Establishment of the absolute configuration of the bioactive marine alkaloid eudistomin X by stereospecific synthesis

Article abstract of DOI:10.1016/j.tetlet.2010.12.052

The marine β-carboline alkaloid eudistomin X has been synthesised stereospecifically, using d-phenylalanine as the chiral pool starting material, establishing the absolute configuration of the natural product as (10R). A stereospecific synthesis of both e

Full text of DOI:10.1016/j.tetlet.2010.12.052

Tetrahedron Letters 52 (2011) 837–840
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Establishment of the absolute configuration of the bioactive marine alkaloid
eudistomin X by stereospecific synthesis
Rhys Finlayson a, Amira Brackovic a, Annabel Simon-Levert b, Bernard Banaigs b, Ronan F. O’Toole c,
Christopher H. Miller c, Brent R. Copp a,
⇑
a Department of Chemistry, University of Auckland, Private Bag 92019, Auckland, New Zealand
b Laboratoire de Chimie des Biomolécules et de l’Environnement, Universite de Perpignan Via Domitia, 52 Avenue Paul Alduy, 66860 Perpignan, France
c School of Biological Sciences, Victoria University of Wellington, PO Box 600, Wellington, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history:
A stereospecific synthesis of both enantiomers of the marine alkaloid eudistomin X using the amino acid
chiral pool is achieved. Comparison of 1H and 13C NMR chemical shifts of the synthetic product as either
the free base, mono-salt or disalt with those reported for the natural product established that the ascidian
metabolite was originally characterised as a mono-salt and that the absolute configuration was (10R).
Ó 2010 Elsevier Ltd. All rights reserved.
Received 18 November 2010
Revised 2 December 2010
Accepted 10 December 2010
Available online 16 December 2010
Keywords:
Marine alkaloid
b-Carboline
Ascidian
Stereospecific synthesis
Chiral pool
Biological activity
Marine organisms of the class Ascidiacea (ascidians) are noted
for their abilities to biosynthesise secondary metabolites derived
from amino acids.1,2 After cyclic peptides, one of largest sub-
classes of metabolites reported are the b-carboline alkaloids,
exemplified by eudistomins C (1),3 G (2), H (3) and I (4),4 woodi-
nine (5),5 eudistalbin A (6)6 and eudistomidin B (7)7 ( Fig. 1).
Biosynthetically, these alkaloids are derived from tryptophan/
tryptamine condensation with a second amino acid, as evidenced
by 3H-labelled -proline incorporation into eudistomins G and H.8
L
The eudistomin alkaloids appear to serve ecological roles related
to inhibition of larval settlement9 though much of the discovery
and synthetic chemistry attention paid to this class of alkaloid
derives from their potent pharmacological activities.10 Of the 65
b-carboline-containing alkaloids reported from ascidians to date,11
only 13 contain an asymmetric centre at C-10 with defined config-
uration, potentially suggestive of the configuration of the second
amino acid utilised in the biosynthetic pathway. The oxathiazepine
ring containing examples, eudistomins C (1), E, F, K, K-sulfoxide,
debromo K and L have (10S) configuration consistent with biosyn-
thetic incorporation of D
-cysteine,3 as do the structurally-simpler
analogues, eudistomidin C7a and an N-methyl analogue.12
The (10S) absolute configuration of woodinine (5),5 eudistalbin
A (6)6 and eudistomidins B (7)7 and G7c is consistent with the
⇑
Corresponding author. Tel.: +64 9 373 7599x88284; fax: +64 9 373 7422.
E-mail address: b.copp@auckland.ac.nz (B.R. Copp).
Figure 1. Structures of eudistomins C (1), G (2), H (3) and I (4), woodinine (5),
eudistalbin A (6), eudistomidin B (7) and eudistomin X (8).
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.12.052

838
R. Finlayson et al. / Tetrahedron Letters 52 (2011) 837–840
incorporation of
L-proline,
L-leucine and
L
-phenylalanine, respec-
the next step without purification. Pictet-Spengler reaction of chi-
tively, during the biosynthesis of these natural products.
ral aldehyde 12 with 5-methoxytryptamine yielded the expected
tetrahydro-b-carboline (13) as a yellow foam in 60% yield. The
subsequent sequence of oxidation with DDQ in THF (27% yield),
de-O-methylation using BBr3 in dichloromethane (70% yield), and
finally silica gel column chromatography using 5% MeOH/CH2Cl2
with 2% NH3 as eluent afforded (10R)-eudistomin X as the free base
The 2003 publication of eudistomin X (8) from a Micronesian
collection of Eudistoma sp. was of interest to us due to it being
chiral, but with undefined configuration and that it exhibited rela-
tively potent in vitro antimicrobial properties.13 We undertook the
stereospecific synthesis of both enantiomers of 8, using the amino
acid phenylalanine as the chiral pool source, to address two ques-
tions: what is the C-10 configuration of eudistomin X, and is there
any influence of the C-10 configuration on the biological activity of
the alkaloid? Our synthesis of eudistomin X made use of the clas-
sical Pictet-Spengler b-carboline ring-forming reaction between an
appropriately substituted (1H-indol-3-yl)ethanamine and a chiral
aldehyde, derived from phenylalanine.14 (R)-(À)-2-(N,N-dimethyl-
(15a). The optical rotation observed for 15a ([
a
]
À106.1 (c 0.49,
D
MeOH)) was substantially different from that reported for
the natural product ([
a
]
D
À7.0 (c 0.49, MeOH)).13 Comparison of
the 1H and 13C NMR data observed for our synthetic 15a18 with
amino)-3-phenyl-propionic acid methyl ester (10) ([
a
a
]
À5.0 (c
] +4.7 (c
D
D
2.84, ethyl acetate), lit.15 data for the (S) enantiomer [
2.84, ethyl acetate)) was prepared in 77% yield by reaction of the
amino acid methyl ester (9) with formaldehyde and sodium cyano-
borohydride in methanol (Scheme 1).16 Reduction of the ester
functionality present in 10 using LiAlH4 in THF afforded chiral
aminoalcohol 11 ([
enantiomer, also as the free base [
93% yield. Oxidation under Swern conditions yielded unstable
a]
+19.3 (c 1.00, CH2Cl2), lit.17 data for the (S)
D
a]
À18.4 (c 1.00, CH2Cl2)) in
D
(R)-2-(dimethylamino)-3-phenylpropanal (12) that was used in
Figure 2. Plots of difference observed for 1H and 13C NMR data
(Dd =
dliterature À dsynthetic) varying by alkaloid numbered position for (a) free base 15a,
(b) mono-salt 15b, and (c) dihydrochloride 15c compared to published data for
eudistomin X.13 All data acquired in the same solvent (CD3OD) at 6 mg/mL
concentration. Note that
for clarity.
Dd
1H values have been arbitrarily scaled by a factor of 10
Scheme 1. Total synthesis of (10R)-eudistomin X (15a).

R. Finlayson et al. / Tetrahedron Letters 52 (2011) 837–840
839
the literature data for the natural product13 indicated major chem-
ical shift differences centred upon C-1, H-10 and the dimethyl-
amino group ( Fig. 2a). We speculated that the differences
between these two data sets were the result of differing proton-
ation states, at the more basic tertiary alkyl amine functionality,
between our synthetic sample and the characterised natural prod-
uct. Sub-samples of synthetic (10R)-eudistomin X free base (15a)
were converted either into a mono-deuterochloride salt 15b by
careful titration with 1 mol equiv of 2HCl (monitored by 1H NMR
spectroscopy) or to the dihydrochloride salt (15c), by dissolution
in excess methanolic HCl.
In summary, we have completed the first synthesis of eudisto-
min X in a stereoselective manner that defines the natural product
as bearing (10R) configuration. Prior to this study, ascidian-derived
b-carboline alkaloids bearing a stereogenic centre at C-10, the con-
figuration of which had been rigorously determined by synthesis,
were limited to those putatively derived from the biosynthetic
incorporation of
nine.7 We can now expand this series to include
The wide variation in [ data observed for various protonation
D
-cysteine,3
L
-proline,5
L
-leucine6 and
-phenylalanine.
L-phenylala-
D
a]
D
states of the same alkaloid (15a, 15b and 15c) should serve as an
extra caution to natural product chemists who attempt to assign
configuration solely by analogy.22 We are currently expanding
our library of natural (10R) and unnatural (10S) b-carboline alka-
loids related to eudistomin X and are evaluating them for a wider
range of biological properties to determine more categorically the
influence of C-10 configuration on ecological and potential
pharmaceutical activities.
Confirmation of the free base, dimethylammonium salt and di-
salt natures of 15a, 15b and 15c, respectively, were made by exam-
ination of the 15N NMR chemical shifts indirectly acquired with the
aid of 1H–15N HMBC NMR experiments (Table 1).
As expected, only minor deshielding of the 15N NMR resonance
observed for the dimethylamino group was detected for the mono-
salt 15b,19 but the pyridine 15N resonance N-2 shifted significantly
upon protonation (15c).20 Re-acquisition of the 1H and 13C NMR
data for 15b and 15c18 and comparison with published data estab-
lished that the natural product had in fact been characterised as a
mono-salt (of unknown counter-ion) (Fig. 2). This conclusion was
further supported by comparison of the optical rotation data ob-
Acknowledgements
We thank Auckland Medical Research Foundation for
a
grant-in-aid (no. 81547) and research support from the University
of Auckland. R.F. acknowledges The University of Auckland for a
doctoral scholarship. We thank the DTP branch of the NCI for
antitumour data, Dr. M. Schmitz for assistance with NMR data
acquisition and Ms. R. Imatdieva for MS data.
served for 15a ([
15c ([
with the reported rotation of the natural product ([
was observed again for the mono-salt 15b. This confirmed the
structure of eudistomin X and established the absolute configura-
tion of the natural product as (10R).
a
]
À106.1), 15b ([
a
]
À7.2 (c 0.49, MeOH)) and
D
D
a
] +10.5 (c 0.49, MeOH)), for which excellent agreement
D
a]
À7.0)13
D
References and notes
In order to realise our second goal of investigating the influence
of the C-10 configuration on the antibacterial activity of eudisto-
min X, we repeated the synthesis utilising S-phenylalanine as the
chiral pool starting material. As expected, unnatural (10S)-eudisto-
min X exhibited identical 1H and 13C NMR data, in all protonation
states, to those observed for (10R)-eudistomin X, and showed sim-
ilar magnitude but opposite sign optical rotation data to those ob-
served earlier for the natural stereoisomer series.21
We evaluated penultimate methyl ether 14 and natural prod-
uct 15a in both the natural (10R) and unnatural (10S) configura-
tions for in vitro antibacterial activity towards three species of
Gram-negative marine bacteria (Aeromonas salmonicida, Listonella
anguillarum and Shewanella baltica), Gram-negative (Escherichia
coli DH5a) and Gram-positive bacteria (Mycobacterium smegmatis
mc2155 and Staphylococcus aureus Newman). Of the four com-
pounds tested, natural (10R)-configuration of methyl ether
14 inhibited all three marine bacteria with modest potency
1. Davidson, B. S. Chem. Rev. 1993, 93, 1771–1791.
2. Wang, W.; Namikoshi, M. Heterocycles 2007, 74, 53–88.
3. Rinehart, K. L.; Kobayashi, J.; Harbour, G. C.; Hughes, R. G.; Mizsak, S. A.; Scahill,
T. A. J. Am. Chem. Soc. 1984, 106, 1524–1526.
4. Kobayashi, J.; Harbour, G. C.; Gilmore, J.; Rinehart, K. L. J. Am. Chem. Soc. 1984,
106, 1526–1528.
5. (a) Debitus, C.; Laurent, D.; Païs, M. J. Nat. Prod. 1988, 51, 799–801; (b)
Mahboobi, S.; Burgemeister, T.; Wiegrebe, W. Arch. Pharm. 1993, 326, 33–37;
(c) McNulty, J.; Still, I. W. J. J. Chem. Soc., Perkin Trans. 1 1994, 1329–1337; (d)
Mahboobi, S.; Dove, S.; Bednarski, P. J.; Kuhr, S.; Burgemeister, T.; Schollmeyer,
D. J. Nat. Prod. 1997, 60, 587–591.
6. (a) Adesanya, S. A.; Chbani, M.; Païs, M.; Debitus, C. J. Nat. Prod. 1992, 55, 525–
527; (b) Zhang, P. Y.; Wang, J. L.; Wan, S. B.; Jiang, T. Chin. Chem. Lett. 2010, 21,
889–891.
7. (a) Kobayashi, J.; Cheng, J.-F.; Ohta, T.; Nozoe, S.; Ohizumi, Y.; Sasaki, T. J. Org.
Chem. 1990, 55, 3666–3670; (b) Ito, T.; Kitajima, M.; Takayama, H. Tetrahedron
Lett. 2009, 50, 4506–4508; (c) Takahashi, Y.; Ishiyama, H.; Kubota, T.;
Kobayashi, J. Bioorg. Med. Chem. Lett. 2010, 20, 4100–4103.
8. Shen, G. Q.; Baker, B. J. Tetrahedron Lett. 1994, 35, 1141–1144.
9. Davis, A. R.; Wright, A. E. J. Chem. Ecol. 1990, 16, 1349–1357.
10. Cao, R.; Peng, W.; Wang, Z.; Xu, A. Curr. Med. Chem. 2007, 14, 479–500.
11. MarinLit database; Department of Chemistry, University of Canterbury: http://
www.chem.canterbury.ac.nz/marinlit/marinlit.shtml.
12. Rashid, M. A.; Gustafson, K. R.; Boyd, M. R. J. Nat. Prod. 2001, 64, 1454–1456.
13. Schupp, P.; Poehner, T.; Edrada, A.; Ebel, R.; Berg, A.; Wray, V.; Proksch, P. J. Nat.
Prod. 2003, 66, 272–275.
14. Larghi, E. L.; Amongero, M.; Bracca, A. B. J.; Kaufman, T. S. ARKIVOC 2005, xii,
98–153.
(25–30
(MIC P250
inhibition of M. smegmatis (MIC 250
was 1000 M), methyl ether 14 was the more active growth inhibi-
tor against S. aureus (MIC 250 M)—the other test compounds
proved inactive. None of the test compounds were active towards
E. coli (MIC P1000 M) and they were also inactive towards a panel
l
M), while the remaining compounds were inactive
M). While all four compounds exhibited weak growth
M; highest test concentration
l
l
l
l
15. Da, C.-S.; Ni, M.; Han, Z.-J.; Yang, F.; Wang, R. J. Mol. Catal. A: Chem. 2006, 245,
1–7.
16. Jeffs, P. W.; Yellin, B.; Mueller, L.; Heald, S. L. J. Org. Chem. 1988, 53, 471–477.
17. Diego-Castro, M. J.; Hailes, H. C.; Lawrence, M. J. J. Colloid Interface Sci. 2001,
234, 122–126.
l
of human tumour cell lines in testing by the Developmental
Therapeutics Program, NCI. These results fail to provide any clear
evidence of a C-10 stereochemical bias for biological activity.
18. Compound characterisation data for 15a, 15b and 15c. (10R)-Eudistomin X free
base (15a): [a]D À106.1 (c 0.49, MeOH); IR mmax (neat) 3221, 1824, 1567, 1493,
1434, 1196, 1025 cmÀ1; Rf (5% MeOH/CH2Cl2) 0.29; 1H NMR (400 MHz, CD3OD)
d 8.07 (1H, d, J = 5.4 Hz, H-3), 7.77 (1H, d, J = 5.4 Hz, H-4), 7.45 (1H, d, J = 2.4 Hz,
H-5), 7.38 (1H, d, J = 8.8 Hz, H-8), 7.07 (1H, dd, J = 8.8, 2.4 Hz, H-7), 6.92 (3H, m,
H-14/H-16, H-15), 6.82 (2H, m, H-13/H-17), 4.16 (1H, dd, J = 10.4, 4.4 Hz, H-
10), 3.47 (1H, dd, J = 13.1, 10.4 Hz, H-11a), 3.33 (1H, dd, J = 13.1, 4.4 Hz, H-11b),
2.37 (6H, s, NMe2); 13C NMR (100 MHz, CD3OD) d 152.2 (C-6), 144.7 (C-1),
139.9 (C-12), 137.3 (C-9a), 136.9 (C-3), 136.8 (C-8a), 130.2 (C-13/C-17), 130.1
(C-4a), 129.0 (C-14/C-16), 126.9 (C-15), 122.9 (C-4b), 119.5 (C-7), 114.9 (C-4),
113.5 (C-8), 106.6 (C-5), 71.4 (C-10), 43.5 (NMe2), 38.0 (C-11); 15N NMR
(40.6 MHz, CD3OD) d 292.9 (N-2), 111.4 (N-9), 37.9 (NMe2); (+)-ESIMS m/z 332
[M+H]+; (+)-HRESIMS [M+H]+ 332.1751 (calcd for C21H22N3O, 332.1757).
Table 1
15N NMR (40.6 MHz) chemical shifts observed for 15a, 15b and 15ca
N-2
N-9
NMe2
15a
15b
15c
292.9
289.3
189.3
111.4
111.1
120.4
37.9
47.5
51.6
a
Determined indirectly from 1H–15N HMBC NMR experiments, optimised for xJHN
(10R)-Eudistomin X mono-salt (15b): [
a
]
D
À7.2 (c 0.49, MeOH) [lit.13 À7.0 (c
6 Hz, acquired in CD3OD solvent and referenced to liquid NH3 using urea as an
external standard.
0.49, MeOH)]; 1H NMR (400 MHz, CD3OD) d 8.42 (1H, d, J = 5.4 Hz, H-3), 7.97

840
R. Finlayson et al. / Tetrahedron Letters 52 (2011) 837–840
(1H, d, J = 5.4 Hz, H-4), 7.45 (1H, d, J = 2.4 Hz, H-5), 7.31 (1H, d, J = 8.8 Hz, H-8),
H-11b), 3.20 (6H, br s, NMe2); 13C NMR (100 MHz, CD3OD) d 154.7 (C-6), 140.6
(C-8a), 136.9 (C-4a), 134.3 (C-12), 131.0 (C-3), 130.0 (C-13/C-17 and C-14/C-
16), 128.9 (C-15), 128.7 (C-1), 125.5 (C-7), 121.7 (C-4b), 119.3 (C-4), 114.8 (C-
8), 107.1 (C-5), 66.4 (C-10), 42.8 (NMe2), 35.2 (C-11), C-9a not observed;
15N NMR (40.6 MHz, CD3OD) d 189.3 (N-2), 120.4 (N-9), 51.6 (NMe2).
19. Naulet, N.; Tome, D.; Martin, G. J. Org. Magn. Reson. 1983, 21, 564–566.
20. Appleton, D. R.; Pearce, A. N.; Lambert, G.; Babcock, R. C.; Copp, B. R.
Tetrahedron 2002, 58, 9779–9783.
7.08 (1H, dd, J = 8.8, 2.4 Hz, H-7), 6.94 (5H, m, H-13/H-17, H-14/H-16, H-15),
5.20 (1H, dd, J = 11.2, 4.3 Hz, H-10), 3.70 (1H, dd, J = 12.1, 4.3 Hz, H-11a), 3.59
(1H, dd, J = 12.1, 11.2 Hz, H-11b), 2.98 (6H, s, NMe2); 13C NMR (100 MHz,
CD3OD) d 152.8 (C-6), 138.0 (C-9a, C-3), 137.1 (C-8a), 136.6 (C-1), 136.1 (C-12),
131.4 (C-4a), 130.4 (C-13/C-17), 129.4 (C-14/C-16), 128.1 (C-15), 122.7 (C-4b),
120.5 (C-7), 116.7 (C-4), 113.5 (C-8), 106.7 (C-5), 68.4 (C-10), 42.5 (NMe2), 37.6
(C-11); 15N NMR (40.6 MHz, CD3OD) d 289.3 (N-2), 111.1 (N-9), 47.5 (NMe2).
(10R)-Eudistomin
X
dihydrochloride salt (15c):
[
a
]
+10.5 (c 0.49, MeOH);
21. (10S)-Eudistomin X free base [
mono hydrochloride salt
dihydrochloride salt [
a] +91.0 (c 0.49, MeOH); (10S)-eudistomin X
D
D
1H NMR (400 MHz, CD3OD) d 8.59 (1H, d, J = 6.2 Hz, H-4), 8.52 (1H, d, J = 6.2 Hz,
H-3), 7.62 (2H, m H-5, H-8), 7.33 (1H, dd, J = 8.8, 2.5 Hz, H-7), 7.25 (2H, d,
J = 7.5 Hz, H-13/H-17), 7.04 (2H, m, H-14/H-16), 6.96 (1H, m, H-15), 6.13 (1H,
br m, H-10), 4.09 (1H, dd, J = 13.8, 12.4 Hz, H-11a), 4.02 (1H, dd, J = 13.8, 5.5 Hz,
[
a
]
+7.3 (c 0.49, MeOH); (10S)-eudistomin X
D
a
]
D
À9.8 (c 0.49, MeOH).
22. Stephens, P. J.; Pan, J. J.; Devlin, F. J.; Cheeseman, J. R. J. Nat. Prod. 2008, 71, 285–
288.