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Formyleleganolone and bibifuran, two metabolites from the brown alga Bifurcaria bifurcata
Phytochemistry Letters 5 (2012) 693–695
Contents lists available at SciVerse ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Formyleleganolone and bibifuran, two metabolites from the brown alga Bifurcaria bifurcata ˜ oz, Matthias Ko¨ck * Qun Go¨thel, Julie Mun Alfred-Wegener-Institut fu¨r Polar- und Meeresforschung in der Helmholtz-Gemeinschaft, Am Handelshafen 12, 27570 Bremerhaven, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 April 2012 Received in revised form 16 June 2012 Accepted 20 June 2012 Available online 7 July 2012
Two new diterpenoids formyleleganolone (1) and bibifuran (2) were isolated from a sample of the brown alga Bifurcaria bifurcata collected near Roscoff (Brittany, France). The structures of the new compounds were established on the basis of 1D and 2D NMR spectroscopic and mass spectrometric (ESI and EI) analyses. Formyleleganolone (1) is derived from eleganolone (4) by formylation of the hydroxy group. Bibifuran (2) has been identified as a new furan derivative of 4. Additionally, the diterpenoid 16hydroxygeranylgeraniol (3) is reported for the first time from this species. ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Sargassaceae Bifurcaria bifurcata Diterpenoids NMR Structure elucidation
1. Introduction
2. Results and discussion
Various linear diterpenes have been isolated from the brown alga Bifurcaria bifurcata (Francisco et al., 1978; Culioli et al., 1999, 2001, 2004; Ortalo-Magne´ et al., 2005). We recently reported four new metabolites from this alga collected near Roscoff, France (Go¨thel et al., 2012). Further re-examination of minor lipophilic secondary metabolites of B. bifurcata yielded two new diterpenes (1 and 2) together with a known compound (3) described for the first time from this species. The isolation and structural elucidation of the new compounds are reported in this paper.
The ethyl acetate extract of freeze-dried B. bifurcata was fractionated by liquid chromatography using silica gel with a stepwise gradient from dichloromethane (DCM) to DCM/methanol (MeOH) (80:20) to yield fractions 1–8. Further purification of fraction 4 eluted with DCM/MeOH (99.5:0.5) resulted in subfraction 4–2 containing compound 2 as an impure oil which was purified by a further silica gel column chromatography. Fraction 5 eluted with DCM/MeOH (98.5:1.5) was subjected to reversed phase HPLC to yield compounds 1 and 3. The molecular formula of the new compound 1 was deduced as C21H32O3 by HRESI(+)MS exhibiting six double bond equivalents. Its acyclic nature was established by the presence of four olefinic protons and five olefinic methyl groups in the 1H NMR spectrum (Table 1). The structure of 1 is supported by comparison of the 1H and 13C NMR chemical shifts with those of eleganolone (4) and related compounds (Francisco et al., 1978; Ortalo-Magne´ et al., 2005). The terminal formate carbon signal appears at d 161.2 (C-21), its proton signal at d 8.09 (s) is correlated with the carbon signal at d 60.7 (C-1) in the 1H,13C-HMBC spectrum. Moreover, the proton signal at d 4.70 (H-1, d) shows correlations to C-21 as well as to C-2 and C-3. These correlations further support the proposed structure of 1, which is probably derived from eleganolone (4) by terminal formylation and is therefore named as formyleleganolone (1). The HREI(+)MS of compound 2 showed a molecular ion at m/z 298.1928 corresponding to the molecular formula C20H26O2 (calcd. m/z 298.1932). The 1H NMR data of 2 revealed the presence of
16
14 15
12
10
13
O
17
O
11
18
8
6 7
9
4
2
5
3
19
1 7
11
O
21
H
O
20 2
4 13
1
3
16
1
15
18
14
19
20
O
2
17 16
1
HO 17
3
OH
20
* Corresponding author. Tel.: +49 471 4831 1497; fax: +49 471 4831 1425. E-mail addresses: [email protected], [email protected] (M. Ko¨ck).
1874-3900/$ – see front matter ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.phytol.2012.06.010
Q. Go¨thel et al. / Phytochemistry Letters 5 (2012) 693–695
694 Table 1 NMR data of compounds 1–5 (600 MHz CDCl3).a,b No.
1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
2
dC
dH
60.7 117.6 143.2 39.5 26.2 123.7 135.3 39.4 26.8 129.2 129.6 55.5 199.5 122.8 155.7 27.8 20.7 16.4 16.0 16.5 161.2
4.70 5.38 – 2.07 2.13 5.12 – 2.03 2.13 5.26 – 3.05 – 6.13 – 1.89 2.16 1.63 1.62 1.74 8.09
(d, 7.2) (t, 7.2) (m) (m) (t, 6.8) (t, 7.2) (m) (t, 6.8) (s) (s) (s) (s) (s) (s) (s) (s)
4c
3
dC
dH
142.6 111.1 125.0 25.0 28.5 123.9 135.6 39.5 26.6 126.6 131.8 38.5 154.5 108.8 120.5 137.7 9.9 15.9 16.1 138.8
7.36 6.30 – 2.47 2.26 5.19 – 2.04 2.13 5.23 – 3.25 – 5.89 – 7.09 2.00 1.61 1.62 7.23
(t, 1.5) (s) (t, 7.6) (q, 7.5) (t, 7.0) (t, 7.6) (q, 7.6) (t, 6.8) (s) (s) (s) (s) (s) (s) (s)
dC
dH
59.4 123.3 139.8 39.3 26.3 123.9 134.6 39.7 26.6 124.5 135.3 39.6 26.2 126.2 134.7 69.1 13.7 16.0 16.0 16.3
4.17 5.44 – 2.07 2.14 5.13 – 2.02 2.14 5.13 – 2.04 2.14 5.41 – 4.01 1.68 1.62 1.62 1.70
(d, 6.8) (t, 6.8) (m) (m) (t, 6.8) (m) (m) (t, 6.8) (s) (m) (t, 6.9) (s) (s) (s) (s) (s)
dC
dH
59.2 123.4 139.4 39.4 26.2 124.0 135.0 39.3 26.7 129.1 129.5 55.3 199.5 122.8 155.6 27.7 20.6 16.3 15.9 16.2
4.13 5.39 – 2.02 2.09 5.10 – 2.02 2.09 5.22 – 3.01 – 6.10 – 1.86 2.12 1.59 1.59 1.66
a
d are given in ppm (reference: CDCl3, dH 7.26 ppm and dc 77.0 ppm), coupling constants are given in Hz.
b
Assignments were confirmed by 2D NMR experiments (1H,1H-COSY, HSQC, HMBC). Added for comparison. Recorded in CDCl3 at 400 MHz.
c
2
O 16 15
3
11 14
20
1
O
17
Fig. 1. Selected HMBC correlations of bibifuran (2).
seven olefinic protons. The two proton signals at d 5.19 (H-6, t) and d 5.23 (H-10, t) show correlations with H-5 (d 2.26, q) and H-9 (d 2.13, q) in the 1H,1H-COSY spectrum, respectively. Additional 1 H,13C-HMBC correlations H-6/C-19 and H-10/C-18 along with the 13 C NMR data indicated that the signals of C-6 to C-10 in compound 2 are comparable to those of eleganolone (4). The remaining olefinic protons, except H-1(t, 1.5), are all singlets: H-2(s), H-14(s), H-16(s), and H-20(s). The proton H-20 (d 7.23) shows correlations to C-1 and C-2 in the 1H,13C-HMBC spectrum. In the same experiment H-1 (d 7.36, t) was observed to correlate with C-2, C-3,
2a
6b
7c
8c
9d
C-8 C-9 C-10 C-11 C-12 C-13 C-14 C-15 C-16 C-17 C-18
39.5 26.6 126.6 131.8 38.5 154.5 108.8 120.5 137.7 9.9 15.9
40.7 26.0 125.4 133.0 38.7 154.5 109.2 120.7 138.2 9.8 15.9
39.0 26.2 125.5 132.2 38.2 153.9 108.6 120.3 137.4 9.6 15.9
39.6 26.5 126.2 132.3 38.4 154.3 108.8 120.5 137.6 9.7 16.0
41.3 26.7 126.7 133.4 38.9 155.0 109.6 121.2 138.7 9.8 15.9
a
Recorded in CDCl3 at 150 MHz. Recorded in C6D6 at 20 MHz (Hochlowski and Faulkner, 1981; Sullivan and Faulkner, 1983). c Recorded in CDCl3 at 20 MHz (Sullivan and Faulkner, 1983). d Recorded in acetone-d6 at 75.5 MHz (Fisch et al., 2003).
(d, 6.5) (t, 6.5) (m) (m) (t, 6.5) (m) (m) (t, 6.5) (s) (s) (s) (s) (s) (s) (s)
dC
dH
142.4 111.1 125.0 25.3 28.5 124.0 135.5 39.2 26.6 128.9 130.0 55.3 198.9 122.8 155.1 27.2 20.3 15.6 16.1 138.9
7.39 6.35 – 2.50 2.31 5.25 – 2.10 2.21 5.29 – 3.06 – 6.17 – 1.92 2.17 1.66 1.66 7.27
(t, 1.5) (d, 0.8) (t, 7.4) (q, 7.4) (dt, 1.2, 6.9) (t, 7.9) (q, 7.7) (dt, 1.2, 6.9) (s) (qui, 1.2) (d, 1.1) (d, 1.2) (s) (s) (qui, 1.1)
and C-20. Moreover, H-2 (d 6.30) shows correlations to C-1, C-3, and C-20. These data revealed the presence of a furan-3-yl ring in the eastern part of compound 2. Thus, the partial structure of 2, from C-1 to C-11, including C-20 is identical to that of the known compound 5 isolated from the same organism (Hougaard et al., 1991; Go¨thel et al., 2012). In a similar manner, the presence of the other methyl-furanyl ring was established by the 1H NMR signals of H-14 (5.89, s), H-16 (d 7.09, s), and H-17 (2.00, s) and confirmed by the 1H,13C-HMBC experiment (Fig. 1). Comparison of the 1H and 13 C NMR data of 2 with those of marislin (6), previously isolated from the nudibranch Chromodoris marislae (Hochlowski and Faulkner, 1981) indicated that 2 and 6 were identical in the region between C-8 and C-18. This substructure is also present in the natural products siphonodictidine (7), hydroquinone 8 (Sullivan and Faulkner, 1983), and 5-oxo-cystofuranoquinol (9) (Fisch et al., 2003) (Table 2). OH
Table 2 13 C NMR data of compounds 2, 6, 7, 8, and 9. Atom
5c
O
O
4
O
5
13
O 7
11
R
16
15 17
O
6: R =
19
18
14
O
H N
NH2
7: R =
OH
O
NH
HO 8: R =
9: R =
b
OH
O
OH
Q. Go¨thel et al. / Phytochemistry Letters 5 (2012) 693–695
O
OH
oxidation
O
3.3. Isolation of secondary metabolites dehydration
O
HO
OH lactonisation
oxidation
O
OH
O
H
oxidation
OH
OH
ethyl acetate at room temperature. After filtration, the filtrate was evaporated to yield 12 g of crude extract.
cyclisation
oxidation
OH
695
O
O H
O
oxidation
The crude extract (12 g) was subjected to silica gel column chromatography eluted with a stepwise gradient: DCM, DCM/ MeOH (99.5:0.5), DCM/MeOH (98.5:1.5), DCM/MeOH (98:2), DCM/ MeOH (95:5), and DCM/MeOH (80:20) to yield several fractions. Further purification of the fraction eluted with DCM/MeOH (99.5:0.5) gave a fraction containing compound 2, which was further purified by silica gel column chromatography [DCM/MeOH (99.5:0.5), isocratic] to afford pure compound 2 (2 mg). The other two fractions eluted with DCM/MeOH (98.5:1.5) were subsequently purified by HPLC yielding compounds 1 (4 mg) and 3 (1 mg). Acknowledgments
OH
reduction O
OH O
Fig. 2. Hypothetical biogenesis of the methyl-furan ring and the furan ring in bibifuran (2) from Bifurcaria bifurcata (Hougaard et al., 1991).
Compound 2 is named bibifuran and it differs from all other metabolites isolated from the alga B. bifurcata by the presence of a furan ring on both sides of the molecule. A possible metabolic pathway of the two-furan rings in bibifuran (2) is shown in Fig. 2. The HRESI(+)MS of compound 3 indicated the molecular formula as C20H34O2. Supported by the 1D and 2D experiments, we conclude that compound 3 is 16-hydroxygeranylgeraniol, which was isolated as its diacetate from an Asteraceae (Mikania sp.) by Bohlmann (Bohlmann et al., 1982). However, this is the first report of 16-hydroxygeranylgeraniol (3) from B. bifurcata. 3. Experimental 3.1. General 1 H and 13C NMR spectra were recorded on a Bruker Avance 600 MHz NMR spectrometer equipped with a cryo platform. All experiments were measured at 293 K. HPLC separation was achieved by a Kromasil RP18 column (16 mm 250 mm, 5 mm) applying a H2O/CH3CN/MeOH gradient. UV spectra were recorded during HPLC separation with a DAD detector (Jasco MD-2010 Plus). Accurate mass spectra were acquired using a Bruker micrOTOFLC mass spectrometer equipped with an ESI source. Mass calibration was performed using sodium formate cluster.
3.2. Material and extraction Bifurcaria bifurcata was collected at about 1 m depth in September 2006 in Roscoff (Brittany, France). A voucher specimen of this species is deposited in our lab (voucher number: BIBI_200609). Freeze-dried material (250 g) was ground and extracted with
We would like to thank Dr. Klaus-Ulrich Valentin for collection and identification of the plant material from Roscoff, France. We further thank Christiane Rudolph (TU Darmstadt) for the HRMS measurement of compound 2. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytol.2012.06.010. References Bohlmann, F., Adler, A., Jakupovic, J., Kings, R.M., Robinsons, H., 1982. A dimeric germacranolide and other sesquiterpene lactones from Mikania species. Phytochemistry 21, 1349–1355. Culioli, G., Daoudi, M., Mesguiche, V., Valls, R., Piovetti, L., 1999. Geranylgeraniolderived diterpenoids from the brown alga Bifurcaria bifurcata. Phytochemistry 52, 1447–1454. Culioli, G., Daoudi, M., Ortalo-Magne´, A., Valls, R., Piovetti, L., 2001. (S)-12-Hydroxygeranylgeraniol-derived diterpenes from the brown alga Bifurcaria bifurcata. Phytochemistry 57, 529–535. Culioli, G., Ortalo-Magne´, A., Daoudi, M., Thomas-Guyon, H., Valls, R., Piovetti, L., 2004. Trihydroxylated linear diterpenes from the brown alga Bifurcaria bifurcata. Phytochemistry 65, 2063–2069. Fisch, K.M., Boehm, V., Wright, A.D., Ko¨nig, G.M., 2003. Antioxidative meroterpenoids from the brown alga Cystoseira crinita. J. Nat. Prod. 66, 968–975. Francisco, C., Combaut, G., Teste, J., Prost, M., 1978. Eleganolone nouveau cetol diterpenique lineaire de la pheophycee Cystoseira elegans. Phytochemistry 17, 1003–1005. Go¨thel, Q., Lichte, E., Ko¨ck, M., 2012. Further eleganolone-derived diterpenes from the brown alga Bifurcaria bifurcata. Tetrahedron Lett. 53, 1873–1877. Hochlowski, J.E., Faulkner, D.J., 1981. Chemical constituents of the nudibranch Chromodoris marislae. Tetrahedron Lett. 22, 271–274. Hougaard, L., Anthoni, U., Christophersen, C., Nielsen, P.H., 1991. Eleganolone derived diterpenes from Bifurcaria bifurcata. Phytochemistry 30, 3049–3051. Ortalo-Magne´, A., Culioli, G., Valls, R., Pucci, B., Piovetti, L., 2005. Polar acyclic diterpenoids from Bifurcaria bifurcata (Fucales Phaeophyta). Phytochemistry 66, 2316–2323. Sullivan, B., Faulkner, D.J., 1983. Siphonodictidine, a metabolite of the burrowing sponge Siphonodictyon sp. that inhibits coral growth. Science 221, 1175–2117. Formyleleganolone (1): colorless oil; 1H NMR (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz) see Table 1; HRESI(+)MS m/z calcd for C21H32O3K; [M+K]+ 371.1983; found: 371.2131. Bibifuran (2): colorless oil; 1H NMR (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz) see Table 1; HREI(+)MS m/z calcd for C20H26O2; [M]+ 298.1932; found: 298.1928. 16-Hydroxygeranylgeraniol (3): colorless oil; 1H NMR (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz) see Table 1; HRESI(+)MS m/z calcd for C20H34O2Na; [M+Na]+ 329.2457; found: 329.2478.
Contents lists available at SciVerse ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Formyleleganolone and bibifuran, two metabolites from the brown alga Bifurcaria bifurcata ˜ oz, Matthias Ko¨ck * Qun Go¨thel, Julie Mun Alfred-Wegener-Institut fu¨r Polar- und Meeresforschung in der Helmholtz-Gemeinschaft, Am Handelshafen 12, 27570 Bremerhaven, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 April 2012 Received in revised form 16 June 2012 Accepted 20 June 2012 Available online 7 July 2012
Two new diterpenoids formyleleganolone (1) and bibifuran (2) were isolated from a sample of the brown alga Bifurcaria bifurcata collected near Roscoff (Brittany, France). The structures of the new compounds were established on the basis of 1D and 2D NMR spectroscopic and mass spectrometric (ESI and EI) analyses. Formyleleganolone (1) is derived from eleganolone (4) by formylation of the hydroxy group. Bibifuran (2) has been identified as a new furan derivative of 4. Additionally, the diterpenoid 16hydroxygeranylgeraniol (3) is reported for the first time from this species. ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Sargassaceae Bifurcaria bifurcata Diterpenoids NMR Structure elucidation
1. Introduction
2. Results and discussion
Various linear diterpenes have been isolated from the brown alga Bifurcaria bifurcata (Francisco et al., 1978; Culioli et al., 1999, 2001, 2004; Ortalo-Magne´ et al., 2005). We recently reported four new metabolites from this alga collected near Roscoff, France (Go¨thel et al., 2012). Further re-examination of minor lipophilic secondary metabolites of B. bifurcata yielded two new diterpenes (1 and 2) together with a known compound (3) described for the first time from this species. The isolation and structural elucidation of the new compounds are reported in this paper.
The ethyl acetate extract of freeze-dried B. bifurcata was fractionated by liquid chromatography using silica gel with a stepwise gradient from dichloromethane (DCM) to DCM/methanol (MeOH) (80:20) to yield fractions 1–8. Further purification of fraction 4 eluted with DCM/MeOH (99.5:0.5) resulted in subfraction 4–2 containing compound 2 as an impure oil which was purified by a further silica gel column chromatography. Fraction 5 eluted with DCM/MeOH (98.5:1.5) was subjected to reversed phase HPLC to yield compounds 1 and 3. The molecular formula of the new compound 1 was deduced as C21H32O3 by HRESI(+)MS exhibiting six double bond equivalents. Its acyclic nature was established by the presence of four olefinic protons and five olefinic methyl groups in the 1H NMR spectrum (Table 1). The structure of 1 is supported by comparison of the 1H and 13C NMR chemical shifts with those of eleganolone (4) and related compounds (Francisco et al., 1978; Ortalo-Magne´ et al., 2005). The terminal formate carbon signal appears at d 161.2 (C-21), its proton signal at d 8.09 (s) is correlated with the carbon signal at d 60.7 (C-1) in the 1H,13C-HMBC spectrum. Moreover, the proton signal at d 4.70 (H-1, d) shows correlations to C-21 as well as to C-2 and C-3. These correlations further support the proposed structure of 1, which is probably derived from eleganolone (4) by terminal formylation and is therefore named as formyleleganolone (1). The HREI(+)MS of compound 2 showed a molecular ion at m/z 298.1928 corresponding to the molecular formula C20H26O2 (calcd. m/z 298.1932). The 1H NMR data of 2 revealed the presence of
16
14 15
12
10
13
O
17
O
11
18
8
6 7
9
4
2
5
3
19
1 7
11
O
21
H
O
20 2
4 13
1
3
16
1
15
18
14
19
20
O
2
17 16
1
HO 17
3
OH
20
* Corresponding author. Tel.: +49 471 4831 1497; fax: +49 471 4831 1425. E-mail addresses: [email protected], [email protected] (M. Ko¨ck).
1874-3900/$ – see front matter ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.phytol.2012.06.010
Q. Go¨thel et al. / Phytochemistry Letters 5 (2012) 693–695
694 Table 1 NMR data of compounds 1–5 (600 MHz CDCl3).a,b No.
1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
2
dC
dH
60.7 117.6 143.2 39.5 26.2 123.7 135.3 39.4 26.8 129.2 129.6 55.5 199.5 122.8 155.7 27.8 20.7 16.4 16.0 16.5 161.2
4.70 5.38 – 2.07 2.13 5.12 – 2.03 2.13 5.26 – 3.05 – 6.13 – 1.89 2.16 1.63 1.62 1.74 8.09
(d, 7.2) (t, 7.2) (m) (m) (t, 6.8) (t, 7.2) (m) (t, 6.8) (s) (s) (s) (s) (s) (s) (s) (s)
4c
3
dC
dH
142.6 111.1 125.0 25.0 28.5 123.9 135.6 39.5 26.6 126.6 131.8 38.5 154.5 108.8 120.5 137.7 9.9 15.9 16.1 138.8
7.36 6.30 – 2.47 2.26 5.19 – 2.04 2.13 5.23 – 3.25 – 5.89 – 7.09 2.00 1.61 1.62 7.23
(t, 1.5) (s) (t, 7.6) (q, 7.5) (t, 7.0) (t, 7.6) (q, 7.6) (t, 6.8) (s) (s) (s) (s) (s) (s) (s)
dC
dH
59.4 123.3 139.8 39.3 26.3 123.9 134.6 39.7 26.6 124.5 135.3 39.6 26.2 126.2 134.7 69.1 13.7 16.0 16.0 16.3
4.17 5.44 – 2.07 2.14 5.13 – 2.02 2.14 5.13 – 2.04 2.14 5.41 – 4.01 1.68 1.62 1.62 1.70
(d, 6.8) (t, 6.8) (m) (m) (t, 6.8) (m) (m) (t, 6.8) (s) (m) (t, 6.9) (s) (s) (s) (s) (s)
dC
dH
59.2 123.4 139.4 39.4 26.2 124.0 135.0 39.3 26.7 129.1 129.5 55.3 199.5 122.8 155.6 27.7 20.6 16.3 15.9 16.2
4.13 5.39 – 2.02 2.09 5.10 – 2.02 2.09 5.22 – 3.01 – 6.10 – 1.86 2.12 1.59 1.59 1.66
a
d are given in ppm (reference: CDCl3, dH 7.26 ppm and dc 77.0 ppm), coupling constants are given in Hz.
b
Assignments were confirmed by 2D NMR experiments (1H,1H-COSY, HSQC, HMBC). Added for comparison. Recorded in CDCl3 at 400 MHz.
c
2
O 16 15
3
11 14
20
1
O
17
Fig. 1. Selected HMBC correlations of bibifuran (2).
seven olefinic protons. The two proton signals at d 5.19 (H-6, t) and d 5.23 (H-10, t) show correlations with H-5 (d 2.26, q) and H-9 (d 2.13, q) in the 1H,1H-COSY spectrum, respectively. Additional 1 H,13C-HMBC correlations H-6/C-19 and H-10/C-18 along with the 13 C NMR data indicated that the signals of C-6 to C-10 in compound 2 are comparable to those of eleganolone (4). The remaining olefinic protons, except H-1(t, 1.5), are all singlets: H-2(s), H-14(s), H-16(s), and H-20(s). The proton H-20 (d 7.23) shows correlations to C-1 and C-2 in the 1H,13C-HMBC spectrum. In the same experiment H-1 (d 7.36, t) was observed to correlate with C-2, C-3,
2a
6b
7c
8c
9d
C-8 C-9 C-10 C-11 C-12 C-13 C-14 C-15 C-16 C-17 C-18
39.5 26.6 126.6 131.8 38.5 154.5 108.8 120.5 137.7 9.9 15.9
40.7 26.0 125.4 133.0 38.7 154.5 109.2 120.7 138.2 9.8 15.9
39.0 26.2 125.5 132.2 38.2 153.9 108.6 120.3 137.4 9.6 15.9
39.6 26.5 126.2 132.3 38.4 154.3 108.8 120.5 137.6 9.7 16.0
41.3 26.7 126.7 133.4 38.9 155.0 109.6 121.2 138.7 9.8 15.9
a
Recorded in CDCl3 at 150 MHz. Recorded in C6D6 at 20 MHz (Hochlowski and Faulkner, 1981; Sullivan and Faulkner, 1983). c Recorded in CDCl3 at 20 MHz (Sullivan and Faulkner, 1983). d Recorded in acetone-d6 at 75.5 MHz (Fisch et al., 2003).
(d, 6.5) (t, 6.5) (m) (m) (t, 6.5) (m) (m) (t, 6.5) (s) (s) (s) (s) (s) (s) (s)
dC
dH
142.4 111.1 125.0 25.3 28.5 124.0 135.5 39.2 26.6 128.9 130.0 55.3 198.9 122.8 155.1 27.2 20.3 15.6 16.1 138.9
7.39 6.35 – 2.50 2.31 5.25 – 2.10 2.21 5.29 – 3.06 – 6.17 – 1.92 2.17 1.66 1.66 7.27
(t, 1.5) (d, 0.8) (t, 7.4) (q, 7.4) (dt, 1.2, 6.9) (t, 7.9) (q, 7.7) (dt, 1.2, 6.9) (s) (qui, 1.2) (d, 1.1) (d, 1.2) (s) (s) (qui, 1.1)
and C-20. Moreover, H-2 (d 6.30) shows correlations to C-1, C-3, and C-20. These data revealed the presence of a furan-3-yl ring in the eastern part of compound 2. Thus, the partial structure of 2, from C-1 to C-11, including C-20 is identical to that of the known compound 5 isolated from the same organism (Hougaard et al., 1991; Go¨thel et al., 2012). In a similar manner, the presence of the other methyl-furanyl ring was established by the 1H NMR signals of H-14 (5.89, s), H-16 (d 7.09, s), and H-17 (2.00, s) and confirmed by the 1H,13C-HMBC experiment (Fig. 1). Comparison of the 1H and 13 C NMR data of 2 with those of marislin (6), previously isolated from the nudibranch Chromodoris marislae (Hochlowski and Faulkner, 1981) indicated that 2 and 6 were identical in the region between C-8 and C-18. This substructure is also present in the natural products siphonodictidine (7), hydroquinone 8 (Sullivan and Faulkner, 1983), and 5-oxo-cystofuranoquinol (9) (Fisch et al., 2003) (Table 2). OH
Table 2 13 C NMR data of compounds 2, 6, 7, 8, and 9. Atom
5c
O
O
4
O
5
13
O 7
11
R
16
15 17
O
6: R =
19
18
14
O
H N
NH2
7: R =
OH
O
NH
HO 8: R =
9: R =
b
OH
O
OH
Q. Go¨thel et al. / Phytochemistry Letters 5 (2012) 693–695
O
OH
oxidation
O
3.3. Isolation of secondary metabolites dehydration
O
HO
OH lactonisation
oxidation
O
OH
O
H
oxidation
OH
OH
ethyl acetate at room temperature. After filtration, the filtrate was evaporated to yield 12 g of crude extract.
cyclisation
oxidation
OH
695
O
O H
O
oxidation
The crude extract (12 g) was subjected to silica gel column chromatography eluted with a stepwise gradient: DCM, DCM/ MeOH (99.5:0.5), DCM/MeOH (98.5:1.5), DCM/MeOH (98:2), DCM/ MeOH (95:5), and DCM/MeOH (80:20) to yield several fractions. Further purification of the fraction eluted with DCM/MeOH (99.5:0.5) gave a fraction containing compound 2, which was further purified by silica gel column chromatography [DCM/MeOH (99.5:0.5), isocratic] to afford pure compound 2 (2 mg). The other two fractions eluted with DCM/MeOH (98.5:1.5) were subsequently purified by HPLC yielding compounds 1 (4 mg) and 3 (1 mg). Acknowledgments
OH
reduction O
OH O
Fig. 2. Hypothetical biogenesis of the methyl-furan ring and the furan ring in bibifuran (2) from Bifurcaria bifurcata (Hougaard et al., 1991).
Compound 2 is named bibifuran and it differs from all other metabolites isolated from the alga B. bifurcata by the presence of a furan ring on both sides of the molecule. A possible metabolic pathway of the two-furan rings in bibifuran (2) is shown in Fig. 2. The HRESI(+)MS of compound 3 indicated the molecular formula as C20H34O2. Supported by the 1D and 2D experiments, we conclude that compound 3 is 16-hydroxygeranylgeraniol, which was isolated as its diacetate from an Asteraceae (Mikania sp.) by Bohlmann (Bohlmann et al., 1982). However, this is the first report of 16-hydroxygeranylgeraniol (3) from B. bifurcata. 3. Experimental 3.1. General 1 H and 13C NMR spectra were recorded on a Bruker Avance 600 MHz NMR spectrometer equipped with a cryo platform. All experiments were measured at 293 K. HPLC separation was achieved by a Kromasil RP18 column (16 mm 250 mm, 5 mm) applying a H2O/CH3CN/MeOH gradient. UV spectra were recorded during HPLC separation with a DAD detector (Jasco MD-2010 Plus). Accurate mass spectra were acquired using a Bruker micrOTOFLC mass spectrometer equipped with an ESI source. Mass calibration was performed using sodium formate cluster.
3.2. Material and extraction Bifurcaria bifurcata was collected at about 1 m depth in September 2006 in Roscoff (Brittany, France). A voucher specimen of this species is deposited in our lab (voucher number: BIBI_200609). Freeze-dried material (250 g) was ground and extracted with
We would like to thank Dr. Klaus-Ulrich Valentin for collection and identification of the plant material from Roscoff, France. We further thank Christiane Rudolph (TU Darmstadt) for the HRMS measurement of compound 2. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytol.2012.06.010. References Bohlmann, F., Adler, A., Jakupovic, J., Kings, R.M., Robinsons, H., 1982. A dimeric germacranolide and other sesquiterpene lactones from Mikania species. Phytochemistry 21, 1349–1355. Culioli, G., Daoudi, M., Mesguiche, V., Valls, R., Piovetti, L., 1999. Geranylgeraniolderived diterpenoids from the brown alga Bifurcaria bifurcata. Phytochemistry 52, 1447–1454. Culioli, G., Daoudi, M., Ortalo-Magne´, A., Valls, R., Piovetti, L., 2001. (S)-12-Hydroxygeranylgeraniol-derived diterpenes from the brown alga Bifurcaria bifurcata. Phytochemistry 57, 529–535. Culioli, G., Ortalo-Magne´, A., Daoudi, M., Thomas-Guyon, H., Valls, R., Piovetti, L., 2004. Trihydroxylated linear diterpenes from the brown alga Bifurcaria bifurcata. Phytochemistry 65, 2063–2069. Fisch, K.M., Boehm, V., Wright, A.D., Ko¨nig, G.M., 2003. Antioxidative meroterpenoids from the brown alga Cystoseira crinita. J. Nat. Prod. 66, 968–975. Francisco, C., Combaut, G., Teste, J., Prost, M., 1978. Eleganolone nouveau cetol diterpenique lineaire de la pheophycee Cystoseira elegans. Phytochemistry 17, 1003–1005. Go¨thel, Q., Lichte, E., Ko¨ck, M., 2012. Further eleganolone-derived diterpenes from the brown alga Bifurcaria bifurcata. Tetrahedron Lett. 53, 1873–1877. Hochlowski, J.E., Faulkner, D.J., 1981. Chemical constituents of the nudibranch Chromodoris marislae. Tetrahedron Lett. 22, 271–274. Hougaard, L., Anthoni, U., Christophersen, C., Nielsen, P.H., 1991. Eleganolone derived diterpenes from Bifurcaria bifurcata. Phytochemistry 30, 3049–3051. Ortalo-Magne´, A., Culioli, G., Valls, R., Pucci, B., Piovetti, L., 2005. Polar acyclic diterpenoids from Bifurcaria bifurcata (Fucales Phaeophyta). Phytochemistry 66, 2316–2323. Sullivan, B., Faulkner, D.J., 1983. Siphonodictidine, a metabolite of the burrowing sponge Siphonodictyon sp. that inhibits coral growth. Science 221, 1175–2117. Formyleleganolone (1): colorless oil; 1H NMR (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz) see Table 1; HRESI(+)MS m/z calcd for C21H32O3K; [M+K]+ 371.1983; found: 371.2131. Bibifuran (2): colorless oil; 1H NMR (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz) see Table 1; HREI(+)MS m/z calcd for C20H26O2; [M]+ 298.1932; found: 298.1928. 16-Hydroxygeranylgeraniol (3): colorless oil; 1H NMR (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz) see Table 1; HRESI(+)MS m/z calcd for C20H34O2Na; [M+Na]+ 329.2457; found: 329.2478.