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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
A new diketopiperazine from South China Sea marine sponge Callyspongia sp. a
b
c
a
Yinning Chen , Yan Peng , Chenghai Gao & Riming Huang a
Key Laboratory of Plant Resource Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510-650, China b
Life Science & Technology School, Zhanjiang Normal University, Zhanjiang 524-048, China c
Guangxi Key Laboratory of Marine Environmental Science, Guangxi Academy of Sciences, Nanning 530-007, China Published online: 04 Apr 2014.
To cite this article: Yinning Chen, Yan Peng, Chenghai Gao & Riming Huang (2014) A new diketopiperazine from South China Sea marine sponge Callyspongia sp., Natural Product Research: Formerly Natural Product Letters, 28:13, 1010-1014, DOI: 10.1080/14786419.2014.903397 To link to this article: http://dx.doi.org/10.1080/14786419.2014.903397
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
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Natural Product Research, 2014 Vol. 28, No. 13, 1010–1014, http://dx.doi.org/10.1080/14786419.2014.903397
A new diketopiperazine from South China Sea marine sponge Callyspongia sp. Yinning Chena, Yan Pengb, Chenghai Gaoc and Riming Huanga* a
Downloaded by [Tulane University] at 00:12 04 September 2014
Key Laboratory of Plant Resource Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510-650, China; bLife Science & Technology School, Zhanjiang Normal University, Zhanjiang 524-048, China; cGuangxi Key Laboratory of Marine Environmental Science, Guangxi Academy of Sciences, Nanning 530-007, China (Received 3 January 2014; final version received 6 March 2014) Further chemical investigation on the marine sponge Callyspongia sp. collected from South China Sea led to the isolation of a new diketopiperazine, named callysponine A (1), as well as four known diketopiperazines, namely cyclo-(Gly-Pro) (2), cyclo-(ThrPro) (3), cyclo-(Ile-Pro) (4) and cyclo-(Pro-Pro) (5). The new structure was determined on the basis of NMR and MS analysis, and the absolute stereochemistry was defined by analysis of the coupling constants and optical rotation. The structures of the known compounds were identified by comparing their spectroscopic data with those reported in the literature. Compounds 1 – 5 did not inhibit the growth of HepG2 (hepatoma carcinoma cell), A549 (lung carcinoma cell) and HeLa (cervical cancer cell) cell lines. Keywords: Callyspongia sp.; diketopiperazine; callysponine A
1. Introduction Till date, there are a few reports on diketopiperazines from marine sponges (Huang, Zhou et al. 2010). The genus Callyspongia (order Haplosclerida, family Callyspongiidae) is widely distributed and contains various bioactive constituents (Yang et al. 2012). In the course of our investigation on the marine sponge Callyspongia species, we have reported some metabolites (Huang, Ma et al. 2010; Huang et al. 2011). Further investigation on the marine sponge Callyspongia species has led to the isolation and characterisation of a new diketopiperazine, named callysponine A (1), as well as four known diketopiperazines, namely cyclo-(Gly-Pro) (2) (Xie et al. 2013), cyclo-(Thr-Pro) (3) (Stark & Hofmann 2005), cyclo-(Ile-Pro) (4) (Adamczeski et al. 1995) and cyclo-(Pro-Pro) (5) (Stark & Hofmann 2005). 2. Results and discussion Compound 1, colourless oil, was established as C14H14N2O2S (nine units of unsaturation) based on the HR-ESI-MS ([M þ H]þ at m/z 275.0852, calcd for C14H15N2O2S, 275.0854). The NMR data for 1 confirmed the presence of 14 carbon signals, including 3 sp3 methylenes, 2 sp3 methines, 5 sp2 methines and 4 sp2 quaternary carbons. 1H NMR chemical shifts of two a-methine protons at dH 4.73 (1H, dd, J ¼ 9.5, 4.5 Hz, H-9) and 4.15 (1H, dd, J ¼ 9.0, 4.0 Hz, H-6) and 13C NMR chemical shifts of two amides at dC 174.6 (C-7) and 174.9 (C-1) supported the presence of a diketopiperazine ring system (Li et al. 2008). The fact that compound 1 was negative to the ninhydrin test suggested a cyclic or an N-terminus-blocked peptide (Shigemori
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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et al. 1998; Huang, Ma et al. 2010; Gao, et al. 2013). Five aromatic proton resonances at dH 7.31 (2H, d, J ¼ 7.5 Hz, H-20 /60 ), 7.26 (2H, dd, J ¼ 7.5, 7.1 Hz, H-30 /50 ) and 7.22 (1H, d, J ¼ 7.1 Hz, H-40 ) and four aromatic carbon resonances at dC 138.5 (C-10 ), 130.4 (C-30 /50 ), 129.6 (C-20 /60 ) and 124.4 (C-40 ) suggested the presence of an alkylbenzene. Given that the molecular formula afforded nine degrees of unsaturation, therefore the molecule was satisfied by the tricyclic skeleton with one thione (dC 181.6). The whole structure was further established by the aid of COSY and HMBC experiments (Figure 1). From the 1H – 1H COSY spectrum of 1, it was possible to differentiate between the key separate spin systems of H2-4/H2-5/H-6 and H-9/H2-10. These data, together with the key HMBC correlations between H2-10/C-1, C-20 and C-60 ; H2-5/C-3 and C-7; H-9/C-10 and C-7 and H-6/C-1 and C-3 (Figure 1), permitted the elucidation of the carbon skeleton of 1. The absolute configuration of compound 1 was determined by the optical rotation and analysis of the coupling constants (J). The coupling patterns of the H-9 (dd, J ¼ 9.5, 4.5 Hz) and Hb-10 (d, J ¼ 9.5 Hz) led to confirmation of the cis-orientation of H-9/Hb-10 and possessed b-orientation (Wang et al. 2008), and thus the configuration of C-9 was determined to be R*. The sign for [a ]D for proline-containing diketopiperazines is either negative or positive, depending only on the absolute configuration of Pro (Adamczeski et al. 1995; Huang, Ma et al. 2010). On the basis of the ½a20 D (2 57) and by comparing the NMR data of the proline residue with those of proline-containing diketopiperazines (Adamczeski et al. 1995; Jayatilake et al. 1996; Huang, Ma et al. 2010), which suggested C-6 has (S)-configuration, Pro in 1 has therefore (S)-configuration, and the above-mentioned data approved the absolute configuration of 1 as 6S and 9R. Four known diketopiperazines were identified as cyclo-(Gly-Pro) (2), cyclo-(Thr-Pro) (3) (Stark & Hofmann 2005), cyclo-(Ile-Pro) (4) (Adamczeski et al. 1995) and cyclo-(Pro-Pro) (5) (Stark & Hofmann 2005) by comparing with previously reported data. Compounds 1– 4 were evaluated for cytotoxicity by the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide method (Mosmann 1983), and exhibited a marginal activity against a small panel of three human tumour HepG2 (hepatoma carcinoma cell), A549 (lung carcinoma cell) and HeLa (cervical cancer cell) cell lines, their inhibition ratios were lower than 10% at a concentration of 100 mg/mL with the IC50 values of the positive control compound 5-Fu 13.70, 2.13 and 3.83 mg/mL, respectively. 3. Experimental 3.1. General experimental procedures NMR spectra were recorded on a Bruker AC 500 NMR spectrometer with TMS as an internal standard (Bruker, Fa¨llanden, Switzerland). ESI-MS data were measured on an Agilent 1200 LC – MS spectrometer (Agilent, Palo Alto, CA, USA). HR-ESI-MS data were obtained from a Bruker Maxis mass spectrometer (Bruker, Bremen, Germany). The silica gel GF254 plates used for TLC were supplied by the Qingdao Marine Chemical Factory (Qingdao, China). Analytical HPLC was performed on a Hitachi L-2400 HPLC system, using a YMC ODS-H80 column
O 3′
HN 5′
1′ 9
7 1
10
O 1
O
O
O
O
6
HN N
HN
HN
3
N S
O 2
Figure 1. Structures of compounds 1 – 5.
N N
N OH
N
O
O
O
3
4
5
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(250 £ 4.6 mm i.d., 4 mm) coupled to an Alltech ELSD 800 detector (Hitachi, Tokyo, Japan); semi-preparative HPLC was performed on a Hitachi L-2400 HPLC system, using a YMC ODSH80 column (250 £ 10 mm i.d., 4 £ mm) coupled to an Alltech ELSD 800 detector with flowsplitter valve (Parker: NS) set at a split ratio of 20:1 (collector:detector) (Hitachi, Tokyo, Japan). Optical rotation data were measured by Perkin-Elmer Model 341 polarimeter (Perkin Elmer, Waltham, MA, USA). Spots were detected on TLC under UV light or by heating after spraying with 5% H2SO4 in EtOH (v/v). 3.2. Animal material The sponge is an undescribed species of Callyspongia (order Haplosclerida, family Callyspongiidae) collected off the coast of Hainan Island, South China Sea, in January 2007. The specimen was identified by Dr Kyung Jin Lee. A voucher specimen (No. 20070101) has been deposited at the Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, China. 3.3. Extraction and isolation The wet sponges (10 kg) were extracted three times with EtOH/H2O (90:10, 20 L). Ethanol was evaporated in vacuo to afford a syrupy residue that was suspended in distilled water and fractionated successively with petroleum ether, ethylacetate and n-butanol. The EtOAc soluble part (28 g) was partitioned between petroleum ether and EtOH/H2O (7:3). The EtOH/H2O soluble part (8.0 g) was chromatographed on a silica gel column (80 g) eluted with petroleum ether/EtOAc/MeOH to yield eight fractions (A – H). Fraction C was subjected to column chromatography with petroleum ether/EtOAc (from 3:7 to 0:10, v/v) as eluent to afford three subfractions (C1 – C3). The subfraction C2 was purified by Sephadex LH-20 (CHCl3/MeOH, 2:8, v:v) to afford 4 (8.2 mg). Fraction F was subjected to column chromatography with EtOAc/ acetone (1:1, v:v) as eluent to afford four subfractions (F1 – F4). The subfraction F3 was subjected to column chromatography with EtOAc/acetone (1:1, v:v) as eluent, then purified by Sephadex LH-20 (CHCl3/MeOH, 2:8, v:v) to afford 3 (17.3 mg). Fraction G was subjected to column chromatography with EtOAc/acetone (1:1, v:v) as eluent to afford two subfractions (G1 and G2). The subfraction G1 was purified by Sephadex LH-20 (CHCl3/MeOH, 2:8, v:v) to afford 2 (5.8 mg). The n-butanol soluble part (80.1 g) was subjected to reversed-phase flash column ˚ , 230 mesh), using EtOH/H2O (from 90:10 to 0:100, chromatography (YMC Gel ODS-A, 60 A v:v) as eluent, yielding six fractions (An –Fn). Fraction Bn was subjected to column chromatography with CHCl3/MeOH (from 8:2 to 4:6, v:v) as eluent to afford three subfractions (Bn-1 – Bn-3). The subfraction Bn-1 was separated by HPLC, using the mixtures of MeOH/H2O (25:75, v:v) to yield 5 (8.9 mg). Fraction En was subjected to column chromatography with CHCl3/MeOH (from 10:0 to 8:2, v:v) as eluent to afford three subfractions (En-1 and En-2). The subfraction En-1 was further subjected to CC with CHCl3/MeOH (95:5), purified by Sephadex LH-20 (CHCl3/MeOH, 0:10, v:v), then separated by HPLC, using the mixtures of MeOH/H2O (15:85, v:v) to yield 1 (5.5 mg). Callysponine A (1), a colourless oil; ½a20 D 2 57 (c 0.31, MeOH). HR-ESI-MS m/z: 275.0852 ([M þ H]þ, calcd for C14H15N2O2S, 275.0854). 1H NMR (500 MHz, CDCl3) dH: 7.31 (2H, d, J ¼ 7.5 Hz, H-20 /60 ), 7.26 (2H, dd, J ¼ 7.5, 7.1 Hz, H-30 /50 ), 7.22 (1H, d, J ¼ 7.1 Hz, H-40 ), 4.73 (1H, dd, J ¼ 9.5, 4.5 Hz, H-9), 4.15 (1H, dd, J ¼ 9.0, 4.0 Hz, H-6), 3.29 (1H, dd, J ¼ 11.0, 9.5 Hz, Hb-10), 3.02 (1H, dd, J ¼ 11.0, 4.5 Hz, Ha-10), 2.40 (1H, m, Hb-4), 2.24 (2H, m, H-5) and 1.95 (1H, m, Ha-4). 13C NMR (125 MHz, CDCl3) dC: 181.6 (C-3), 174.9 (C-1), 174.6 (C-7), 138.5 (C-10 ), 130.4 (C-30 /50 ), 129.6 (C-20 /60 ), 124.4 (C-40 ), 58.1 (C-6), 55.1 (C-9), 38.2 (C-10), 30.1 (C-4) and 26.7 (C-5).
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Cyclo-(Gly-S- Pro) (2), ½a20 D : 2 38.3 (c 0.23, MeOH). Cyclo-(R-Thr-S- Pro) (3), ½a20 D : 2 72.4 (c 1.34, MeOH). Cyclo-(R- Ile-S- Pro) (4), ½a20 D : 2 40.4 (c 0.65, MeOH). Cyclo-(R- Pro-R- Pro) (5), ½a20 D : þ 15.3 (c 0.92, MeOH). 3.4. Cytotoxicity assay The cytotoxicity assay was carried out by the reported method (Mosmann 1983).
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4. Conclusion Diketopiperazines, which are a relatively unexplored class of bioactive peptides that may hold great promise for the future, have been detected in a variety of natural resources (Huang, Zhou et al. 2010). In the course of our investigation on the marine sponge Callyspongia species, we have reported some metabolites (Huang, Ma et al. 2010; Huang et al. 2011). Further investigation on the marine sponge Callyspongia species has led to the isolation and characterization of five diketopiperazines (1– 5), including a new one, callysponine A (1). Compounds 1– 5 did not inhibit the growth of HepG2 (hepatoma carcinoma cell), A549 (lung carcinoma cell) and HeLa (cervical cancer cell) cell lines. Supplementary material Supplementary material relating to this article is available online, alongside Figures S2 – S6. Acknowledgements This study was supported by grants from the National Natural Science Foundation of China (No. 31100260), Knowledge Innovation Program of Chinese Academy of Sciences (No. KSCX2-EW-J-28), Program of Guangzhou City (No.12C14061559) and Foundation of Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences (No. 201210ZS).
References Adamczeski M, Reed AR, Crews P. 1995. New and known diketopeperazines from the Caribbean sponge, Calyx cf. podatypa. J Nat Prod. 58:201–208. Gao CH, Lin L, Long B, Chen YN, He BJ, Sun HY, Huang RM. 2013. A new diketopiperazine from the gorgonian coral Menella kanisa. Nat Prod Res. doi:10.1080/147864192013879134. Huang RM, Ma W, Dong JD, Zhou XF, Xu TH, Lee KJ, Yang XW, Xu SH, Liu YH. 2010. A new 1,4-diazepine form South China Sea marine sponge Callyspongia species. Molecules. 15:871–877. Huang RM, Zhou XF, Xu TH, Yang XW, Liu YH. 2010. Diketopiperazines from marine organisms. Chem Biodivers. 7:2809–2829. Huang RM, Zhou XF, Peng Y, Yang XW, Xu TH, Liu YH. 2011. Nucleosides from the marine sponge Callyspongia sp. Chem Nat Compd. 46:1010– 1011. Jayatilake GS, Thornton MP, Leonard AC, Grimwade JE, Baker BJ. 1996. Metabolites from an Antarctic spongeassociated bacterium, Pseudomonas aeruginosa. J Nat Prod. 59:293–296. Li ZY, Peng CS, Shen Y, Miao XL, Zhang HJ, Lin HW. 2008. L,L-diketopeperazines form Alcaligenes faecalis A72 associated with South China Sea sponge Stelletta tenuis. Biochem Syst Ecol. 36:230–234. Mosmann T. 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assay. J Immumol Methods. 65:55–63. Shigemori H, Tenma M, Shimazaki K, Kobayashi J. 1998. Three new metabolites from the marine yeast Aureobasidium pullulans. J Nat Prod. 61:696–698. Stark T, Hofmann T. 2005. Structures, sensory activity, and dose/response functions of 2,5-diketopiperazines in roasted cocoa nibs Theobroma cacao. J Agric Food Chem. :7222–7231.
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Wang YL, Li ZL, Zhang HL, Sha Y, Pei YH, Hua HM. 2008. New germacrane sequiterpenes from Salvia chinensis. Chem Pharm Bull. 56:843–863. Yang B, Hu J, Lei H, Chen XQ, Zhou XF, Liu YH. 2012. Chemical constituents of marine sponge Callyspongia sp. from the South China Sea. Chem Nat Compd. 48:350–351. Xie LR, Li DY, Wang PL, Hua HM, Wu X, Li ZL. 2013. New 3,4-seco-lanostane triterpenoid from a marine-derived fungus Ascotrich sp. zi-m-5. Yaoxue XueBao. 48:89–93.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
A new diketopiperazine from South China Sea marine sponge Callyspongia sp. a
b
c
a
Yinning Chen , Yan Peng , Chenghai Gao & Riming Huang a
Key Laboratory of Plant Resource Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510-650, China b
Life Science & Technology School, Zhanjiang Normal University, Zhanjiang 524-048, China c
Guangxi Key Laboratory of Marine Environmental Science, Guangxi Academy of Sciences, Nanning 530-007, China Published online: 04 Apr 2014.
To cite this article: Yinning Chen, Yan Peng, Chenghai Gao & Riming Huang (2014) A new diketopiperazine from South China Sea marine sponge Callyspongia sp., Natural Product Research: Formerly Natural Product Letters, 28:13, 1010-1014, DOI: 10.1080/14786419.2014.903397 To link to this article: http://dx.doi.org/10.1080/14786419.2014.903397
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
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Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 Vol. 28, No. 13, 1010–1014, http://dx.doi.org/10.1080/14786419.2014.903397
A new diketopiperazine from South China Sea marine sponge Callyspongia sp. Yinning Chena, Yan Pengb, Chenghai Gaoc and Riming Huanga* a
Downloaded by [Tulane University] at 00:12 04 September 2014
Key Laboratory of Plant Resource Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510-650, China; bLife Science & Technology School, Zhanjiang Normal University, Zhanjiang 524-048, China; cGuangxi Key Laboratory of Marine Environmental Science, Guangxi Academy of Sciences, Nanning 530-007, China (Received 3 January 2014; final version received 6 March 2014) Further chemical investigation on the marine sponge Callyspongia sp. collected from South China Sea led to the isolation of a new diketopiperazine, named callysponine A (1), as well as four known diketopiperazines, namely cyclo-(Gly-Pro) (2), cyclo-(ThrPro) (3), cyclo-(Ile-Pro) (4) and cyclo-(Pro-Pro) (5). The new structure was determined on the basis of NMR and MS analysis, and the absolute stereochemistry was defined by analysis of the coupling constants and optical rotation. The structures of the known compounds were identified by comparing their spectroscopic data with those reported in the literature. Compounds 1 – 5 did not inhibit the growth of HepG2 (hepatoma carcinoma cell), A549 (lung carcinoma cell) and HeLa (cervical cancer cell) cell lines. Keywords: Callyspongia sp.; diketopiperazine; callysponine A
1. Introduction Till date, there are a few reports on diketopiperazines from marine sponges (Huang, Zhou et al. 2010). The genus Callyspongia (order Haplosclerida, family Callyspongiidae) is widely distributed and contains various bioactive constituents (Yang et al. 2012). In the course of our investigation on the marine sponge Callyspongia species, we have reported some metabolites (Huang, Ma et al. 2010; Huang et al. 2011). Further investigation on the marine sponge Callyspongia species has led to the isolation and characterisation of a new diketopiperazine, named callysponine A (1), as well as four known diketopiperazines, namely cyclo-(Gly-Pro) (2) (Xie et al. 2013), cyclo-(Thr-Pro) (3) (Stark & Hofmann 2005), cyclo-(Ile-Pro) (4) (Adamczeski et al. 1995) and cyclo-(Pro-Pro) (5) (Stark & Hofmann 2005). 2. Results and discussion Compound 1, colourless oil, was established as C14H14N2O2S (nine units of unsaturation) based on the HR-ESI-MS ([M þ H]þ at m/z 275.0852, calcd for C14H15N2O2S, 275.0854). The NMR data for 1 confirmed the presence of 14 carbon signals, including 3 sp3 methylenes, 2 sp3 methines, 5 sp2 methines and 4 sp2 quaternary carbons. 1H NMR chemical shifts of two a-methine protons at dH 4.73 (1H, dd, J ¼ 9.5, 4.5 Hz, H-9) and 4.15 (1H, dd, J ¼ 9.0, 4.0 Hz, H-6) and 13C NMR chemical shifts of two amides at dC 174.6 (C-7) and 174.9 (C-1) supported the presence of a diketopiperazine ring system (Li et al. 2008). The fact that compound 1 was negative to the ninhydrin test suggested a cyclic or an N-terminus-blocked peptide (Shigemori
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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et al. 1998; Huang, Ma et al. 2010; Gao, et al. 2013). Five aromatic proton resonances at dH 7.31 (2H, d, J ¼ 7.5 Hz, H-20 /60 ), 7.26 (2H, dd, J ¼ 7.5, 7.1 Hz, H-30 /50 ) and 7.22 (1H, d, J ¼ 7.1 Hz, H-40 ) and four aromatic carbon resonances at dC 138.5 (C-10 ), 130.4 (C-30 /50 ), 129.6 (C-20 /60 ) and 124.4 (C-40 ) suggested the presence of an alkylbenzene. Given that the molecular formula afforded nine degrees of unsaturation, therefore the molecule was satisfied by the tricyclic skeleton with one thione (dC 181.6). The whole structure was further established by the aid of COSY and HMBC experiments (Figure 1). From the 1H – 1H COSY spectrum of 1, it was possible to differentiate between the key separate spin systems of H2-4/H2-5/H-6 and H-9/H2-10. These data, together with the key HMBC correlations between H2-10/C-1, C-20 and C-60 ; H2-5/C-3 and C-7; H-9/C-10 and C-7 and H-6/C-1 and C-3 (Figure 1), permitted the elucidation of the carbon skeleton of 1. The absolute configuration of compound 1 was determined by the optical rotation and analysis of the coupling constants (J). The coupling patterns of the H-9 (dd, J ¼ 9.5, 4.5 Hz) and Hb-10 (d, J ¼ 9.5 Hz) led to confirmation of the cis-orientation of H-9/Hb-10 and possessed b-orientation (Wang et al. 2008), and thus the configuration of C-9 was determined to be R*. The sign for [a ]D for proline-containing diketopiperazines is either negative or positive, depending only on the absolute configuration of Pro (Adamczeski et al. 1995; Huang, Ma et al. 2010). On the basis of the ½a20 D (2 57) and by comparing the NMR data of the proline residue with those of proline-containing diketopiperazines (Adamczeski et al. 1995; Jayatilake et al. 1996; Huang, Ma et al. 2010), which suggested C-6 has (S)-configuration, Pro in 1 has therefore (S)-configuration, and the above-mentioned data approved the absolute configuration of 1 as 6S and 9R. Four known diketopiperazines were identified as cyclo-(Gly-Pro) (2), cyclo-(Thr-Pro) (3) (Stark & Hofmann 2005), cyclo-(Ile-Pro) (4) (Adamczeski et al. 1995) and cyclo-(Pro-Pro) (5) (Stark & Hofmann 2005) by comparing with previously reported data. Compounds 1– 4 were evaluated for cytotoxicity by the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide method (Mosmann 1983), and exhibited a marginal activity against a small panel of three human tumour HepG2 (hepatoma carcinoma cell), A549 (lung carcinoma cell) and HeLa (cervical cancer cell) cell lines, their inhibition ratios were lower than 10% at a concentration of 100 mg/mL with the IC50 values of the positive control compound 5-Fu 13.70, 2.13 and 3.83 mg/mL, respectively. 3. Experimental 3.1. General experimental procedures NMR spectra were recorded on a Bruker AC 500 NMR spectrometer with TMS as an internal standard (Bruker, Fa¨llanden, Switzerland). ESI-MS data were measured on an Agilent 1200 LC – MS spectrometer (Agilent, Palo Alto, CA, USA). HR-ESI-MS data were obtained from a Bruker Maxis mass spectrometer (Bruker, Bremen, Germany). The silica gel GF254 plates used for TLC were supplied by the Qingdao Marine Chemical Factory (Qingdao, China). Analytical HPLC was performed on a Hitachi L-2400 HPLC system, using a YMC ODS-H80 column
O 3′
HN 5′
1′ 9
7 1
10
O 1
O
O
O
O
6
HN N
HN
HN
3
N S
O 2
Figure 1. Structures of compounds 1 – 5.
N N
N OH
N
O
O
O
3
4
5
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(250 £ 4.6 mm i.d., 4 mm) coupled to an Alltech ELSD 800 detector (Hitachi, Tokyo, Japan); semi-preparative HPLC was performed on a Hitachi L-2400 HPLC system, using a YMC ODSH80 column (250 £ 10 mm i.d., 4 £ mm) coupled to an Alltech ELSD 800 detector with flowsplitter valve (Parker: NS) set at a split ratio of 20:1 (collector:detector) (Hitachi, Tokyo, Japan). Optical rotation data were measured by Perkin-Elmer Model 341 polarimeter (Perkin Elmer, Waltham, MA, USA). Spots were detected on TLC under UV light or by heating after spraying with 5% H2SO4 in EtOH (v/v). 3.2. Animal material The sponge is an undescribed species of Callyspongia (order Haplosclerida, family Callyspongiidae) collected off the coast of Hainan Island, South China Sea, in January 2007. The specimen was identified by Dr Kyung Jin Lee. A voucher specimen (No. 20070101) has been deposited at the Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, China. 3.3. Extraction and isolation The wet sponges (10 kg) were extracted three times with EtOH/H2O (90:10, 20 L). Ethanol was evaporated in vacuo to afford a syrupy residue that was suspended in distilled water and fractionated successively with petroleum ether, ethylacetate and n-butanol. The EtOAc soluble part (28 g) was partitioned between petroleum ether and EtOH/H2O (7:3). The EtOH/H2O soluble part (8.0 g) was chromatographed on a silica gel column (80 g) eluted with petroleum ether/EtOAc/MeOH to yield eight fractions (A – H). Fraction C was subjected to column chromatography with petroleum ether/EtOAc (from 3:7 to 0:10, v/v) as eluent to afford three subfractions (C1 – C3). The subfraction C2 was purified by Sephadex LH-20 (CHCl3/MeOH, 2:8, v:v) to afford 4 (8.2 mg). Fraction F was subjected to column chromatography with EtOAc/ acetone (1:1, v:v) as eluent to afford four subfractions (F1 – F4). The subfraction F3 was subjected to column chromatography with EtOAc/acetone (1:1, v:v) as eluent, then purified by Sephadex LH-20 (CHCl3/MeOH, 2:8, v:v) to afford 3 (17.3 mg). Fraction G was subjected to column chromatography with EtOAc/acetone (1:1, v:v) as eluent to afford two subfractions (G1 and G2). The subfraction G1 was purified by Sephadex LH-20 (CHCl3/MeOH, 2:8, v:v) to afford 2 (5.8 mg). The n-butanol soluble part (80.1 g) was subjected to reversed-phase flash column ˚ , 230 mesh), using EtOH/H2O (from 90:10 to 0:100, chromatography (YMC Gel ODS-A, 60 A v:v) as eluent, yielding six fractions (An –Fn). Fraction Bn was subjected to column chromatography with CHCl3/MeOH (from 8:2 to 4:6, v:v) as eluent to afford three subfractions (Bn-1 – Bn-3). The subfraction Bn-1 was separated by HPLC, using the mixtures of MeOH/H2O (25:75, v:v) to yield 5 (8.9 mg). Fraction En was subjected to column chromatography with CHCl3/MeOH (from 10:0 to 8:2, v:v) as eluent to afford three subfractions (En-1 and En-2). The subfraction En-1 was further subjected to CC with CHCl3/MeOH (95:5), purified by Sephadex LH-20 (CHCl3/MeOH, 0:10, v:v), then separated by HPLC, using the mixtures of MeOH/H2O (15:85, v:v) to yield 1 (5.5 mg). Callysponine A (1), a colourless oil; ½a20 D 2 57 (c 0.31, MeOH). HR-ESI-MS m/z: 275.0852 ([M þ H]þ, calcd for C14H15N2O2S, 275.0854). 1H NMR (500 MHz, CDCl3) dH: 7.31 (2H, d, J ¼ 7.5 Hz, H-20 /60 ), 7.26 (2H, dd, J ¼ 7.5, 7.1 Hz, H-30 /50 ), 7.22 (1H, d, J ¼ 7.1 Hz, H-40 ), 4.73 (1H, dd, J ¼ 9.5, 4.5 Hz, H-9), 4.15 (1H, dd, J ¼ 9.0, 4.0 Hz, H-6), 3.29 (1H, dd, J ¼ 11.0, 9.5 Hz, Hb-10), 3.02 (1H, dd, J ¼ 11.0, 4.5 Hz, Ha-10), 2.40 (1H, m, Hb-4), 2.24 (2H, m, H-5) and 1.95 (1H, m, Ha-4). 13C NMR (125 MHz, CDCl3) dC: 181.6 (C-3), 174.9 (C-1), 174.6 (C-7), 138.5 (C-10 ), 130.4 (C-30 /50 ), 129.6 (C-20 /60 ), 124.4 (C-40 ), 58.1 (C-6), 55.1 (C-9), 38.2 (C-10), 30.1 (C-4) and 26.7 (C-5).
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Cyclo-(Gly-S- Pro) (2), ½a20 D : 2 38.3 (c 0.23, MeOH). Cyclo-(R-Thr-S- Pro) (3), ½a20 D : 2 72.4 (c 1.34, MeOH). Cyclo-(R- Ile-S- Pro) (4), ½a20 D : 2 40.4 (c 0.65, MeOH). Cyclo-(R- Pro-R- Pro) (5), ½a20 D : þ 15.3 (c 0.92, MeOH). 3.4. Cytotoxicity assay The cytotoxicity assay was carried out by the reported method (Mosmann 1983).
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4. Conclusion Diketopiperazines, which are a relatively unexplored class of bioactive peptides that may hold great promise for the future, have been detected in a variety of natural resources (Huang, Zhou et al. 2010). In the course of our investigation on the marine sponge Callyspongia species, we have reported some metabolites (Huang, Ma et al. 2010; Huang et al. 2011). Further investigation on the marine sponge Callyspongia species has led to the isolation and characterization of five diketopiperazines (1– 5), including a new one, callysponine A (1). Compounds 1– 5 did not inhibit the growth of HepG2 (hepatoma carcinoma cell), A549 (lung carcinoma cell) and HeLa (cervical cancer cell) cell lines. Supplementary material Supplementary material relating to this article is available online, alongside Figures S2 – S6. Acknowledgements This study was supported by grants from the National Natural Science Foundation of China (No. 31100260), Knowledge Innovation Program of Chinese Academy of Sciences (No. KSCX2-EW-J-28), Program of Guangzhou City (No.12C14061559) and Foundation of Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences (No. 201210ZS).
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