Alkaloids from marine bryozoan Cryptpsula pallasiana

Biochemical Systematics and Ecology 38 (2010) 1250–1252

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Alkaloids from marine bryozoan Cryptpsula pallasiana Xiang-Rong Tian a, b, Hai-Feng Tang a, *, Yu-Shan Li b, **, Hou-Wen Lin c, Xin-Yi Tong a, d, Ning Ma a a

Department of Pharmacy, Xijing Hospital, Fourth Military Medical University, 15 Changle West Rd., Xi’an 710032, PR China School of Traditional Chinese Medicines, Shenyang Pharmaceutical University, 103 Wenhua Rd., Shenyang 110016, PR China c Department of Pharmacy, Changzheng Hospital, Second Military Medical University, Shanghai 200433, PR China d School of Pharmacy, Fudan University, Shanghai 200433, PR China b

a r t i c l e i n f o Article history: Received 15 September 2010 Accepted 21 December 2010 Available online 13 January 2011 Keywords: Marine bryozoan Cryptpsula pallasiana Alkaloids Brominated alkaloids

1. Subject and source Cryptosula pallasiana (Moll, 1803), a marine bryozoan belonging to the order of Cheilostomata and the Family of Cryptosulidae, often occurs along the coast of China. The material used in this experiment was collected in Huang Island of Qingdao, Shandong Province of China, in March 2009, and was identified by Prof. Hou-Wen Lin. A voucher specimen (No: QD-0903-1) was deposited in the herbarium of the Department of Pharmacy, Changzheng Hospital, Second Military Medical University, Shanghai, China.

2. Previous work Our previous work focused on the secondary metabolites of another marine bryozoan Bugula neritina (Tian et al., 2009, 2010). The elemental composition including organic matters, CO2, calcium, magnesium, strontium, barium, phosphorus and iron, have been analyzed from C. pallasiana (Schopf and Manheim, 1967). However, to the best of our knowledge, exact chemical constituents from this genus have not been reported yet.

* Corresponding author. Tel./fax: þ86 29 84775471. ** Corresponding author. Department of Pharmacognosy, School of Traditional Chinese Medicines, Shenyang Pharmaceutical University, 103 Wenhua Rd., Shenyang 110016, PR China. Tel./fax: þ86 24 23986496. E-mail addresses: [email protected] (H.-F. Tang), [email protected] (Y.-S. Li). 0305-1978/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2010.12.019

X.-R. Tian et al. / Biochemical Systematics and Ecology 38 (2010) 1250–1252

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3. Present study The fresh animal of C. pallasiana (about 20 kg, dry weight after extraction) was exhaustively extracted with 95% EtOH at room temperature. The extract solution was filtered and concentrated in vacuum to afford aqueous extract (600 g), which was extracted with EtOAc, and then the EtOAc extract (63.3 g) was partitioned between 90% aqueous MeOH and petroleum ether (1:1). The MeOH solution was adjusted to 80% aqueous MeOH and extracted with CCl4 (1:1) to give CCl4 extract (12.9 g). And then the MeOH solution was adjusted to 60% aqueous MeOH and extracted with CH2Cl2 (1:1) to afford CH2Cl2 extract (8.2 g). The CCl4 extract and the CH2Cl2 extract were subjected to column chromatography (CC) over Sephadex LH-20 column (CHCl3/MeOH, 1:1) to afford three fractions, respectively, and then the obtained six fractions were combined to yield three major fractions (Frs. A–C) based on TLC analysis. Fraction B (0.9 g) was subjected to CC over reversed-phase Si gel (Lichroprep RP-18, 40–63 mm) using MeOH/H2O (30%–70%) to give three major fractions (Frs. B1–B3). Fraction B1 (559.6 mg) was purified by semi-preparative HPLC (YMC-Pack R&D ODS-A, 5 mm, 250 mm  20 mm, UV detection at 206 nm) using MeOH/H2O (45%) as the mobile phase at a flow rate of 6.0 mL/min to afford five sub-fractions (S-Frs. 1–5). Sub-fraction 1 was further purified by above mentioned semi-preparative HPLC using MeOH/H2O (10%) as the mobile phase at a flow rate of 8.0 mL/min to afford aldisin (1, 25.6 mg, tR ¼ 64.6 min) (Schmitz et al., 1985; Li et al., 1998). Sub-fraction 2 was further purified by the same semipreparative HPLC using MeOH/H2O (20%) as the mobile phase at a flow rate of 6.0 mL/min to afford 2-bromoaldisin (2, 11.0 mg, tR ¼ 106.2 min) (Schmitz et al., 1985; Li et al., 1998) and 2,4(1H,3H)-quinazolinedione (6, 7.8 mg, tR ¼ 64.9 min) (Xu et al., 2005). Sub-fraction 2 was further purified by the same mentioned semi-preparative HPLC using MeOH/H2O (35%) as the mobile phase at a flow rate of 6.0 mL/min to afford indole-3-carboxyaldehyde (4, 35.4 mg, tR ¼ 44.9 min) (Chowdhury and Chakraborty, 1971; Aldrich, 1992; Hassan et al., 2004) and indole-3-oxoacetamide (5, 7.2 mg, tR ¼ 48.6 min) (Bao et al., 2007). Fraction C (176.3 mg) was purified by the above mentioned semi-preparative HPLC using MeOH/H2O (55%) as the mobile phase at a flow rate of 7.0 mL/min to afford 6-bromoindole-3-carbaldehyde (3, 7.1 mg, tR ¼ 40.3 min) (Rasmussen et al., 1993) and another two sub-fractions C1 and C2, C2 was further purified by the same mentioned semi-preparative HPLC using MeOH/H2O (45%) as the mobile phase at a flow rate of 6.0 mL/min to afford 7-bromo-2,4(1H,3H)-quinazolinedione (7, 3.0 mg, tR ¼ 58.5 min) (Niwa et al., 1988). The structures were established conclusively by MS, extensive 1H NMR, 13C NMR, DEPT, HSQC, HMBC and 1H–1H COSY spectra analysis and comparison with literature data (Fig. 1). Some spectral data and physical constants have not been reported yet these data are presented as follows. 3.1. 2,4(1H,3H)-quinazolinedione (6) Light yellow solid, mp 354–356  C (dec); 1H NMR and 13C NMR data, see Table 1; EI-MS m/z: 162 [M]þ (100),119 [M–CO–NH]þ (92), 92 (65), 64 (37), 53 (16). 3.2. 7-Bromo-2,4(1H,3H)-quinazolinedione (7) Light yellow solid, mp 335–337  C (dec); 1H NMR and 13C NMR data, see Table 1; EI-MS m/z: 242/240 [M þ 2]þ/[M]þ (100), 199/197 [M þ 2–CO–NH]þ/[M–CO–NH]þ (75), 172/170 (47), 144/142 (9), 105 (15), 90 (33), 63 (56), 53 (20). 4. Chemotaxonomic and ecological significance Most bryozoan secondary metabolites are alkaloids and the large majority of them have novel structures. Heterocyclic ring systems which are represented include pyrrole, pyridine, indole, isoquinoline, purine and b-carboline as well as numerous new polycyclic systems (Blackman and Walls, 1995). This study, to the best of our knowledge, is the first report the chemical constituents of seven alkaloids from marine bryozoan C. pallasiana, which correspond to two pyrrole alkaloids (1 and 2), three indole alkaloids (3–5) and two quinazolinedione alkaloids (6 and 7). Pyrrole alkaloids (1 and 2) and indole alkaloids (3–5) have been previously obtained from several marine sponges such as Axinella carteri (Li et al., 1998), Hymeniacidon aldis (Schmitz et al., 1985), Pseudosuberites hyalinus (Rasmussen et al., 1993), Hamigera hamigera (Hassan et al., 2004) and Spongosorites sp. (Bao et al., 2007), respectively. As bryozoans typically have a simple associated microbial community relative to sponges (Haygood et al., 1999), we assume that alkaloids 1–5 as common secondary metabolites between C. pallasiana and related marine sponges are produced by associated microbial community from the ecological perspective.

O R

O

O

O NH2

H N H

1 R=H

NH O 2 R=Br

R

N H 3 R=Br

O

N H 4 R=H

5

Fig. 1. Chemical structures of alkaloids 1–7.

NH R 6 R=H

N H

O

7 R=Br

1252 Table 1 1 H NMR (500 MHz) and

X.-R. Tian et al. / Biochemical Systematics and Ecology 38 (2010) 1250–1252

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No.

1 2 3 4 4a 5 6 7 8 8a a

C NMR (125 MHz) data of compounds 6 and 7 in DMSO-d6a. 6

7

dC, m

dH, m (J in Hz)

dC, m

dH, m (J in Hz)

– 150.3 – 162.8 114.3 126.9 122.3 134.9 115.3 140.9

11.2 – 11.2 – – 7.87 7.16 7.63 7.18 –

– 150.1 – 162.2 113.6 129.0 125.2 128.2 117.8 142.0

11.4 – 11.4 – – 7.79 7.33 – 7.35 –

(s) (s) (s) (d) (d) (d) (d) (s)

(1H, brs) (1H, brs)

(1H, (1H, (1H, (1H,

dd, 8.2, 1.5) m) td, 8.3, 1.6) m)

(s) (s) (s) (d) (d) (s) (d) (s)

(1H, brs) (1H, brs)

(1H, d, 8.4) (1H, dd, 8.4, 1.6) (1H, dd, 1.6)

The resonance assignments were confirmed by DEPT, COSY, HSQC and HMBC spectra.

To the best of our knowledge, the orders Cheilostomata and Ctenostomata belong to the same class of Gymnolaemata, the presence of pyrrole alkaloids (1 and 2) in C. pallasiana (Cheilostomata) may be of chemtaxonomic value for the order of Cheilostomata, as this type of alkaloids have been isolated from the same order of Cheilostomata in marine bryozoans such as Sessibugula translucens (Christophersen, 1985), Bugula dentate (Matsunaga et al., 1986) and Bugula pacifica (Shellenberger and Ross, 1998), but they are not widely distributed in another order of Ctenostomata. However, indole alkaloids and brominated alkaloids have been isolated from the order of Cheilostomata in some marine bryozoans such as Chartella papyracea (Christophersen, 1985), Flustra foliacea (Christophersen, 1985), and Hincksinoflustra denticulata (Blackman et al., 1987), and also isolated from the order of Ctenostomata such as marine bryozoans Zoobotryon verticillatum (Sato and Fenical, 1983), Amathia alternata (Fenical, 1993) and Amathia wilsoni (Morris and Prinsep, 1999). Therefore, the appearance of indole alkaloids (3–5) and brominated alkaloids (2, 3 and 7) in C. pallasiana confirms that indole alkaloids and brominated alkaloids are characteristic chemical composition in the class of Gymnolaemata. Actually, the natural occurrence of 2,4(1H,3H)-quinazolinedione compounds are quite rare (Johne, 1986). To date, although 6 has been isolated from a terrestrial plant Isatis indigotica (Xu et al., 2005), and 7 has been isolated from a marine tunicate Pyura sacciformis (Niwa et al., 1988), the alkaloids (6 and 7) with a nucleus of 2,4(1H,3H)-quinazolinedione are isolated for the first time from C. pallasiana in the marine bryozoans, which implies that they might be specific chemical constituents for this species. Interestingly, since the alkaloids with a nucleus of 2,4(1H,3H)-quinazolinedione are distributed both in a terrestrial plant and marine sources, further research between those compounds and related organisms should be studied. Acknowledgements Thanks are due to Mr. Min-Chang Wang and Mrs. Hui-Min Wang for the measurements of NMR and MS spectra. This work was financially supported by National High Technology Research and Development Program Project of China (863 Project, 2007AA09Z401). Appendix. Supplementary data Supplementary data associated with this article can be found in the on-line version, at doi:10.1016/j.bse.2010.12.019. References Aldrich Library of 13C and 1H FT NMR Spectra, 1992. Sigma Aldrich. Bao, B., Zhang, P., Lee, Y., Hong, J., Lee, C.O., Jung, J.H., 2007. Mar. Drugs 5, 31. Blackman, A.J., Hambley, T.W., Picker, K., Taylor, W.C., Thirasasana, N., 1987. Tetrahedron Lett. 28, 5561. Blackman, A.J., Walls, J.T., 1995. Stud. Nat. Prod. Chem. 17, 73. Chowdhury, B.K., Chakraborty, D.P., 1971. Phytochemistry 10, 481. Christophersen, C., 1985. Acta Chem. Scand. B 39, 517. Fenical, W., 1993. Private Commun. Hassan, W., Edrada, R.A., Ebel, R., Wray, V., Proksch, P., 2004. Mar. Drugs 2, 88. Haygood, M.G., Schmidt, E.W., Davidson, S.K., Faulkner, D.J., 1999. J. Mol. Microbiol. Biotechnol. 1, 33. Johne, S., 1986. In: Brossi, A. (Ed.), The Alkaloids. Academic Press, New York, pp. 99–135. Li, C.-J., Schmitz, F.J., Kelly-Borges, M., 1998. J. Nat. Prod. 61, 387. Matsunaga, S., Fusetani, N., Hashimoto, K., 1986. Experientia 42, 84. Morris, B.D., Prinsep, M.R., 1999. J. Nat. Prod. 62, 688. Niwa, H., Yoshida, Y., Yamada, K., 1988. J. Nat. Prod. 51, 343. Rasmussen, T., Jensen, J., Anthoni, U., Christophersen, C., Nielsen, P.H., 1993. J. Nat. Prod. 56, 1553. Sato, A., Fenical, W., 1983. Tetrahedron Lett. 24, 481. Schmitz, F.J., Gunasekera, S.P., Lakshni, V., Tillekeratne, L.M.V., 1985. J. Nat. Prod. 48, 47. Schopf, T.J.M., Manheim, F.T., 1967. J. Paleontol. 41, 1197. Shellenberger, J.S., Ross, J.R.P., 1998. Northwest Sci. 72, 23.