 s 3.76 s ----
146.0 qC 113.0 CH2
-17
-1, 15, 17
22.3 CH3 175.5 qC 21.0 CH3 52.6 CH3 ----
16 -------
1, 15, 16 -6, 7, 8 14 ----
6
Tetrahedron
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The 1H-1H DQF-COSY spectral data revealed only a single linear correlation between 13-H, 14-H, 1-H and 2-H. The HMBC spectral data correlations (Table 3) established a complete structure for 3. The HMBC correlations suggested that compound 3 was a bicyclic structure, which contained a trisubstituted furan ring (H/C: 2-H/3, 4, 1; 5-H/3, 4, 6, 7; 7-H/5) and a 13-membered macrocyclic ring (H/C: 7-H/8, 9; 9-H/7, 8, 10; 11-H/12, 13; 13H/11, 12, 14, 1; 14-H/12, 13, 1, 2; 1-H/2, 13; 2-H/1, 3, 4) bridging the furan ring at C3 and C6. The C1 methine proton 1-H showed an HMBC correlation with C15, which belonged to an isopropenyl group (H/C: 16-H/15, 17; 17-H/15, 16). The protons 14-H and 2-H also showed HMBC cross peaks with C15. Thus, the isopropenyl group was located on the C1 methine carbon. In the HMBC spectrum, C23-OMe protons (δH 3.25, s) showed a cross peak with the C7 methine (δC 83.7), the 7-H showed correlations with C5, C8, C9, C19 and C23 on the 14-membered ring (Figure 6). Thus, from the foregoing data analysis and an exhaustive literature survey,8,9 the structure of 3 was assigned as shown in Figure 1. Both 3 and 8 exhibited the same molecular formula deduced from mass spectrometry (ESI, m/z 457 [M+Na]+) and each 13C NMR spectrum showed 23 carbon resonances with similar functionalities, including an isopropenyl group, two α,β-unsaturated methyl esters, one ketone, a quaternary methyl and a methoxy group.
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measurements and showed an m/z 377.15841 [M+H]+ (calcd for spectral data indicated nine degrees of unsaturation including MANUSCRIPT ACCEPTED four double bonds and three carbonyl groups, it was concluded C20H25O7 is 377.16003 [M+H]+). The NMR (1H, 13C and DEPT) spectral data (Table 4) indicated the presence of 20 carbons, that 3 had a bicyclic skeletal structure. including carbons for one isopropenyl group, one methyl ester, one quaternary methyl group, two ketones, three sp3 methylenes and seven sp3 methines. The molecular formula (C20H24O7) indicated nine degrees of unsaturation, and with one double bond and four carbonyl groups, confirmed that the structure consisted of a tetracyclic carbon framework. In the 1H-1H DQF-COSY spectrum, the presence of a single, wide spin system was noted with eight different kinds of protons (out of 11 total) on carbons C8 to C11 to C3 to C1 to C12 in a linear connection and one proton on C6 that branched off of C10 (see Figure 7 for number scheme). In the 1H-NMR spectrum, proton 4-H (δH 5.20) appeared as a singlet instead of the expected doublet from coupling with its neighboring proton 3-H (δH 2.40), suggesting that they showed a dihedral angle of 90o to each other.8 Figure 6. Selected HMBC ( ), COSY ( ) and NOESY ( ) Consequently protons 3-H and 4-H did not show any correlation correlations for pambanolide C (3) in the COSY spectral experiment.
Scheme 2. A proposed pathway for formation of pambanolide C (3) from sethukarailin (8)
The NOEDIFF spectrum for 3 showed clear NOEs from 1-H to 19-CH3, and from 19-CH3 to 7-H. Generally, 14-membered cembrane type diterpenoids9 exhibit an α isopropenyl group attached to the C1 stereocenter. In addition, 3 could represent an artifact derived from sethukarailin (8) (Scheme 2). According to the results of 2D NMR studies described here and information found in the literature, the relative stereochemistry of 3 is assigned as shown in Figure 6. 4,5-Secosinulochmodin C (4) was obtained as a white solid. It had a molecular formula of C20H24O7 according to HR ESI-MS
Figure 7. Selected HMBC ( ), COSY ( correlations for 4,5-secosinulochmodin C (4)
) and NOESY (
)
Table 4. NOESY correlations for compounds 3 and 4 recorded in CDCl3 1
H Site 1 2 3 6 7 8 9 10 11 17 18 19
Pambanolide C (3)
4,5-Secosinulochmodin C (4)
NOESY 19-CH3
NOESY 2-H (1.90) 1-H, 11-H 11-H, 10-H, 19-CH3 -9-H, 19-CH3 8-H (2.17), 10-H 9-H, 11-H, 6-H 1-H, 3-H,10-H 1-H -6-H, 8-H (2.17)
--19-CH3 -19-CH3 ----1-H, 7-H, 9-H (3.16)
The total HMBC correlations of 4 restricted the tetracyclic formulation to be made up of a cyclohexanone ring (H/C: 3-H/4, 5, 11; 4-H/3, 5, 6, 11; 6-H/4, 5, 10; 10-H/11; 11-H/3, 6), which was fused with a cyclopentane ring (H/C: 6-H/10; 8-H/6, 7, 8, 10;
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Scheme 3. A possible pathway for the formation of compound 4 from sinulochmodin C (12)
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The structures of known compounds 5-9 (Figure 1) were established by interpretation of the 1D and 2D NMR spectral data. Among these known compounds, isomandapamate (5), rameswaralide (6) and sethukarailin (8) have been previously reported from our group. 6,7 The revised and exact stereochemical structure of rameswaralide11 was confirmed for the first time by single crystal X-ray crystallographic studies (Figure 9). Water in methanol was the solvent system used for the crystallization of 6. Compounds 7 and 9 are known and have been described in an earlier report.12
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10-H/8) at the C6 and C10 carbon centers. A γ-lactone ring (H/C: 9-H/11, 18; 10-H/11, 18; 11-H/18), fused to the cyclohexanone at C10 and C11, was also observed from HMBC correlations of 4. From the HMBC correlations (H/C: H-4/C5, C6; H-6/C4, C5 and H-19/C6, C7) and the 13C chemical shift values for C4 and C7, it was obvious that, an oxygen atom had to be placed between these two carbons to form a tetrahydrofuran ring. The 1H and 13C NMR chemical shifts of the C4 methine group (δH 5.20; δC 79.5) and the downfield 13C chemical shift value for C7 (δC 90.8) provided strong evidence for a tetrahydrofuranone ring in the structure of 4. By extensive analysis of HMBC spectral data in combination with DQF-COSY spectral data correlations, it was determined that the structure of 4 appears to be the product of a redox bond cleavage at C4-C5 bond in sinulochmodin C,10 and consists of a tetracyclic (5/5/5/6) core with an eight-carbon side chain residue incorporating an α-keto ester functionality (H/C: 12-H/C13, C14) and an isopropenyl group. The 1-H showed an HMBC correlation with C15, which is part of the isopropenyl substituent (H/C: 16H/15, 17; 17-H/15, 16). Protons 2-H and 12-H also showed HMBC cross peaks with C15. Thus, C15 of the isopropenyl group is connected to the C1 methine center. From the above data analysis and information taken from the literature,9 it is clear that 4 is a tetracyclic norditerpenoid (19 carbon skeleton) bearing an additional methoxy group.
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Figure 9. The crystal structure of rameswaralide (6) [CCDC No. 933433]
2.2. Postulated Biogenetic Pathway to Compounds 1, 4 and 6
Figure 8. One energy minimized structure of compound 4
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The NOESY spectrum of 4 displayed a clear correlation (Figure 7) between H-6 and H-19. On the other hand, H-19 showed an NOE correlation with one of the methylene protons at C8 (δΗ 2.17 (Ηα)) and C8-Hα showed a clear correlation with H9, which in turn, showed the correlation with H-10. In the NOESY spectrum of 4, the NOE interaction between H-6, H-19, H-8α, H-9 and H-10 reflected the α orientation of H-6 and subsequently the β-orientation of the “C4-O-C7” ethereal bridge. Proton 1-H did not show any NOEs in the NOEDIFF spectrum with protons at the other stereocenters. Rather, the proton attached to C2 (δΗ 1.90, dm) showed NOE correlations with the 1-H and 11-H protons, indicating that they were on the same side of the structure. The stereochemistry of 4 appeared to be analogous to sinulochmodin C (12) except for the chiral center at C1. The energy-minimized structure as shown in Figure 8 (Table 2 in the SI) was also in agreement with the assigned structure of 4 from the NMR data. On the basis of the above observations and consideration of the proposed biosynthetic pathway (Scheme 3), the structure of 4 was fully established and found to possess the 1S, 5S, 7S, 8R, 10S, 11S, 12R, 13S absolute configuration at the respective stereocenters. Thus, 4,5-secosinulochmodin C (4) was assigned the norditerpenoid tetracyclic (5/5/5/6) core skeleton shown in Figure 8.
To the best of our knowledge, compound 1 is an unprecedented skeleton with an unusual pentacyclic (5/5/5/5/10) framework. A conceivable biogenetic pathway has been proposed for compounds 1 and 6 in Scheme 1. They could have a common precursor, probably mandapamate (10). Cleavage of the C6-C14 bond in mandapamate (10) or cleavage of the C5-C14 bond in rameswarlide (6) could lead to unstable intermediate (11), which could undergo a ring closing oxa-Michael reaction to afford 1. The new furanocembranoid 3 seems to be an artifact of the known compound sethukarailin (8) (Scheme 2). The previously unreported tetracyclic (5/5/5/6) norditerpenoid (4) with an eightcarbon side chain bearing an isopropenyl substituent and terminated by an α-keto ester functionality, appears to be a C4C5 redox bond cleavage product of sinulochmodin C (12). 2.3. Biological Activity In vitro cytotoxicity for seven selected compounds (1-3, 5-7 and 9) isolated from Sinularia inelegans was assayed. For these in vitro studies, we used four human cancer cell lines, which included A549 (human epithelial lung carcinoma), DU145 (human prostate cancer), HeLa (human epithelial cervical cancer) and MCF-7 (human breast adenocarcinoma).13 Each cell line was treated with four different concentrations of each compound ranging from 1 to 100 µM concentration for 48 h and the obtained IC50 values are summarized in Table 3 (SI). All these compounds showed weak cytotoxic activity towards the four
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compounds: 5 (38 mg), 6 (17 mg), 7 (130 mg), 8 (1.8 g), and 9 cancer cell lines tested. Among these compounds, 2 and 3 MANUSCRIPT ACCEPTED (12 mg). showed slightly higher activity against the DU145 and A549 cancer cell lines when compared to the other compounds tested. While compound 3 exhibited IC50 values of 41 µM and 69 µM, 3.2.1. Pambanolide A (1) was a white solid, mp 231-235 oC. compound 2 showed IC50 values of 63 µM and 55 µM against the [α]D25 –24.39 (c 0.82, CHCl3); for the 1H and 13C NMR, see Table DU145 and A549 cell lines, respectively. Among all of the 1 and Figures S1 and S2 (SI); EI-MS m/z = 411.2 [M+Na]+; HR compounds tested, cembranoid diterpene 3 showed the best ESI-MS, m/z = 411.14036 [M+Na]+ (calcd for C21H24NaO7 is cytotoxic activity across the entire panel of cell lines. Cell 411.14197); IR (neat): 3403, 1745 and 1710 cm-1; UV: 256 nm proliferation studies indicated that growth of these cell lines was (MeOH). The detailed molecular structure was deduced by slowed significantly by these same seven compounds. interpretation of the NMR spectroscopic data, which included 1H, 13 C, DEPT, HSQC, COSY, HMBC and NOESY spectra. 3. Experimental Section 3.2.2. Pambanolide B1 (2a) and Pambanolide B2 (2b), as a diastereomeric mixture, were obtained as a white solid, mp 189Commercially available chemicals were used for the isolation 196 oC. [α]D25 –9.17 (c 0.71, CHCl3); for the 1H and 13C NMR, protocols without further purification. The 1D and 2D 1H and 13C see Table 1 and Figures S6 and S7 in the SI; EI-MS m/z 425 NMR spectra of compounds were recorded on Bruker 300, [M+Na]+; HR ESI-MS m/z 425.15685 ([M+Na]+) C22H26O7 Avance 500 and Avance 600 instruments in CDCl3 solvent at 25 (calcd for C22H26NaO7 is 425.15762); IR (neat): 1768, 1745, 1710 °C; chemical shifts are reported in δ (ppm) and are referenced to cm-1; UV: 226 nm (MeOH). The detailed molecular structure was the CHCl3 solvent peak at δ 7.25 and the CDCl3 solvent peak at elucidated by interpretation of the NMR spectroscopic data, δ 77.00. FT-IR spectra for the compounds were measured as which included 1H, 13C, DEPT, HSQC, COSY, HMBC and solutions (CHCl3) or liquid films between KBr plates. Mass NOESY spectra. spectra were calculated from ESI and HR ESI-MS (Q-TOF, Agilent tech. (Mass Spectroscopy Division IICT-Hyderabad)) spectrometers. Optical rotations were measured in CHCl3 solvent. 3.2.3. Pambanolide C (2b) crystallography data: C22H26O7, M = Isolation and purification of compounds were done by silica gel 402.43, colorless block, 0.34 × 0.24 × 0.20 mm3, monoclinic, (100-200 and 230-400 mesh) column chromatography. Further space group P2 (1), a = 9.3368(13) Å, b = 10.7714(12) Å, c = purification, if necessary, was performed by reversed phase 0.5965(17) Å, V = 1017.0(16) Å3, Z = 2, Dc = 1.314 g/cm3, F000 = HPLC. Analytical TLC was carried out on pre-coated plates 428, CCD area detector, MoKα, radiation, λ = 0.71073 Å, T = (Merck, silica gel 60 PF254), and spots were visualized with UV 298(2) K, 2θmax = 50.0º, 4340 reflections collected, 1957 unique light, I2, or by charring after treatment with anisaldehyde (or (Rint = 0.0173). Final GoF = 1.036, R1 = 0.0446, wR2 = 0.0968, sulfuric acid). All chromatographic solvents were distilled prior final R indices (I > 2σ(I)), R1=0.0563, WR2 = 0.1063, R indices to use. (all data). Crystallographic data have been deposited for compound 2b with the Cambridge Crystallographic Data Centre 3.1. Collection of Soft Coral [CCDC No. 941403]. Copies of the data can be obtained free of charge at www.ccdc.cam.ac.uk/conts/retrieving.html [or from the Cambridge Crystallographic Data Centre (CCDC), 12 Union The soft coral Sinularia inelegans was collected from the sea at a depth of 20-30 m by skin diving along the Mandapam coast Road, Cambridge CB2 1EZ, UK; fax: +44(0) 1223 336 033; email: [email protected]]. (N 9° 18'; E 79° 08') in the Gulf of Mannar on the southern coast of India, during April 2011. The specimens (ca 3 kg wet) were preserved in methanol until worked up. Voucher material has 3.2.4. Pambanolide D (3) was obtained as viscous colorless oil. been deposited at the INO in Goa, India. [α]D25 –103.29 (c 0.82, CHCl3); for 1H and 13C NMR, see Table 3 and Figures S12 and S13 in the SI); HR ESI-MS m/z = 457.18179 [M+Na]+ (calcd for C23H30NaO8 is 457.18384). IR 3.2. Purification of compounds (neat): 3434, 1714 and 1666 cm-1; UV: 216 nm (MeOH). The detailed molecular structure was determined by interpretation of Immediately after collection, the wet samples were soaked in the NMR spectroscopic data, which included 1H, 13C, DEPT, methanol. After five days, the methanol was decanted and the HSQC, COSY, HMBC and NOESY spectra. sample was extracted with a 1:1 mixture of MeOH and DCM for three days. The later extraction was repeated three times. The 3.2.5. 4,5-Secosinulochmodin C (4) was a white solid, 208-214 resulting extracts were combined with the initial methanol o C. [α]D25 –27.39 (c 0.1, CHCl3); for 1H and 13C NMR, see Table preservative and concentrated under reduced pressure. 2 and Figures S17 and S18 in the SI); EI-MS m/z = 377 [M+Na]+; Subsequently, the residue (94 g) was suspended in 125 mL water HR ESI-MS (calcd for C20H25O7 is 377.16003; found 377.15841 and extracted with EtOAc (4 × 250 mL). The combined organic [M+H]+). The detailed molecular structure was deduced by layers were concentrated to afford 37 g of residue. The final interpretation of the NMR spectroscopic data, which included 1H, crude extract was directly separated by silica gel (100-200 mesh) 13 C, DEPT, HSQC, COSY, HMBC and NOESY spectra. column chromatography by using EtOAc in n-hexane (0%, 5%, 10%, 20%, 40%, 60%, 80% and 100%) and methanol in EtOAc 3.2.6. Rameswaralide (6) crystallography data: C21H24O7, M = (5%, 10% and 20%) as 24 fractions. On the basis of a preliminary bioassay and 1H-NMR spectral data, some of the 24 fractions (11, 388.40, colorless block, 0.22 × 0.17 × 0.11 mm3, orthorhombic, 12, 15, 16, 18, 19, 20, 21. 23 and 24) were purified by repeated space group P212121, a = 10.6679(10), b = 11.7688(11), c = silica gel (230-400 mesh) column chromatography and further 15.4535(15) Å, V = 1940.2(3) Å3, Z = 4, Dc = 1.330 g/cm3, F000 = purified (when necessary) by preparative HPLC using a reversed 824, CCD area detector, MoKα radiation λ = 0.71073 Å, T = phase C-18 column (5 µM particle size) eluted with 20% water in 294(2) K, 2θmax = 50.0º, 18622 reflections collected, 1957 unique acetonitrile to give five new compounds: 1 (8 mg), 2a and 2b (Rint = 0.0228). Final GoF = 1.036, R1 = 0.0285, wR2 = 0.0778, (mixture, 11.2 mg), 3 (16 mg), and 4 (3 mg); and five known R indices based on 1878 reflections with I > 2σ(I) (refinement on F2), 264 parameters, 0 restraints µ = 0.100 mm-1.
Tetrahedron
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6.
3.3. Bioassays Methods and procedures are outlined in the SI. 4. Conclusion
Corresponding Author: Dr. Chitturi Bhujanga Rao, c/o Dr. U. V. Mallavadhani, Principal Scientist, Natural Products Chemistry Division, Indian Institute of Chemical Technology, Taranaka, Hyderabad, A.P. India-500007. Corresponding author: E-Mail: [email protected].
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Acknowledgments
The authors are sincerely thankful to the CSIR, New Delhi and the Ministry of Earth Sciences, SMiLE project, CSIR, India for financial support and to the Director, IICT for his constant encouragement.
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(a) Harvey, A. L. Drug Discovery Today. 2008, 13, 894-901. b) Chin, Y. W.; Balunas, M. J.; Chai, H. B.; Kinghorn, A. D. The AAPS Journal. 2006, 8, 239-253. (a) Sarma, N. S.; Krishna, M. S.; Pasha, Sk. G.; Rao, T. S. P.; Venkateswarlu, Y.; Parameswaran, P. S. Chem. Rev. 2009, 109, 2803-2828. (b) Butler, M. S. Nat. Prod. Rep., 2005, 22, 162-195. (c) Mahido, C.; Ruchirawat, S.; Prawat, H.; Pisutjaroenpong, S.; Engprasert, S.; Chumsri, P.; Tengchaisri, T.; Siriinha, S.; Pichas, P. Pure Appl. Chem. 1998, 70, 20652072. (d) Newman, D. j.; Cragg, G. M. J. Nat. Prod. 2007, 70, 461-477. (e) Gerwick, W. H.; Simmons, T. L.; Andrianasolo, E.; McPhail, K.; Flatt, P. Mol. Cancer Ther. 2005, 4, 333-342. (f) Supriya, J.; Yogesh, C. IJRAP. 2010, 1, 55-62. (g) Boopathy, N. S.; Kathiresan, K. J. Oncology 2010, DOI:10.1155/2010/214186. (a) Sawadogo, W. R.; Schumacher, M.; Teiten, M. H.; Cerella, C.; Dicato, M.; Diederich, M. Molecules. 2013, 18, 36413673. (b) Ovenden, S. P. B.; Nielson, J. L.; Liptrot, C. H.; Willis, R. H.; Tapiolas, D. M.; Wright, A. D.; Motti, C. A. Molecules. 2012, 17, 2929-2938. (c) Kumar, S. V.; Parameswaran, P. S. Biotechnol. Adv. 2013, 31, 1826-1845. (a) Paul, V. J.; Arthur, K. E.; Williams, R. R.; Ross, C.; Sharp, K. Biol. Bull. 2007, 213, 226-251. (b) Mollica, A.; Locatelli, M.; Stefanucci, A.; Pinnen, F. Molecules. 2012, 17, 60836099.
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References and notes
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9. 10.
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We have isolated five new compounds (three pure and two as a mixture) and five known compounds from the marine soft coral Sinularia inelegans. The structures of the new compounds have been confirmed on the basis of 1D and 2D NMR spectroscopic data. Single crystal X-ray diffraction studies have also been carried out for compounds 2b and 6. The in vitro biological activity studies demonstrated that all compounds exhibited weak cytotoxic activity (IC50 values > 50 µM) against a panel of four human cancer cell lines. The observed antiproliferative effects of the compounds, however, suggest that several may serve as lead molecules for the subsequent development of new synthetic analogs with enhanced therapeutic values.
(a) Harvey, A. L. Drug Discovery Today. 2008, 13, 894-901. (b) Kinghorn, A. D.; Salim, A. A.; Chin, Y. W. “Bioactive Molecules and Medicinal Plants”, Chapter 1, Springer-Verlag Berlin Heidelberg, 2008, pp.1-24. (c) Kinghorn, A. D.; Chin, Y. W.; Balunas, M. J.; Chai, H. B. The AAPS Journal. 2006, 8, 239-253. (a) Sarma, N. S.; Krishna, M. S.; Pasha, Sk. G.; Rao, T. S. P.; Venkateswarlu, Y.; Parameswaran, P. S. Chem. Rev. 2009, 109, 2803-2828. (b) Ramesh, P.; Reddy, N. S.; Reddy, M. V. R.; Venkateswarlu, Y.; Faulkner, D. J. Tetrahedron Lett. 1998, 39, 8217-8220. (c) Venkateswarlu, Y.; Biabani, M. A. F.; Reddy, M. V. R.; Rao, T. P.; Kunwar, A. C. ; Faulkner, D. J. Tetrahedron Lett. 1994, 35, 2249-2252. (d) Reddy, M. V. R.; Lakshman, S.; Rama Rao, A. V.; Rao, J. V.; Venkateswarlu, Y. J. Nat. Prod. 1993, 56, 970-972. (e) Reddy, N. S.; Goud, T. V.; Venkateswarlu. Y. J. Nat. Prod. 2002, 65, 1059-1060. (a) Ravinder, K.; Reddy, A. V.; Krishnaiah, P.; Ramesh, P.; Ramakrishna, S.; Laatsch, H.; Venkateswarlu, Y. Tetrahedron Lett. 2005, 46, 5475-5478. (b) Krishnaiah, P.; Reddy, V. L. N.; Ravinder, K.; Venkateswarlu, Y. J. Nat. Prod., 2004, 67, 1168-1171. (a) Venkateswarlu, Y.; Sridevi, K. V.; Rama Rao. M. J. Nat. Prod. 1999, 62, 756-758. (b) Kamel, H. N.; Ferreira, D.; Fernandez, L. F. G.; Slattery, M. J. Nat. Prod. 2007, 70, 12231227. Li, Y.; Pattenden, G. Nat. Prod. Rep. 2011, 28, 1269-1310. Tseng, Y. J.; Ahmed, A. F.; Dai, C. F.; Chiang, M. Y.; Sheu, J. H. Org. Lett. 2005, 7, 3813-3816. (a) Earlier we reported rameswaralide (6)6b from the soft coral Sinularia dissecta with the relative stereochemistry C1-Hβ, C5-Hβ, C7-Hβ, C8-OHα, C10-β, C11-Hβ and C14-Hβ. In the present work, we have reported the absolute stereochemistry (C1-Hα, C5-Hα, C7-Hα, C8-OHβ, C10-α, C11-Hα, and C14Hα) of rameswaralide (6) which has been confirmed by the single crystal X-Ray crystallography (Figure 6). (a) Sato, A.; Fenical, W. Tetrahedron. 1985, 41, 4303-4308. (b) Yang, B.; Zhou, X.; Huang, H.; Yang, X. W.; Liu, J.; Lin, X.; Li, X.; Peng, Y.; Liu, Y. Mar. Drugs, 2012, 10, 20232032. (a) Reddy, M. A.; Jain, N.; Yada, D.; Kishore, C.; Reddy, V. J.; Reddy, P. S.; Addlagatta, A.; Kalivendi, S. V.; Sreedhar, B. J. Med. Chem. 2011, 54, 6751- 6760.
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Crystallographic data has been deposited for compound 6 with the Cambridge Crystallographic Data Centre [CCDC No. 933433]. Copies of the data can be obtained free of charge at www.ccdc.cam.ac.uk/conts/retrieving.html [or from the Cambridge Crystallographic Data Centre (CCDC), 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44(0) 1223 336 033; email: [email protected]].
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