Xiamycin, a pentacyclic indolosesquiterpene with selective anti-HIV activity from a bacterial mangrove endophyte

Article abstract of DOI:10.1016/j.bmcl.2010.09.010

A novel pentacyclic indolosesquiterpene, named xiamycin (1), and its methyl ester (2) have been obtained from Streptomyces sp. GT2002/1503, an endophyte from the mangrove plant Bruguiera gymnorrhiza. The structures were established by 1D and 2D NMR, MS, and X-ray crystallography, and the absolute configuration of 1 was elucidated by the modified Mosher method. Compound 1 exhibits selective anti-HIV activity; it specifically blocks R5 but has no effects on X4 tropic HIV-1 infection. In a panel of cytotoxicity assays, compound 2 showed to be more potent (geometric mean IC₅₀ = 10.13 μM) compared to compound 1 (geometric mean IC₅₀ >30 μM), with antitumor potency being generally less pronounced. Xiamycin represents one of the first examples of indolosesquiterpenes isolated from prokaryotes.

Full text of DOI:10.1016/j.bmcl.2010.09.010

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6685–6687
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Xiamycin, a pentacyclic indolosesquiterpene with selective anti-HIV activity
from a bacterial mangrove endophyte
Ling Ding a, Jan Münch b, Helmar Goerls c, Armin Maier d, Heinz-Herbert Fiebig d, Wen-Han Lin e,
Christian Hertweck a,
⇑
a Leibniz Institute for Natural Product Research and Infection Biology, HKI, Beutenbergstr. 11a, 07745 Jena, Germany
b Institute of Molecular Virology, University Hospital Ulm, Meyerhofstr. 1, 89081 Ulm, Germany
c Institute for Inorganic and Analytical Chemistry, Friedrich Schiller University, Lessingstr. 8, 07743 Jena, Germany
d Oncotest GmbH, Am Flughafen 12-14, 79108 Freiburg, Germany
e State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 July 2010
Revised 30 August 2010
Accepted 1 September 2010
Available online 9 September 2010
A novel pentacyclic indolosesquiterpene, named xiamycin (1), and its methyl ester (2) have been
obtained from Streptomyces sp. GT2002/1503, an endophyte from the mangrove plant Bruguiera
gymnorrhiza. The structures were established by 1D and 2D NMR, MS, and X-ray crystallography, and
the absolute configuration of 1 was elucidated by the modified Mosher method. Compound 1 exhibits
selective anti-HIV activity; it specifically blocks R5 but has no effects on X4 tropic HIV-1 infection. In a
panel of cytotoxicity assays, compound 2 showed to be more potent (geometric mean IC50 = 10.13
lM)
Keywords:
compared to compound 1 (geometric mean IC50 >30 M), with antitumor potency being generally less
l
Indolosesquiterpene
Natural products
HIV
Endophytes
Streptomyces
pronounced. Xiamycin represents one of the first examples of indolosesquiterpenes isolated from
prokaryotes.
Ó 2010 Published by Elsevier Ltd.
Endophytes are a prolific source of pharmacologically active nat-
ural products with potential medicinal or agrochemical applica-
tions.21,19,18,28,16 In recent years, it has been shown that the
mangrove plants harbor various fungal25,14,23,11,24,13,26 and bacte-
rial2,1,12,7 endophytes, a largely unexplored source for novel natural
products. The large-leafed mangrove (Bruguiera gymnorrhiza) is one
of the most important and widespread mangrove species in the
Pacific. While several compounds have been isolated from this
plant,3–5,8 to our knowledge, no investigations on metabolites from
its endophytes have been reported. Here we describe the isolation,
structure elucidation, and biological activity of two novel indoloses-
quiterpenes from an endophytic actinomycete from B. gymnorrhiza.
Streptomyces sp. GT20021503 was isolated from the stem of B.
gymnorrhiza. HPLC-UV/MS analysis of the crude extract from the
bacterial broth indicated the presence of diverse secondary metab-
olites. To obtain sufficient material for their isolation and full char-
acterization, the fermentation was scaled up. Broth and mycelium
from a 300 L fermentation were separated and individually ex-
tracted using an XAD-161M resin column and ethyl acetate,
respectively. The combined extracts were subjected to flash chro-
matography on silica, followed by a Sephadex LH-20 column. Final
purification was achieved by preparative reversed phase HPLC to
yield compounds 1 (50 mg) and 2 (1.5 mg). Compound 1 was ob-
tained as a pale-yellow powder, which gives a strong violet color
reaction with anisaldehyde–sulfuric acid. The HRESIMS established
the molecular formula of C23H25NO3 (m/z 362.177 [MÀH]À). In the
aromatic region of the 1H NMR spectrum four proton signals (d
7.96, 7.34, 7.27, 7.08) were observed that are diagnostic for a 1,2-
disubstituted benzene ring. Furthermore, two singlets (d 7.92,
7.05) suggested the presence of a carbazole ring.22 The aliphatic re-
gion of the 1H NMR spectrum revealed signals for one oxygenated
methine (d 4.09, H-15) and for two methyl groups (appearing as
singlets, d 1.29, Me-22, and d 1.23, Me-23), as well as various com-
plicated coupling systems. The 13C NMR spectrum showed 23 car-
bon signals including one indicating a carbonyl group (d 181.3), 12
aromatic carbon signals, one oxygenated carbon signal (d 76.3) and
another nine aliphatic carbons. The 1,2-disubstituted benzene ring
was readily confirmed by H,H COSY correlations. In the HMBC
spectrum, correlations between H-5, H-21 and C-3; H-10 and
C-2, and H-21 and C-10, 11 substantiated the structure of the car-
bazole ring (Fig. 1). The H,H COSY spectrum revealed the connec-
tivities for H-13, H-14, and H-15, as well as for H-17, H-18, and
H-19. Further HMBC correlations observed for H-17 and C-17,
Me-22, 23 and C-24, H-14 and C-12, 16 corroborated the partial
structure for the aliphatic part as a decaline-like substructure.
Based on the HMBC correlations between H-17, H-19, Me-22 and
⇑
Corresponding author. Tel.: +49 3641656700; fax: +49 3641656705.
E-mail address: christian.hertweck@hki-jena.de (C. Hertweck).
0960-894X/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2010.09.010

6686
L. Ding et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6685–6687
-0.20 -0.06
OH
OR
-0.08
H H
H
-0.02H
OMTPA
-0.02
OH
-0.05
H
O
N
H
H
+0.03O
1 (R = H)
N
H
2 (R = CH3)
3 (R-MTPA)
4 (S-MTPA)
14
OH
23
22
10
5
Figure 3. Elucidation of the absolute configuration using the advanced Mosher
method. d values ( R) obtained for (R)- and (S)-MTPA esters of 1.
S À
OH
12
16
H
D
D
D
24
3
1
7
18
O
9
20
N
H
COSY
HMBC
Compounds 1 and 2 represent unusual pentacyclic indoloses-
quiterpenes. Indole terpene alkaloids are typical plant and fungal
metabolites, such as the well-known ergot alkaloids from Claviceps
purpurea and the anticancer agents vincristine and vinblastine
from the tropical plant Catharanthus roseus.17 Indolosesquiterpenes
constitute a rather small group, with only few examples reported
with plants origin, such as polyalthenol from Polyalthia oliveri,10
isopolyalthenol and neopolyalthenol from Polyalthia suaveolens,9
polyveoline from P. suaveolens and suaveolindole6 isolated from
Greenwayodendron suaveolens.27 Tubingensin A, an indoloditerpene
from the fungus Aspergillus tubingensis has been the only known
natural product sharing the pentacyclic carbazole skeleton of 1.22
Surprisingly, there have been no reports of indolosesquiterpenes
from bacteria. However, during the preparation of this Letter,
Imamura and co-workers published the isolation and structure
NOE
Figure 1. Structure of 1. Selected COSY, HMBC, and NOE correlations for compound
1. (Arrows point from proton to carbon.)
C-11, and H-21 and C-19, we could connect the aliphatic part to the
carbazole ring system. Compound 1, named xiamycin, was thus
established as a novel pentacyclic carbazole derivative (Fig. 1).
For compound 2, the congener of 1 that is produced only in
minute amounts, the molecular formula of C24H27NO3 was estab-
lished through HRESIMS (m/z [M+Na]+ 400.1846). The NMR spectra
were similar to that of 1 except for one additional methyl signal
from one methoxy group (d 3.70, d 52.6). Consequently, compound
2 represents the methyl ester of 1.
elucidation of the structurally related oridamycin
A from a
Streptomyces sp.20
To evaluate its bioactivity profile, 1 was subjected to a panel of
biological assays involving antimicrobial, cytotoxic (compounds 1
and 2) and antiviral test systems. In the cytotoxicity assays (mod-
ified propidium iodide assay), 1 exhibited only weak to moderate
activities, while compound 2 showed to be more potent (geometric
The relative configuration of 1 was elucidated by NOESY exper-
iments. The NOE effect was observed between H-15 and H-17, as
well as between Me-22 and Me-23, thus providing strong evidence
for the substitution pattern shown in Figure 1. To unequivocally
prove the proposed configuration, we crystallized compound 1
from a mixture of CHCl2/MeOH and subjected a single crystal to
X-ray crystallography.
The crystal structure (CCDC-780372) fully confirmed the consti-
tution of the pentacyclic ring system and the relative configuration
of 1 (Fig. 2). To establish the absolute configuration of 1, the mod-
ified Mosher method was applied.15 Through analysis of the spe-
cific 1H NMR shifts (Fig. 3 and Table S2) we were able to
determine the absolute configuration of 1 as 12S, 15S, 16S, 17R.
mean IC50 = 10.13 lM) (see Supplementary data). Compound 1 was
also tested for its antiviral activity against entry by CXCR4 (X4) and
CCR5 (R5) tropic HIV-1. Interestingly, 1 specifically blocks R5 but
has no effects on X4 tropic HIV-1 infection (Fig. 4).
Albeit only being moderate, the antiviral activity of 1 is intrigu-
ing in light of the reported activity of the related fungal metabolite
tubingensin against herpes simplex virus type 1.22 Thus, it seems
that the pentacyclic carbazole system could be a suitable scaffold
for antiviral agents.
HIV-1 X4
HIV-1 R5
120
100
80
60
40
20
0
0
0.288
1.44
c [µg mL-1]
7.2
36
Figure 2. Molecular structure of 1. The ellipsoids represent a probability of 40%, H
atoms are drawn with arbitrary radii.
Figure 4. Anti-HIV activity of 1 against X4 and R5 tropic HIV.

L. Ding et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6685–6687
6687
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sp. from the large-leafed mangrove, B. gymnorrhiza, and identified
a novel indolosesquiterpene, xiamycin (1), along with its methyl
ester (2). The structure of 1 was fully elucidated by HR-MS, various
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Financial support by the BMBF (Intercommunicate) is gratefully
acknowledged. We thank Mrs. U. Valentin for screening and sam-
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Mrs. Heike Heinecke for HPLC/MS and preparative HPLC; Dr. F. A.
Gollmick and Mrs. F. Rhein for NMR measurements; Dr. H.-M.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.09.010.
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