Leptosins, antitumour metabolites of a fungus isolated from a marine alga

摘要

J. CHEM. SOC. PERKIN TRANS. 1 1994 Leptosins, Antitumour Metabolites of a Fungus Isolated from a Marine Alga Chika Takahashi,a Atsushi Numata,*na Yoshinori Ito,a Eiko Matsumura,a Hiromasa Araki,b Hideo lwakib and Katsuhiko KushidaC a Osaka University of Pharmaceutical Sciences, Matsubara, Osaka 580, Japan Fuso Pharmaceutical Industries, Ltd, Jot0 -ku, Osaka 532, Japan Varian Instruments Ltd, Shinjuku-ku, Tokyo 169, Japan Leptosins A B, C, D, E and F, chaetocin derivatives, have been isolated from the mycelium of a strain of Leptosphaeria sp. attached to the marine alga Sargassum tortile. Their stereostructures, with a different configuration from that of related compounds, have been elucidated by spectroscopic analyses using various 1 D and 2D NMR techniques and some chemical transformations.All the compounds showed potent cytotoxicity against cultured P388 cells, and leptosins A and C exhibited significant antitumour activity against Sarcoma 180 ascites. Studies on the natural-product chemistry of marine animals have illustrated that they are prolific sources for structurally unique, highly bioactive and biomedically utilitarian secondary metabolites. Of the many bioactive compounds found in marine animals, toxic principles of several animals (tetrodotoxin, neosurugatoxin, saxitoxin and palytoxin) have proven to be produced by ba~teria.'~~ This has evoked wide interest in marine microorganisms because of the potential for the development of new pharmaceutical agents and also in the search for the origin of marine animal metabolites.We have focussed our attention on antineoplastic and/or cytotoxic metabolites from microorganisms which inhabit the marine environment. As part of this programme, we previously reported that cytotoxic substances, three fumiquinazolines and two communesins, were produced by a strain of Aspergillus fumigatus, isolated from the gastrointestinal tract of the saltwater fish Pseudolabrus japonicu~,~ and by a strain of Penicillium sp. isolated from the marine alga Enteromorpha respectively. In the present study, we examined secondary metabolites from a strain of Leptosphaaeria sp. isolated from the marine alga Sargassum tortile, and isolated six novel antitumour and cytotoxic metabolites, designated leptosins A-F 1-6, which belong to a series of dimeric epipolysulfanyldioxopiperazinessuch as chaetocin and chetor- acin A.7-9We describe herein the structure elucidation and cytotoxic activity of these metabolites.The fungal strain was cultured at 27 "C for 3 weeks in a medium containing 2 glucose, 1 peptone and 0.5 yeast extract in artificial seawater. The MeOH extract of the mycelium was purified by bioassay-directed fractionation employing a combination of Sephadex LH-20 and silica gel column chromatographies and high-performance liquid chrom- atography (HPLC) to afford leptosins A 1, B 2, C 3, D 4, E 5 and F 6. Leptosin A 1 had the molecular formula C32H32N607S6 established by high-resolution fast atom bombardment mass spectrometry (HRFABMS) m/z 805.0740 (MH'), A +0.5 mmu.Its IR spectrum exhibited bands at 3412, 1686, 1664, 1608 and 1593 cm-l, characteristic of an alcohol, an amine, an amide and an aromatic ring. A close inspection of the 'H and '3C NMR spectra of 1(Table 1) by distortionless enhancement by polarization transfer (DEPT) and 'H-'H and 'H-13C correlation spectroscopy (COSY) experiments and comparison with spectral data for related compounds revealed signals for two hydroxy methine groups (C-31 and C-11') linked to two quaternary sp3-hybridized carbons, two methines (C-5a and C-5'a) bearing two nitrogens and a quaternary sp3-carbon, four quaternary sp3-carbons (C-3, C-12, C-3' and C-12') each 1 x=4 2 x=3 3 x=2 7 R~,R~=+-8 R', R2=SMe 15A16 4 x=2 5 x=3 6 x=4 9 bearing a nitrogen and a sulfur, four amides (C- 1, C-4, C- 1 'and C-4'), two N-methyl groups (C-13 and C-13'), isopropyl (C-14, C-15 and C-16) and hydroxymethyl C-14') groups each linked to a quaternary sp3-carbon, and two 1 ,Zdisubstituted benzenes (C-6a to 10a and C-6'a to C-lO'a). The signals for one quaternary sp2-carbon (C-6a and C-6'a) of each of the two aromatic rings appeared lowfield (S148.3 and 149.9) in the 13C NMR spectrum, indicating that one substituent on each benzene is an amino group.The connection of the functional groups was demonstrated J. CHEM. soc. PERKIN TRANS. 1 1994 Table 1 'H (300 MHz) and I3C (75.4 MHz) NMR data of leptosin A 1in CDCl, Position 6 'Ha NOES 6 I3C HMBCd I 3 4 5a 6 6.43 s 5.25 br s 1 1-OH, 5'a-H, 11'-H 167.1(9)' 80.3 (9)160.9 (q) 80.3 (t) 13-H 13-H, 14-H, 15-H, 16-H 14-H 11-H 6a 7 8 9 10 1Oa 10b 11 12 13 14 15 16 1 1-OH 6.64 d (8.0) 7.10 t (8.0) 6.46 t (8.0) 5.59 d (8.0) 4.95 s 3.04 s 2.64 heptet (6.8) 1.42 d (6.8) 1.42 d (6.8) 5.69 s 11-H 10-H, 10'-H 15-H, 16-H 13-H 13-H 5a-H 148.3(q) 110.4 (t) 130.0 (t) 119.7 (t) 125.7 (t) 126.0 (q) 62.7 (q) 80.9 (t) 75.5 (9) 27.8 (P) 17.9 (P) 18.6 (P) 32.3 (t) 5a-H, 8-H, 10-H 9-H 10-H 7-H 8-H 5a-H, 6-H, 7-H, 9-H, 11-H 6-H, 11-OH, 5'a-H, 11'-H 5a-H 15-H, 16-H 16-H 15-H 1' 3' 4' 5'a 6' 5.42 s 3.90 br s 5a-H 168.2(q) 79.0 (q) 169.3(q) 80.2 (t) 13'-H, 1 1'-H 13'-H, 14'-H 14'-H, 15'-H 11'-H 6'a 7' 8' 9' 10' IO'a 10'b 11' 12' 13' 14'A 14'B 6.55 d (7.8) 7.24 t (7.8) 6.95 t (7.8) 7.87 d (7.8) 5.43 s 3.00 s 3.93 d (12.0) 4.10 m 11-H 5a-H 149.9 (q) 109.8 (t) 130.0 (t) 119.0 (t) 130.3 (t) 123.1(q) 64.5 (9)79.3 (t) 80.2(9) 28.9 (P) 63.6 (s) 5'a-H, 8'-H, 10'-H 9'-H 10'-H 7'-H 8'-H 5'a-H, 7'-H, 9'-H, 11'-H 5a-H, 11'-OH 5'a-H I 1'-OH 5.30 br se 14'-OH 3.27 br se a 'H Chemical shift values (6 ppm from SiMe,) followed by multiplicity and then the coupling constant (J/Hz) in parentheses.* Observed in the NOESY experiment. Letters, p. s, t and q, in parentheses indicate respectively primary, secondary, tertiary and quaternary carbons, assigned by DEPT. Long range 'H-13C correlation from H to C observed in the HMBC experiment. Interchangeable. 3) showed close correspondence with those of 1 (Table 1).Formation of 7 from 1and the molecular formula of 1indicated that the tetrasulfide and disulfide bridges existed in the hydroxymethyl- and isopropyl-bearing dioxopiperazine rings of 1, respectively. This was supported by the FABMS fragments at m/z 428 (a+) and 377 (b+ HI'), corresponding to the hydroxymethyl- and isopropyl-bearing halves of the molecule of 1, respectively, as well as five other ions at m/z 282 (a -4s -H20+), 312 (b -2S+), 493 (e + HI+), 429 (eH -w 2S+) and 41 1 (eH -2s -H20+) (see structure 1 for a, b Fig. 1 NOESY data summary for 8 and e). The FABMS of 1also exhibited the fragment peaks at m/z 232 (bis-indol-3-y1+), 197 (eH -2s -232+), 677 (MH -421') and 659 (MH -4s -H20+), the last two on the basis of heteronuclear multiple-bond connectivity fragments showing that the sulfur atoms of the hydroxymethyl- (HMBC) correlations (Table 1).The principal correlations are bearing dioxopiperazine ring are eliminated more easily than as follows: 13-H to C-1 and C-3,14-H to C-4, 11-H to C-5a and those of the isopropyl-bearing ring. C-1Oa, 5a-H to C-6a, C- 12 and C- 1 O'b, 13'-H to C- 1' and C-3', The relative configuration of 1 was deduced from detailed 14'-H to C-4', 11 '-H to C- 1',C-Sa, C- 1O'a and C- 1Ob, and 5'a- nuclear Overhauser enhancement (NOE) spectral analysis of 1 H to C-lob, C-4', C-6'a and C-lO'a. This evidence led to planar (Table 1) and 8 (Fig. 1). NOE observed between 11-H and structure of 1for leptosin A. The number of sulfur atoms in the 12-SMe in 8 was indicative of their cis configuration while polysulfide bridges of 1were determined by chemical and mass NOES between 10-H and 12-SMe, and 10-H and 11-H indicated spectral evidence as follows. Leptosin A 1was transformed into that 1 1 -H and the C- 1 OM-1O'b bond, and 5a-H and 1 1-H have bis(methylsulfany1) and tetrakis(methylsulfany1) derivatives 7 trans configurations.If 5a-H and 11-H have a cis configuration, and 8 by treatment with NaBH, and Me1 in pyridine. The then no NOE between 10-H and 12-SMe should be observed. position of the two methylsulfanyl groups in 7 was established The trans configuration of 11-H and 5a-H was supported by an from the fact that the NMR signals of all the carbons (C-l-C- NOE between 5a-H and 5'a-H.On the other hand, the NOE 16) on the isopropyl-bearing half of the molecule of 7 (Table between 11'-H and 12-SMe was indicative of their cis J. CHEM. soc. PERKIN TRANS. 1 1994 Table 2 'H (300 MHz) NMR data of leptosins EF 26 and derivatives 7 and 8 in CDCl,' ~~~~ ~ ~ Position 2 3 4 5 6 7 8 5a 6.48 s 6.47 br s 6.34 br s 6.20 s 6.51 s 6.71 s 6.96 s 6 5.30 s C 5.40 s 5.62 s 5.15 s 5.35 s 5.30 sb 7 8 9 10 11 13 6.66 d (8.0) 7.11 t (8.0) 6.50 t (8.0) 5.66 d (8.0) 4.99 s 3.03 s 6.56 d (8.0) 7.14 t (8.0) 6.47 br s 5.68 br s 4.80 s 3.05 s 6.72 dd (7.8, 1.0) 7.16 td (7.8, 1.0) 6.84 td (7.8, 1.0) 7.45 dd (7.8, 1.0) 5.37 s 3.08 s 6.76 d (7.8) 7.15 t (7.8) 6.74 t (7.8) 7.33 d (7.8) 5.41 d (2.0) 3.23 s 6.52 d (7.8) 7.04 t (7.8) 6.72 t (7.8) 7.32 d (7.8) 5.21 d (2.3) 3.05 s 6.65 d (7.8) 7.09 t (7.8) 6.45 t (7.8) 5.60 d (7.8) 4.97 s 3.05 s 6.54 d (7.8) 7.06 t (7.8) 6.33 t (7.8) 5.58 d (7.8) 4.84 d (2.8) 3.07 s 14 15 16 11-OH 3-SMe 2.65 heptet (6.8) 1.416 d (6.8) 1.424 d (6.8) 5.65 br s 2.67 heptet (7.0) 1.41 d (7.0) 1.42 d (7.0) 5.73 br s 2.72 heptet (7.0) 1.47 d (7.0) 1.49 d (7.0) 5.22 br s 2.52 heptet (6.8) 1.22 d (6.8) 1.48 d (6.8) 3.71 d (2.0) 2.73 heptet (6.8) 1.19 d (6.8) 1.53 d (6.8) 3.43 br s 2.67 heptet (7.0) 1.44 d (7.0) 1.45 d (7.0) 5.62 br s 2.63 heptet (7.0) 1.12d(7.0) 1.20 d (7.0) 3.63 d (2.8) 2.17 s 12-SMe 1.84s 1' 8.01 br s 8.05 br s 8.12 br s 2' 4' 5' 5'a 5.62 s 5.97 br s 7.02 d (2.7) 7.96 dd (7.5, 1 .O) 7.18 td (7.5, 1.0) 7.12 d (3.0) 7.87 dd (7.5, 1 .O) 7.18 td (7.5, 1.0) 7.09 d (3.0) 7.87 dd (7.5, 1.0) 7.12 td (7.5, 1.0) 5.57 s 5.49 s 6' 7' 8' 9' 10' 11' 3.08 br s 6.56 d (7.8) 7.25 t (7.8) 6.95 t (7.8) 7.91 d (7.8) 5.44 s 6.26 br s 7.14 br s 6.88 br s 7.80 br s 5.36 br s C 7.19 td (7.5, 1.0) 7.30 dd (7.5, 1.0) 7.20 td (7.5, 1 .O) 7.34 dd (7.5, 1.0) 7.20 td (7.5, 1.0) 7.33 dd (7.5, 1.0) 4.97 br sb 6.50 d (7.8) 7.18 t (7.8) 6.91 t (7.8) 7.85 d (7.8) 5.38 s 5.02 br s 6.50 d (7.8) 7.19 t (7.8) 6.89 t (7.8) 7.72 d (7.8) 5.13 d (2.6) 13' 2.99 s 2.94 br s 3.02 s 3.02 s 14'A 14'B 1 1 '-OH 14'-OH 3.73 d (12.8) 4.53 br d (12.8) 4.51 s 2.67 br s 4.18 m 4.34 br d 3.35 br s C 3.71 d (12.0) 4.03 d (12.0) 3.73 br s* 2.69 br s* 3.70 br s (12.0) 4.02 br s (12.0) 3.75 d (2.6) 1.84 br sb 3'-SMe 2.22 s 2.22 s 12'-SMe 2.43 s 2.37 s a 'H Chemical shift values (8 ppm from SiMe,) followed by multiplicity and then the coupling constant (H/Hz) in parentheses.Assignments interchangeable. Not detected. +80-+60-+40-2 +20--204 Fig. 2 CD spectra of leptosins C 3 (-acetylchaetocin9 (-. -) in EtOH ---), D 4 (-) and di-0- configuration while NOES between 5'a-H and 5a-H and 1 1 '-H and 5a-H showed that 11'-H and 5'a-H, and 11'-H and the C-10-C-lob bond have cis configurations. Because of the presence of sulfide bridges between C-3 and C-12, and C-3' and C-12' in 1, the two S-Me groups at C-3 and C-12, and the two S-Me groups at C-3' and C- 12' in 8 should have cis orientations. The above summarized evidence, supported by an NOE experiment of 1, allowed assignment of the relative configur- ation of 1, and also suggested that 1 and 8 exist in the conformation shown in Fig.1 in CHCl, solution. The relative configuration of C-5'a and C-lO'b in 1 was different from that of related compounds, chaetocin and chetracin A,' and this is the first isolation of a dimeric epipolysulfanyldioxopiperazine with such a configuration. Leptosins B 2 and C 3were assigned the molecular formulae C32H32N60,S5and C32H,2N,0,S,, respectively, as deduced from MH+ peaks in HRFABMS (m/z773.1024, A + 0.9 mmu and 741.13 16, A + 2.2mmu, respectively). The general features of their UV, IR and NMR spectra (Tables 2 and 3) closely resembled those of 1except that some 13CNMR signals (C-4, C- 14', C- 1O'b, etc.) for their hydroxymethyl-bearing dioxo- piperazine rings exhibited a chemical shift difference relative to those of 1.Both 2 and 3 afforded 7 and 8 on treatment with NaBH, and MeI. In the FABMS, 2 exhibited two fragments m/z396 (c+) and 377 (bH+), corresponding to the two halves of the molecule, together with two fragments m/z282 (c -3s -H20+) and 312 (b -2S+), while 3 showed two fragments m/z 364 (d+) and 377 (bH+), corresponding to the two halves of the molecule, together with two other fragments m/z 282 (d -2s -H20+) and 312 (b -2S+). In addition, both the compounds showed other fragment ions at m/z 493 (eH+),429 (eH -2S+), 411 (eH -2s -H20+), 232 (@is-indol-3-y1+), 197 (eH -2s -232+), 677 (MH -3s or 2S+) and 659 (677 -H20+), the last two fragments illustrating that the hydroxymethyl-bearing dioxo- piperazine rings in 2 and 3 have three and two sulfide bridges, respectively.This evidence led to relative stereostructures 2 and 3for leptosins B and C, respectively. In the circular dichroism (CD) spectrum of leptosin C 3, there was a negative band at 271 nm due to an S+CO charge-transfer transition, comparable in strength to that of the related compound, di-0-acetylchaetocin 9 (Fig. 2),**" showing the asymmetric centres of the dioxopiperazine ring in 3 to have the J. CHEM. soc. PERKIN TRANS. 1 1994 Table 3 13C(75.4 MHz) NMR data of leptosins ELF 2-6 and derivatives 7and 8 in CDCI, Position 2 3 4 5 6 7 8 1 3 4 5a 6a 7 8 9 10 1 Oa 10b 11 12 13 14 15 16 3-SMe 12-SMe 1' 1 'a 2' 3' 3'a 4' 5' 5'a 6' 6'a 7' 8' 9' 10' 1 O'a 1 O'b 11' 12' 13' 14' 167.1 (9)" 160.9 (9) 80.7 (t) 148.5 (q) 110.6 (t) 130.0 (t) 119.8 (t) 125.7 (t) 126.3 (q) 80.8 (t) 27.8 (p) 32.3 (t) 18.0 (p) 18.6 (p) 80.2 (4) 62.6 (9) 75.5 (s) 167.6 (q) 80.6 (9) 165.7 (q) 79.4 (t) 150.0 (q) 109.7 (t) 130.0 (t) 118.8 (t) 130.4 (t) 123.4 (q) 65.5 (9)79.9 (t) 75.8 (9)28.4 (p) 62.0 (s) 167.3 (9) 160.7 (q) 79.8 (t) 149.0 (4) 109.4 (t) 130.1 (t) 119.0 (t) 126.6 (t) 126.8 (q) 81.4 (t) 27.8 (p) 32.3 (t) 80.4 (9) 63.2 (4) 75.6 (4) 18.0 (PI 18.6 (PI 167.3 (q) 79.2 (9) 164.7 (q) 81.4 (t) 149.2 (q) 109.9 (t) 129.7 (t) 119.2 (t) 130.0 (t)b 122.3 63.2 (4) 75.6 (9) 26.5 (P) 79.2 (t) 60.5 (s) 167.6 (q) 161.3 (q) 82.6 (t) 147.2 (q) 110.6 (t) 129.2 (t) 119.7 (t) 124.4 (t) 130.9 (9) 81.3 (t) 80.5(4) 60.7 (9) 76.2 (4) 27.8 (P) 18.7 (PI 18.1 (P) 32.4 (t) 136.9 (q) 123.4 (t) 1 13.2 (q) 126.2 (q) 121.5 (t) 119.8 (t) 122.3 (t) 11 1.5 (t) 170.2 (q) 78.0 (9)165.4 (q) 81.7 (t) 149.7 (q) 110.9 (t) 130.1 (t) 119.6 (t) 125.2 (t) 128.6 (q) 58.6 (9)83.5 (t) 83.9 (4) 28.1 (P) 35.5 (t) 19.0 (PI 18.2 (P) 136.9 (q) 122.9 (t) 113.5 (q) 125.7 (q) 120.9 (t) 120.0 (t) 122.4 (t) 1 1 1.6 (t) 168.2 (q)* 81.6 (q)' 167.8 (q)* 83.0 (t) 147.4 (q) 109.0 (t) 129.3 (t) 119.4 (t) 124.5 (t) 131.2 (q) 83.1 (t) 80.2 (9)" 30.1 (p) 36.0 (t) 18.3 (p) 59.0 (4) 18.4 (P) 137.0 (q) 123.5 (t) 1 14.0 (q) 125.8(q) 121.0 (t) 120.0 (t) 122.5 (t) 11 1.7 (t) 167.2 (q) 161.0 (q) 80.8 (t) 148.7 (q) 110.1 (t) 129.9 (t) 119.4 (t) 125.7 (t) 126.5 (q) 81.3 (t) 80.3 (9) 62.8 (9) 75-8(9) 27.8 (PI 18.7 (P) 18.0 (P) 32.3 (t) 165.2 (q) 73.3 (9) 165.2(q) 80.2 (t) 150.1 (q) 109.5 (t) 129.8 (t) 118.9 (t) 130.2 (t) 123.7 (q) 64.9 (9)79.1 (t) 70-0(9) 64.9 (s) 28.4 (p) 165.9 (q) 165.3 (q) 80.7 (t) 149.6 (q) 108.8 (t) 129.5 (t) 118.2 (t) 124.2 (t) 127.0 (q) 61.2 (9)79.7 (t) 72.9 (q) 30.2 (p) 37.0 (t) 18.3 (p) 18.3 (p) 13.9 (p) 165.0 (9) 77.8 (9) 16.4 (P) 73.8 (9) 165.1 (9) 80.5 (t) 150.3 (4) 109.6 (t) 129.7 (t) 118.9 (t) 130.0 (t) 123.9 (q) 66.1 (q) 78.8 (t) 69.6 (9) 28.4 (P) 65.0 (s) 3'-SMe 12'-SMe 13.5 (P) 15.4 (P) 13.4 (p) 15.2 (p) Letters, p, s, t and q, in parentheses indicate respectively primary, secondary, tertiary and quaternary carbons, assigned by DEPT.b~cAssignments interchangeable.same configuration (S) as those of 9.'O A relatively weak negative band (Aamp; -17.3) at 233 nm due to the indolinyl chromophore was observed in the tetrakis(methylsulfany1) derivative 8, which has no 231 nm band due to disulfide no* transitions and exist in the same conformation as that of 3. This evidence suggests that the contribution of the disulfide no* transitions to the 231 nm band (Aamp;+ 62.6) in the CD spectrum of 3 is more important than that of the indolinyl chromophore and the weak band due to the latter is hidden by overlapping with the strong band attributable to the former in the CD spectrum of 3. Therefore, it is assumed that the stereochemical difference between 3 and 9 at C-5'a does not appear in their CD spectra.Based on the above evidence, the absolute stereostructures of leptosin C and, consequently, leptosins A and B are represented as 3,l and 2, respectively. Leptosin D 4 was assigned the molecular formula C25H24N403S2 deduced from HRFABMS m/z 493.1366 (MH+), A -0.2 mmu. A close inspection of its 'H and 13C NMR spectra (Tables 2 and 3) revealed that the hydroxymethyl- bearing half of the molecule of 1 was replaced by a 3-substituted indole moiety in 4. A chemical shift difference of the I3C NMR signals for C-Sa, C-lOa and C-lob of 4 relative to those of 1 revealed that C-lob was linked at C-3 (namely C-3' of 4) of the indole moiety in 4. In addition, the FABMS of 4 exhibited a fragment ion at m/z 429 (MH -2S+), arising from desulfurization of MH+, together with other fragments at m/z 411 (MH -2s -H20+), 232 (bis-indol-3-yl+), 197 (MH -2s -232+), 154 (197 -isopropyl+) and 136 (154 -H20+).For the purpose of desulfurization, 2 was treated with triphenylphosphine 'to afford 4 together with 3. It has been reported that the tetradesulfanyl-derivative 10 of verticillin A was treated with methanolic potassium hydroxide to give compound 11.' It is considered that a similar reaction took place on treatment of 2 with triphenylphosphine as a nucleophile to give 4. Formation of 4 from 2 showed the absolute configuration of 4 to be the same as that of 1-3. This was supported by CD spectral comparison of 4 with 1 (Fig. 2). The above-mentioned evidence allowed assignment of stereo- structure 4 to leptosin D.Leptosins E 5 and F 6 were shown to have molecular formulae of C25H24N403S3 respectively,and C25H24N403S4, by HRFABMS (MH', m/z 525.1061, A -2.8 mmu; 557.0828, A + 1.8 mmu). The general features of their UV,IR and NMR spectra (Tables 2 and 3) closely resembled those of 4 except that some I3C NMR signals for their dioxopiperazine rings exhibited a chemical shift difference relative to those of 4. Desulfurization of 6 and 5 with triphenylphosphine afforded 4 and 5, and 4, respectively. In addition, the FABMS of both 5 and 6 showed a fragment peak at m/z 429 (MH -3S+ or MH -4S+), arising from desulfurization of MH+, together J. CHEM. soc. PERKIN TRANS. 1 1994 Table 4 Cytotoxicity of compounds 1-6 against tumour cells Cell line Compound P-388(ED5, pg ~m-~) Leptosin A 1 1.85 x 10-3 Leptosin B 2 2.40 x 10-3 Leptosin C 3 1.75 x 10-3 Leptosin D 4 8.60 x Leptosin E 5 4.60 x Leptosin F 6 5.60 x Mitomycin C (standard) 4.40 x Me Me 10 11 with the same fragments (m/z 41 1, 232, 197, 154 and 136) as those of 4.This evidence led stereostructures 5 and 6 for leptosins E and F, respectively. The cytotoxic activities of compounds 1-6 were examined in the P-388 lymphocytic leukemia test system in cell culture, according to the method reported previously.'' As shown in Table 4, all the compounds tested exhibited potent cytotoxic activity. Among them, dimeric epipolysulfanyldioxopiperazines 1-3 showed more potent activity than the monomeric epi- polysulfanyldioxopiperazines 44 with the indole moiety, and the number of sulfur atoms in dioxopiperazine rings were found not to influence the activity.The antitumour activity of compounds 1 and 3 was also examined against Sarcoma-180 ascites tumour. The ascites cells (about 1 O6 cells per mouse) were inoculated intraperitoneally into ICR mice. The test sample was injected intraperitoneally once 24 h after the inoculation of ascitic cells. Prolongation of survival of mice bearing the Sarcoma- 180 ascites was evaluated by the ratio of the mean survival time of the treated animal (7') to that of control animals (C) (TIC ). As the result, both compounds 1 and 3 were found to show significant antitumour activity (TIC 260 and 293, respectively) at doses of 0.5 mg kg-' and 0.25 mg kg-', respectively. The cytotoxicity of 1 and 3 to other tumour cells and the detailed results on their in uiuo screening will be reported elsewhere.Experimental General Procedures.-M.p.s were obtained on a Yanagimoto micromelting point apparatus and are uncorrected. UV spectra were recorded on a Shimadzu spectrophotometer and IR spectra on a Perkin-Elmer FT-IR spectrometer 1720X. Optical rotations were obtained on a JASCO ORD/UV-5 spectro- polarimeter and are given in units of lo-' deg cm2 g-'. CD spectra were recorded on a JASCO J-500A spectrometer. NMR spectra were recorded at 27OC on a Varian XL-300 spectrometer, operating at 300 and 75.4 MHz for 'H and "C, respectively, in CDCI, with tetramethylsilane (TMS) as an 1863 internal reference.The 'H-'H and 'H-I3C COSY spectra were recorded on a Varian XL-300 spectrometer, and the HMBC and NOESY spectra on a Varian UNITY400 spectrometer with the usual parameters. FABMS was determined using a VG ZAB-SE mass spectro- meter (low resolution) and a JEOL JMS-HX 100/110A mass spectrometer (high resolution) in 3-nitrobenzyl alcohol matrix. Liquid chromatography over silica gel (mesh 230-400) was performed at medium pressure. HPLC was run on a Waters ALC-200 instrument equipped with a differential refractometer (R 401) and Shim-pack PREP-SIL (25 cm x 20 mm i.d.). Analytical TLC was performed on precoated Merck aluminium sheets (DC-Alufolien Kieselgel 60 F254, 0.2 mm) with CH,Cl,-MeOH (97 :3), and compounds were viewed under a UV lamp and sprayed with 10H2S04 followed by heating.Culturing and Isolation of Metabolites.-A strain of Leptosphaeria sp. was initially isolated from the marine alga Sargassum tortile C. Agaroh (Sargassaceae), collected in the Tanabe Bay of Japan. The marine alga was homogenized with sterile artificial seawater and applied onto the surface of nutrient agar layered in a Petri dish. Serial transfers of one of the resulting colonies provided a pure strain of Leptosphaeria sp. The fungal strain was grown in a liquid medium (20 dm3) containing 2 glucose, 1 peptone and 0.5 yeast extract in artificial seawater adjusted to pH 7.5 for three weeks at 27 "C.The culture was filtered under suction and the mycelium collected was extracted 'thrice with MeOH. The combined extracts were evaporated under reduced pressure to give a mixture of crude metabolites, the CH,C12-MeOH (1 :1) soluble fraction (21.5 g) of which exhibited cytotoxicity (EDso c 1 pg cm-,). This fraction was passed through Sephadex LH-20, using CH,C12-MeOH (1 :1) as the eluent. The second fraction (8.4 g) was chromatographed on a silica gel column with a hexane- CH2C12 gradient as the eluent. The hexane-CH,Cl, (6 :4) and (4:6) and CH2C12 eluates were collected as 4 fractions Fr. 1 (469 mg), Fr. 2 (78 mg), Fr. 3 (139 mg) and Fr. 4 (1 17 mg), 4 fractions Fr. 5 (965 mg), Fr. 6 (106 mg), Fr. 7 (141 mg) and Fr. 8 (210 mg) and 2 fractions Fr.9 (142 mg) and Fr. 10 (45 mg), respectively. Fr. 2 and Fr. 9 were purified by HPLC (SIL) using CH,C12 and MeOH-CH,Cl, (1 :99) as the eluents, respectively, to afford 4 (1 1 mg) and 1(3 1 mg), respectively. Fr. 4, Fr. 3 and Fr. 7 afforded 3 (47 mg) and 6 (6 mg), 5 (5 mg) and 2 (80 mg), respectively, after purification by HPLC using acetonsCH,Cl, (2 :98) as the eluent. Leptosin A 1.This was obtained as a pale yellow powder, m.p. 216-218 OC, aD +237 (c 0.49 in CHCI,); A,,(EtOH)/nm 209 (log e 4.61), 242 (4.19) and 298 (3.83); v,,(KBr)/cm-' 3412 (OH, NH), 1686, 1664 (CON), 1608 and 1593 (Ar C-C); m/z (FAB) 805 (2, MH+), 677 (18, MH+ -4S), 659 (3, MH+ -4s -H20), 493 (7, eH+), 429 (13, eH+ -2S), 428 (3, a+), 41 1 (9, eH+ -2s -H20), 377 (3, bH+), 312 (15, b+ -2S), 296 (16), 282 (6, a+ -4s -H,O), 232 (100, bis-indol-3-yl +),197 (64, eH+ -2s -232) and 185 (29) (Found: MH', 805.0740.C32H33N607S6requires MH', 805.0735); CD A(c 0.93 x lo-' mol dm-, in EtOH)/nm 240 (Aamp; +46.0),287 (+ 13.2) and 321 (-1.62). 'H and I3C NMR data are listed in Table 1. Leptosin B 2. This was obtained asa pale yellow powder, m.p. 21amp;213*C, amp;ID +392 (c 0.50 in CHCI,); A,,(EtOH)/nm 208 (log E 4.43, 244 (4.00) and 299 (3.68); v,,(KBr)/cm-' 3394 (OH, NH), 1687, 1666 (CON), 1608 and 1593 (Ar C-C); m/z (FAB) 773 (8, MH'), 677 (8, MH+ -3S), 659 (2, MH+ -3s -H20), 512 (4), 493 (5, eH+), 429 (1 1, eH+ -2S), 41 1 (9, eH+ -2s -H,O), 396 (3, c+), 377 (3, bH+), 312 (13, bH+ -2S), 296 (19), 282 (6, c+ -3s -H20), 232 (100, bis-indol-3- yl'), 197 (63, eH+ -2s -232) and 185 (29) (Found: MH+, 773.1024.C32H33N607S5 requires MH+, 773.1014); CD A(c 2.07 x lo-' mol dm-, in EtOH)/nm 234 (Aamp; +45.3), 286 (+12.1) and 322 (-1.32). 'H and 13C NMR data are listed in Tables 2 and 3. Leptosin C3. This was obtained as a pale yellow powder, m.p. 2O8-21O0C, aID +237 (c 0.36 in CHCl,); A,,(EtOH)/nm 206 (log E 4.78), 2.40 (4.23) and 301 (3.71); v,,(KBr)/cn-' 3406 (OH, NH), 1685, 1665 (CON), 1610 and 1593 (Ar C-C); m/z (FAB) 741 (3, MH'), 677 (6, MH' -2S), 659 (2, MH' -2s -H20), 493 (5, eH'), 429 (11, eH' -2S), 411 (8, eH+ -2s -HZO), 397 (28), 395 (35), 377 (57, bH'), 364 (2, d'), 312 (12, b+ -2S), 296 (8), 282 (3, d+ -2s -HZO), 232 (100, bis-indol-3-yl+), 197 (51, eH' -2s -232) and 185 (17) (Found: MH', 741.1316.C32H33N607S4 requires MH', 741.1294); CD A(c 2.57 x lo-' mol dmP3 in EtOH)/nm 231 (Aamp; +62.6), 271 (-9.3), 301 (+3.1) and 357 (-0.71). 'H and 13C NMR data are listed in Tables 2 and 3. Leptosin D 4. This was obtained as a pale yellow powder, m.p. 19amp;192OC, aID +436 (c 0.51 in CHCl,); A,(EtOH)/nm 206 (log E 4.60), 219 (4.62), 240 (4.06), 272 (3.78), 282 (3.83) and 290 (3.83); v,,,(KBr)/cm-' 3404 (OH, NH), 1688, 1665 (CON), 1607 and 1595 (Ar GC); m/z (FAB) 493 (lo, MH'), 429 (8, MH' -2S), 41 1 (3, MH' -2s -H2O), 307 (19), 289 (lo), 232 (100, bis-indol-3-yl'), 197 (23, MH' -2s -232), 154 (77, 197-isopropyl') and 136 (49, 154 -H 0 ' (Found: MH', 493.1366.CisHz5N4O3S2 requires MH I+, 493.1368); CD A(c 3.92 x lo-' mol dm-3 in EtOH/nm 229 (Aamp;+41.7), 264 (-1.66), 294 (+8.50) and 367 (-0.70). 'H and "C NMR data are listed in Tables 2 and 3. Leptosin E 5. This was obtained as a pale yellow powder, m.p. 229-231 "C, alD +563 (c 0.32 in CHCl,); A,,(EtOH)/nm 206 (log E 4.63), 218 (4.65), 240 (4.12), 273 (3.83), 282 (3.84) and 291 (3.81); v,(KBr)/cm-' 3406 (OH, NH), 1676, 1655 (CON) 1608 and 1595 (Ar C-C);m/z (FAB) 525 (40, MH+), 460 (51, MH' -2S), 429 (11, MH' -3S), 411 (10, MH' -3s -H20), 307 (50), 289 (25), 232 (30, bis-indol-3-yl+), 197 (8, MH' -3s -232), 154 (99, 197 -isopropyl') and 136 (100, 154 -H20') (Found: MH', 525.1061. CZSHZ5N4- oamp; requiresMH', 525.1089); CDA(c3.85 x 10-5moldm-3in EtOH)/nm 225 (Aamp; +29.9), 255 (+17.0) and 305 (+9.0).'H and 13C NMR data are listed in Tables 2 and 3. Leptosin F6. This was obtained as a pale yellow powder, m.p. 219-221 OC, aID +452 (c 0.39 in CHC1,); Amax(EtOH)/nm 206 (log E 4.66), 216 (4.69), 240 (4.19), 272 (3.88), 281 (3.90) and 290 (3.90); v,(KBr)/cm-' 3408 (OH, NH), 1677,1655 (CON), 1609 and 1595 (Ar C-C); m/z (FAB) 557 (21, MH'), 460 (13, MH+ -3S),429(1l,MH+ -4S),411(4,MH' -4s -H2O), 307 (35), 289 (21), 232 (93, bis-indol-3-yl'), 197 (31, MH' -4s -232), 154 (100, 197 -isopropyl') and 136 (100, 154 -H20+) (Found: MH', 557.0828. C25H2sN403S4 requires MH', 557.0810); CD A(c 3.24 x lo-' mol dm-' in EtOH)/nm 232 (Aamp; +24.2), 253 (+13.6), 290 (+11.8) and 344 (-2.1 1).'H and 13C NMR data are listed in Tables 2 and 3. Formation of the Bis(methylsulfany1) and Tetrakis(methy1- sulfanyl) Derivatives 7 and 8from Leptosins A 1, B 2 and C 3.-Leptosin C 3 (12 mg) was dissolved in a solution (0.26 cm3) of pyridine and MeOH (5 :8). Me1 (1 cm3) and NaBH, (4.8 mg) were added, and the mixture was stirred for 20 min at room temperature. The reaction mixture was then diluted with water and extracted with diethyl ether. The solvent was evaporated off under reduced pressure, and the residue was chromatographed on a silica gel column with a CH,Cl,-MeOH gradient as the eluent. The MeOH-CH2C12 (1:99) eluate afforded 7 (5.0 mg) and 8 (4.6 mg). 7 was obtained as a pale yellow oil; vmaX(KBr)/m-' 3529 (OH, NH), 1680, 1658 (CON), 1608 and 1593 (Ar C-C); m/z (FAB) 771 (20,MH').'H and ',C NMR data are listed in Tables 2 and 3.8 was obtained as a pale yellow oil; A,,(EtOH)/nm 213 (log E 4.70), 238 (4.23) and 303 (3.76); v,,,(KBr)/cm-' 3527 (OH, NH), 1658, 1641 (CON), J. CHEM. soc. PERKIN TRANS. 1 1994 1610 and 1593 (Ar C-C); m/z (FAB) 801 (6, MH+); CD A(c 1.31 x lo-' mol dm-, in EtOH)/nm 233 (AE -17.3), 252 (+12.0), 270sh (+3.5) and 296 (-4.6). 'H and 13C NMR data are listed in Tables 2 and 3. The same reaction with leptosin A 1 (3 mg) and B 2 (3 mg) gave 7 (0.2 mg and 0.8 mg, respectively) and 8 (1.9 mg and 1.1 mg, respectively). Formation of Leptosins C 3 and D 4 from Leptosin B 2.-Triphenylphosphine (10 mg) was added to a CHCIJ solution (5 cm3)of leptosin B 2 (28 mg), and the reaction mixture was left at room temperature for 2 h.The solvent was evaporated off under reduced pressure, and the residue was purified by silica gel column chromatography using MeOH-CH2C12 (1 :99) to afford leptosins C 3 (2.6 mg) and D 4 (2.2 mg), which were identified by IR, 'H NMR, CD and TLC. Formation of Leptosins D 4 and E 5from Leptosin F6.-Using the same procedure as above with leptosin B 2, leptosin F 6 (14 mg) was treated with triphenylphosphine (5 mg) to yield leptosins D 4 (1.2 mg) and E 5 (1.4 mg), which were identified by 'H NMR, CD and TLC. Formation of Leptosins D 4 from Leptosin E 5.-Using the same procedure as above, leptosin E 5 (7 mg) was treated with triphenylphosphine (2.5 mg) to yield leptosin D 4 (1.1 mg), which was identified by 'H NMR, CD and TLC.Acknowledgements We are grateful to Drs. T. Hasegawa and T. Ito, Institute for Fermentation, Osaka, for the identification of the fungus, and to Dr. S. Hagishita, Shionogi Co., Ltd. for valuable discussions on CD spectra. Thanks are also due to Drs. H. Tanaka and N. Shigematsu, Fujisawa Pharmaceutical Co. Ltd. and Dr. K. Nomoto, Suntory Institute for Bioorganic Research, for the FABMS measurements, and to Dr. Y. Usami, of this university, for the NMR measurements. References 1 M. Yotsu, T. Yamazaki, Y. Meguro, A. Endo, M. Murata, H. Naoki and T. Yasumoto, Toxicon, 1987, 25, 225; T. Yasumoto, D. Yasumura, M. Yotsu, T. Michishita, A. Endo and Y. Kotaki, Agric. Biol. Chem., 1986, 50, 793; T. Noguchi, J. K. Jeon, 0. Arakawa, H. Sugita, Y. Deguchi, Y. Shida and K. Hashimoto, J. Biochem. (Tokyo), 1986,99,331. 2 T. Kosuge, K. Tsuji,K. Hirai and T. Fukuyama, Chem. Pharm. Bull., 1985,33,3059. 3 M. Kodama, T. Ogataand S. Sato,Agric. Biol. Chem., 1988,52,1075. 4 R.E. Moore, P. Helfrich and G. M. L. Patterson, Oceunus, 1989,25, 54. 5 A. Numata, C. Takahashi, T. Matsushita, T. Miyamoto, K. Kawai, Y. Usami, E. Matsumura, M. Inoue, H. Ohishi and T. Shingu, Tetrahedron Lett., 1992,33, 1621. 6 A. Numata, C. Takahashi, Y. Ito, T. Takada, K. Kawai, Y. Usami, E. Matsumura, M. Imachi, T. Ito and T. Hasegawa, Tetrahedron Lett., 1993,34, 2355. 7 T. Saito, Y. Suzuki, K. Koyama, S. Natori, Y. Iitaka and T. Kinoshita, Chem. Pharm. Bull., 1988,36, 1942. 8 D. Hauser, H. P. Weber and H. P. Sigg, Helv. Chim. Acta, 1970,53, 1061. 9 H. Minato, M. Matsumoto and T. Katayama, J. Chem. SOC.,Perkin Trans. I, 1973, 1819. 10 R.NagarajanandR. W. Woody, J. Am. Chem. SOC.,1973,95,7212. 11 A. Numata, P. Yang, C. Takahashi, R. Fujiki, M. Nabae and E. Fujita, Chem. Pharm. Bull., 1989,37,648. Paper 3/07436F Received 17th December 1993 Accepted 18th March 1994