Chartarolides A-C, novel meroterpenoids with antitumor activities

Article abstract of DOI:10.1016/j.tetlet.2017.03.079

Three phenylspirodrimane-based meroterpenoids with novel scaffolds, namely chartarolides A-C (1–3), were isolated from a sponge (Niphates recondite) associated fungus Stachybotrys chartarum WGC-25C-6. Their structures were determined on the basis of comprehensive analyses of the spectroscopic data (IR, 1D and 2D NMR, HRESIMS), including the TDDFT-ECD calculation for the assignments of absolute configurations. The biogenetic generation of 1–3 through the conjunction of phenylspirodrimane and mollicellin-type analogues as the precursors was postulated. Chartarolides A-C exhibited significant cytotoxic activities against a panel of human tumor cell lines, and showed strong inhibitory activities against the human tumor related protein kinases of FGFR3, IGF1R, PDGFRb, and TrKB.

Full text of DOI:10.1016/j.tetlet.2017.03.079

Tetrahedron Letters xxx (2017) xxx–xxx
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Tetrahedron Letters
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Chartarolides A-C, novel meroterpenoids with antitumor activities
Dong Liu a,c, Yong Li a,c, Xiaodan Li a, Zhongbin Cheng a, Jian Huang a, Peter Proksch b, Wenhan Lin a,
⇑
a State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, PR China
b Institute of Pharmaceutical Biology and Biotechnology, Heinrich-Heine University, 40225 Duesseldorf, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Three phenylspirodrimane-based meroterpenoids with novel scaffolds, namely chartarolides A-C (1–3),
were isolated from a sponge (Niphates recondite) associated fungus Stachybotrys chartarum WGC-25C-6.
Their structures were determined on the basis of comprehensive analyses of the spectroscopic data
(IR, 1D and 2D NMR, HRESIMS), including the TDDFT-ECD calculation for the assignments of absolute
configurations. The biogenetic generation of 1–3 through the conjunction of phenylspirodrimane and
mollicellin-type analogues as the precursors was postulated. Chartarolides A-C exhibited significant cyto-
toxic activities against a panel of human tumor cell lines, and showed strong inhibitory activities against
the human tumor related protein kinases of FGFR3, IGF1R, PDGFRb, and TrKB.
Received 18 February 2017
Revised 18 March 2017
Accepted 21 March 2017
Available online xxxx
Keywords:
Fungus
Stachybotrys chartarum
Chartarolides A-C
Structure elucidation
Antitumor effects
Ó 2017 Elsevier Ltd. All rights reserved.
Phenylspirodrimane-type derivatives are a profile of unique
natural meroterpenoids, structurally characterized by the fusion
of
fraction (MCF), which exerted significant inhibition against a panel
of human tumor cell lines. These findings encouraged us for further
chemical examination of the unknown natural products with anti-
tumor activities. Chromatographic separation including semi-
preparative HPLC approach of MCF fractionated from the solid rice
fermentation resulted in the isolation of five compounds, including
three new phenylspirodrimane-type meroterpenoids, namely
chartarolides A-C (1–3) (Fig. 1), along with stachybotrylactam1
and mollicellin D.3 This paper describes the structure determina-
tion and antitumor activities of 1–3, while the plausible biogenetic
pathway was postulated.
Chartarolide A (1) is a light yellow amorphous powder. Its
molecular formula was determined as C44H49O10Cl on the basis
of the HRESIMS (m/z 773.3098 [M+H]+) and NMR data, requiring
20 degrees of unsaturation. The IR absorptions as 3368, 1708,
and 1622 cmꢀ1 suggested the presence of hydroxy, carbonyl, and
aromatic functionalities. The 1H NMR spectrum exhibited seven
methyl singlet resonances and a methyl doublet, two aromatic sin-
glet protons, and a number of multiple-coupling alkyl protons. The
13C NMR spectrum displayed a total of 44 carbon resonances,
involving 18 aromatic resonances for three phenyl groups, a car-
bonyl carbon, and two olefinic carbons for a double bond (Support-
ing Information, Table S1). Diagnostic 2D NMR data (1H–1H COSY,
HMQC, and HMBC) established the planar structure of 1, which
consists of three moieties (units A-C). In regard to unit A, the
1H–1H COSY couplings assigned the segments of C-1 to C-3 and
C-5 to C-8 on the basis of the spin systems from H2-1 (dH 0.92,
1.78) to H-3 (dH 3.20) and H-5 (dH 2.03) to H-8 (dH 1.76). The HMBC
correlations from both H3-13 (dH 0.90, s) and H3-14 (dH 0.82, s) to
a drimane-based sesquiterpene with a
phenyl moiety.1–5
Biogenetically, mevalonate pathway is involved in the generation
of the partial structure of acyclic or cyclic sesquiterpene (dri-
mane),5 while a polyketide biosynthesis conducted to produce
the aromatic moiety.6 These drimane-based aromatic sesquiterpe-
nes could be classified into four subtypes based on the positions of
the ether bridge across drimane and aromatic moieties, including
phomoarcherin-type,7 stachyflin-type,5 grifolin-type,8 and stachy-
botryamide (phenylspirodrimane)-type,9 of which phenylspirodri-
mane analogues are the dominant class produced by
microorganisms. It is noted that the structural variety directly
affected their biological activities, such as stachyflin exhibited
potent inhibition against flu virus, stachybotryamides showed
tumor-related kinase inhibition, whereas phomoarcherins exhib-
ited antibiotic activities. Our previous studies revealed that the
sponge-derived fungus Stachybotrys chartarum WGC-25C-6 is a
rich source of meroterpenoids with diverse scaffolds.1 Subsequent
analyses of the HPLC-ESIMS/MS profile of the fungal fermentation
indicated that the minor components in the same strain (WGC-
25C-6) showed the molecular weights around two fold than
phenylspirodrimane analogues, whereas the NMR data were char-
acteristic of the meroterpenoid analogues. Chromatographic frac-
tionation allowed to collect the minor component-containing
⇑
Corresponding author.
E-mail address: whlin@bjmu.edu.cn (W. Lin).
Contribution in equal.
c
http://dx.doi.org/10.1016/j.tetlet.2017.03.079
0040-4039/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Liu D., et al. Tetrahedron Lett. (2017), http://dx.doi.org/10.1016/j.tetlet.2017.03.079

2
D. Liu et al. / Tetrahedron Letters xxx (2017) xxx–xxx
11"
'
10"
'
11" '
10" '
8" '
8" ' H
HO
H
H
12"
1" '
'
6" '
HO
HO
H
3'
12" '
6" '
HO
7" O
O
HO
1" '
O
O
O
3" '
5'
O
H
7" O
O
6"
1"
H
O
6'
O
O
H
HO
6'O
1"
1'
11
H
H
O
O
9"
3" '
HO
1"
HO
O
H
3'
5"
8"
O
7" O
7' 9" 5"
15
7'
9" O
8"
O
1
3'
7'
3"
N
12
1"
3"
O
O
5' 8'
HO
Cl
HO
HO
10
HO
HO
8
5"
O
Cl
O
Cl
O
Cl
8'
5' 8'
8"
5
3
1'
11
1'
1'
6'
6'
11
15
1
HO
15
O
H
O
6
1
12
12
H
3"
O
Cl
10
14
10
8
8
un1it3 A
3
5
unit C
unit B
3
HO
HO
HO
6
H
H
13
H
2
3
1
14 13
14
Fig. 2. Key 1H–1H COSY and HMBC correlations of units A-C in 1.
Fig. 1. Structures of chartarolides A-C (1–3).
C-3 (dC 73.6), C-4 (dC 37.9), and C-5 (dC 40.8), H3-15 (dH 0.94, s) to
C-1 (dC 24.9), C-5, C-9 (dC 98.3) and C-10 (dC 42.0), established a
6,6-bicyclic moiety with the substitution of methyl groups H3-
13/H3-14 at C-4 and H3-15 at C-10. A hydroxy group at C-3 was
evident from the 1H–1H COSY correlation between H-3 and a D2O
exchangeable proton at dH 3.76 (d, J = 4.0 Hz), while an additional
methyl group locating at C-8 was clarified by the HMBC correla-
tions of a methyl doublet H3-12 (dH 0.64, d, J = 6.5 Hz) with C-7
(dC 30.9), C-8 (dC 37.6), and C-9. Beside, a dihydrobenzofuran ring
fused to C-9 with a spiro ring was ascribed to the HMBC correla-
tions from H2-11 (dH 2.65, 3.02) to C-8, C-9, and C-10, and to the
aromatic carbons at C-10 (dC 113.3), C-20 (dC 154.9) and C-60 (dC
159.0). Thus, unit A was characterized as a phenylspirodrimane-
based moiety, closely related to chartarlactams, which were previ-
ously isolated from the same fungal strain.1 Investigation of the 2D
NMR (1H–1H COSY, HMQC, and HMBC) data of the remaining NMR
resonances in association with the comparison of the spectroscopic
data revealed the second partial structure (unit B in Fig. 2) to be
closely related to the coexisted mollicellin D.3 The distinction
was found by the substitution at C-400 (dC 150.1) and C-500 (dC
111.8). Although the HMBC data were unable to distinguish the
positions of the ether or ester connection of the aromatic rings in
the mollicellin D moiety, the obvious NOE interaction between
H-900 (dH 5.43, s) and H-4000 (dH 6.90, s) supported the ether bond
occurred between C-600 and C-3000 rather than C-600 and C-2000. In
addition, a 6,6-heterocycle unit (unit C) was established by addi-
tional 1H–1H COSY and HMBC relationships. The 1H–1H COSY cor-
Fig. 3. Key NOE correlations of 1.
be approximated to 90o, which was characteristic of an equatorial-
equatorial coupling (erythro orientation for 70S⁄ and 900R⁄ configu-
rations). This assignment was supported by the NOE interactions
between OH-70/H-900 and H-70/H-4000, whereas threo-configuration
with the same heterocycle unit in integrastatin derivative afforded
the coupling constant of the vicinal protons to be 5.9 Hz.10
Since the modified Mosher method11,12 was failed due to minor
amount which was unable to obtain qualified 1H NMR spectra for
configurational analyses, an alternative approach such as the
TDDFT-ECD method was used for the configurational assignment.
The ECD calculation of the model molecules of 1 was performed
at the B3LYP/6-311++G(2d, p) level in the gas phase using the
B3LYP/6-31G(d) optimized geometries after conformational
searches via the MMFF94S force field.13 Comparison of the com-
puted ECD spectra for (3R, 5S, 8R, 9R, 10S, 70S, 80S, 900R)-1 and (3R,
5S, 8R, 9R, 10S, 70R, 80R, 900S)-1 with the experimental ECD spectrum
(Fig. 4) reflected the configurations of 1 to be 3R, 5S, 8R, 9R, 10S, 70S,
80S, and 900R. These assignments were also supported by the nega-
tive Cotton effect at ca. 290 nm for 1Lb transition, which agreed the
M-helicity of the aromatic moieties using the benzene sector and
benzene chirality rules,14,15 resulting in 80S and 900R configurations.
In addition, the weak NOE interaction between H3-12 and H-80 in
association with the NOE correlations in the drimane moiety, con-
ducted the absolute configurations of the phenylspirodrimane
moiety to be in agreement with those assigned by the calculated
ECD data.
Chartarolide B (2) has the same molecular formula as that of 1
as determined by the HRESIMS and NMR data. Diagnostic 2D NMR
data in association with the comparison of the NMR data (SI,
Table S1) revealed that both 1 and 2 possess the same planar
structure.
The NOE correlations of the phenylspirodrimane moiety for
both 1 and 2 were closely similar, indicating that they have the
same configuration of unit A. However, compound 2 displayed
the specific rotation in opposite phase to that of 1, indicating that
2 is a stereoisomer of 1 regarding the stereogenic centers in unit C.
The singlet proton of H-900 and the NOE correlations between OH-
70/H-900 and H-70/H-4000 as the case of 1 were indicative of the ery-
thro orientation of the dioxabicyclononane unit, while the weak
relation between H-70 (dH 4.56, d, J = 6.0 Hz) and
a D2O
exchangeable proton OH-70 (dH 6.13, d, J = 6.0 Hz) was attributed
to a hydroxymethine, which was positioned at the aromatic carbon
C-40 (dC 134.0) of unit A, according to the HMBC correlations
between OH-70/C-40 and H-30 (dH 6.24, s)/C-70 (dC 67.6). Additional
HMBC correlations between H-70 and C-500 (dC 111.8) of unit B and
from H-900 to C-40, C-400 (dC 150.1), C-500, and C-600 (dC 155.9) indi-
cated a dioxyethyl bridge across C-40 and C-500. Moreover, an acetal
proton H-80 (dH 6.65, s) correlated to C-40, C-50, and C-60 of unit A
and to C-400 of unit B in the HMBC spectrum clarified the acetal car-
bon linked to C-50 directly and connected to C-400 of unit B via an
ether bond. The linkage of C-80 and C-900 through an ether bond
resulting in a dioxabicyclononane unit (unit C) was assigned by
the HMBC correlations between H-900/C-80 (dC 90.6), and in turn
between H-80/C-900 (dC 72.0). Therefore, the conjunction of units A
and B via unit C was clarified (Fig. 2).
The NOE correlations between H3-14/H3-15, H3-15/H-8, H-5/
OH-3, and H3-14/H-3 (Fig. 3), established the trans fusion of rings
A and B, while H3-15, H-3 and H-8 were in the same orientation.
Additional NOE interaction between H3-15 and H2-11 indicated
the methylene group of the dihydrobenzofuran ring oriented in
the same face as H3-15 group (Fig. 3). The singlet proton H-900 sug-
gested the dihedral angle of the vicinal protons of H-900 and H-70 to
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D. Liu et al. / Tetrahedron Letters xxx (2017) xxx–xxx
3
O
CHO
NH
CHO
NH2
CHO
NH
HO
HO
HO
HO
CHO
H
O
O
O
O
HO
HO
O
HO
HO
stachybotrydial
H
H
H
H
stachybotrylactam
O
O
O
Cl
Cl
-H2O
OH
O
OH
O
OHC
HO
HO
OH
mollicellin D
mollicellin J
HO
N
HO
HO
O
O
O
O
O
O
O
O
O
O
HO
O
+
HO
OH
O
OH
O
HO
Cl
HO
HO
HO
Cl
Cl
OH
OH
O
O
-H2O
HO
-H2
O
HO
HO
H
H
H
3
2
1
Scheme 1. Plausible biosynthetic pathway of 1–3.
Fig. 4. Experimental ECD spectra of 1 and 2 in MeOH and the calculated ECD
spectra of model molecules at the B3LYP/6-311++G(2d,2p) level.
stachybotrydial and mollicellin J3 was postulated, while the
in situ nucleophilic addition of the phenol group of mollicellin J
with the second formaldehyde group of stachybotrydial to gener-
ate a hemiacetal. Subsequent dehydration to form an ether bridge
across C-80 and C-900 resulted in the production of 1 and 2
(Scheme 1). Compound 3 was postulated to be derived by the N-
alkylation of stachybotrylactam with the primary alcohol of molli-
cellin D through dehydration.
Chartarolides A-C (1–3) exerted significant inhibition against
the tumor cell lines, including human colon carcinoma (HCT-
116), human liver hepatocellular carcinoma (HepG2), human gas-
tric carcinoma (BGC-823), non-small cell lung carcinoma (NCI-
H1650), human ovarian carcinoma (A2780), and human ovarian
carcinoma (MCF7) with the IC50 values ranging from 1.4 to
NOE interaction between H-80 and H-900 assumed the cis-fusion of
the ether bridge across C-80 and C-900. The experimental ECD data
of 2 showed positive Cotton effect at ca. 290 nm for 1Lb transition,
which was in opposite to that of 1 but was in agreement with the
ECD data calculated for (3R, 5S, 8R, 9R, 10S, 70R, 80R, 900S)-2 (Fig. 4).
Thus, the absolute configurations of the stereogenic centers at C-70,
C-80, and C-900 were determined to be 70R, 80R, and 900S, respectively.
The molecular formula of chartarolide C (3) was determined as
C
44H50NO9Cl based on the HRESIMS (m/z 794.3334 [M+Na]+) and
NMR data, requiring 20 degrees of unsaturation. The IR absorptions
at 3398 and 1704 cmꢀ1 suggested the presence of hydroxy and car-
bonyl functionalities. Examination of 2D NMR data revealed the
partial structure of 3 closely related to stachybotrylactam,1,14
which was co-isolated from the same fungal strain. This assign-
ment was evident from the similar 1H–1H COSY and HMBC corre-
lations of the phenylspirodrimane moiety as observed in the
known analogue. The HMBC correlations of H-30 (dH 6.62, s) with
C-10 (dC 117.5), C-50 (dC 112.5), and C-70 (dC 168.7) in association
with the correlations from the methylene protons H2-80 (dH 4.19,
4.33) to C-40 (dC 133.5), C-50, C-60 (dC 154.3), and C-70 indicated
12.5
stachybotrylactam, showed weak cytotoxic effects with IC50 values
>25 M. Comparison of cytotoxic effects revealed that 2 possesses
lM (Table 1), whereas the monomers, mollicellin D and
l
the weaker inhibition than 1, indicating the configuration of the
dioxabicyclononane unit directly affects the cytotoxicity. In addi-
tion, compounds 1–3 exhibited inhibitory effects against tumor-
related kinases FGFR3 (fibroblast growth factor receptor 3), IGF1R
(insulin-like growth factor I receptor), PDGFRb (platelet derived
growth factor receptor-b), and TrKB (tropomyosin receptor kinase
the presence of a c-lactam unit, which was fused to the aromatic
ring across C-40 and C-50. Diagnostic 2D NMR data of the remaining
NMR resonances afforded the second building block, whose struc-
ture was almost identical to mollicellin D.3 The distinction was
attributed to the substituent at C-500 (dC 114.2), where a methylene
group H2-900 (dH 4.91, 4.96; dC 36.3) of 3 to replace the hydrox-
ymethylene group of the known analogue, was recognized by the
HMBC correlations from H2-900 to the aromatic carbons C-400, C-500,
B), with IC50 values ranging 2.6–25 lM (Table 2). FGFR represents
an attractive therapeutic target in oncology to regulate cell growth
and proliferation for controlling cell proliferation, migration, apop-
tosis, and differentiation.18 PDGFR is an attractive target related to
tumor growth, angiongenesis and fibrosis.19 Kinase TrkB also plays
an important role in tumor progression in various kinds of cancers,
whereas little is known about biological significance of TrkB in
human lung and neuroendocrine carcinoma.20
and C-600. The linkage of CH2-900 to the nitrogen atom of the
c-lac-
tam was demonstrated by the HMBC correlations from H2-900 to C-
70 and C-80. The similar NOE relationships of 1 and 3 in regard to
the phenylspirodrimane moiety indicated both compounds sharing
the same relative configurations. Acidic hydrolyses15 of 3 gener-
ated two products. Comparison of the NMR, HRESIMS, and specific
rotation data with those of the authentic samples revealed that the
hydrolyzed products were identical to stachybotrylactam and mol-
licellin D, respectively.
Table 1
Inhibition of 1–3 against tumor cells.
IC50 (lM)
HCT-116
HepG2
BGC-823
NCI-H1650
A2780
MCF7
1
2
3
1.9
2.3
7.8
0.03
1.8
2.8
8.9
0.02
1.3
1.6
5.4
0.001
5.5
4.8
11.3
0.07
1.5
3.2
12.5
0.03
1.4
3.8
8.7
0.09
Biogenetically, stachybotrydial2,16 was considered as a precur-
sor to generate stachybotrylactam via series enzyme-mediated
steps.6 A pinacol coupling reaction17 to generate a vicinal diol
bridge for the conjunction of the formaldehyde groups between
Taxola
a
Taxol: positive control.
Please cite this article in press as: Liu D., et al. Tetrahedron Lett. (2017), http://dx.doi.org/10.1016/j.tetlet.2017.03.079

4
D. Liu et al. / Tetrahedron Letters xxx (2017) xxx–xxx
Table 2
A. Supplementary data
Inhibition of 1–3 toward protein kinases.
IC50
(
lM)
Supplementary data (1H, 13C, 1H–1H COSY, HMBC, 2D NOESY,
and HSQC NMR spectra of 1–3) associated with this article can
be found, in the online version, at http://dx.doi.org/10.1016/j.tet-
let.2017.03.079.
FGFR3
IGF1R
PDGFRb
TrKB
1
2
3
2.6
4.9
21.4
<0.5
6.8
8.4
>25
<0.5
9.1
8.0
20.3
>25
<0.5
11.3
18.8
<0.5
Satratoxin Ha
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dial with mollicelin
J (1 and 2) or stachybotrylactam with
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D
dioxabicyclononane-bearing compounds were only found in inte-
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Acknowledgments
This work was supported by the grants from 973 program
(2015CB755906), and NSFC (41376127, 21302005).
Please cite this article in press as: Liu D., et al. Tetrahedron Lett. (2017), http://dx.doi.org/10.1016/j.tetlet.2017.03.079