J. Org. Chem. 1990,55, 223-227 Duke University Spectroscopy Center, funded by NSF Grant DMB8501010, NIH Grant RR62780, and NC Biotechnology Grant 86U02151. We thank Mr. Larry Dillard for carrying out the MNDO calculations, T r i p s Associates for allowing us to participate in their Academic Users Program, and Professor Don Chesnut for helpful discus-
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sions. Supplementary Material Available: Experimental procedures and characterization data for imides 7a-c, 8a,b, hydroxy lactams 9a-c, 10a,b, and allyl lactams lla,b, 12a-c, 13a,b,and 14a,b (8 pages). Ordering information is given on any current masthead page.
Antiviral and Antitumor Agents from a New Zealand Sponge, Mycale sp. 2. Structures and Solution Conformations of Mycalamides A and B Nigel B. Perry, John W. Blunt,* Murray H. G. Munro,* and Andrew M. Thompson Department of Chemistry, University of Canterbury, Christchurch, New Zealand Received M a r c h 21, 1989
Mycalamide B (2) has been found to occur with mycalamide A (1) in a New Zealand sponge of the genus Mycale. Both are potent antiviral and antitumor agents. T h e solution conformations of mycalamide A (1) have been explored in detail by analysis of 'H-lH coupling constants and NOE interactions and modeled by molecular mechanics calculations. Significant populations of several solution conformations were indicated. A comparison of 'H NMR data showed t h a t analogous conformations were present in solutions of mycalamide B (2) and the structurally similar compounds pederin (4) and onnamide A (6).
The isolation of mycalamide A (I), an antiviral agent from a New Zealand sponge of the genus Mycale, has been described recently.' This multifunctional heterocyclic compound is also being evaluated as an antitumor agent on the basis of its in vivo activity against P388 murine leukemia and a variety of solid tumor model systems, including Lewis Lung, M5076, Burkitt's Lymphoma, and MX-1 and CX-1 human tumor xenografts.2 To provide further mycalamide A (1) for this biological testing, more of the active Mycale species (family Mycalidae, order Poecilosclerida)' was collected and extracted. During this processing a set of 'H HMR peaks close to those of mycalamide A were noticed in the spectra of some samples. Further chromatography gave a less polar compound, mycalamide B (2), which was also antiviral and had significant in vivo antitumor activity.2
1 R7 L R77
2 3
L
R7 L RIB L R7 L R1, L
RIB L H H, R17 L CHI CO.CHa, R17 L CH:,
The 'H NMR spectrum of mycalamide B was very similar to that of mycalamide A, but contained three, rather than two, methoxyl signals (Table I). High-resolution mass spectroscopy gave a molecular formula C2,H4,NOlo (mycalamideA is CuH,lNOlo), and chemical ionization MS with NDBas the reagent gas indicated the presence of just three exchangeable protons,3 thus implying that one of the (1) Perry, N. B.; Blunt, J. W.; Munro, M. H. G.; Pannell, L. K. J. Am. Chem. SOC. 1988,110,4850. (2) (a) Burres, N. S.; Clement, J. J. Cancer Res., in press. (b) Burres, N. S., personal communication.
three hydroxyl groups of mycalamide A (1) is replaced by a methoxyl group in mycalamide B. The 13C NMR spectrum, assigned by heteronuclear correlation experiments, showed that the greatest difference between A and B was associated with the C17 signal (71.51 ppm in A, 78.84 ppm in B), indicating the presence of the additional methoxyl group at C17.4 The locations of the two hydroxyl groups of mycalamide B were confirmed by preparing mycalamide B diacetate (3), in which the H7 and H18 NMR signals were each shifted more than 0.6 ppm downfield. Thus mycalamide B (2) is the 17-methoxyl derivative of mycalamide A ( l).5 To eliminate the possibility that the methoxyl groups in these compounds were solventderived, an extraction of the Mycale sponge was carried out with ethanol substituted for methanol. This led to the isolation of mycalamides A and B as before. No other compounds in this series have been detected in any extracts of this sponge species. Mycalamide B (2) was present in the sponge at less than half the level of mycalamide A (l),but it was a more potent antiviral agent in vitro than A (minimum active dose 1-2 ng/disk for B, 3.5-5.0 ng/disk for AI). The partially purified extract which had in vivo antiviral activity' contained both mycalamides A and B (no in vivo antiviral data have been obtained on the pure compounds). Mycalamide B was more cytotoxic than A (P388 IC50)s0.7 f 0.3 and 3.0 f 1.3 ng/mL, respectively), and B was active in vivo against P388 leukemia at a lower optimum dose than mycalamide A.2 The natural products most similar in structure to mycalamides A (1) and B (2) are the insect toxin pederin (4)6 (3) Daly, J. W.; Spande, T. F.; Whittaker, N.; Highet, R. J.; Feigl, D.; Nishimori, N.; Tokuyama, T.; Meyers, C. W. J. Nat. Prod. 1986,49,265. (4) Stothers, J. B. Carbon-I3 NMR Spectroscopy; Academic Press: New York, 1972; p 143. (5) Further work is underway to establish the absolute stereochemistries of C2 to C7, C10 to C15, and C17, which are drawn as for pederin (4) for convenience. (6) (a) Cardani, C.; Ghiringhelli, D.; Mondelli, R.; Quilico, A. Tetrahedron Lett. 1965, 2537. (b) Bonamartini Corradi, A.; Mangia, A.; Nardelli, M.; Pelizzi, G. Gazz. Chim. Ital. 1971,101, 591. (c) Matsumoto, T.; Yanagiya, M.; Maeno, S.; Yasuda, S. Tetrahedron Lett. 1968,6297. (d) Furusaki, A.; Watanabe, T.; Mataumoto, T.; Yanagiya, M. Tetrahedron Lett. 1968,6301.
0022-3263/90/1955-0223$02.50/0 0 1990 American Chemical Society
224 J . Org. Chem., Vol. 55, No. 1, 1990
proton 2CH3 H2 H3 3CH3 4=CHZ 4=CHE H5a H5e 60CH3 H7 NH H10 lOOCHa lOOCHe H11 H12a H12e H13 130CH3 14CH3e 14CH3a H15 H16 H16 H17 170CH3 H18 H18
Perry et al.
Table I. 'H NMR Dataa for Mycalamides A and B. Pederin. and Onnamide A mycalamide A mycalamide B pederinb mycalamide A' 1.19 (6.6) 1.20 (6.6) 1.20 (6.6) 1.16 (6.6) 4.01 (2.8, 6.6) 3.88 (2.6, 6.6) 3.98 (2.7, 6.6) 4.02 (2.8, 6.6) 2.24 (2.4, 6.9) 2.26 (2.8, 7.0) 2.19 (2.7, 7.0) 2.24 (2.7, 7.0) 1.01 (7.1) 1.03 (7.0) 0.95 (7.0) 0.99 (7.0) 4.79 (2.1, 2.1) 4.85 (2.0, 2.0) 4.86 (1.9, 1.9) 4.84 (m) 4.64 (2.2, 2.2) 4.72 (1.9, 1.9) 4.75 (1.9, 1.9) 4.73 (m) 2.39 (2.1, 2.1, 14.3) 2.22 (2.0, 2.0, 13.5) 2.36 (1.9, 1.9, 14.2) 2.36 (m) 2.30 (14.2) 2.36 (13.9) 2.45 (14.2) 2.36 (m) 3.24 3.29 ?3.35 3.29 4.28 4.31 (2.2, 2.2) 4.29 4.30 7.54 (10.0) 7.16 (9.7) 7.49 (9.8) 5.81 (9.5) 5.79 (9.7, 9.7) 5.39 (8.0, 9.7) 5.87 (9.8, 9.8) 5.21 (6.9) 5.12 (7.0) 5.13 (6.9) 4.80 (6.8) 4.84 (6.9) 4.87 (6.9) 3.81 (2.5, 6.3, 8.0) 3.98 (6.5, 9.4) 3.79 (6.78 9.7) 3.86 (6.7, 9.8) 4.21 (6.7, 10.4) 1.76 (6.2, 10.9, 13.4) 4.17 (6.5, 9.9) 4.22 (6.7, 10.3) 2.05 (2.5, 4.6, 13.4) 3.44 (10.5) 3.66 (10.1) 3.65 (4.6, 10.9) 3.46 (10.3) 3.55 3.56 3.55 1.00 0.95 0.97 0.98 0.86 0.88 0.85 0.87 3.25 (2.0, 10.2) 3.5 (2.3, 10.3) 3.41 (3.3, 8.3) 3.64 (5.1, 7.5) 1.66 (2.3, 7.0, 14.2) 1.73 (3.0, 10.2, 14.0) 1.55 (m) 1.54 (m) 1.49 (5.6, 10.3, 14.2) 1.61 (2.0, 9.3, 14.0) 1.55 (m) 1.54 (m) 3.7 (m) 3.5-3.3 (m) 3.2 (m) 3.74 (m) ?3.34 3.24 3.49 (3.8, 11.3) 3.5-3.3 (m) 3.55 (m) 3.65 (3.3, 11.9) 3.5-3.3 (m) 3.38 (6.2, 11.2) 3.47 (5.7, 11.9) 3.38 (6.2, 11.4)
onnamide 1.17 (6.5) 3.87 (2.4, 6.5) 2.18 (m) 0.96 (6.9) 4.79 4.63 2.40 (14.4) 2.32 (14.4) 3.22 4.23 5.79 (9.3) 5.18e (6.9) 4.80 (6.9) 3.98 (6.5, 9.3) 4.16 (6.5, 9.7) 3.62 (9.6) 3.55 1.00 0.85 3.47 (3.6, 8.1) 1.53 (m) 1.53 (m) 3.64 (m) 1.49 (m) 1.28 (m)
"In CDC13 unless otherwise stated; shift in ppm (coupling in hertz, m = multiplet). bSee ref 16, lOOCH, and 180CH3 omitted. CIn CD30D. dSee ref 7, remainder of side chain omitted. eCorrected by reference to the original spectrum.ls
saturated chains and six-membered rings, appear to allow many different conformations. Their solution conformations have therefore been examined to establish if there are similarities in shape which could account for the common mode of action.
A
B
Figure 1. The two solution conformations proposed from the NMR data for 01-C10 of mycalamide A (1).
and onnamide A (6).7 Pederin (41, the vesicatory principle from blister beetles of the genus Puederus,6 is a potent inhibitor of protein synthesis,8 and its mode of action has been studied in some detail.g Until the discovery of the mycalamides and onnamide A (6), an antiviral compound from a sponge of the genus Theonella, the only other pederin-like natural products were found with pederin and were very similar in structure.'O Mycalamides A (1) and B (2) and onnamide A (6) are also protein synthesis inhibitors (potency 4 = 2 > 1 > 6), and it is this property that is thought to be the basis of biological activity in these compounds.2 In view of their structural similarities, an obvious hypothesis is that some common substructure in the mycalamides, pederin (4) and onnamide A (6), is interacting with the same active site to inhibit protein synthesis. The structures of these inhibitors, made up of
HO2C
0 I
NH
H
CH2
(7) Sakemi, S.;Ichiba, T.; Kohmoto, S.; Saucy, G.; Higa, T. J. Am. Chem. SOC. 1988,110,4851. (8) Brega, A.; Falaschi, A,; de Carli, L.; Pavan, M. J. Cell Biol. 1968, 36, 485. (9) Vazquez, D. Inhibitors of Protein Biosynthesis; Springer-Verlag: Berlin. 1979. (10) (a) Cardani, C.; Ghiringhelli, D.; Mondelli, R.; Quilico, A. Gazz. Chim. Ital. 1966,96,3. (b) Cardani, C.; Ghiringhelli,D.; Quilico, A.; Selva, A. Tetrahedron Lett. 1967, 4023.
6
The conformations of mycalamide A (1) in both CDC1, and CD30D solutions were established from proton-proton couplings (Table I) and difference NOE NMR experiments (Table 11). The 'H NMR data in CDBODsolution were obtained for direct comparison with onnamide A (6) d a h 7 There were no major differences in the IH NMR data for
J. Org. Chem., Vol. 55, No. 1, 1990
Antiviral and Antitumor Agents from Mycale Sp.
225
Table 11. *H-'H NOE Interactions for Mycalamide A signal enhanced (% enhancement) 1
signal irradiated 2-CHq H2 H3 3-CH3 4=CHZ 4=CHE H5a H5e 6-OCH, H7 NH H10 10-OCHa 10-OCHe H11 H12 H13 13-OCH3 14-CH3e 14-CH3a H15 H16 H16C H17 H18 H18'
" Omitted.
in CDC1, solution
in CD30D solution ND" 6-OCH3 (21, H3 (lo), 2-CH3 (2) 4=CHZ (6), H2 (lo), 2-CH3 (l), 3-CH3 (2) H5a (4), H3 (lo), 2-CH3 (2)
H2 (10) 6-OCH3 (2), H3 (7), 2-CH3 (1) 4=CHZ (6), H2 (ll),3-CH3 (2) H15 (3),b13-OCH3 (l),b H13 (4): H5 (l), H3 (8), H16 (2)b 10-CHa (7): 4=CHE (21), H3 (7) 4=CHZ (19), H5 (1) NH (2), 4=CHE (6),bH7 (2) NH (2),b4=CHE (6), H7 (2)b H7 (71, H2 (7) NH (l),6-OCH3 (3) H7 (2), H11 (7) 10-OCHa (4),H15 (5), H13 (3) H10 (5), 10-OCHe (16), 13-OCH3 (l),H13 (5) 10-OCHa (14), 4=CHE (6)b NH (5), H12 (11) H11 (lo), 14-CH3a (1) H10 (4), 10-OCHa (5), H15 (2), 14-CH3e (1) 10-OCHa (1) H15 (4), 13-OCH3 (l), H13 (5), H5 (l),bH3 (4),bH16 (2) H12 (lo), H16 (2) H10 (lo), H18 (15),b H16 (l),14-CH3e (1) H17 (5), 14-CH3e (l),14-CH3a (1) H17 (5), 14-CH3e (l),14-CH3a (1) H16 (1) H10 (10): H18 (151, H16 (l), 14-CH3e ( l ) b H18 (16)
ND 4=CHZ (18), H5e (4) H7 (4), H5e (30), 3-CH3 (2) 4=CHE (7), H7 (l)* H7 (8), H2 (8) 6-OCH3 (3) ND ND ND ND ND ND 14-CH3e (1) ND ND ND 14-CH3e (2) 14-CH3a (1) ND ND H18 (>lo)
Enhancement interpreted as being due to irradiation of an overlapping signal.
these two solvents (Tables I and 11). The 01-C6 ring is in a chair conformation, with 6-OCH, axial and O-CH, anti to C5 (Figure 1) as expected from the anomeric and exo-anomeric effects, respectively." This was shown by NOE interactions between the two pairs of 1,3-diaxial substituents: 6-OCH3 and H2, and 3-CH3 and H5a (Table 11). This chair conformation accounts for the allylic couplings which were observed for H5a, but not for H5e (Table I). This effect, also found for pederin (4), is due to the dependence of the allylic coupling on the H-C5-C4=C dihedral angle.loaJ2 There was a strong NOE interaction between 6-OCH3 and H7. The H7 singlet was also enhanced strongly upon irradiation of the H5a signal, but only marginally upon irradiation of the H5e signal (these signals were just resolved in CD30D, but were coincident in CDC1,). Irradiation of the C5H2signal gave an enhancement of the NH signal (only present in CDC1,). These NOE interactions are not consistent with the predominance of a single conformation about the C6-C7 bond of mycalamide A (1)and are best explained by the presence of the two rotamers shown in Figure 1. An NOE interaction between H7 and NH in mycalamide A (1) showed that the amide bond had the usual trans configuration and that H7 and N were in a gauche relationship (Figure 1). An anti relationship for NH and H10 was suggested by the observed coupling constant (9.8 Hz)13 and supported by the absence of any NOE interaction between these protons. The couplings and NOE interactions were consistent with the trioxadecalin system in mycalamide A (1) being cis fused with the two rings in chair conformations and the N9 and C16 side chains equatorial.' However, the line widths of some of the 13CNMR signals of this ring system (11)Deslongchamps, P. Stereoelectronic Effects in Organic Chemis-
Higher field signal of a geminal pair.
were temperature dependent, which has been interpreted as being due to conformational equilibration. The 13C NMR signals of 14-CH3(axial)and C11 were broader than the other signals a t 296 K (in both CDC1, and CD,OD), but these signals were sharp for a solution of mycalamide A (1) in CDCl, at 323 K. Cooling to 273 K or 248 K led to further broadening, but the 14-CH3(axial)signal was sharp a t 223 K. The C11 signal seemed to show parallel behavior, but the situation was confused by changes in chemical shifts and other signals beginning to broaden at 223 K. As no extra signals were observed in the 13C NMR spectrum at 223 K, the proposed other conformation could only be present at a low level (
