MXPA99009507A - Crambescidin compounds - Google Patents

Abstract

Junto con las crambescidinas (1-5) y ptilomicalina A (6) conocidas,se han obtenido dos nuevas crambescidinas menores 673 (9),687 (10), 657 (11) y 13,14,15-isocrambescidina 657 (12) sin una unidad derivada de espermicida,mediante el aislamiento guiado por FABMS de extractos de la esponja mediterranea Crambe. Sus estructuras se dilucidaron mediante la interpretación de datos de espectros (ver figuras 4-9). En un bioanalisis paralelo contra células de leucemia de murinos L1210, se mostróque las crambescidinas 834(7), 818 (8) y 657 (11) son cinco veces tan citotoxicas como las crambescodinas conocidas 816 (1). Las crambescidinas con una espermicida, o unidad derivada de espermicida, también exhibieron actividad antimicrobiana contra Rhodotorula glutinis.

Description

COMPOSITES OF CRAM BESCIDINA BACKGROUND OF THE INVENTION In the course of the classification of new bioactive agents from marine sponges, a new group of potent antiviral and cytotoxic compounds has been described, the crambescidins (1-5) 1"4 of the Crambe crambe sponge and ptilomicalin A ( 6) of the sponge Ptilocaulis spiculifer and a Hemimycale sp., 5'7 which possesses a complex of pentacyclic guanidines linked by a linear α-hydroxy f acid to a unit of spermidine or hydroxyespermidine Extensive NMR studies have shown that the relative stereochemistry of the portions of pentacyclic guanidines of crambescidins (1-4) and ptilomicalin A (6) is identical, while the oxidative degradation of crambescidin 816 (1) 2 and total enantioselective synthesis of ptilomicalin A (6) 8 have been rigorously established its identical absolute configuration of the central guanidine portions Recently, the co-occurrence of crambescidins and ptilomicalin A was found in the spo nja Batzella sp.9. Anti-fungal, antiviral, and substantial cytotoxic activities have been described for crambescidin 1 '4 and ptilomicalin A5"7, and crambescidin 816 has been shown to be a potent calcium channel blocker, in order to obtain substantial amounts of crambescidin 816 (1) for testing At the pre-clinical and clinical levels, three Crambe crambe samples were subjected to an isolation procedure similar to that previously described (see Experimental Section) .1"2 1.48 g of total crambescidin 81 6 (1) were isolated together with the crambescidins (2-5), ptilomicalin A (6) and six newly discovered crambescidin compounds (7-1 2) by FABMS-guided isolation.

BRIEF DESCRIPTION OF THE INVENTION The structures and bioactivities of the new crambescidins (7-12) are the objective of the present invention. The chlorinated spermidine unit of the crabscidins 834 (7) and 818 (8) is unprecedented from a natural source. The structures of the crambescidins are as follows: 9 R = OH 11 R = H 12 Structures of the new crambescidins (7-12) To the crambescidin 834 (7) was assigned the molecular formula C45H79N6O6CI using high resolution fast atom bombardment mass spectrometry data (HRFABMS) [m / z 835.5821 (M + H,? 0.7 mmu)]. The presence of a chlorine and a free hydroxyl group was evident, since the ion fragments for losses of hydrochloride (M + H-HCL, 799.6075,? -1 .4 mmu) and water (M + H-HCL-H20, 781.5984,? -2.9 mmu) were observed in the FABMS and FABMS spectra / collision induced dissociation (CI D) / MS of 7. The structure of the pentacyclic hydroxyl guanidine portion of 7 was assigned as the same as that of the of 1 based on FABMS and NMR data. The fragment ions in m / z 420, 374, 358, 264 and 246, which are characteristic of the pentacyclic guanidine portion of hydroxyl in crambescidins (1-4), 1 were observed in the FAMBS and FAMBS spectra. CI D / MS of 7. The position of the hydroxyl group in the pentacyclic guanidine unit was assigned by COZY, HMQC and HMBC NMR experiments, and were almost identical to those in crambescidins (1-4), suggesting that the group position hydroxyl and the stereochemistry of the pentacyclic hydroxyl guanidine portion is the same in all these crambescidins. The ions of fragments of the side chain from C-23 to -45 were observed as intense peaks in m / z 398.3729 (C2sH48N302,? 1.8 mmu) and 380.3636 (C23H46N3O? 0.5 mmu) in FABMS and FABMS / CI D / MS of 7, are absent in the spectrum of other chlorinated crambescidins. A polymethylene chain of C-23 to -37 was indicated by NMR and especially, by data from FABMS / CI D / MS, which provided an almost unbroken series of ions of cutting fragments in successive methylene groups, of / z 380 to 1 98.1616 (C10H48N3O,? -1 .0 mmu). The ester bond between the side chain and the pentacyclic hydroxyl guanidine unit, suggested by the fragment ion in m / z 614.4549 (C36H48N3O,? -1 .6 mmu), was confirmed by the long-range correlates between the carboxyl carbon in d 1 68. 1 2 (C-22) and protons in d 3.48 (s, H-14) and 4.16 (t, H2-23) in the HMBC spectrum of 7. Two isolated spin systems (from H-39b to H2-41 and H-42a, - 42b to H2-45) and the position of the chlorine in the chlorinated spermidine unit from the COZY and HMQC data. The two rotation systems were connected to each other by long-range CH correlations, between C-42 (d 57.83) and H-39b (d 3.62) and between C-39 (547.19) and H-42a, -42b (d 3.36, 3.62) observed in the H MBC spectrum. Moreover, the amide bond in 7 was established by observing the correlations between the second carboxyl carbon in d 165.49 (C-38) and protons in d 3.36 (H-42a), 2.49 (H-37a), and 2.76 (H-37b) in the HMBC spectrum. Crambescidin 818 (8), assigned to the molecular formula C45H79N605CI by HRFABMS (M + H, 819.5889,? -1 .0 mmu), has an oxygen (hydroxyl group) less than crambescidin 834 (7). The FABMS and FABMS / CI D / MS spectra of 8 show the fragment ion in m / z 783.6145 (? -3.3 mmu) corresponding to M + H-HCL, identifying the presence of a chlorine. At the same time, the presence of m / z 404 and 358 and the absence of m / z 420, 374, 264 and 246 indicated that crambescidin 818 (8) has the same proportion of pentacyclic guanidine as that of crambescidin 800 (4). ), which was confirmed by comparing the data of 1 H and 13C of 8 with those of 4. The chemical shifts of 1 H and 13C NM R of the chlorinated spermidine unit assigned by the COZY data in 8 were almost identical to those in 7, suggesting that the position and stereochemistry of chlorine in crambescidins 81 8 and 834 are the same. Crambescidin 673 (9) was assigned to the molecular formula C38Hß3N307 by H RFABMS data (M + H, 674.4734,? 1.0 mmu). The FABMS and FABMS / CI D / MS spectra of 9 showed the characteristic peaks for the pentacyclic hydroxyl guanidine unit in crambescidins at m / z 420, 358, 264 and 246 and an almost unbroken series of ion homolog fragments cut in successive methylene groups from m / z 628 to 420, suggesting that crambescidin 673 (9) has a carboxylic acid terminus, lacking a spermidine unit. The proposed structure was fully supported by the data of 1 H and C N MR for 1 0 and 9 indicated that the crambescidin 687 (10) is the methyl ester of crambescidin 673 (9). The methoxyl group was observed at dH 3.65 and dc 51.44 in the NMR spectrum for 10. The signal C-38 (d 1 74.39) at 10 was displaced upfield compared to the corresponding signal (d 181.60) at 9. The crambescidin 687 (10) (HRFABMS, 688.4907, M + H; ? -0.6 mmu for CasHßeNsOy by data HRFABMS (M + H, 674.4734,? 1 .0 mmu). The FABSMS and FABMS / CI D / MS spectra of 9 showed the characteristic peaks for the pentacyclic hydroxyl guanidine unit in crambescidins in m / z 420, 358, 264 and 246 and an almost unbroken series of ion homolog fragments. cutting in successive methylene groups from m / z 628 to 420, suggesting that crambescidin 673 (9) has a carboxylic acid terminal, lacking a spermidine unit. The proposed structure was fully supported by the data of 1 H and 13C N MR (see Tables 1 and 2) that were assigned with the help of COZY data. The carboxyl carbonyl signal was observed in d 1 81.60.

Crabscidin 687 (10) (HRFABMS, 688.4907, M + H;? -0.6 mmu for C39H66N307), which differs from 9 for a CH group, showed ions of FABMS and FABMS / CI D / MS fragments similar to those of 9. Comparison of 1 H and 13 C NMR data for 1 0 and 9 indicated that crambescidin 687 (10) is crambescidin methyl ester 673 (9). The methoxyl group was observed in dH 3.65 and dc 51.44 in the NMR spectra for 1 0. The signal of C-38 (d 1 74.39) in 10 was displaced upfield compared to the corresponding signal (d 1 81 .60 ) in 9.

Crambescidin 657 (1 1), assigned to the molecular formula C38H6ßN3O7), which differs from 9 for a C38H63N3O6 by H RFABMS (M + H, 658.4797,? -0.2 mmu), which differs from 9 by an oxygen (hydroxy group) . The spectra of FABMS and FABMS / CI D / MS for 11 showed ions of fragments in m / z 404 and 358, and an almost unbroken series of ions of cutting fragments in successive methylene groups of m / z 612 1 404, indicating the absence of an oxygen in C-1 3 in the pentacyclic guanidine portion, as confirmed by 1 H and 13 C NMR data in Tables 1 and 2. At 1 3, 14, 1 5-isocrambescidin 657 (12) the identical molecular formula C38H63N3O6 was assigned to that of 1 1 by HRFABMS data (m / z 658.4790, M + H,? 0.5 mmu). The spectral data of FABMS and FABMS / CI D / MS for 12 were also identical to those of 1 1. However, the chromatographic properties and NMR patterns of 1 1 and 12 were similar but clearly distinguishable, indicating that they are isomers of one another. The chemical shifts of 1 H and 13 C NMR and coupling constants in the pentacyclic guanidine unit at 12, assigned by COZY and TOCSY experiments, were very similar to those at 5.2 suggesting that the stereochemistry for the pentacyclic guanidines portion of 1 2, was further tested by the NOESY experiment, in which NOE's were observed between H-1 0 and CH3-1 and between H-14 and H-1 3, H-19, while NOE between H-1 0 and H-1 3 were absent.1 * 2 Crambescidins 9, 11 or 12 had an acid carboxylic acid end and a portion of strongly basic pentacyclic guanidine, 6 so they occur as the inner salt form. Two interchangeable downstream protons, correlated with H-9b and H-14 respectively by the COZY experiment, were observed in the spectrum of 1 H NMR (in CDCI3) of 7, 8 or 10, indicating that the pentacyclic guanidine portion of These crambescidins are in the salt form. The nature of the "counterion" is not determined, but presumably it is Cr1 due to the contact involved in the various isolation steps with NaCl.

DETAILED DESCRIPTION OF THE INVENTION As described above, the present invention is directed to the isolation and characterization (ie, structures and bioactivities) of six new crambescidins (Compounds 7-12). These were determined as follows: General. The NMR spectra were obtained with U-500 or U-500 spectrometers (500-MHz, 1 H, 1 25-MHz, 13C); chemical shifts (d) are reported in ppm with reference to solvent peaks. The high and low resolution fast atom bombardment (FAB) mass spectra were measured on a ZAB-SE spectrometer, and the FABMS / CID / MS spectra on a 70 SE-4F instrument using dithiothreitol-dithioerythritol as matrix.1 0 A column of C-1 8 (25 X 0.8 cm, 5-μm particle size) and solvent CH 3 OH: NaCl 0.1 M (8: 2) were used for HP LC separation. Extraction and isolation. The isolation was guided by measurement of FABMS in all the extracts and separated fractions. Three samples of Crambe crambe were involved.

The first sample was collected by SCUBA in Murcia, Spain, and was identified by Dr. M. J. Uriz-Lespe (Centred 'Estudis Avancats de Blanes, Spain). The frozen sample (100.2 g) was extracted with CHCl3: toluene (3: 1). The extract was evaporated in vacuo to give a residue (6.5 g), which was divided between CHCl 3 and 1.0 M NaCl (1: 1, 50 ml X 3). The organic layer (3.2 g) was further divided between the lower phasewas purified by HPLC to give 1 (12.0 mg), 2 (7.0 mg), 3 (2.4 mg), 4 (9.4 mg) and 5 (5.4 mg). The second frozen sample (500.0 g), collected in Ibiza, Spain, was isolated by following the same procedure to give crambescidin 816 (1, 104.5 mg). The third sample (3208.0 g) was collected on the Island of Formentor (Cave) Palma de Mallorca, Spain. The frozen sample was extracted with CHCl3: toluene (3: 1) to obtain an extract (143.0 g), which was divided between CHCl3 and 1.0 M NaCI (1: 1, 1000 mlX3). The CHCl3 layer (55.4 g) was further partitioned with hexane: EtOAc: MeOH: H20 (4: 7: 4: 3). The lower phase (18.2 g), shown by the main peaks in m / z 817, 801, was subjected to flash chromatography on a C-18 column (200 g). The column was levigated with the lower layer of the mixed solvent [hexane: EtOAC: MeOH: H20 (4: 7: 4: 3)] to give two fractions, which were purified by HPLC to give the crabmescidins 816 (1, 1367.4 mg ), 843 (7, 4.4 mg), 818 (8, 3.1 mg) and ptilomicalin A (6, 2.9 mg), The upper phase (37.2 g), shown by small peaks at m / z 658 and 674, was separated by flash chromatography on a silica gel column (300 g, 230-400 mesh), levigating with a solvent gradient system increasing methanol (0% -100%) in CHCl3 (100% -0%). The fractions shown in the peaks in m / z 658, 674 and 688 were further purified by repeated silica gel column chromatography (230-400 mesh) using CHCl3: EtOAc: MeoH (9: 9: 1), CHCl3: MeOH (15: 1) and CHCl3: MeOH (9: 1) as solvent systems to produce crambescidins 673 (9, 23.8 mg), 687 (10, 14.7 mg), 657 (11, 3.4 mg) and 13,14,15 -iso-crambescidin 657 (12, 6.6 mg). Crabscidin 834 (7): colorless gum [a] 25D -24.7 ° (c 0.44, MeOH); FABMS m / z 835 (M + H), 799 (M + H-HCl C45H79N606, HRFABMS 799.6075,? -1.4 mmu), 781 (M + H-HCI-H2O, C45H79N6O6l HRFABMS 799.6075,? -2.9 mmu), 694 , 655, 614 (C36H6oN3O5, HRFABMS 781.5984,? -2.9 mmu), 694, 655, 614 (C3ßH6oN305, HRFABMS 614.4549,? -1.6 mmu), 426, 420, 398 (C23H48N302, HRFABMS 398.3729,? 1.8 mmu), 380 (C23H46N30, HRFABMS 380.3636,? 0.5mmu), 374, 358, 314, 264, 246, 198 (C ^ HzoNaO, HRFABMS 198.1616,? -1.0 mmu); 1 H NMR (CD3OD) see Table 1; 13C NMR (CD3OD) see Table 1; 13C NMR (CD3OD) see Table 2; 1H NMR (CDCl 3) d: 0.88 (t, J = 7, H3-1), 1.46 (m, H-2a), 1.54 (m, H-2b), 4.51 (br d, J = 10, H-3 ), 5.49 (br d, = 11, H-4), 5.67 (br dd, J = 11, 7.5, H-5), 2.19 (m, H-9a), 2.34 (m, H-6b), 1.94 (m, H-7a), 2.47 (br t, J = 14, H-7b), 1.42 (t, J = 12.5, H-9a), 2.56 (dd, 12.5, 5, H-9b), 4.32 ( , H-10), 1.57 (m, H-11a), 2.33 (m, H-11b), 2.04 (ddd, J = 14, 10, 4.5, H-12a), 2.16 (m, H-12b), 3.36 (s, H-14), 1.62 (m, H-16a), 1.77 (ddd, J = 14, 14, 4.5, H-16b), 1.76 (m, H-17a), 2.32 (m, H- 17b), 1.23 (M, H-18a), 1.76 (dddd, J = 14, 7, 7, 2, H-18b), 4.09 (m, H-19), 1.10 (d, J = 6, H- 20), 4.09 (m, H2-23), 1.61 (m, H2-40), 3.43 (br, H2-41), 3.27 (br, H2-42), 3.79 (br, H-43), 1.68 ( m, H-44a), 2.15 (m, H-44b), 2.75 (br, H-45a), 2.86 (br, H-45B), 5.83 (s, H-130H), 10.01 (br s, H- 8N), 10.07 (br s, H-15N); 13 C NMR (CDCl 3) d: 10.16 (C-1), 29.09 (c-2), 71.29 (C-3), 133.66 (C-4), 129.84 (C-5), 23.44 (C-6), 36.90 (C-7), 83.56 (C-8), 37.03 (C-9), 52.41 (C-10), 29.53 (C-11), 37.19 (C-12), 88.68 (C-13), 54.55 ( C-14), 83.04 (C-15), 32.09 (C-16), 18.00 (C-17), 31.56 (C-18), 68.84 (C-19), 21.43 (C-20), 148.13 (C -21), 167.17 (C-22), 65.93 (C-23), 28.38 (C-24), 25.78 (C-36), 32.19 (C-37), 165.01 (C-38), 47.21 (C-) 39), 19.10 (C-40), 38.74 (C-41), 57.50 (C-42), 65.05 (C-43), 31.71 (C-44), 37.04 (C-45); HRFABMS cale Mr for C45H8oN606CI 835.5828 (M + H) +, found Mr 835.5821. Crambescidin 818 (8): colorless gum, [a] 25D -11.4 ° (c 0.31, MeOH); FABMS m / z 819 (M + H), 783 (M + H-HCl, C45H79N6O5, HRFABMS 783.6145,? -3.3 mmu), 696 (C14H70N5O4, HRFABMS 696.5437,? -.09 mmu) 639, 612, 598, 404 , 430, 398, 380, 358, 288, 260, 206; 1H NMR (CD3OD) see Table 1; 13C NMR (CD3OD) see Table 2; 1 H NMR (CDCl 3) d: 0.83 (t, J = 7, H-1), 1.42 (m, H-2a), 1.53 (m, H-2b), 4.50 (br d, J = 9.5, H-3). 5.48 (br d, J = 11, H-4), 5.68 (br dd, J = 11, 7, H-5), 2.18 (m, H-6a), 2.34 (m, H-6b), 1.69 ( m, H-7a), 2.46 (br t, J = 13, H-7b), 1.41 (t, J = 12.5, -9a), 2.55 (dd, J = 12.5, 4.5, H-9b), 3.96 ( M, H-10), 1.61 (m, H-11a), 2.21 (m, H-11b), 1.79 (m, H-12a), 2.27 (m, H-12b), 4.28 (ddd, 10, 5 , 5, H-13), 2.94 (d, J = 5, H-14), 1.79 (m, H2-16), 1.79 (M, H2-17), 1.20 (m, H-18a), 1.71 ( m, H-18b), 3.96 (m, H-19), 1.05 (d, J = 6, H-20), 4.09 (M, H2-23), 1.61 (m, H2-24), 1.60 (m , H2-36) 2.02 (m, H2-37), 3.48 (br, H2-40), 3.43 (br, H2-41), 3.28 (br, H2-42), 3.80 (br, H-43), 1.75 (m, H-44b), 2.77 (br, H-45a), 2.85 (br, H-45b), 9.53 (br s, H-8n), 9.77 (br s, H-15N); 13 C NMR (CDCl 3) d: 10.09 (C-1), 29.11 (C-2), 71.03 (C-3), 133.69 (C-4), 129.94 (C-5), 23.42 (C-6), 36.97 (C-7), 83.59 (C-8), 36.99 (C-9), 53.95 (C-10), 30.66 (C-1 I), 26.84 (C-12), 51.84 (C-13), 49.67 (C-14), 80.71 (C-15), 31.92 (C-16), 18.40 (C-17), 31.96 (C-18), 67.29 (C-19), 21.46 (C-20), 148.85 ( C-21), 168.39 (C-22), 65.46 (C-23), 28.47 (C-24), 25.79 (C-36), 31.92 (C-37), 165.04 (C-38); HRFABMS caled Mr for C45H80N6? 5 Cl 819.5879 (M + H) +, found Mr 819.5889. Crambescidin 673 (9): colorless gum [a] 25D-16.6 (c 0.50, MeOH); FABMS m / z 674 (M + H), 576, 420, 358, 314 (C19H28N3O, HRFABMS 314. 2240,? -0.8 mmu), 264, 246, 168; 1 H NMR see Table 1; 13C NMR see Table 2; HRFABMS caled Mr for C38H64N3O7 674.4744 (M + H) +, found Mr 674.4734. Crambescidin 687 (10): colorless gum, [α] 25 D d -18.2 ° (c 0.52, MeOH); FABMS m / z 688 (M + H), 630, 590, 420, 374, 358, 314, 264, 246, 168; 1 H NMR see Table 1; 13C NMR see Table 2; HRFABMS caled Mr for C39H66N307688.4901 (M + H) +, found Mr 688.4907. Crambescidin 657 (11): colorless gum, [] 25D- -12.1 ° (c 0.34, MeOH); FABMS m / z 658 (M + H), 612, 560, 404, (C 22 H 34 N 3 O 4, HRFABMS 4042541, δ 0.8 mmu), 360, 358, 288, 206, (C 13 H 20 NO, HRFABMS 206.1547, λ-0.2 mmu); H NMR see Table 1; 13C NMR see Table 2; HRFABMS caled Mr for C38H64 3? 6 658.4795 (M + H)? Found Mr 658.4797. Crambescidin 657 methanation: A mixture of 11 (1 mg) dissolved in MeOH (1 ml) and diazomethane in Et20 (2 ml) was kept at room temperature for 24. The solvents were removed (N2) and the residue was subjected to chromatography on silica gel using CHCl3: MeOH (9: 1) as a solvent system to produce the methyl ester of 11 (0.7 mg). Colorless gum, FABMS m / z 672 (M + H), 574, 404, 358, 288, 206; HRFABMS caled Mr for C39H66N306 672.4952 (M + H) +, found Mr 672.4984. 13,14,15-lsocrambescidin 657 (12): colorless gum, [a] 25D -32.7 ° (c 0.29, MeOH); FABMS m / z 658 (M + H), 612, 560 (C32H5.IN3O2, HRFABMS 560.4034,? 2.9 mmu), 404, 360, 358 (C21H32N3O2, HRFABMS 358.2494,? 0.1 mmu), 288, 206; 1 H NMR see Table 1; 13C NMR see Table 2; HRFABMS cale Mr C38H64N306658.4795 (M + H) +, found Mr 658.4790.

Table 1 provides 1 H NMR data for compounds 7-12. Table 2 provides 13C NMR data for compounds 7-12.

Table 1. 1H NMR data for compounds 7-12 HNo 7 (CD3OD) 8 (CD3OD) 9 (CDCb) 10 (CDCb) 11 (CDCb) 12 (CDCb) 1 0.89, t, 7 0.85, t, 7 0.86, t, 7 0.87, t, 7 0.82, t, 7 0.94, t, 7.5 2a 1.49, m 1.48, m 1.45, tn 1.46, m 1.42, m i.47, m 2b 1.57, m l.55, m 1.54, m 1.55, ml, 53, m 1.51, m 3 4.46, br d, 10 4.42, brd, 9.5 4.52, brd, 10 4.51, brd, 1 LO 4.38, brd9.5 4.63, br s 4 5.52, brd, 11 5.51, brd, 11 5.48, brd, 11 5.48, br d, 11 5.48, brd, 11 5.50, brd, 11 5.72, brdd, 11.7 5.72, brdd, 11.7 5.64, brdd, 11.7 5.66, br dd, 11.7 5.64, brdd, 11.7 5.62, brdd, 11.7 6a 2.18, m 2.18, m 2.12, m 2.18, m 2.14, m 2.10, m 6b 2.44, m 2.42, m 2.33, m 2.34, m 2.32, m 2.30, m 7a 2.02, m 2.02, m 1.81, m 1.92, m 1.84, m 1.78, m 7b 2.37, brt, 13.5 2.33, br t, 13.5 2.73 brt, 14 2.51, brt, 14.5 2.74, br t, 14 2.73, br t, 13.5 9a 1.44, t, 12.5 1.44, t, 13 1.32, t, 12.5 1.41, t, 12.5 1.31, t, 12.5 1.35, t, 12.5 4.32, m 4.05, m 4.32, m 43.2, m 3.98, m 3.89, lia 1.61, m 1.60, m 1.55, m 1.57, m 1.61, m 1.55, 11b 2.43, m 2.29, m 2.30, m 2.33, m 2.14, m 2.19, 12a 2.06, m 1.86, m 1.98, m 2.03, m 1.78, m 1.62, m 12b 2.18, m 2.31, m 2.14, m 2.16, m 2.24, m 2.15, m 13 4.35, m 4.26, 3.98, m 14 3.48, s 3.08, d, 5 3.31.3 3.35, s 2.89, d, 4 3.48, d, 3.5 16a 1.65, m 1.68, m 1.68, m 1.62, m 1.62, 1.62, 16b l, 87, m 1.76, m 1.74, m 1.76, m 2.02, 1.71, -n 17th 1.84, m 1.75, m 1.67, m 1.76, m 1.67, m 1.69, m Table 2. C NMR data for compounds 7-12 C No. 7 (CD3OD) 8 (CD3OD) 9 (CDCb) 10 (CDCb) 11 (CDCb) 12 (CDCb) 1 10.26 10.12 10.22 10.14 10.17 10.30 2 29.70 29.57 29.06 29.10 29.18 29.14 3 71. 83 71.64 70.70 71.26 70.57 70.36 4 133.54 133.62 133.84 133.67 133.91 133.89 130.59 130.66 129.78 129.75 129.88 129.62 6 23.73 23.74 23.72 23.45 23.77. 24.37 7 36.88 37.19 36.78 36.88 36.83 37.41 8 84.44 84.41 83.53 83.60 83.66 85.00 9 38.98 37.67 37.77 37.1 1 37.78 37.34 53.41 54.90 52.21 52.40 53.80 52.88 11 30.00 30.78 29.66 * 29.67. 30.66 * 30.26 12 37.52 26.88 35.70 36.88 26.85 29.64 * 13 89.86 53.47 88.44 88.69 51.58 51.99 14 55.17 49.97 55.59 54.64 50.64 41.48 83.82 81.48 83.15 83.08 80.64 83.20 16 31.90 31.91 29.45 * 32.08 31.91 * 29.68 * 17 18.46 18.77 17.60 17.94 17.95 19.90 18 31.76 32.27 * 31.67 * 31.53 32.19 ** 31.58 ** 19 69.34 67.75 68.09 68.82 66.71 68.81 21.00 21.09 21.49 21.41 21.59 22.25 21 148.86 149.54 148.85 148.12 149.48 149.74 Biological activity Crambescidins (1-4) inhibited the growth of L121 cells 0, 1 crambescidin 816 (1) also exhibited antiviral activity against Herpes simplex virus type I (HSV-1) and was shown to be a channel blocker. Ca2 + strong.4 1 3, 14, 15-Isocrambescidin 800 (5) was substantially less cytotoxic for L1 210 cells, and did not observe any antiviral activity.2 Ptilomicalin A (6) showed cytotoxicity against L1 210, P388, and KB cells, antifungal activity against Candida albicans, as well as antiviral activity (HSV) .5'6 In a parallel assay against murine L1210 leukemia cells (see Table 3), using crambescidin 816 (1) as a standard, crabscidin 834 (7) and 81 8 (8) with a chlorinated spermidine unit are approximately 5 times more active than 1. However, crambescidins 674 (9) and 687 (10) without a unit derived from spermidine are less than 5 times as active as 1. As expected, 1 3, 14, 1 5-isocrambescidin 657 (1 2) is substantially less active (no inhibition at 5 μg / ml) than other crambescidins. Ptilomicalin A (6) is slightly more active than 1. Meanwhile, in an antimicrobial assay against Rhodotorula glutinis, the crambescidins with one unit derived from spermidine and ptilomicalin A showed to be active at 2 μg / well, other crambescidins showed no activity at 20 μg / well. These observations revealed that both the cage-like structure of the pentacyclic portions and the spermidine or its derived unit in the crambescidins and ptilomicalin A play important roles in their strong biological activities. Interestingly, crambescidin 657 (11) shows that it is the most cytotoxic compound in the test. See especially Table 4. Activity is significantly decreased by methanation with diazomethane. Because the acid terminal of the side chain is bent toward the basic pentacyclic guanidine portion in the inner salt form of 1 1, and that the conformation of the inner salt is different from the other crambescidins, the cytotoxicity of 1 1 could come from a different mechanism of action for the cells. The new crambescidin compounds will have pharmaceutical uses comparable to the previously known crambescidin compounds, particularly as antitumor compounds, as shown in Tables 3 and 4.

Table 3. Cytotoxicities against L121 0 cells for compounds 1, 6-12, and methyl 1 1 Concentration I nhibition (%) (μg / ml) 8 10 11 12 Methyl 11 1. 0 100 100 100 100 100 100 100 100 0 0.5 97 99 95 95 0 0 100 0 0 0.25 70 60 95 95 0 0 100 0 0 0.1 0 20 90 90 0 0 93 0 0 Table 4. Cytotoxicities against the cell lines of tumor Crambescidins μg / ml P-388 A-549 HT-29 MEL-28 834 (7) 0.05 0.05 0.05 0.05 818 (8) 0.1 0.1 0.1 0.1 673 (9) Nd Nd Nd Nd 687 (10) Nd Nd Nd Nd 657 (1 1) 0.25 0.05 0.05 0.05 81 6 (1) 0.5 0.5 0.4 Nd: Activity not determined.

The compounds of the present invention have been isolated (or semi-synthetically prepared) in substantially pure form, ie, at a level of purity sufficient to permit physical and biological characterization thereof. As described above, it has been found that these compounds possess specific antitumor activities, and as such will be useful as medicinal agents in mammals, particularly in humans. Thus, another aspect of the present invention concerns pharmaceutical compositions containing the active compounds identified herein and methods of treatment employing such pharmaceutical compositions. The antitumor activities of the compounds have been determined in vitro in cell cultures of mouse leukemia P-388, human lung carcinoma A-549, human colon carcinoma HT-29 and human melanoma M EL-28. The procedure was carried out using the methodology described by Bergeron, et a !. , Biochem. Biophys. Res. Comm. , 1 21: 848, 1884 and by Schroeder, et al. , J. Med. Chem., 24: 1078, 1981. The active compounds of the present invention exhibit antitumor activity against mammalian tumors, such as murine leukemia P-388, human lung carcinoma A-549, human colon carcinoma HT-29 and human melanoma MEL-28. The present invention thus includes a method for treating any mammal affected by a malignant tumor sensitive to these compounds, which comprises administering to the affected individual a therapeutically effective amount of an active compound or mixture of compounds, or pharmaceutical compositions thereof. The present invention also relates to pharmaceutical compositions containing as active ingredient one or more of the compounds of this invention, as well as the processes for their preparation. Examples of pharmaceutical compositions include any solid (tablets, pills, capsules, granules, etc.) or liquid (solutions, suspensions or emulsions) with suitable composition or oral, topical or parenteral administration, and may contain the pure compound or in combination with any carrier or other pharmacologically active compounds. These compositions may need to be sterile when they are to be administered parenterally. The correct dosage of a pharmaceutical composition comprising the compounds of this invention will vary according to the particular formulation, the mode of application and the particular situs, host and bacteria or tumor being treated. Other factors such as age, body weight, sex, diet, time of administration, speed of excretion, condition of the host, combinations of medications, reaction sensitivities and severity of the disease should be considered. The administration can be carried out continuously or periodically within the maximum tolerated dose.

References The following references provide support information related to this invention. (1) Jares-Erijiman, E.A. Sakai, R; Rinehart, K.L. J. Org. Chem. 1991, 56, 5712-5715. (2) Jares-EriJiman, E.A .; Ingrum, A.L .; Carney, J.R .; Rinehart, K.L .; Sakai, R. J. Org. Chem. 1993, 58, 4805-4808. (3) Taveras, R .; Daloze, D .; Braekman, J.C .; Hajdue, E. Biochem. Syst. Ecol. 1994, 22, 645. (4) Berlinck, R. G. S .; Braekman, J.C. Daloze, D .; Bruni, I .; Riccio, R .; Ferri, S., Spampinato, S .; Speroni, E. J. Nat. Prod. 1993, 56, 1007-1015. (5) Kashman, Y .; Hirsch, S .; McConnell, O.J .; Ohtani, I .; Kusumi, T .; Kakisawa, H. J. Am. Chem. Soc. 1989, 111, 8925-8926. (6) Ohtani, I .; Kusumi, T .; Kakisawa, H .; Kasman, Y .; Hirsh, S. J. Am. Chem. Soc. 1992, 114, 8472-8479. (7) Ohtani, l .; Kusumi, T .; Kakisawa, H. Tetrahedron Lett. 1992, 33, 2525-2528. (8) Overman, L.E .; Rabinowitz, M.H .; Renhowe, P.A. J. Am. Chem. Soc. 1995, 117, 2657-2658. (9) Patil, A.D .; Kumar, N.V .; Kokke, W.C .; Bean, M.F .; Freyer, A.J .; Brosse, C.D .; Mai, S .; Truneh, A .; Faulkner, D.J .; Carte, B .; Breen, A.L .; Hertzberg, R.P .; Johnson, R.K .; Westley, J.W .; Potts B.C.M. J. Org. Chem. 1995, 60, 1182-1188. (10) Witten, J.L .; Schaffer, M.H .; O'Shea, M .: Cook, J.C .; Hemling, M.E .; Rinehart, K.L., Jr. Biochem, Biophys. Res. Commun. 1984, 124, 350-358.

The present invention has been described in detail, including preferred embodiments thereof. However, it will be appreciated that those skilled in the art, upon consideration of the present disclosure, may make modifications and / or improvements in this invention and still be within the scope and spirit of this invention as set forth in the following claims.

Claims (9)

REVIVAL NAME IS

1 . Crabscidin 834 substantially pure. 2. Crambescidin 81 8 substantially pure. 3. Crambescidin 673 substantially pure. 4. Crambescidin 687 substantially pure. 5. Crambescidin 657 substantially pure. 6. 13, 14, 1 5 Isocrambescidin 657 substantially pure. 7. Metii crambescidin 657 substantially pure. 8. A pharmaceutical or veterinary composition comprising an effective anti-tumor amount of the substantially pure compound designated herein as Crambescidin 834 and a pharmaceutically acceptable carrier, diluent or excipient. 9. A pharmaceutical or veterinary composition comprising an effective antitumor amount of the substantially pure compound designated herein as Crambescidin 81 8 and a pharmaceutically acceptable carrier, diluent or excipient. 1 0. A pharmaceutical or veterinary composition comprising an effective anti-tumor amount of the substantially pure compound designated herein as Crambescidin 673 and a pharmaceutically acceptable carrier, diluent or excipient. eleven . A pharmaceutical or veterinary composition comprising an effective anti-tumor amount of the substantially pure compound designated herein as Crambescidin 687 and a pharmaceutically acceptable carrier, diluent or excipient.

2. A pharmaceutical or veterinary composition comprising an effective antitumor amount of the substantially pure compound designated herein as Crambescidin 657 and a pharmaceutically acceptable carrier, diluent or excipient. 1

3. A pharmaceutical or veterinary composition comprising an effective anti-tumor amount of the substantially pure compound designated herein as 1 3, 14, 1 Isocrambescidin 657 and a pharmaceutically acceptable carrier, diluent or excipient. 1

4. A pharmaceutical or veterinary composition comprising an effective antitumor amount of the substantially pure compound designated herein as methyl crambescidin 657 and a pharmaceutically acceptable carrier, diluent or excipient. 1

5. The use for the manufacture of a medicament for the therapeutic or prophylactic treatment of a patient suffering from a mammalian tumor, of an effective antitumor amount of the substantially pure compound designated herein as Crambescidin 834 and a carrier, diluent or pharmaceutically acceptable excipient. 1

6. The use for the manufacture of a medicament for the therapeutic or prophylactic treatment of a patient suffering from a mammalian tumor, of an effective antitumor amount of the substantially pure compound designated herein as Crambescidin 81 8 and a carrier, diluent or pharmaceutically acceptable excipient.

7. The use for the manufacture of a medicament for the therapeutical or prophylactic treatment of a patient suffering from a myelodic tumor, of an effective antitumor amount of the substantially pure compound designated herein as Crambescidin 673 and a carrier, pharmaceutically acceptable diluent or excipient. 1

8. The use for the manufacture of a medicament for the therapeutic or prophylactic treatment of a patient suffering from a mammalian tumor, of an effective antitumor amount of the substantially pure compound designated herein as Crambescidin 687 and a carrier, diluent or pharmaceutically acceptable excipient. 1

9. The use for the manufacture of a medicament for the therapeutic or prophylactic treatment of a patient suffering from a mammalian tumor, of an effective antitumor amount of the substantially pure compound designated herein as Crambescidin 657 and a carrier, diluent or pharmaceutically acceptable excipient. 20. The use for the manufacture of a medicament for the therapeutic or prophylactic treatment of a patient suffering from a mammalian tumor, of an effective antitumor amount of the substantially pure compound designated herein as 1 3, 14, 15 Isocrambescidin 657 and a pharmaceutically acceptable carrier, diluent or excipient. twenty-one . The use for the manufacture of a medicament for the therapeutic or prophylactic treatment of a patient suffering from a mammalian tumor, of an effective antitumor amount of the substantially pure compound designated herein as Methyl Crambescidin 657 and a carrier, diluent or excipient pharmaceutically acceptable. SUMMARY Together with the known crambescidins (1 -5) and ptilomicalin A (6), two new minor crambescidins 673 (9), 687 (10), 657 (11) and 1 3, 14, 15 -socrambescidin have been obtained. 756 (12) without a unit derived from spermidine, by FABMS-guided isolation of extracts from the Mediterranean sponge Crambe. Their structures were elucidated through the interpretation of spectral data (see Figures 4-9). In a parallel bioassay against murine leukemia cells L1 21 0, crambescidins 834 (7), 818 (8) and 657 (11) were shown to be five times as cytotoxic as the known crambescidins 816 (1). Crambescidins with a spermidine, or unit derived from spermidine, also exhibited antimicrobial activity against Rhodotorula glutinis.