Prepatellamide A, a new cyclic peptide from the ascidian Lissoclinum patella

Vol. 43 No. 6 SCIENCE IN CHINA (Series B) December 2000 Prepatellamide A, a new cyclic peptide from the ascidian Lissoclinum patella FU Xiong ( ), SU Jingyu ( ) & ZENG Longmei ( ) Department of Chemistry, Zhongshan University, Guangzhou 510275, China Correspondence should be addressed to Zeng Longmei (email: ceszlm@zsu.edu.cn) Received June 27, 2000 Abstract A new cyclic peptide, prepatellamide A (1), along with three known cyclic peptides (2) (4), was isolated from the ascidian Lissoclinum patella. The structure of prepatellamide A was determined from one- and two-dimensional 1 H and 13 C NMR spectra. The known cyclic peptides were identified as patellamides A (2), B (3) and C (4). Keywords: ascidian, Lissoclinum patella, cyclopeptide, cytotoxicity. Ascidians have been proven to be rich sources of biologically active cyclic peptides [1]. A variety of cyclic peptides have been isolated from the ascidian Lissoclinum patella Gottshaldt (Didemnidae) and some of them exhibit strong in vitro cytotoxicity [2 15]. In the continuation of our search for bioactive compounds from marine organisms, we have examined the cytotoxic extracts of L. patella, and isolated a new cyclic peptide, prepatellamide A (1), along with three known ones, patellamides A (2), B (3) and C (4) [11, 12] (see scheme 1). We report here the isolation, structural determination of these compounds. 1 Experimental 1.1 General experimental procedures All solvents were redistilled. Merck Si gel 60 (230 240 mesh) was used for vacuum flash chromatography. HPLC was conducted by a UV detector (235 nm). Optical rotation was taken on a Rudolph Autopol III automatic polarimeter. NMR experiments were conducted with varian VXR-500 instrument, and signals are reported in parts per million (δ ). FABMS were measured on a VG ZAB E mass spectrometer. 1.2 Animal material The ascidian Lissoclinum patella was collected in 1995, in Indonesia, and frozen shortly after collection. 1.3 Extraction and isolation Freshly thawed specimens (487 g in wet wt.; 49 g in dry wt. after extraction) were cut into small pieces and extracted with MeOH (2 1 L) and then MeOH-CH 2 Cl 2 (1 1). The extracts were concentrated in vacuo and combined to give a residue which was subjected to solvent parti-

644 SCIENCE IN CHINA (Series B) Vol. 43 Scheme 1 tioning as described previously [16]. Three organic fractions were obtained after removal of solvents: hexane (0.50 g); CH 2 Cl 2 (1.07 g), and n-buoh (0.62 g). The CH 2 Cl 2 solubles showed cytotoxicity against P388 cell lines and were therefore fractionated on an open column of silica gel using increasing amount of EtOAc in hexane (40% to 100% EtOAc) as eluent. Six fractions were collected. Predominant compounds patellamide A (2) and patellamide B (3) were obtained from the third (60% EtOAc in hexane eluate) and fifth (80% EtOAc in hexane eluate) fractions, respectively, after removal of the solvents. Reversed-phase HPLC of fraction six (EtOAc eluate) using 25% H 2 O in MeOH as eluent afforded patellamide C (4) as the major component in addition to small amount of patellamide B(3), and the new peptide prepatellamide A (1). Prepatellamide A (1) (3.0 mg): amorphous solid, [α] D +80.1 (c 0.1, MeOH); 1 H and 13 C NMR, see tables 1 and 2; FABMS m/z 761 [M + H] + ; HRFABMS m/z 761.348 9 [M + H] +, calcd. for C 35 H 53 N 8 O 7 S 2 761.347 9. Patellamide A (2) (45 mg): amorphous powder, [α] D +114.0 (c 1.0, CH 2 Cl 2 ), literature +113.9 [11] ; FABMS m/z 743 [M + H] + ; NMR data (see tables 1 and 2) identical to literature [11]. Patellamide B (3) (37 mg): amorphous solid, [α] D +43.0 (c 0.86, CH 2 Cl 2 ), literature

No. 6 PREPATELLAMIDE A, A NEW CYCLIC PEPTIDE FROM ASCIDIAN 645 +29.4 [11] ; +50.6 (synthetic) [17] ; FABMS m/z 777 [M + H] + ; NMR data (see tables 1 and 2) identical to literature [11]. Patellamide C (4)(12 mg): amorphous solid, [α] D +23.7 (c 3.04, CH 2 Cl 2 ), literature +19 [11] ; +32 (synthetic) [17] ; FABMS m/z 763 [M + H] +, 785 [M + Na] + ; NMR data (see tables 1 and 2) identical to literature [11]. Table 1 1 H NMR data for compounds 1 4 a) Position Prepatellamide A (1) b) Patellamide A (2) b) Patellamide B (3) b) Patellamide C (4) b) 2 4.73(m) 4.73(dd, 10.8, 6.0) 4.34(d, 2.5) 4.35(d, 2.6) 3 4.84(dd, 9.5, 4.0) 4.56(dd, 8.8, 6.0) 4.49(dd, 9.5, 9.0) 4.48(dd, 10.8, 8.8) 4.97(m) 4.98(m) 5 4.63(m) 4.72(m) 1.42(d, 6.0) 1.46(d, 6.3) 6 2.17(m) 1.90(m) 4.96(m) 4.52(dd, 7.4, 11.6) 7 1.60(m); 1.17(m) 1.25(m);1.10(m) 2.13(m); 1.57(m) 2.31(m) 8 0.83(t, 7.5) 0.67(t, 7.5) 1.66(m) 1.09(d, 6.9) 9 0.84(d, 6.5) 0.75(d, 6.0) 1.06(d, 6.5) 1.08(d, 6.3) 10 1.00(d, 6.5) 12 7.61(br s) 7.74(s) 7.43(s) 13 7.40(s) 14 5.27(m) 5.14(m) 5.49(dt, 5.8, 10.0) 15 2.34(m) 2.24(m) 5.45(dt, 6.0, 10.0) 3.44(dd,14.3,10.0) 3.29(dd, 14.3, 5.8) 16 1.08(d, 7.0) 1.02(d, 6.5) 3.40(dd,14.0,10.0) 3.27(dd, 14.0, 6.0) 17 1.12(d, 7.0) 1.07(d,7.0) 7.39(br d, 7.4) 18 7.38(br d, 7.5) 7.36(t, 7.9) 19 4.14(m) 4.22(d, 5.7) 7.33(t, 7.5) 7.28(br t, 7.4) 20 4.73(m) 4.80(dq, 5.7, 6.5) 7.25(br t, 7.5) 21 4.26(d, 3.2) 22 1.27(d, 7.0) 1.42(d, 6.5) 4.23(br d, 2.5) 4.98(m) 23 4.14(m) 4.72(m) 4.96(m) 24 2.07(m) 1.90(m) 1.42(d, 6.3) 25 1.60(m); 1.17(m) 1.25(m); 1.10(m) 1.42(d,6.0) 4.76(dd, 7.4, 11.6) 26 0.86(t, 7.5) 0.69(t, 7.5) 4.76(dd, 8.0, 11.5) 2.24(m) 27 0.89(d, 6.5) 0.74(d, 6.0) 2.26(m) 1.65(m); 1.43(m) 28 1.66(m); 1.43(m) 0.90(t, 7.4) 29 0.90(t, 7.0) 1.04(d, 6.9) 30 7.56(br s) 7.74(s) 1.04(d, 6.5) 32 5.24(dd, 10.0, 4.5) 5.14(m) 7.49(s) 33 2.34(m) 2.24(m) 7.48(s) 34 1.12(d, 7.0) 1.03(d, 6.5) 5.36(dq, 9.5, 7.4) 35 1.15(d, 7.0) 1.07(d, 7.0) 5.33(dq, 10.0, 7.0) 1.73(d, 7.4) 36 1.70(d, 7.0) N-1 7.51(d, 10.0) 7.30(d, 10.5) 7.61(d, 10.0) 7.58(d, 9.5) N-2 8.30(br) 7.86(br) 7.62(d, 6.5) 7.62(d, 7.4) N-3 7.67(d, 9.5) 7.30(d, 10.5) 7.59(d, 10.0) 7.63(d, 10.0) N-4 6.88(br) 7.86(br) 7.57(d, 8.0) 7.58(d, 9.5) N-5 8.04(br) a) Spectra were recorded in CDCl 3 at 500 MHz, referenced to CDCl 3 (δ 7.26), coupling constant J in Hz; b) assignments assigned by COSY and RCT-COSY. 2 Results and discussion Compounds 1 4 were isolated from the CH 2 Cl 2 -solubles of MeOH and MeOH-CH 2 Cl 2 extracts of Lissoclinum patella by open column chromatography over silica gel, followed by rever-

646 SCIENCE IN CHINA (Series B) Vol. 43 Table 2 13 C NMR data for compounds 1 4 a) Carbon Prepatellamide A (1) b) Patellamide A (2) c) Patellamide B (3) c) Patellamide C (4) c) 1 170.0(s) 171.8(s) 173.0(s) 173.1(s) 2 68.2(d) 67.4(d) 73.6(d) 73.7(d) 3 72.9(t) 72.2(t) 82.4(d) 82.5(d) 4 169.6(s) 169.5(s) 168.3(s) 168.1(s) 5 53.7(d) 52.5(d) 20.9(q) 21.2(q) 6 33.1(d) 36.8(d) 47.6(d) 56.0(d) 7 25.4(t) 24.7(t) 38.9(t) 27.9(s) 8 10.5(q) 10.6(q) 24.9(d) 19.3(q) 9 15.1(q) 14.9(q) 23.1(q) 19.8(q) 10 162.1(s) 160.5(s) 21.7(q) 161.9(s) 11 148.7(s) 149.4(s) 161.5(s) 147.4(s) 12 123.5(d) 123.1(d) 147.1(s) 123.8(d) 13 169.3(s) 169.1(s) 123.6(d) 170.9(s) 14 56.32(d) 55.0(d) 170.7(s) 52.4(d) 15 31.9(d) 33.4(d) 53.1(d) 40.8(t) 16 22.7(q) 19.3(q) 40.6(t) 136.3(s) 17 17.4(q) 17.9(q) 136.2(s) 129.3(d)(2C) 18 173.9(s) 171.6(s) 129.2(d)(2C) 128.8(d)(2C) 19 60.0(d) 73.5(d) 128.7(d)(2C) 127.2(d) 20 66.0(d) 81.6(d) 127.1(d) 173.3(s) 21 175.6(s) 168.4(s) 173.3(s) 73.7(d) 22 20.1(q) 21.7(q) 73.7(d) 82.5(d) 23 53.7(d) 52.1(d) 82.0(d) 168.2(s) 24 33.1(d) 37.1(d) 168.0(s) 21.0(q) 25 25.5(t) 24.9(t) 20.9(q) 53.3(d) 26 10.3(q) 11.0(q) 52.3(d) 32.8(d) 27 15.3(q) 15.0(q) 32.8(d) 25.0(t) 28 161.4(s) 160.5(s) 24.9(t) 8.7(q) 29 148.0(s) 149.5(s) 8.7(q) 15.1(q) 30 123.3(d) 123.1(d) 14.9(q) 161.9(s) 31 169.6(s) 169.1(s) 161.8(s) 147.7(s) 32 56.26(d) 55.0(d) 147.6(s) 123.7(d) 33 32.6(d) 33.3(d) 123.5(d) 172.7(s) 34 19.9(q) 19.3(q) 172.7(s) 46.7(d) 35 17.5(q) 17.9(q) 46.6(d) 20.8(q) 36 20.7(q) a) Spectra were recorded in CDCl 3 at 125 MHz, referenced to CDCl 3 (δ 77.0); b) assignments assigned by analogy to patellamide A (2); c) multiplicities were determined by DEPT experiments and assignments confirmed by HMQC and HMBC experiments. sed-phase HPLC on a C18 column. The known patellamides A (2), B (3), and C (4) [11,12] were identified by comparison of their FABMS, 1 H and 13 C NMR data (tables 1 and 2) with those reported in literature. Prepatellamide A (1) has the molecular formula C 35 H 52 N 8 O 7 S 2 based on HRFABMS analysis, m/z 761.348 9 [M+H] + ( 1.0 mmu), and NMR data (tables 1 and 2). This formula differs from that of patellamide A (2) by addition of H 2 O indicating that prepatellamide A might be a hydrolysate of patellamide A (2). The peptide property of 1 was readily recognized by eight 13 C NMR

No. 6 PREPATELLAMIDE A, A NEW CYCLIC PEPTIDE FROM ASCIDIAN 647 signals ranging from 161 to 176, and five NH signals at δ 6.88, 7.51, 7.67, 8.04, and 8.30 which coupled to signals in the right region for α-protons of amino acids. The partial structures corresponding to a free threonine, an oxazoline, two isoleucine, and two valine residues, and two thiazole rings were identified by comparison of the NMR data of 1 with appropriate patellamide [2, 12 models 14] and confirmed by COSY, and relayed coherence transfer (RCT)-COSY experiments. Inspection of the 1 H NMR data (table 1) of prepatellamide A (1) reveals striking similarities to those of patellamide A (2) [11,12], but the former has an additional NH signal at δ 6.88 correlating with a signal at δ 4.14. The signal at δ 4.14 further couples to a signal at δ 4.73 which in turn couples to a methyl doublet at δ 1.27. This spin system was confirmed by COSY and RCT-COSY experiments. 13 C NMR data (table 2) of 1 are nearly identical with those of patellamide A (2) except for C-19 (δ 60.0 in 1 vs. 73.5 in 2), C-20 (δ 66.0 in 1 vs. 81.6 in 2), and C-21 (δ 175.6 in 1 vs. 168.4 in 2). The chemical shifts for C-19, C-20, and C-21 are indicative of the presence of a methyl-substituted free threonine residue [2, 12]. Therefore, prepatellamide A is assigned as structure 1 which might be a precursor of patellamide A (2). Prepatellamide A(1), patellamides A(2), B(3), and C(4) are responsible for the cytotoxicity against P388 murine leukemia cell lines observed in the crude extract with IC 50 = ~5 µg/ml. Acknowledgements We gratefully acknowledge Dr. F. Monniot, Laboratoire de Biologie des Invertebres Marins et Malacologie, Museum National d Histoire Naturelle, Paris, France, for ascidian identification. This work was supported by the National Natural Science Foundation of China (Grant Nos. 29572088 and 29932030). References 1. Davidson, B. S., Ascidians producter of amino acid derived metabolites, Chem. Rev., 1993, 93: 1771. 2. Fu, X., Do, T., Schmitz, F. J. et al., New cyclic peptides from the ascidian Lissoclinum patella, J. Nat. Prod., 1998, 61: 1547. 3. Wasylyk, J. M., Biskupiak, J. E., Costello, C. E. et al., Cyclic peptide structure from the tunicate Lissoclinum patella by FAB mass spectrometry, J. Org. Chem., 1983, 48: 4445. 4. Ireland, C., Scheuer, P. J., Ulcyclamide and ulthiacycliamide, two new small peptides from a marine tunicate, J. Am. Chem. Soc., 1980, 102: 5688. 5. Williams, D. E., Moore, R. E., The structure of ulthiacycliamide B, antitumor evaluation of cyclic peptides and macrolides from Lissoclinum patella, J. Nat. Prod., 1989, 52: 732. 6. Degnan, B. M., Hawkins, C. J., Lavin, M. F. et al., New cyclic peptides with cytotoxic activity from the ascidian Lissoclinum patella, J. Med. Chem., 1989, 32: 1349. 7. Hawkins, C. J., Lavin, M. F., Marshall, K. A. et al., Structure-activity relationship of the lissoclinamides cytotoxic cyclic peptides from the ascidian Lissoclinum patella, J. Med. Chem., 1990, 33: 1634. 8. McDonald, L. A., Foster, M. P., Phillips, D. R. et al., Tawicyclamides A and B, new cyclic peptides from the ascidian Lissoclinum patella, studies on the solution and solid-state conformation, J. Org. Chem., 1992, 57: 4616. 9. Zabriskie, T. M., Foster, M. P., Stout, T. J. et al., Studies on the solution and solid-state structure of patellin 2, J. Am. Chem. Soc., 1990, 112: 8080. 10. In, Y., Doi, M., Inoue, M. et al., Molecular conformation of patellamide A, a cytotoxic cyclic peptide from the assidian Lissoclinum patella by X-ray crystal analysis, Chem. Pharm. Bull., 1993, 41: 1686. 11. Ireland, C. M., Durso, A. R., Newman, R. A. et al., Antineoplastic cyclic peptides from the marine tunicate Lissoclinum patella, J. Org. Chem., 1982, 47: 1807.

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