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Marine alkaloids—5. Flustramide A and 6-bromo-Nb-methyl-Nb-formyltryptamine from the marine bryozoan Flustra foliacea
Comp. Biochem. Physiol. Vol. 71B, No. 3, pp. 523 to 524, 1982 Printed in Great Britain.
0305-0491/82/030523-02803.00/0 © 1982 Pergamon Press Ltd
MARINE ALKALOIDS 5. FLUSTRAMIDE A AND 6-BROMO-Nb-METHYL-Nb-FORMYLTRYPTAMINE FROM THE MARINE BRYOZOAN F L U S T R A FOLIACEA P. WULFF,J. S. CARLr~and C. CHRISTOPHERSEN Marine Chemistry Section, Department of General and Organic Chemistry, the H. C. Orsted Institute, University of Copenhagen, Univergitetsparken 5, DK-2100 Copenhagen, Denmark
(Received 22 July 1981) Abstract--1. Two novel bromosubstituted alkaloids, flustramide A [2-oxoflustramine A (2)] and 6-bromo-Nb-methyl-Nb-formyltryptamine~) have been isolated from the marine bryozoan Flustra foliacea.
2. The structures have been elucidated using spectroscopic methods.
INTRODUCTION
From the marine bryozoan Flustra foliacea (L.) six bromo-substituted alkaloids have so far been isolated and identified (Carl6 & Christophersen, 1979, 1980 and 1981). All of them except one are formally derived from the tricyclic physostigmine skeleton. One of them, flustrabromine ~) is a simple tryptamine derivative (Wulff et al., 1981). We now wish to report the isolation and structure determination of two new alkaloids. One of them, flustramide A (_1)belongs to the tricyclic group and is formally closely related to flustramine A (_4)(Carl6 & Christophersen, 1979, 1980) while the other, 6-bromo-Nb-methyl-Nb-formyltryptamine (2) bears strong resemblance to flustrabromine (3) (Wulff et al., 1981).
C•H3 N--CHO
17((ls L ..0
B
)o/9 12
B
13 1
H 2
C•H3
Br~~'~OBr~N~cH3 3
~
MATERIALS AND METHODS
Flustramide A (!) was isolated from a sample of Flustra foliacea (100g) collected directly from the shore. This sample was used without exhaustive drying. Successive extraction with petroleum ether and methylene chloride left extracts, which following column chromatography (sil-
ica gel eluted with ethyl acetate) gave fractions which in the case of the methylene chloride were further purified by the same procedure. Fractions containing flustramide A from the petroleum ether and methylene chloride extracts were combined and subjected to column chromatography (silica gel, A60 Lobar, Merck) with CHCI3: EtOAc, 85:15, as eluent. From these separations a yield of 5 mg of flustramide A (_1)(oil) and 5 mg flustrabromine (3) (amorphous) was obtained. 6-Bromo-Nb-methyl-Nb-formyltryptamine (2) was isolated as described (Carl6 & Christophersen, 1981) for flustraminol A, B and ftustramine C. One of the most polar fractions was further purified by column chromatography (silica gel, A60 Lobar, Merck) using MeOH:EtOAc, 10:90, as eluent. Yield: 2 mg pure amorphous solid. RESULTS AND DISCUSSION
High resolution mass spectrometry of flustramide A revealed the composition of base peak m/e 265 as CllHloBrN20 (calc. 265.0008 for 79Br, found 264.9977). The base peak represents the loss of two isoprene units from the molecular ion, in close analogy with the findings reported for flustramine A. According to this analogy flustramide A is to be formulated C21H27BrN20. The other alkaloid is C12HI3BrN20 (calc. 280.0212 for 79Br, found 280.0209). Comparison of NMR data of flustramide A with those of flustramine A indicates close resemblance of the two structures except for the ~3C NMR signal appearing at 3 172.6 ppm in flustramide A assigned to an amide carbon atom. The presence of the amide functionality was further substantiated by a 1680 cm-1 (CDC13) IR absorption band. A consistent assignment of the ~3C NMR data was only possible for the isomer 1 (see Table 1). To secure the assigned location of the bromine atom a aH-[1H] NOE difference spectrum was built up by a sequence of alternate irradiations of the two methyl groups at C-14 resonating at 0.94 and 1.03 ppm, respectively. An enhancement of 2.1~o of the signal at 3 6.94 ppm identified this signal as originating from the proton attached at position 4, unambiguously placing the bromine atom at position 6. A large enhancement of 10.5% of the signal at
523
524
P. WULFF, J. S. CARLEand C. CHRISTOPHERSEN Table 1. 13C NMR of flustramide A (isomer 1)
Carbon 2 3 3a 3b 4 5 6
6 ppm 172.6 28.0 55.5 132.0' 126.2 120.5+ 122.6
Carbon 7 7a 8a 9 10 11 12
6 ppm 111.0 n.o, 86.0 46.6 120.8t 135.7' 18.1
Carbon 6 ppm 13 14 15 16 17 18 19
25.7 zx 41.2 21.8 zx 22.6 ix 143.4 114.6 34.4
Assignments for values marked with the same symbols may be interchanged. Spectrum measured at 22.5 MHz in CDC13 (10 mg/ml).
6 4.83 ppm (H at 8a) not only proves the cis fusion around C-3a/C-8a but also indicates a closer spatial relationship of the C-15/C-16 protons and the C-8a proton as compared to flustramine A (corresponding enhancements 3,6% and 3.1~o for protons at position 4 and 8a respectively). Based on the evidence presented above we conclude that flustramide A must have structure of 1-methyl-2-oxo-3a-(2-methyl3 -buten- 2 - yl) - 6 - bromo - 8 - ( 3 - methyl - 2 - butenyl ) 1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole (1). In the case of the other alkaloid the mass spectrum (160°C: 280/282 (20); 221/223 (10); 208/210 (80)) strongly suggests the presence of a brominated Nb-methyl-Nb-formyltryptamine (debromoanalog: M + 202(12), M + - N H ( C H 3 ) C H O 143(71), M + - C H 2 N ( C H a ) C H O 130(100), Lauwers et al., 1975). Inspection of the 2 7 0 M H z 1H N M R spectrum (2 mg in 400 gl CDC13) and comparison with a spectrum of authentic Nb-methyl-Nb-formyltryptamine (NMR (270 MHz, 30 mg in 400 #1 CDC13) (6 ppm, Z, E), 2.93, 2.88 (s, N-CH3); 3.01 (overlapping triplets, CH2); 3.53, 3.66 (t, N-CH2); 6.95, 7.04 (d, 2-H); 7.10-7.65 (aromatic signals, 4 H); 7.76, 8.07 (s, CHO); 8.30, 8.24 (bs, N-H) further substantiates this structure and leaves little doubt that the two compounds are identical except for the bromosubstitution of the former.
Furthermore, the N M R data reveals the natural products to be a roughly equal mixture of two rotational isomers, a Z and an E form, in close analogy with the findings for the unbrominated compound and for flustrabromine (3) (Wulff et al., 1981). N M R : (6 ppm, Z, E), 2.92, 2.89 (s, N-CHa); 2.99 (overlapping triplets, CH2); 3.53, 3.64 (t, N-CH2); 6.96, 7.08 (d, 2-H); 7.41, 7.50 (d, 4-H); 7.22 (m, 5-H); 7.53 (m, 7-H); 7.73, 8.07 (s, CHO); 8.15, 8.08 (bs, NH). On addition of C6D 6 the expected benzene shift (Wulff et al., 1981) was observed. We therefore conclude that the natural product is a mixture of Z and E, 6-bromo-Nbmethyl-Nb-formyltryptamine (2). Acknowledoements- The chromatographic equipment (J.nr. 511-6810) and the Bruker HX 270 NMR spectrometer was purchased by the Danish Natural Science Research Council, to whom we are also grateful for a fellowship (J.nr. 511-100166) to one of us (J.S.C.). Finally we wish to thank Dr. K. Schaumburg for obtaining the NOE difference data.
REFERENCES CARLI~ J. S. & CHRISTOPHERSENC. (1979) Bromo-substituted physostigmine alkaloids from a marine Bryozoa Flustra foliacea. J. Amer. Chem. Soc. 101,401~4013. CARLI~J. S. • CHRISTOPHERSENC. (1980) Marine alkaloids. 2. Bromo alkaloids from the marine Bryozoan Flustra foliacea. Isolation and structure elucidation. J, Org. Chem. 45, 1586-1589. CARLEJ. S, t~ CHRISTOPHERSENC. (1981) Marine alkaloids. 3. Bromosubstituted alkaloids from the marine Bryozoan Flustra foliacea, Flustramine C, Flustraminol A and B. J. Org. Chem. 46, 3440-3443. LAUWERS W., LEYSEN J., VERHOEVENH. & LADURON P. (1975) Identification of alkaloids; the condensation products of biogenic amines with formaldehyde enzymatically formed from 5-methyltetrahydrofolic acid Biomed. Mass Spectrum. 2, 15-22. WULFF P., CARL~J. S. & CHRISTOPHERSENC. (1981) Marine alkaloids. 4. A formamide, flustrabromine, from the marine Bryozoan Flustra foliacea. J. Chem. Soc. Perkin Trans. I. In press.
0305-0491/82/030523-02803.00/0 © 1982 Pergamon Press Ltd
MARINE ALKALOIDS 5. FLUSTRAMIDE A AND 6-BROMO-Nb-METHYL-Nb-FORMYLTRYPTAMINE FROM THE MARINE BRYOZOAN F L U S T R A FOLIACEA P. WULFF,J. S. CARLr~and C. CHRISTOPHERSEN Marine Chemistry Section, Department of General and Organic Chemistry, the H. C. Orsted Institute, University of Copenhagen, Univergitetsparken 5, DK-2100 Copenhagen, Denmark
(Received 22 July 1981) Abstract--1. Two novel bromosubstituted alkaloids, flustramide A [2-oxoflustramine A (2)] and 6-bromo-Nb-methyl-Nb-formyltryptamine~) have been isolated from the marine bryozoan Flustra foliacea.
2. The structures have been elucidated using spectroscopic methods.
INTRODUCTION
From the marine bryozoan Flustra foliacea (L.) six bromo-substituted alkaloids have so far been isolated and identified (Carl6 & Christophersen, 1979, 1980 and 1981). All of them except one are formally derived from the tricyclic physostigmine skeleton. One of them, flustrabromine ~) is a simple tryptamine derivative (Wulff et al., 1981). We now wish to report the isolation and structure determination of two new alkaloids. One of them, flustramide A (_1)belongs to the tricyclic group and is formally closely related to flustramine A (_4)(Carl6 & Christophersen, 1979, 1980) while the other, 6-bromo-Nb-methyl-Nb-formyltryptamine (2) bears strong resemblance to flustrabromine (3) (Wulff et al., 1981).
C•H3 N--CHO
17((ls L ..0
B
)o/9 12
B
13 1
H 2
C•H3
Br~~'~OBr~N~cH3 3
~
MATERIALS AND METHODS
Flustramide A (!) was isolated from a sample of Flustra foliacea (100g) collected directly from the shore. This sample was used without exhaustive drying. Successive extraction with petroleum ether and methylene chloride left extracts, which following column chromatography (sil-
ica gel eluted with ethyl acetate) gave fractions which in the case of the methylene chloride were further purified by the same procedure. Fractions containing flustramide A from the petroleum ether and methylene chloride extracts were combined and subjected to column chromatography (silica gel, A60 Lobar, Merck) with CHCI3: EtOAc, 85:15, as eluent. From these separations a yield of 5 mg of flustramide A (_1)(oil) and 5 mg flustrabromine (3) (amorphous) was obtained. 6-Bromo-Nb-methyl-Nb-formyltryptamine (2) was isolated as described (Carl6 & Christophersen, 1981) for flustraminol A, B and ftustramine C. One of the most polar fractions was further purified by column chromatography (silica gel, A60 Lobar, Merck) using MeOH:EtOAc, 10:90, as eluent. Yield: 2 mg pure amorphous solid. RESULTS AND DISCUSSION
High resolution mass spectrometry of flustramide A revealed the composition of base peak m/e 265 as CllHloBrN20 (calc. 265.0008 for 79Br, found 264.9977). The base peak represents the loss of two isoprene units from the molecular ion, in close analogy with the findings reported for flustramine A. According to this analogy flustramide A is to be formulated C21H27BrN20. The other alkaloid is C12HI3BrN20 (calc. 280.0212 for 79Br, found 280.0209). Comparison of NMR data of flustramide A with those of flustramine A indicates close resemblance of the two structures except for the ~3C NMR signal appearing at 3 172.6 ppm in flustramide A assigned to an amide carbon atom. The presence of the amide functionality was further substantiated by a 1680 cm-1 (CDC13) IR absorption band. A consistent assignment of the ~3C NMR data was only possible for the isomer 1 (see Table 1). To secure the assigned location of the bromine atom a aH-[1H] NOE difference spectrum was built up by a sequence of alternate irradiations of the two methyl groups at C-14 resonating at 0.94 and 1.03 ppm, respectively. An enhancement of 2.1~o of the signal at 3 6.94 ppm identified this signal as originating from the proton attached at position 4, unambiguously placing the bromine atom at position 6. A large enhancement of 10.5% of the signal at
523
524
P. WULFF, J. S. CARLEand C. CHRISTOPHERSEN Table 1. 13C NMR of flustramide A (isomer 1)
Carbon 2 3 3a 3b 4 5 6
6 ppm 172.6 28.0 55.5 132.0' 126.2 120.5+ 122.6
Carbon 7 7a 8a 9 10 11 12
6 ppm 111.0 n.o, 86.0 46.6 120.8t 135.7' 18.1
Carbon 6 ppm 13 14 15 16 17 18 19
25.7 zx 41.2 21.8 zx 22.6 ix 143.4 114.6 34.4
Assignments for values marked with the same symbols may be interchanged. Spectrum measured at 22.5 MHz in CDC13 (10 mg/ml).
6 4.83 ppm (H at 8a) not only proves the cis fusion around C-3a/C-8a but also indicates a closer spatial relationship of the C-15/C-16 protons and the C-8a proton as compared to flustramine A (corresponding enhancements 3,6% and 3.1~o for protons at position 4 and 8a respectively). Based on the evidence presented above we conclude that flustramide A must have structure of 1-methyl-2-oxo-3a-(2-methyl3 -buten- 2 - yl) - 6 - bromo - 8 - ( 3 - methyl - 2 - butenyl ) 1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole (1). In the case of the other alkaloid the mass spectrum (160°C: 280/282 (20); 221/223 (10); 208/210 (80)) strongly suggests the presence of a brominated Nb-methyl-Nb-formyltryptamine (debromoanalog: M + 202(12), M + - N H ( C H 3 ) C H O 143(71), M + - C H 2 N ( C H a ) C H O 130(100), Lauwers et al., 1975). Inspection of the 2 7 0 M H z 1H N M R spectrum (2 mg in 400 gl CDC13) and comparison with a spectrum of authentic Nb-methyl-Nb-formyltryptamine (NMR (270 MHz, 30 mg in 400 #1 CDC13) (6 ppm, Z, E), 2.93, 2.88 (s, N-CH3); 3.01 (overlapping triplets, CH2); 3.53, 3.66 (t, N-CH2); 6.95, 7.04 (d, 2-H); 7.10-7.65 (aromatic signals, 4 H); 7.76, 8.07 (s, CHO); 8.30, 8.24 (bs, N-H) further substantiates this structure and leaves little doubt that the two compounds are identical except for the bromosubstitution of the former.
Furthermore, the N M R data reveals the natural products to be a roughly equal mixture of two rotational isomers, a Z and an E form, in close analogy with the findings for the unbrominated compound and for flustrabromine (3) (Wulff et al., 1981). N M R : (6 ppm, Z, E), 2.92, 2.89 (s, N-CHa); 2.99 (overlapping triplets, CH2); 3.53, 3.64 (t, N-CH2); 6.96, 7.08 (d, 2-H); 7.41, 7.50 (d, 4-H); 7.22 (m, 5-H); 7.53 (m, 7-H); 7.73, 8.07 (s, CHO); 8.15, 8.08 (bs, NH). On addition of C6D 6 the expected benzene shift (Wulff et al., 1981) was observed. We therefore conclude that the natural product is a mixture of Z and E, 6-bromo-Nbmethyl-Nb-formyltryptamine (2). Acknowledoements- The chromatographic equipment (J.nr. 511-6810) and the Bruker HX 270 NMR spectrometer was purchased by the Danish Natural Science Research Council, to whom we are also grateful for a fellowship (J.nr. 511-100166) to one of us (J.S.C.). Finally we wish to thank Dr. K. Schaumburg for obtaining the NOE difference data.
REFERENCES CARLI~ J. S. & CHRISTOPHERSENC. (1979) Bromo-substituted physostigmine alkaloids from a marine Bryozoa Flustra foliacea. J. Amer. Chem. Soc. 101,401~4013. CARLI~J. S. • CHRISTOPHERSENC. (1980) Marine alkaloids. 2. Bromo alkaloids from the marine Bryozoan Flustra foliacea. Isolation and structure elucidation. J, Org. Chem. 45, 1586-1589. CARLEJ. S, t~ CHRISTOPHERSENC. (1981) Marine alkaloids. 3. Bromosubstituted alkaloids from the marine Bryozoan Flustra foliacea, Flustramine C, Flustraminol A and B. J. Org. Chem. 46, 3440-3443. LAUWERS W., LEYSEN J., VERHOEVENH. & LADURON P. (1975) Identification of alkaloids; the condensation products of biogenic amines with formaldehyde enzymatically formed from 5-methyltetrahydrofolic acid Biomed. Mass Spectrum. 2, 15-22. WULFF P., CARL~J. S. & CHRISTOPHERSENC. (1981) Marine alkaloids. 4. A formamide, flustrabromine, from the marine Bryozoan Flustra foliacea. J. Chem. Soc. Perkin Trans. I. In press.