Bioactive nonanolide derivatives isolated from the endophytic fungus Cytospora sp.

Article abstract of DOI:10.1021/jo201755v

Cytospolides F - Q (6 - 17) and decytospolides A and B (18 and 19), 14 unusual nonanolide derivatives, were isolated from Cytospora sp., an endophytic fungus from Ilex canariensis. The structures were elucidated by means of detailed spectroscopic analysis

Full text of DOI:10.1021/jo201755v

Article
pubs.acs.org/joc
Bioactive Nonanolide Derivatives Isolated from the Endophytic
Fungus Cytospora sp.
Shan Lu,† Peng Sun,† Tiejun Li,† Tibor Kurtan,‡ Attila Mandi,‡ Sandor Antus,‡ Karsten Krohn,§
́
́
́
Siegfried Draeger,∥ Barbara Schulz,∥ Yanghua Yi,† Ling Li,*,† and Wen Zhang*,†
†Research Center for Marine Drugs, and Department of Pharmacology, School of Pharmacy, Second Military Medical University,
325 Guo-He Road, Shanghai 200433, P. R. China
‡Department of Organic Chemistry, University of Debrecen, POB 20, H-4010 Debrecen, Hungary
§Department of Chemistry, Universitat Paderborn, Warburger Straße 100, 33098 Paderborn, Germany
̈
∥Institut fur Mikrobiologie, Technische Universitat Braunschweig, Spielmannstraße 7, 31806 Braunschweig, Germany
̈
̈
S
* Supporting Information
ABSTRACT: Cytospolides F−Q (6−17) and decytospolides A and B (18 and 19), 14
unusual nonanolide derivatives, were isolated from Cytospora sp., an endophytic fungus
from Ilex canariensis. The structures were elucidated by means of detailed spectroscopic
analysis, chemical interconversion, and X-ray single crystal diffraction. The solution-
and solid-state conformers were compared by the combination of experimental
methods (X-ray, NMR) supported by DFT calculations of the conformers. Absolute
configurations were assigned using the modified Mosher’s method and solution- and
solid-state TDDFT ECD calculations. In an in vitro cytotoxicity assay toward the tumor cell lines of A549, HCT116, QGY, A375,
and U973, the γ-lactone 17 demonstrated a potent growth inhibitory activity toward the cell line A-549, while nonanolide 16
with (2S) configuration showed the strongest activity against cell lines A-549, QGY, and U973. A cell cycle analysis indicated that
compound 16 can significantly mediate G1 arrest in A549 tumor cells, confirming the important role of the C-2 methyl in the
growth inhibition toward the tumor line. The discovery of an array of new nonanolides demonstrates the productivity of the
fungus, and it is an example of chemical diversity, extending the nonanolide family by derivatives formed by ring cleavage,
oxidation, esterification, and Michael addition.
publications. These scientific results were subsequently
summarized in several reviews,1,19−21 followed by an increasing
number of reports.22−42 The chemistry of some nonanolides
has been studied intensively, and their total synthesis was
reported repeatedly. Some examples include the cholesterol
biosynthesis inhibitor decarestrictine,1,19,20,22−25 the antimicro-
filament agent microcarpalide,1,19,21 the phytotoxin herbar-
umins,1,19,21,23,26−30 and the bacterial DNA primase inhibitor
Sch 642305.1,19,21,31
During our ongoing screening for biologically active
secondary metabolites from fungi,43−46 we recently investigated
Cytospora sp., an endophytic fungus from Ilex canariensis
(Aquifoliaceae, Aquifoliales), an evergreen shrub from the
island of Gomera, Spain. The fungus was cultivated on biomalt
agar medium. The crude extract of the culture showed
pronounced antifungal activity against Microbotryum violaceum
and moderate algicidal activity against Chlorella fusca.
Preliminary investigation of the crude acetone extract in our
laboratory led to the isolation and structural elucidation of five
novel 10-membered macrolides, cytospolides A−E (1−5).46
The skeleton of cytospolides A−E (1−5) was constructed of 15
carbons and extended the nonanolide family by a novel carbon
INTRODUCTION
■
Secondary metabolites belonging to the family of nonanolides
(synonym decanolides) are constructed of a 10-membered
macrolide core with a C-9 alkyl chain. Structurally more
complex nonanolides with additional rings are sometimes also
included in the family.1 The early investigations on nonanolides
can be traced back to 1942 when the first example, jasmine
ketolactone, was isolated as a component of the essential oil of
Jasminum grandiflorum.2 The structure of this jasmine
ketolactone was fully elucidated in 1964.3 In 1975, diplodialide
A was isolated as a steroid hydroxylase inhibitor from Diplodia
pinea, representing the second report of such a structure.4
The discovery of a series of new nonanolides from a variety
of natural sources, mainly from microorganisms,1,5−18 has
continuously extended the knowledge on this group of
metabolites. Nonanolides were also isolated from several
marine invertebrates1 and plants1 and were found to have
various biological activities, including cytotoxic,1 phyto-
toxic,1,5−8 antimalarial,9 antifungal,1,10,11 antibacterial,1,12−14
and antimicrofilament activities,15 as well as inhibitory effects
against fish embryo larval development,16 AChE,17 and
calmodulin-dependent cAMP phosphodiesterase.18 The broad
spectrum of bioactivity and the intriguing structure of the
medium-sized ring in nonanolide analogues have drawn the
attention of synthetic chemists, resulting in a great number of
Received: August 23, 2011
Published: October 19, 2011
© 2011 American Chemical Society
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skeleton with a unique structural feature of a C-2 methyl group.
Both C-2 epimers were isolated from the same fungal extract,
and the epimers differed considerably in their cytotoxic
activities against the A549 cell line.46 Our investigation on
the trace compounds of the crude extract has now resulted in
the isolation of additional new members of this family, namely
the cytospolides F−Q (6−17) and decytospolides A and B
(18 and 19). Compounds 6−12 and 16 are analogues of
cytospolides A−D (1−4) and cytospolide E (5), respectively.
The cyclization products 13−15 and rearrangement products
17 are furan containing lactones, while the lipid ketones 18 and
19 are decarboxylated derivatives of cytospolides. Herein, we
report the isolation, structural elucidation, including relative and
absolute configuration, and bioactivities of these compounds.
values for the protons flanking the C-8 chirality center
suggested (8S) absolute configuration in accordance with the
CD result. In contrast, protons flanking the C-13 chirality
center showed positive Δδ (δS − δR) values at both sides
making the assignment ambiguous, which is presumably due to
the presence of the C-8 MTPA ester group. However, 14-H
had much smaller positive value (+15.4) than 10−12-H
(+112.5, +141.7, +58.8, respectively), which is most likely
governed by the C-13 MTPA ester group and suggested (13S)
absolute configuration (Figure 1). The chemical shift difference
data of MTPA diester and monoester derivatives of related
derivatives 11 and 18 corroborate well our assignation (vide
infra). The absolute configuration of compound 6 was thus
elucidated as (−)-(2R,3R,8S,9R,13S)-6.
Cytospolide G (7), an optically active colorless oil, had the
molecular formula C17H28O6 as deduced from HRESIMS.
The 1H and 13C NMR spectra of 7 (Tables 1 and 2) resembled
those of 6, revealing their similar structures. The presence of
signals at δH = 2.14, δC = 170.4, and 20.9 in combination with
the typical downfield shift of H-3 from 4.30 in 6 to 5.34 in 7
indicated the presence of an acetyl group at C-3. Detailed 2D
RESULTS AND DISCUSSION
■
The fungus Cytospora sp. was cultivated on biomalt agar
medium for 4 weeks and then extracted with acetone. The
crude extract was fractionated on a silica gel column, followed
by Sephadex LH-20 column chromatography and RP-HPLC, to
yield pure cytospolides F−Q (6−17) and decytospolides A and
B (18 and 19). Structures of these metabolites were elucidated
by a combination of detailed spectroscopic analysis, chemical
interconversion, X-ray single crystal diffraction, and comparison
with data of reported cytospolides A−E (1−5).46 The absolute
configurations were determined by the modified Mosher’s
method and TDDFT calculations of ECD spectra, including the
solid-state TDDFT ECD approach.
1
NMR analysis, including HSQC, HMBC, H−1H COSY, and
NOESY, supported the above conclusion. Acetylation of 6
and 7 gave the triacetyl derivative 20 with identical data in all
respects, including MS, NMR, and [α]20 (Experimental
D
section). The structure and absolute stereochemistry of 7 was
thus determined as the 3-acetyl derivative of 6 with the
(−)-(2R,3R,8S,9R,13S) absolute configuration.
Cytospolide H (8) was isolated as an optically active
colorless oil. Its molecular formula C19H30O7 was established by
HRESIMS. Again, the 1H and 13C NMR spectra of 8 (Tables 1
and 2) showed similarity to those of 7 with the exception of an
additional acetyl group (δH = 2.03, δC = 170.8, and 21.4). The
location of the acetyl group at C-13 was shown by the typical
downfield shift of the proton signal at C-13 from 3.80 in 7 to
4.89 in 8 (Tables 1 and 2). Acetylation of 8 resulted in the
same product (20) (Experimental Section) as that of 6 and 7.
The structure of 8 was then determined as shown above with
(−)-(2R,3R,8S,9R,13S) absolute configuration.
Cytospolides I (9) and J (10) were isolated as a 7:3 mixture
that could not be separated using normal-phase column
chromatography or reversed-phase HPLC techniques. How-
ever, the structural elucidation of both compounds from the
mixture was possible because of the well-separated resonance
Cytospolide F (6) was obtained as an optically active
colorless oil. Its molecular formula C15H26O5 was established by
HRESIMS, indicating an additional oxygen atom in comparison
to that of 4. The 1H and 13C NMR spectra of 6 showed signals
identical to those of 4 in the 10-membered lactone ring, except
for the signals of the side chain, in which a hydroxyl group was
introduced at C-13 (Tables 1 and 2). The location of 13-OH
was deduced by the doublet signal of H3-14 and confirmed by
the correlations from H3-14 to H3-15 established by the 1H−1H
COSY and HMBC spectra. The NOESY spectrum of 6
suggested the same relative configuration of the 10-membered
lactone ring as that of 4. Similarly to the ECD spectrum of 4,46
the ECD spectrum of 6 showed a negative Cotton effect (CE)
at 195 nm, indicating the same (2R) absolute configuration. On
the basis of the relative configuration, the absolute configuration
of the lactone ring of 6 was then assigned as (2R,3R,8S,9R).
In order to determine the absolute configuration of the
secondary C-13 hydroxyl group by the Mosher’s method,47−49
diastereomeric triesters were prepared with (R)- and (S)-MTPA.
As shown in Figure 1, the positive and negative Δδ (δS−δR)
1
signals observed in their H and 13C NMR spectra (Tables 1
and 2). Compounds 9 and 10 had the same molecular formula
of C17H28O6 as established by ESIMS, 13C NMR, and DEPT
1
spectra. The H and 13C NMR spectra of both compounds
showed patterns similar to that of 6 in the 10-membered
lactone ring. Differences were only recognized in the sub-
stituent pattern of an acetoxyl group at the side chain. The
position of the acetoxy group in 9 was obviously the same
as that in 8 due to their identical 13C NMR data from C-10 to
1
C-15 (Table 2). In the H and 13C NMR spectra of 10, the
appearance of a primary oxygenated carbon (δ H = 4.06, t, J =
6.7; δC = 64.4, t) and a lack of the 14-methyl signals led the
location of the acetoxyl group at C-14. The established
structures were fully supported by 2D NMR experiments.
Cytospolide K (11) was also obtained as an optically active
colorless oil, with the molecular formula of C15H26O4, as
1
deduced from HRESIMS. The H and 13C NMR spectra of 11
were closely related to those of 6, except that the secondary
alcohol at C-8 (δH = 3.65, δC = 73.6/73.8) in 6 was replaced by
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a
1
Table 1. H NMR Data for Cytospolides F−J (6−10)
no.
6
7
8
9
10
2
3
4
5
2.68, qd, 6.9, 3.1
4.30, brs
5.60, ov
5.60, ov
2.14, m
2.73, qd, 7.2, 3.6
5.34, brs
2.73, qd, 7.0, 3.1
5.34, brs
2.70, qd, 7.0, 3.0
4.32, brs
5.62, ov
2.70, qd, 7.0, 3.0
4.32, brs
5.62, ov
5.59, dd, 16.2, 2.4
5.59, d, 16.1
5.55, ddd, 16.1, 9.2, 4.3
2.11, m
5.52, ddd, 16.2, 9.6, 4.2
2.09, m
5.62, ov
5.62, ov
6α
2.16, m
2.16, m
6β
2.39, m
2.35, m
2.36, m
2.40, m
2.40, m
7α
1.99, m
1.97, m
1.98, m
2.00, m
2.00, m
7β
1.83, m
1.79, m
1.83, m
1.85, m
1.85, m
8
3.65, t, 7.3
4.76, m
3.64, t, 7.2
4.76, dt, 7.2, 3.6
1.78, m
3.62, m
3.65, t, 7.0
4.77, dt, 7.2, 3.6
1.73, m
3.65, t, 7.0
4.77, dt, 7.2, 3.6
1.75, m
9
4.75, dt, 7.2, 3.6
1.55, m
10a
10b
11a
11b
12a
12b
13a
13b
14
1.81, m
1.63, m
1.60, m
1.55, m
1.55, m
1.75, m
1.42, m
2.07, m
1.33, m
1.30, m
1.32, m
1.42, m
2.01, m
1.33, m
1.30, m
1.32, m
1.45, m
1.45, m
1.55, m
1.45, m
1.33, m
1.45, m
1.40, m
1.55, m
1.45, m
1.33, m
3.80, m
3.80, m
4.89, m
4.91, m
1.62, m
1.62, m
1.18, d, 6.0
1.28, d, 7.0
1.18, d, 6.1
1.17, d, 7.0
2.14, s
1.19, d, 6.2
1.18, d, 7.0
2.14, s
1.21, d, 6.2
1.30, d, 7.0
4.06, t, 6.7
1.30, d, 7.0
15
3-OAc
13-OAc
14-OAc
2.03, s
2.04, s
2.06, s
a
1
In CDCl3, assignments made by DEPT, H−1H COSY, HSQC, HMBC, and NOESY.
a methylene single (δH = 1.50 and 1.81; δC = 33.5) in 11
(Table 3). Analysis of the 1H−1H COSY and HMBC spectra of
11 supported the proposed planar structure. On the basis of the
corresponding chirality centers of the lactone ring, the NOESY
experiment suggested that 11 has the same relative
configuration as 6. The ECD curve of 11 had a negative CE
at 195 nm in agreement with those of 4 and 6 indicating the
same (2R) absolute configuration. The absolute configuration
of the three chirality centers of the lactone ring was thus
assigned as (2R,3R,9S).
and 21.4, q) (Table 3). The location of the acetyl group at C-13
was assured by the downfield shift of the H-13 (δ H = 4.87 in
12 and 3.78 in 11), so that compound 12 was determined as the
13-acetyl derivative of 11. The long-range correlation from
H-13 to the acetyl carbonyl atom (δ C = 170.8) in the HMBC
spectrum further confirmed the proposed structure. Acetylation
of 11 and 12 resulted in the same product 21 (Experimental
Section). The absolute stereochemistry of compound 12 was
thus determined to be the same as that of 11.
Interestingly, carbon atoms of C-8, C-12, C-13, and C-14 of
compounds 6−9 were observed to resonate as two signals when
at least one of the C-8 and C-13 carbons has a free hydroxyl
group. The two series of signals may be due to two different
intramolecular hydrogen-bonding interactions. These dupli-
cated signals change into single ones when intramolecular
hydrogen bonds are broken, i.e., if one of the oxygenated
groups at C-8 and C-13 was absent (e.g., in 1−546 and 10−12
see Table 2) or none of the C-8 and C-13 substituents was a
free hydroxyl group (e.g., acetylating 6−8 to 20).
Cytospolide M (13) was isolated as optically active colorless
crystals. The HRESIMS of 13 established the molecular
formula C15H26O5, indicating three double bond equivalents.
The IR absorption showed the presence of hydroxy (3346
cm−1) and ester carbonyl (1731 cm−1) groups. The 13C NMR
and DEPT spectra revealed one sp2 carbon of an ester carbonyl
atom at lower field and fourteen sp3 carbons (1 × CH, 6 ×
CH2, 2 × CH3, and 5 × OCH at higher field, which were
assigned to their corresponding proton signals by HSQC
experiments (Table 4). Obviously, there are two rings present
in the structure based on the established molecular formula and
the relevant NMR data.
The absolute configuration of C-13 was determined by the
modified Mosher’s method.47−49 The negative Δδ (δS − δR)
value of H-14 and the positive ones of H-8−H-12 allowed
determination of the absolute configuration at C-13 as (13S)
(see the Supporting Information, Figure S1). Moreover, the
negative Δδ (δS − δR) value of 15-H and the positive ones of
H-4−H2-6 corroborated the absolute configuration determined
by ECD. The absolute configuration of compound 11 was thus
elucidated unambiguously as (−)-(2R,3R,9S,13S)-11.
Cytospolide L (12) was isolated as an optically active
colorless oil with its molecular formula of C17H28O5 being
1
established by HRESIMS. The H and 13C NMR spectra of 12
displayed a great similarity to those of 11, except for the
presence of an additional acetyl group (δ H = 2.02, s; δC = 170.8, s
The planar structure and (2R*,3S*,4R*,5R*,8S*,9R*) relative
stereochemistry of 13 were determined by both NMR (Figure 2
and 3) and single-crystal X-ray analysis (Figure 4), the geometry
of the latter was then used to determine the absolute
Figure 1. Δδ (δS-δR) values (in Hz) for the MTPA esters of 6.
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a
Table 2. 13C NMR Data for Cytospolides F−J (6−10)
no.
6
7
8
9
10
1
2
3
4
5
6
7
8
9
174.3/174.4, C
46.7, CH
172.4, C
172.3, C
174.3, C
174.4, C
44.9/45.0, CH
72.5/72.6, CH
128.9, CH
45.0, CH
72.5, CH
46.7, CH
46.7, CH
72.0, CH
133.2, CH
127.5, CH
28.5, CH2
38.6, CH2
73.4, CH
77.6, CH
32.1, CH2
24.3, CH2
25.9, CH2
31.7, CH2
64.4, CH2
12.4, CH3
72.0 CH
72.0, CH
133.2, CH
128.9, CH
129.6, CH
29.3, CH2
133.2, CH
127.5, CH
129.5, CH
127.5, CH
28.6/28.9, CH2
38.6, CH2
29.4, CH2
28.4, CH2
38.8, CH2
38.6, CH2
38.5, CH2
73.6/73.8, CH
77.6, CH
73.6/73.8, CH
77.4/77.5, CH
32.1, CH2
73.6/73.9, CH
77.3, CH
73.6/73.8, CH
77.4, CH
10
31.5/32.1, CH2
20.3/20.8, CH2
38.8/38.9, CH2
67.5/68.0, CH
23.5/23.6, CH3
12.5, CH3
32.0, CH2
32.0, CH2
11
20.7, CH2
20.2/20.6, CH2
35.7/35.8, CH2
70.6/70.8, CH
19.9/20.1, CH3
12.2, CH3
20.2/20.6, CH2
35.7/35.8, CH2
70.6/70.7, CH
19.9/20.1, CH3
12.4, CH3
12
38.5/38.6, CH2
67.5/68.1, CH
23.5/23.7, CH3
12.2, CH3
13
14
15
3-OAc
170.4, C
170.4, C
20.9, CH3
20.9, CH3
13-OAc
14-OAc
170.8, C
170.8/170.9, C
21.4, CH3
21.4, CH3
171.3, C
21.0, CH3
a
1
In CDCl3, assignments made by DEPT, H−1H COSY, HSQC, and HMBC.
a
Table 3. NMR Data for Cytospolides K and L (11 and 12)
11
12
no.
δH, m, J (Hz)
δC, m
δH, m, J (Hz)
δC, m
1
2
3
4
5
174.4, C
174.3, C
2.69, qd, 6.9, 3.1
4.30, brs
47.1, CH
72.8, CH
133.4, CH
127.3, CH
32.7, CH2
2.68, qd, 6.8, 3.3
4.31, brs
47.1, CH
72.8, CH
133.4, CH
127.4, CH
32.6, CH2
5.62, d, 17.4
5.57 (ddd, 17.4, 9.5, 3.7)
1.95, m
5.62, d, 17.5
5.57, ddd (17.5, 9.0, 3.0)
1.95, m
6α
6β
2.30, m
2.30, m
7α
1.84, m
28.1, CH2
33.5, CH2
1.84, m
28.0, CH2
33.4, CH2
7β
1.45, m
1.44, m
8α
1.50, m
1.47, m
8β
1.81, m
1.78, m
9
4.75, q, 6.0
1.55, m
76.2, CH
35.8, CH2
4.74, q, 6.0
1.55, m
76.0, CH
35.5, CH2
10a
10b
11a
11b
12a
12b
13
1.45, m
1.47, m
1.40, m
21.5, CH2
38.9, CH2
1.31, m
21.1, CH2
35.6, CH2
1.40, m
1.31, m
1.45, m
1.55, m
1.45, m
1.55, m
3.78, m
67.9, CH
23.6, CH3
12.5, CH3
4.87, m
70.7, CH
19.9, CH3
12.5, CH3
170.8, C
14
1.18, d, 6.1
1.28, d, 7.0
1.19, d, 6.5
1.28, d, 7.0
2.02, s
15
13-OAc
21.4, CH3
a
1
In CDCl3, assignments made by DEPT, H−1H COSY, HSQC, HMBC, and NOESY.
configuration of 13 by the solid-state TDDFT ECD approach.50
In this method, the DFT-optimized X-ray structure is used as
input for TDDFT ECD calculations,51,52 and the resultant
computed ECD spectrum is compared with the experimental
solid-state ECD measured as a KCl disk (Figure 5). The
solution- and solid-state ECD spectra of 13 showed a negative
band at 214 nm and a positive one below 200 nm. Their good
agreement suggested that the solid-state X-ray conformer is also
prevalent in solution. This was confirmed by a conformational
analysis on the truncated 9-methyl model compound of 13,
which showed that the solid-state conformer had 94.8%
population (see the Supporting Information, Figure S2). The
ECD spectra calculated for the optimized (2R,3S,4R,5R,8S,9R)
enantiomer of the X-ray structure reproduced well the
experimental solid-state ECD, allowing the determination of
absolute configuration.
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a
Table 4. NMR Data for Cytospolides M, N, and Q (13, 14, and 17)
13
14
17
δH, m, J (Hz)
δC, m
δH, m, J (Hz)
δC, m
δH, m, J (Hz)
δC, m
1
175.1, C
45.6, CH
81.9, CH
76.5, CH
80.6, CH
29.5, CH2
175.3, C
45.5, CH
81.8, CH
76.4, CH
80.6, CH
29.5, CH2
177.9, C
2
2.75, qd, 6.9, 2.5
3.88, brs
2.75, qd, 7.0, 2.3
3.88, brs
2.80, m, 7.4
4.51, dd, 7.0, 3.6
4.12, dd, 8.0, 3.6
3.94, dt, 7.5, 4.0
2.09, m
39.2, CH
71.1, CH
85.2, CH
78.5, CH
28.9, CH2
3
4
3.55, d, 9.1
4.10, dd, 9.1, 7.0
2.08, m
3.53, t, 7.2
4.10, dd, 8.4, 7.2
2.08, m
5
6α
6β
7α
7β
8
2.00, m
2.00, m
1.95, m
1.87, m
23.4, CH2
1.86, m
23.5, CH2
1.83, m
23.1, CH2
1.84, m
1.84, m
1.94, m
4.03, dt, 8.3, 3.8
5.36, dt, 9.1, 3.8
1.54, m
81.2, CH
75.0, CH
29.1, CH2
4.02, dt, 7.9, 3.8
5.34, br d, 8.8
1.54, m
81.1, CH
74.9, CH
28.9, CH2
3.90, dt, 8.0, 3.0
3.82, dt, 7.8, 3.6
1.38, m
83.4, CH
71.5, CH
33.1, CH2
9
10a
10b
11a
11b
12a
12b
13a
13b
14
1.45, m
1.45, m
1.38, m
1.35, m
25.6, CH2
31.6, CH2
22.4, CH2
1.43, m
25.6, CH2
25.7, CH2
28.4, CH2
1.50, m
25.6, CH2
31.8, CH2
22.6, CH2
1.35, m
1.43, m
1.33, m
1.32, m
1.43, m
1.30, m
1.32, m
1.43, m
1.30, m
1.31, m
1.64, m
1.32, m
1.31, m
1.64, m
1.32, m
0.90, t, 6.6
1.30, d, 6.9
14.0, CH3
14.6, CH3
4.06, t, 6.8
1.29, d, 6.9
2.04, s
64.3, CH2
14.6, CH3
171.2, C
0.90, t, 6.5
1.27, d,7.5
14.0, CH3
8.8, CH3
15
14-OAc
21.0, CH3
a
1
In CDCl3, assignments made by DEPT, H−1H COSY, HSQC, HMBC, and NOESY.
Cytospolide N (14) was isolated as an optically active
which afforded the absolute configuration as (2R,3S,4R,-
5R,8S,9R)-14.
colorless gum. Its molecular formula C17H28O7 was established
1
by HRESIMS. The H and 13C NMR spectra of 14 (Table 4)
Cytospolide O (15), an optically active colorless oil, has the
molecular formula C15H24O4 as deduced from HRESIMS. A
comparison of the 1H and 13C NMR spectra of 15 with those of
13 revealed similarity. However, the 3-OH and 4-OH groups of
13 were replaced by a carbonyl oxygen and a proton,
respectively (Table 5). The C-4 methylene group was assigned
by the proton spin system from H2-4 to H3-14 as deduced from
the 1H−1H COSY spectrum (Figure 6). The C-3 ketone
carbonyl group was confirmed by the observation of long-range
correlation of both H-2 and H3-15 with C-1 and C-3, H-4 with
C-2 and C-3 in the HMBC spectrum. The diagnostic NOE
cross peaks between H-4α and H-2, H-6α, and H-7α, and
between H-7α and H-10b indicated an α configuration of these
protons, and H-6β, H-8 and H-9 were therefore identified as β
protons. The observed NOE effects between H-8 and both
H-6β and H-9 supported the established (2R*,5R*,8S*,9R*)
were almost identical to those of 13, except for replacement of
signals for the 14-methyl group (δC = 14.0; δH = 0.9) in 13 by
signals of an oxygenated methylene group (δ C = 64.3; δH
=
4.06) in 14. Moreover, signals of an acetyl group (δ C 171.2,
21.0 and δH 2.04) indicated that compound 14 is the 14-
acetoxy derivative of 13. A detailed 2D NMR analysis, including
1H−1H COSY, HMBC, and NOESY, supported the established
structure and relative configuration. The absolute configuration
at C-2 of 14 was proven to be the same as that of 13 by its ECD
spectrum, showing a similar negative band above 200 nm,
Figure 2. 1H−1H COSY (bond) and selected HMBC (arrow)
correlations of 13.
Figure 3. Key NOESY correlations of 13 indicated on the lowest
energy DFT solution conformer of the truncated 9-methyl derivative.
Figure 4. Single-crystal X-ray structure of 13 (ORTEP drawing).
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negative band below 250 nm. The conformational analysis of
the truncated C-9 methyl derivative of 15 afforded three
conformers with 79.0%, 17.1% and 3.9% populations (see
Supporting Information, Figure S4). In the two high-energy
conformers, the C-3 carbonyl group was rotated compared to
that of the lowest-energy one. The ECD spectrum calculated
for the (2R,5R,8S,9R) enantiomer of the solution conformers of
truncated 15 reproduced well the experimental solution ECD
(see Supporting Information, Figure S5) curve of 15, allowing
determination of the absolute configuration as (2R,5R,8S,9R).
Cytospolide P (16) was obtained as an optically active
colorless crystal. The HRESIMS showed the molecular formula
of C17H28O6, indicating four double bond equivalents. The
presence of a ketone and two ester functionalities in the
molecule was deduced from the IR absorption (1736, 1708
cm−1) and the 13C NMR and DEPT spectra (δC = 201.2, 170.1,
and 170.3) (Table 5), accounting for three degrees of
unsaturation. The remaining double bond equivalent was
attributed to the presence of the lactone ring. The detailed
NMR (Figures 7 and S6) and single crystal X-ray diffraction
Figure 5. Experimental solution (MeCN) and solid-state (KCl) ECD
spectra of cytospolide M (13) compared with the TDDFT ECD
spectra calculated for the optimized X-ray geometry of the
(2R,3S,4R,5R,8S,9R) enantiomer of 13. Bars represent the calculated
rotatory strength (R) obtained with the PBE0/TZVP method.
a
Table 5. NMR Data for Cytospolides O and P (15 and 16)
15
δH, m, J (Hz)
16
δH, m, J (Hz)
no.
δC, m
δC, m
1
2
3
170.6, C
57.2, CH
203.6, C
170.3, C
55.7, CH
201.2, C
41.8, CH2
3.73, q, 6.7
3.46, q, 7.0
4α
3.14, dd, 11.7, 4.4
2.34, t, 11.4
4.56, m
47.1, CH2 2.59, dd, 15.0, 5.3
3.13, dd, 15.0, 11.0
77.2, CH 5.38, m
30.9, CH2 2.05, m
1.38, m
Figure 7. 1H−1H COSY (bond) and selected HMBC (arrow)
correlations of 16.
4β
5
69.9, CH
26.8, CH2
6α
1.78, m
(Figure 8) analysis of cytospolide P (16) afforded its planar
structure and (2S*,5R*,8S*,9R*) relative configuration.
6β
2.00, m
7α
2.10, m
24.0, CH2 1.64, m
1.65, m
28.8, CH2
7β
1.93, m
8
4.07, dt, 7.0, 3.8
5.33, dt, 9.6, 3.6
1.60, m
80.8, CH
75.9, CH
3.55, br t, 8.8
73.5, CH
79.4, CH
32.7, CH2
9
4.89, dt, 8.5, 3.0
10a
10b
11a
11b
12a
12b
13a
13b
14
29.2, CH2 1.83, m
1.57, m
1.47, m
1.47, m
25.6, CH2 1.28, m
1.28, m
24.5, CH2
31.5, CH2
22.4, CH2
1.38, m
1.33, m
31.6, CH2 1.28, m
1.28, m
1.33, m
1.30, m
22.5, CH2 1.28, m
1.28, m
1.30, m
0.90, t, 7.0
1.26, d, 6.7
14.0, CH3 0.88, t, 6.8
10.4, CH3 1.33, d, 7.0
2.01, s
13.9, CH3
11.2, CH3
170.1, C
15
5-OAc
21.1, CH3
Figure 8. Single-crystal X-ray structure of 16 (ORTEP drawing).
a
In CDCl3, assignments made by DEPT, 1H−1H COSY, HSQC,
HMBC, and NOESY.
Although 15 and 16 had the same β-keto lactone
chromophore, the characteristic n→π* CE of 16 was negative
(Δε = −0.88 at 290 nm), opposite to that of 15 (Δε = +1.14 at
296 nm), while their ECD bands above 200 nm (lactone n→
π*) exhibited the same positive sign. For the configurational
assignment of 16, the TDDFT ECD spectra were calculated for
the optimized (2S,5R,8S,9R) enantiomer of the X-ray geometry.
Based on the good agreement with the experimental solid-state
ECD measured as KCl disk, the absolute configuration was
deduced as (2S,5R,8S,9R). Thus, 16 has an opposite absolute
configuration at C-2 compared to that of 15, which was
responsible for the opposite CE of the ketone n→π* ECD
transition. A conformational analysis of 16 was also performed
on a truncated C-9 methyl model compound, which showed
Figure 6. 1H−1H COSY (bond) and selected HMBC (arrow)
correlations of 15.
relative stereochemistry (see the Supporting Information,
Figure S3).
For the configurational assignment of 15, the solution
TDDFT ECD protocol was pursued. The solution ECD spectra
of 15 showed a positive n→π* band around 300 nm and a
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that the solid-state conformer with equatorial 8-OH is the most
abundant conformer represented by six slightly different
conformers with a total population of 58.3% (see the
Supporting Information, Figure S7). The 8-OHax conformer
was represented by four conformers with 38.4% of the total
population. In accordance with the result of the conformational
analysis, the measured solution and solid-state ECD spectra
were quite similar (see the Supporting Information, Figure S8).
Cytospolide Q (17) was isolated as an optically active
colorless gum. Its molecular formula C15H26O5 was established
by HRESIMS. The IR absorption showed absorptions of a
hydroxy (3435 cm−1) and an ester carbonyl (1768 cm−1)
group. This was in agreement with the observation of an ester
carbonyl and fourteen sp3 carbons (1 × CH, 6 × CH2, 2 ×
CH3, and 5 × OCH) in the 13C NMR and DEPT spectra
(Table 4). Three degrees of double bond equivalence were
attributed to one carbonyl group and two rings in the molecule.
The proton spin system from H3-15 to H3-14 was readily
Figure 10. Single-crystal X-ray structure of 17 (ORTEP drawing).
of 17 as depicted in Figure 10 with the convincing absolute
structure parameter 0.1 (2).
Decytospolide A (18) was isolated as an optically active
colorless oil. Its molecular formula C14H26O3 was established by
HRESIMS. The absence of the ester carbonyl signal in the 13C
NMR spectrum suggested a different structure than that of the
above lactones (Table 6). Analysis of 1H−1H COSY data
1
identified by H−1H COSY, and the planar structure was then
established by the HMBC correlations as shown in Figure 9.
Table 6. NMR Data for Decytospolides A and B (18
a
and 19)
18
δH, m, J (Hz)
2.49, m
19
δH, m, J (Hz)
no.
δC, m
δC, m
2
3
37.1, CH2 2.48, m
210.0, C
37.2, CH2
209.9, C
Figure 9. 1H−1H COSY (bond) and selected HMBC (arrow)
correlations of 17.
4a
2.66; dd, 15.0, 7.8;
2.39, dd, 15.0, 4.8
3.74, m
48.4, CH2 2.68; dd, 15.2, 7.9;
2.38, dd, 15.2, 4.2
74.1, CH 3.77, m
31.3, CH2 1.40, m
1.75, m
48.2, CH2
4b
The relative stereochemistry of 17 was elucidated on the basis
of detailed analysis on its NOE data. Concerning the lactone
ring (A), the NOE effect between H-4 and H3-15 indicated a β
configuration of the protons while the NOE effect between H-3
and H-5 indicated their α orientation. As for the tetrahydrofuran
ring (B), the observed NOE effects between H-4 and H-7β, and
between H-5 and H-8 led to the assignment of H-7β as a β
proton and H-5 and H-8 as α protons, respectively. Ring A is
attached to ring B in a β position (see the Supporting
Information, Figure S9). The relative configuration of H-9 was
deduced from the homo- and heteronuclear 3J couplings.53 The
5
74.2, CH
30.8, CH2
6α
1.38, m
6β
1.78, m
7α
2.08, m
32.9, CH2 2.14, m
1.45, m
29.4, CH2
7β
1.46, m
8
3.26, dt, 10.2, 4.8
3.04, dt, 9.0, 2.4
1.79, m
70.5, CH 4.45, dt, 10.0, 4.6
82.1, CH 3.23, dt, 9.3, 2.4
31.9, CH2 1.51, m
1.32, m
72.1, CH
79.3, CH
31.9, CH2
9
10a
10b
11a
11b
12a
12b
13a
13b
14
1.36, m
1.47, m
25.0, CH2 1.40, m
1.25, m
24.8, CH2
31.7, CH2
22.6, CH2
1.29, m
1.30, m
31.8, CH2 1.25, m
1.25, m
3
small coupling constant of JH8,H9 = 3.0 Hz indicated a gauche
1.30, m
arrangement of H-8 and H-9, and the large coupling constant of
3JC7,H9 = 11.5 Hz, measured by means of J-modulated HMBC
experiments,54 indicated antiperiplanar orientation of C-7 and
H-9, which finally led to (2R*,3S*,4S*,5R*,8S*,9R*) relative
configuration.
1.30, m
22.6, CH2 1.28, m
1.28, m
1.30, m
0.89, t, 6.9
1.05, t, 7.2
14.0, CH3 0.87, t, 6.8
7.5, CH3 1.04, t, 7.3
2.04, s
14.0, CH3
7.5, CH3
15
8-OAc
170.3, C
21.2, CH3
A conformational analysis of the solution conformers of
truncated 17 confirmed the NMR coupling pattern. In the
lowest-energy conformer (67.7% population) the ωC7,C8,C9,H9
torsional angle was 174.2°, while the ωH8,C8,C9,H9 was found to
be −60.7°, corroborating the values of experimental homonuclear
and heteronuclear coupling constants (see the Supporting
Information, Figure S10). These torsional angles have similar
values in the higher-energy conformers as well, since they only
differed in the orientation of their OH groups. The 9-OH was
hydrogen-bonded to the oxygen of the 3-OH in conformer A,
B, D with 67.7%, 12.8%, and 5.6% populations, respectively,
and the 3-OH was hydrogen-bonded to the oxygen of 9-OH in
two minor conformers with 8.4% and 2.3% populations.
The structure and relative stereochemistry of 17 were further
confirmed by a single-crystal X-ray analysis (Figure 10). The
X-ray diffraction also established the absolute stereochemistry
a
In CDCl3, assignments made by DEPT, 1H−1H COSY, HSQC,
HMBC and NOESY.
revealed two proton spin systems, i.e., H3-15 to H2-2, and H2-4
to H3-14. The HMBC correlation from protons H2-2 and
H2-4 to C-3 led the connection of the two proton sequence
through a ketone carbonyl. The formation of a pyran ring was
apparent due to the diagnostic long-range correlation from H-5
to C-9, giving the planar structure of 18 (Figure 11). The C-5
and C-9 substituents of the furan ring had cis orientation, since
a NOE effect was observed between the 1,3-diaxial protons of
H-5 and H-9. H-8 had trans-diaxial relationship with H-9 due
to the large coupling constant (3JH8,H9 = 10.2 Hz) and showed a
NOE effect with H-10b (δH = 1.36) (see the Supporting
Information, Figure S11) allowing the assignment of the relative
configuration. The absolute configuration of the secondary
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The discovery of an array of new nonanolides demonstrates
the productivity of the fungus and it is an example of chemical
diversity, extending the nonanolide family by derivatives
formed by ring cleavage, oxidation, esterification and Michael
addition. Cytospolides A−L (1−12) may biogenetically derive
from precursor A by a dehydration involving the 5-OH,
followed by oxidation at C-8, C-13, and/or C-14. Further
oxidation of OH-3 would result in the reactive α,β-unsaturated
ketone precursor B, the C-3 carbonyl and Δ4,5 bond of which
can be found separately in the reported nanolides. An inter-
molecular Michael addition to the β carbon of the α,β-
unsaturated ketone and the acetylation of 5-OH may produce
cytospolide P (16) from precursor B, while an intramolecular
oxa-Michael addition of 8-OH to the α,β-unsaturated ketone
of precursor B may give cytospolide O (15). The subsequent
oxidation of C-4 and reduction of C-3 carbonyl of cytospolide
O (15) may produce cytospolides M and N (13, 14).
Consequently, a transesterification of the lactone acyl group
with 4-OH of cytospolide M (13) would construct the
γ-lactone moiety of cytospolide Q (17). The two decytospo-
lides A and B (18, 19) may be produced from precursor B
by an initiation of a ring cleavage on the lactone bond, followed
by intramolecular oxa-Michael addition of 9-OH to the
α,β-unsaturated ketone and decarboxylation of the β-keto
carboxylic acid derivative (Scheme 1).
Figure 11. 1H−1H COSY (bond) and selected HMBC (arrow)
correlations of 18.
8-OH was determined as (8S) by the modified Mosher’s method
(see Supporting Information, Figure S12),46−49 which in the
Figure 12. Effect of cytospolides E and Q (5, 16) on cell cycle in A549
and QGY cells. Data were obtained by densitometry measurements
(n = 3).
knowledge of the relative configuration afforded the absolute
configuration of 18 as (5R,8S,9R).
The cytotoxic activity of compounds 6−19 toward the tumor
cell lines of A549, HCT116, QGY, A375, and U973 was
evaluated in vitro (Table 7). It had been reported that the
absolute configuration of C-2 has an effect on the cytotoxic
activity; e.g. cytospolide D (4) with (2R) configuration showed
no activity against A-549 cell line at 50 μg/mL, while its C-2
epimer, cytospolide E (5), displayed modest cytotoxic activity
with an IC50 value of 7.09 μg/mL.46 In accordance, 16 with
(2S) configuration showed the strongest activity against cell
lines A-549, QGY and U973.
Furan containing nonanolides 13, 14, 15, and 17 also
showed considerably cytotoxic activity against A-549 cells. The
free hydroxyl groups at C-3 and C-4 seem to play an important
role for the cytotoxicity, since the inhibition remarkably
decreased in compound 15 lacking the two secondary hydroxyl
Decytospolide B (19) was isolated as an optically active
colorless oil. 1H and 13C NMR spectra greatly resembled those
of 18 except for the presence of an additional acetyl group
(δH = 2.04, s; δC = 170.3, s and 21.2, q). This conclusion was
in agreement with its molecular formula of C16H28O4 as
deduced from HRESIMS, showing 42 mass units more than 18.
The location of the acetyl group at C-8 was assured by the
typical downfield shift of the H-8 (δ H = 4.45 in 19 and 3.26 in
18) (Table 6). The long-range correlation from H-8 to the
acetyl carbonyl atom (δC = 170.3) in the HMBC spectrum
further confirmed the proposed structure. The acetylation
product of 18 was identical with 19, which served as further
evidence for the elucidation of the planar structure and the
absolute stereochemistry. Compound 19 was thus determined
as the 8-acetate derivative of 18.
Scheme 1. Proposed Biogenetic Connection of Isolated Cytospolides A−Q (1−17) and Decytospolides A and B (18 and 19)
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Culture, Extraction, and Isolation. The endophytic fungus
Cytospora sp., internal strain No. ZW02, was isolated following surface
sterilization from Ilex canariensis, a woody, evergreen shrub from
Gomera, and was cultivated on biomalt (5% w/v) solid agar medium
at room temperature for 28 days.43−45 The culture medium was then
extracted with acetone to afford a residue (9.98 g) after removal of the
solvent under reduced pressure. The extract was subjected to column
chromatography (CC) on silica gel, eluted with a gradient of CH2Cl2
in acetone (100:1, 80:20, 50:50, 30:70, 1:100 v/v), to give 17
subfractions. Fractions 3 and 4 were first purified on silica gel CC
(400-500 mesh, CH2Cl2/acetone, 100:1) and then fractionated by
semi-preparative RP-HPLC (MeOH/H2O, 8:2, 1.5 mL/min) to give
16 (1.7 mg, 15.8 min, yield 0.17‰), 15 (6.3 mg, 20.3 min, yield
2.03‰), and 19 (1.5 mg, 27.4 min, yield 2.74‰), respectively.
Fractions 8−10 were first purified on silica gel CC (400-500 mesh,
CHCl3/MeOH, 50:1), followed by a purification of semi-preparative
RP-HPLC: fraction 8 afforded 12 (1.7 mg, yield 0.17‰) at 27.7 min
with the elution of MeOH/H2O (8:2, 1.0 mL/min); fractions 9 and 10
yielded pure 7 (5.4 mg, yield 0.54‰), 13 (9.4 mg, yield 0.94‰), 14
(1.0 mg, yield 0.10‰), and 18 (8.1 mg, yield 0.81‰) at 23.2, 18.3,
19.9, and 29.7 min, respectively, eluted with MeOH/H2O (7:3, 1.5
mL/min). Fraction 11 gave 6 (7.7 mg, yield 0.77‰) and 8 (0.9 mg,
yield 0.09‰) by a simple fractionation on silica gel CC (400-500
mesh, CHCl3/MeOH, 30:1). Fractions 14, 15, and 16 were purified on
silica gel CC (400-500 mesh, CHCl3/MeOH, 20:1), and then
separated by semi-preparative HPLC, eluted with MeOH/H2O (7:3,
1.5 mL/min) to yield pure 11 (6.6 mg, yield 0.66‰), 17 (4.5 mg, yield
0.45‰) and the mixture products 9/10 (6.1 mg, yield 0.61‰), at 19.6,
18.6, and 14.9 min, respectively.
Table 7. Cytotoxic Assays toward Different Tumor Cell
Lines
a b
,
no.
A549
HCT116
QGY
A375
U937
6
−
−
−
−
−
20.47
−
−
−
−
−
−
−
−
−
−
−
−
−
−
0.45
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
0.44
−
−
−
−
−
−
−
−
−
7
8
9
10
11
12
13
14
15
16
17
18
19
−
12.29
16.55
39.17
2.05
10.55
−
−
15.82
−
28.26
−
−
46.79
−
−
−
0.23
14.79
adriamycin
0.44
0.28
a
b
IC50 (μg/mL). −: IC50 > 50 μg/mL.
groups. The activity of decytospolide A (18) increased
dramatically when the 8-OH was acetylated.
A cell cycle analysis was executed to further evaluate the
tumor cell growth inhibition of the two potent compounds,
cytospolides E and Q (5, 16), both having (2S) absolute
configuration. Both compounds can significantly mediate G1
arrest in A549 tumor cells at 1 μg/mL. The percentage of cells
at the G1 phase was increased from 58.8% to 82.5% and 83.1%
(P < 0.01), while those at the S phase were decreased from
30.3% to 11.6% and 10.2% (P < 0.01), respectively. Cytospolide
E (5) was not active toward QGY cells, whereas cytospolide P
(16) displayed a significant effect (10 μg/mL), increasing the
percentage at G1 phase from 56.9% to 72.0% (P < 0.01) and
decreasing the percentage of cells at S phase from 30.3% to
11.6% (P < 0.01), respectively (Figure 12).
Cytospolide F (6): colorless oil; Rf = 0.38 (CHCl3/MeOH 10:1);
[α]20 = −72.7 (c 0.08, CHCl3); CD (CH3CN, c 2.0 × 10−4) λmax
D
(Δε) = 195 (−11.48) nm; UV (CH3CN) λmax (ε) 219 (2449) nm; IR
(film) νmax = 3415, 3004, 2920, 1716, 1656, 1436, 1410, 1317, 1185,
1
1022, 953, 903, 707 cm−1; H and 13C NMR spectroscopic data, see
Tables 1 and 2; HRMS (ESI) [M + Na]+ m/z 309.1675 (calcd for
C15H26O5Na, 309.1678).
Cytospolide G (7:). colorless oil; Rf = 0.79 (CHCl3/MeOH 10:1);
[α]20 = −53.7 (c 0.05, CHCl3); UV (CH3CN) λmax (ε) 221 (1797)
D
nm; IR (film) νmax = 3406, 2930, 2857, 1737, 1670, 1457, 1375, 1240,
1
1180, 1106, 1065, 1024, 990, 943 cm−1; H and 13C NMR spectros-
copic data, see Tables 1 and 2; HRMS (ESI) [M + Na]+ m/z 351.1785
(calcd for C17H28O6Na, 351.1784).
EXPERIMENTAL SECTION
■
Cytospolide H (8): colorless oil; Rf = 0.32 (CHCl3/MeOH 10:1);
[α]20 = −88.9 (c 0.01, CHCl3); UV (CH3CN) λmax (ε) 215 (1829)
General Experimental Procedures. Precoated silica gel plates
were used for analytical thin-layer chromatography (TLC). Spots were
detected on TLC under UV or by heating after spraying with 0.5 mL
of anisaldehyde in 50 mL of HOAc and 1 mL of H2SO4. TLC Rf values
are reported. The NMR spectra were recorded at 293K. Chemical
shifts are reported in parts per million (δ), using the residual CHCl3
signal (δH 7.26 ppm) as an internal standard for 1H NMR, and CDCl3
D
nm; IR (film) νmax = 3454, 3360, 3197, 2924, 2853, 1736, 1662, 1462,
1
1440, 1375, 1242, 1176, 1107, 1065, 1023, 989 cm−1; H and 13C
NMR spectroscopic data, see Tables 1 and 2; HRMS (ESI) [M + Na]+
m/z 393.1887 (calcd for C19H30O7Na, 393.1889).
Cytospolide I and J (9 and 10): colorless oil; Rf = 0.31 (CHCl3/
MeOH 5:1); [α]20 = −80.3 (c 0.06, CHCl3); CD (CH3CN, c 2.0 ×
D
(δC 77.0 ppm) for 13C NMR; coupling constant (J) in Hz. 1H and 13
C
10−4) λmax (Δε) = 196 (−15.16) nm; UV (CH3CN) λmax (ε) 220
(2413) nm; IR (film) νmax = 3443, 2929, 2855, 1728, 1668, 1453,
NMR assignments were supported by 1H−1H COSY, HMQC,
3
1
1373, 1247, 1177, 1101, 1068, 1043, 989 cm−1; H and 13C NMR
HMBC, and NOESY experiments. JC,H couplings were measured by
spectroscopic data, see Tables 1 and 2; ESIMS [M + Na]+ m/z 351.13.
Cytospolide K (11): colorless oil; Rf = 0.41 (CHCl3/MeOH 5:1);
means of pulsed field gradient HMBC spectra, recorded by varying
J-refocusing time τ between 0.04 and 0.16 s (10 ms interval),
3
[α]20 = −40.9 (c 0.07, CHCl3); CD (CH3CN, c 2.2 × 10−4) λmax
corresponding to J = 1/(2τ) = 3.1−12.5 Hz. JC,H values were
D
estimated with least-squares sinusoidal fit of the experimental cross-
peak intensities as a function of J.54 The following abbreviations are
used to describe spin multiplicity: s = singlet, d = doublet, t = triplet, q
= quartet, m = multiplet, brs = broad singlet, dd = doublet of doublets,
ddd = doublet of doublets of doublets, dt = doublet of triplets, qd =
quartet of doublets, ov = overlapped signals. Infrared spectra were
recorded in thin polymer films. For the protocol of the solid-state
circular dichroism (CD) protocol, see ref50. The mass spectra and
high-resolution mass spectra were performed on a Q-TOF Micro mass
spectrometer, resolution 5000. An isopropyl alcohol solution of
sodium iodide (2 mg/mL) was used as a reference compound. The
X-ray diffraction study was carried out using Cu Kα radiation (λ =
1.542178 Å). Semi-preparative RP-HPLC was performed using a
refractive index detector and a column with 5 μm, 250 × 10 mm size.
(Δε) = 195 (−10.90) nm; UV (CH3CN) λmax (ε) 221 (1110) nm; IR
(film) νmax = 3429, 2927, 2856, 1714, 1455, 1375, 1242, 1181, 1101,
1
1029, 979, 916 cm−1; H and 13C NMR spectroscopic data, see Table
3; HRMS (ESI) [M + Na]+ m/z 293.1727 (calcd for C15H26O4Na,
293.1729).
Cytospolide L (12): colorless oil; Rf = 0.50 (CHCl3/MeOH
10:1); [α]20 = −75.0 (c 0.01, CHCl3); CD (CH3CN, c 2.0 × 10−4)
D
λmax (Δε) = 196 (−14.48) nm; UV (CH3CN) λmax (ε) 219 (1133)
nm; IR (film) νmax = 3358, 3196, 2924, 2853, 1731, 1660, 1633, 1463,
1
1374, 1245, 1177, 1135, 1099 cm−1; H and 13C NMR spectroscopic
data, see Table 3; HRMS (ESI) [M + Na]+ m/z 335.1831 (calcd for
C17H28O5Na, 335.1834).
Cytospolide M (13): colorless crystals (ethyl acetate/petroleum
ether 2:1); mp 136−137 °C; Rf = 0.57 (CHCl3/MeOH 10:1);
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The Journal of Organic Chemistry
Article
[α]20 = −25.5 (c 0.09, CHCl3); CD (CH3CN, c 5.0 × 10−4) λmax
by recrystallization from ethyl acetate/petroleum ether 2:1. C17H28O6
(Mr = 328.39), orthorhombic, space group C2221 with a = 5.1842(1)
Å, b = 22.7750(4) Å, c = 30.4741(4) Å, α = β = γ = 90.00°, V =
3598.08(11) Å3, Z = 8, Dcalcd = 1.212 g/cm3. Intensity data were
measured using a Cu Kα radiation (graphite monochromator). A total
of 6130 reflections were collected to a maximum 2θ value of 134.92° at
296 (2) K. The structure was solved by direct methods and refined by
full matrix least-squares procedure. All non-hydrogen atoms were
given anisotropic thermal parameters; hydrogen atoms were located
from difference Fourier maps and refined at idealized positions riding
on their parent atoms. The refinement converged at R1(I > 2σ(I)) =
0.0388, wR2 = 0.1035 for 2894 independent reflections and 210
variables. CCDC-838719 (16) contains the supplementary crystallo-
graphic data for this paper. These data can be obtained free of charge
from the Cambridge Crystallographic Data Centre via www.ccdc.cam.
ac.uk/data_request/cif.
D
(Δε) = 190 (+2.35), 214 (−3.07) nm; CD (KCl) λ (mdeg) 155 μg of
13 in 250 mg KCl 215 (−26.06), positive below 196 nm; UV
(CH3CN) λmax (ε) 219 (1828) nm; IR (film) νmax = 3346, 2928,
2856, 1731, 1665, 1459, 1377, 1184, 1131, 1098, 1064, 992, 934 cm−1
;
1H and 13C NMR spectroscopic data, see Table 4; HRMS (ESI) [M +
Na]+ m/z 309.1677 (calcd for C15H26O5Na, 309.1678).
Cytospolide N (14): colorless oil; Rf = 0.71 (CHCl3/MeOH
10:1); [α]20 = −60.0 (c 0.01, CHCl3); CD (CH3CN, c 5.0 × 10−4)
D
λmax (Δε) = 207 (−3.01) nm; UV (CH3CN) λmax (ε) 214 (1130) nm;
IR (film) νmax = 3359, 3205, 2925, 2854, 1733, 1661, 1633, 1462,
1371, 1241, 1179, 1099, 1059, 1028, 989, 935 cm−1; 1H and 13C NMR
spectroscopic data, see Table 4; HRMS (ESI) [M + Na]+ m/z
367.1732 (calcd for C17H28O7Na, 367.1733).
Cytospolide O (15): colorless oil; Rf = 0.47 (CH2Cl2/n-hexane
20:1); [α]20 = −6.3 (c 0.06, CHCl3); CD (CH3CN, c 5.0 × 10−4)
D
λmax (Δε) = 189 (−6.63), 216 (−1.83), 296 (+1.14) nm; UV
(CH3CN) λmax (ε) 226 (2569), 251 (390), 278 (707) nm; IR (film)
νmax = 3359, 3194, 2922, 2852, 1658, 1633, 1465, 1416 cm−1; 1H and
13C NMR spectroscopic data, see Table 5; HRMS (ESI) [M + Na]+
m/z 291.1570 (calcd for C15H24O4Na, 291.1572).
X-ray Crystallographic Studies of Cytospolide Q (17). A
colorless needle crystal of 17 (0.10 × 0.10 × 0.12 mm) was obtained
by recrystallization from CH2Cl2/petroleum ether (1:1). C15H26O5
(Mr = 286.36), orthorhombic, space group P212121 with a = 5.1139(2)
Å, b = 16.2267(5) Å, c = 19.0927(6) Å, α = β = γ = 90.00°, V =
1584.35(9) Å3, Z = 4, Dcalcd = 1.201 g/cm3. Intensity data were
measured using a Cu Kα radiation (graphite monochromator). A total
of 4500 reflections were collected to a maximum 2θ value of 132.02° at
296(2) K. The structure was solved by direct methods and refined
by full matrix least-squares procedure. All non-hydrogen atoms were
given anisotropic thermal parameters; hydrogen atoms were located
from difference Fourier maps and refined at idealized positions riding
on their parent atoms. The refinement converged at R1(I > 2σ(I)) =
0.0311, wR2 = 0.10877 for 2571 independent reflections and 185
variables. Absolute structure parameter 0.1(2). CCDC-838720 (17)
contains the supplementary crystallographic data for this paper. These
data can be obtained free of charge from the Cambridge Crystallo-
graphic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
Esterification of Cytospolide F (6) with (R)-MTPA Chlo-
ride. Treatment of 6 (0.68 mg) with (R)-MTPA chloride (10 μL) in
dry pyridine (0.5 mL), stirring at room temperature overnight, and
purification by a mini silica gel column chromatography (500 mesh,
n-hexane/CHCl3, 3:1) afforded the (S)-MTPA ester of 6 (1.37 mg,
62%): 1H NMR (600 MHz, CDCl3) δ = 2.73 (m, 1H, H-2), 5.57 (brs,
1H, H-3), 5.66 (dd, J = 16.2, 2.4 Hz, 1H, H-4), 5.40 (m, 1H, H-5),
2.14 (m, 1H, H-6α), 2.14 (m, 1H, H-6β), 1.85 (m, 1H, H-7α), 1.78
(m, 1H, H-7β), 4.93 (m, 1H, H-8), 4.88 (m, 1H, H-9), 1.32 (m, 1H,
H-10a), 1.32 (m, 1H, H-10b), 1.37 (m, 1H, H-11a), 1.37 (m, 1H, H-
11b), 1.46 (m, 1H, H-12a), 1.46 (m, 1H, H-12b), 5.02 (m, 1H, H-13),
1.27 (t, J = 6.4, 3H, H-14), 1.07 (d, J = 7.0, 3H, H-15).
Cytospolide P (16): Colorless crystals (ethyl acetate/petroleum
ether 2:1); mp 103−104 °C; Rf = 0.42 (CHCl3); [α]20 = −105.9 (c
D
0.02, CHCl3); CD (CH3CN, c 1.0 × 10−3) λmax (Δε) =198 (−1.94),
213 (−1.57), 290 (−0.88) nm; CD (KCl) λ (mdeg) 123 μg of 16 in
250 mg KCl 289 (−16.61), 214 (−21.93); UV (CH3CN) λmax (ε) 223
(2558) nm; IR (film) νmax = 3408, 2926, 2855, 1736, 1708, 1669,
1
1459, 1372, 1242, 1073, 1030, 987, 953, 890 cm−1; H and 13C NMR
spectroscopic data, see Table 5; HRMS (ESI) [M + Na]+ m/z
351.1787 (calcd for C17H28O6Na, 351.1784).
Cytospolide Q (17): colorless crystals (CH2Cl2/petroleum ether
1:1); mp 97−98 °C; Rf = 0.35 (CHCl3/MeOH 5:1); [α]20D = −4.4 (c
0.04, CHCl3); CD (CH3CN, c 8.0 × 10−4) λmax (Δε) = 194 (−0.86),
217 (−1.48) nm; UV (CH3CN) λmax (ε) 219 (1537) nm; IR (film)
νmax = 3435, 3359, 2925, 2855, 1768, 1667, 1460, 1377, 1178,
1047,1023, 985 cm−1; 1H and 13C NMR spectroscopic data, see
Table 4; HRMS (ESI) [M + Na]+ m/z 309.1677 (calcd for
C15H26O5Na, 309.1678).
Decytospolide A (18): Colorless oil; Rf = 0.48 (CHCl3/MeOH
10:1); [α]20 = +6.1 (c 0.08, CHCl3); UV (CH3CN) λmax (ε) 220
D
(823) nm; IR (film) νmax = 3431, 2929, 2857, 1713, 1671, 1459, 1374,
1
1238, 1078, 1026, 989, 935 cm−1; H and 13C NMR spectroscopic
data, see Table 6; HRMS (ESI) [M + Na]+ m/z 265.1778 (calcd for
C14H26O3Na, 265.1780).
Decytospolide B (19:). colorless oil; Rf = 0.51 (CH2Cl2/n-hexane
20:1); [α]20 = +26.6 (c 0.02, CHCl3); UV (CH3CN) λmax (ε) 222
D
(2210), 267 (543), 276 (559) nm; IR (film) νmax = 3360, 3193, 2923,
Esterification of Cytospolide F (6) with (S)-MTPA Chloride:
1
2852, 1658, 1633, 1465, 1416, 1315, 1235, 1134, 1043, 879 cm−1; H
The same reaction of 6 (1.03 mg) with (S)-MTPA chloride (15 μL)
and 13C NMR spectroscopic data, see Table 6; HRMS (ESI) [M +
Na]+ m/z 307.1888 (calcd for C16H28O4Na, 307.1885).
1
afforded the (R)-MTPA ester of 6 (2.16 mg, 64%): H NMR (600
MHz, CDCl3) δ = 2.69 (m, 1H, H-2), 5.51 (brs, 1H, H-3), 5.62 (dd,
J = 16.2, 2.4 Hz, 1H, H-4), 5.12 (m, 1H, H-5), 2.21 (m, 1H, H-6α),
2.21 (m, 1H, H-6β), 1.92 (m, 1H, H-7α), 1.74 (m, 1H, H-7β), 4.74
(m, 1H, H-8), 4.84 (m, 1H, H-9), 1.14 (m, 1H, H-10a), 1.14 (m, 1H,
H-10b), 1.13 (m, 1H, H-11a), 1.13 (m, 1H, H-11b), 1.36 (m, 1H, H-
12a), 1.36 (m, 1H, H-12b), 4.95 (m, 1H, H-13), 1.24 (t, J = 6.4, 3H,
H-14), 1.14 (d, J = 7.0, 3H, H-15).
X-ray Crystallographic Studies of Cytospolide M (13). A
colorless needle crystal of 13 (0.05 × 0.10 × 0.10 mm) was obtained
by recrystallization from ethyl acetate/petroleum ether 2:1. C15H26O5
(Mr = 286.36), orthorhombic, space group P212121 with a = 5.3240(1)
Å, b = 12.6918(3) Å, c = 23.0937(6) Å, α = β = γ = 90.00°, V =
1560.47(6) Å3, Z = 4, Dcalcd = 1.219 g/cm3. Intensity data were
measured using a Cu Kα radiation (graphite monochromator). A total
of 4890 reflections were collected to a maximum 2θ value of 134.80° at
296 (2) K. The structure was solved by direct methods and refined by
full matrix least-squares procedure. All non-hydrogen atoms were
given anisotropic thermal parameters; hydrogen atoms were located
from difference Fourier maps and refined at idealized positions riding
on their parent atoms. The refinement converged at R1(I > 2σ(I)) =
0.0568, wR2 = 0.1510 for 2552 independent reflections and 182
variables. CCDC-838718 (13) contains the supplementary crystallo-
graphic data for this paper. These data can be obtained free of charge
from the Cambridge Crystallographic Data Centre via www.ccdc.cam.
ac.uk/data_request/cif.
Esterification of Cytospolide H (11) with (R)-MTPA
Chloride. Compound 11 (0.56 mg) was treated with (R)-MTPA
chloride according to the above procedure to afford the (S)-MTPA
1
ester of 11 (0.81 mg, 55%): H NMR (600 MHz, CDCl3): δ = 2.75
(m, 1H, H-2), 5.59 (brs, 1H, H-3), 5.63 (d, J = 16.3 Hz, 1H, H-4),
5.36 (m, 1H, H-5), 1.90 (m, 1H, H-6α), 2.25 (m, 1H, H-6β), 1.85 (m,
1H, H-7α), 1.42 (m, 1H, H-7β), 1.45 (m, 1H, H-8α), 1.72 (m, 1H, H-
8β), 4.66 (m, 1H, H-9), 1.52 (m, 1H, H-10a), 1.52 (m, 1H, H-10b),
1.40 (m, 1H, H-11a), 1.40 (m, 1H, H-11b), 1.67 (m, 1H, H-12a), 1.67
(m, 1H, H-12b), 5.11 (m, 1H, H-13), 1.24 (t, J = 6.2, 3H, H-14), 1.08
(d, J = 6.8, 3H, H-15).
Esterification of Cytospolide H (11) with (S)-MTPA
Chloride. The same reaction of 11 (0.45 mg) with (S)-MTPA
chloride afforded the (R)-MTPA ester of 11 (0.73 mg, 62%):
X-ray Crystallographic Studies of Cytospolide P (16). A
colorless needle crystal of 16 (0.10 × 0.10 × 0.15 mm) was obtained
9708
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The Journal of Organic Chemistry
Article
1H NMR (600 MHz, CDCl3) δ = 2.74 (m, 1H, H-2), 5.54 (brs, 1H, H-3),
5.58 (d, J = 16.3 Hz, 1H, H-4), 5.13 (m, 1H, H-5), 1.82 (m, 1H, H-
6α), 2.14 (m, 1H, H-6β), 1.72 (m, 1H, H-7α), 1.40 (m, 1H, H-7β),
1.35 (m, 1H, H-8α), 1.64 (m, 1H, H-8β), 4.56 (m, 1H, H-9), 1.47 (m,
1H, H-10a), 1.47 (m, 1H, H-10b), 1.22 (m, 1H, H-11a), 1.22 (m, 1H,
H-11b), 1.61 (m, 1H, H-12a), 1.61 (m, 1H, H-12b), 5.12 (m, 1H, H-
13), 1.31 (t, J = 6.2, 3H, H-14), 1.17 (d, J = 6.8, 3H, H-15).
Esterification of Cytospolide R (18) with (R)-MTPA Chlo-
ride. Compound 18 (0.38 mg) was treated with (R)-MTPA chloride
according to the above procedure to afford the (S)-MTPA ester of 18
(0.54 mg, 75%): 1H NMR (600 MHz, CDCl3) δ = 1.04 (t, J = 7.3, 3H,
H-1), 2.47 (m, 1H, H-2a), 2.47 (m, 1H, H-2b), 2.68 (dd, J = 15.2, 7.7
Hz, 1H, H-4a), 2.40 (dd, J = 15.2, 74.9 Hz, 1H, H-4b), 3.77 (m, 1H,
H-5), 1.41 (m, 1H, H-6α), 1.78 (m, 1H, H-6β), 2.23 (m, 1H, H-7α),
1.48 (m, 1H, H-7β), 4.65 (dt, J = 10.2, 2.4 Hz, 1H, H-8), 3.30 (t, J =
9.1 Hz, 1H, H-9), 1.50 (m, 1H, H-10a), 1.32 (m, 1H, H-10b), 1.16 (m,
1H, H-11a), 1.16 (m, 1H, H-11b), 1.22 (m, 1H, H-12a), 1.22 (m, 1H,
H-12b), 1.23 (m, 1H, H-13a), 1.23 (m, 1H, H-13b), 0.86 (t, J = 7.0,
3H, H-14).
Esterification of Cytospolide R (18) with (S)-MTPA
Chloride. The same reaction of 18 (0.40 mg) with (S)-MTPA
chloride afforded the (R)-MTPA ester of 18 (0.49 mg, 65%): 1H
NMR (600 MHz, CDCl3) δ = 1.04 (t, J = 7.3, 3H, H-1), 2.47 (m, 1H,
H-2a), 2.47 (m, 1H, H-2b), 2.68 (dd, J = 15.2, 7.7 Hz, 1H, H-4a), 2.40
(dd, J = 15.2, 74.9 Hz, 1H, H-4b), 3.78 (m, 1H, H-5), 1.46 (m, 1H,
H-6α), 1.82 (m, 1H, H-6β), 2.28 (m, 1H, H-7α), 1.63 (m, 1H, H-7β),
4.68 (dt, J = 10.2, 2.4 Hz, 1H, H-8), 3.26 (dt, J = 9.1, 1.9 Hz, 1H, H-9),
1.30 (m, 1H, H-10a), 1.18 (m, 1H, H-10b), 1.10 (m, 1H, H-11a), 1.10
(m, 1H, H-11b), 1.20 (m, 1H, H-12a), 1.20 (m, 1H, H-12b), 1.18 (m,
1H, H-13a), 1.18 (m, 1H, H-13b), 0.83 (t, J = 7.0, 3H, H-14).
Acetylation of Cytospolide E (5). Treatment of 5 (0.7 mg) with
Ac2O, using the above procedure, afforded an acetate in quantitative
yield, identical with the natural product 2.
(t, C-11), 35.6 (t, C-12), 70.7 (t, C-13), 19.9 (q, C-14), 12.2 (q,
C-15), 170.4 (s, 3-OAc), 20.9 (q, 3-OAc), 170.8 (s, 13-OAc), 21.4 (q,
13-OAc).
Synthesis of 21 by acetylation of cytospolide L (12). The
same reaction was performed with 12 (0.6 mg) to afford an identical
sample of 21 as a colorless oil in quantitative yield.
Acetylation of cytospolide R (18). Treatment of 18 (0.4 mg)
with Ac2O, using the above procedure, afforded an acetate in
quantitative yield, identical with the natural product 19.
Cytotoxicity assay. The cytotoxic activity of tested compounds
against human lung adenocarcinoma (A549), human colon cancer cells
(HCT116), human hepatocarcinoma cells (QGY), human malignant
melanoma cells (A375), and human leukemic cells (U937) was
assayed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide] colorimetric method.55 Adriamycin was used as
standard compound.
Cell cycle test. A549 cells were treated with compounds 5 and 16
at 1 μg/mL for 48 h, and QGY cells were treated with compound 16 at
10 μg/mL for 48 h, respectively. Then the cells were trypsinized,
washed with PBS twice, and then fixed with 70% ethanol on ice
overnight. The fixed cells were spun down, resuspended in PBS at 1 ×
106 cells/mL and incubated with 50 μg/mL propidium idide (PI) and
100 μg/mL ribonuclease A (RNase A) at 4 °C for 30 min, before
measured by flow cytometry. All experiments were performed in
triplicate. The data were expressed as mean ± SD. The significance was
expressed by using one-way analyses of variance (ANOVAs) of the SPSS
13.0 for Windows (SPSS Inc., Chicago, IL, USA), followed by Duncan
post hoc tests. P values less than 0.05 were considered significant.
Computational section. Geometry optimizations [B3LYP/
6-31G(d) level of theory] and TDDFT calculations were performed
with Gaussian 0356 using various functionals (B3LYP, BH&HLYP,
PBE0) and TZVP basis set. CD spectra were generated as the sum of
Gaussians57 with 3000 cm−1 half-height width (corresponding to 12
at 200 nm), using dipole-velocity computed rotational strengths.
Conformational searches were carried out by means of the Macro-
model 9.7.21158 software using Merck Molecular Force Field (MMFF)
with implicit solvent model for chloroform. Boltzmann distributions
were estimated from the ZPVE corrected B3LYP/6-31G(d) energies.
The MOLEKEL59 software package was used for visualization of the
results.
Synthesis of 20 by Acetylation of Cytospolide F (6). To a
solution of 6 (0.5 mg) in dry pyridine (0.5 mL) was added two drops
of Ac2O. The mixture was kept at room temperature for 16 h to afford,
after usual workup, 20 quantitatively as colorless oil: [α]20 = −84.7
D
(c 0.04, CHCl3); 1H NMR (600 MHz, CDCl3) δ = 2.75 (m, 1H, H-2),
5.35 (brs, 1H, H-3), 5.64 (dd, J = 15.6, 2.4 Hz, 1H, H-4), 5.52 (ddd,
J = 15.6, 6.0, 4.8 Hz, 1H, H-5), 2.20 (m, 1H, H-6α), 2.25 (m, 1H,
H-6β), 1.88 (m, 1H, H-7α), 1.83 (m, 1H, H-7β), 4.73 (q, J = 7.2, 1H,
H-8), 4.92 (dt, J = 7.2, 3.6, 1H, H-9), 1.52 (m, 2H, H-10a, H-10b),
1.31 (m, 2H, H-11a, H-11b), 1.28 (m, 2H, H-12a, H-12b), 4.85 (m,
1H, H-13), 1.18 (t, J = 6.8, 3H, H-14), 1.18 (t, J = 6.8, 3H, H-15), 2.14
(s, 3H, 3-OAc), 2.07 (s, 3H, 8-OAc), 2.02 (s, 3H, 13-OAc); 13C NMR
(150 MHz, CDCl3) δ = 171.7 (s, C-1), 45.0 (d, C-2), 72.6 (d, C-3),
129.1 (d, C-4), 129.1 (d, C-5), 29.3 (t, C-6), 35.5 (t, C-7), 75.1 (d,
C-8), 75.6 (d, C-9), 31.8 (t, C-10), 20.4 (t, C-11), 35.6 (t, C-12), 70.6
(t, C-13), 19.9 (q, C-14), 12.3 (q, C-15), 170.2 (s, 3-OAc), 20.9 (q,
3-OAc), 169.8 (s, 8-OAc), 21.1 (q, 8-OAc), 170.7 (s, 13-OAc), 21.3
(q, 13-OAc).
ASSOCIATED CONTENT
■
S
* Supporting Information
MS, one- and two-dimensional NMR spectra for compounds
6−19, X-ray data for crystals 13, 16, and 17 (CIF), and atom
coordinates and absolute energies of the computed structures.
These material are available free of charge via the Internet at
http://pubs.acs.org
AUTHOR INFORMATION
■
Synthesis of 20 by acetylation of cytospolide G (7). The same
reaction was performed with 7 (0.7 mg) to afford an identical sample
of 20 as a colorless oil in quantitative yield.
Synthesis of 20 by acetylation of cytospolide H (8). The same
reaction was performed with 8 (0.4 mg) to afford an identical sample
of 20 as a colorless oil in quantitative yield.
Synthesis of 21 by acetylation of cytospolide K (11). Treatment
of 11 (0.7 mg) with Ac2O according to the above procedure, afforded
21 quantitatively as a colorless oil. [α]20 D= −53.4 (c 0.02, CHCl3).
1H NMR (600 MHz, CDCl3): δ = 2.73 (m, 1H, H-2), 5.37 (brs, 1H,
H-3), 5.62 (d, J = 15.6 Hz, 1H, H-4), 5.49 (m, 1H, H-5), 2.206 (m,
1H, H-6α), 2.28 (m, 1H, H-6β), 1.88 (m, 1H, H-7α), 1.45 (m, 1H,
H-7β), 1.50 (m, 1H, H-8α), 1.77 (m, 1H, H-8β), 4.70 (m, 1H, H-9),
1.55 (m, 1H, H-10a), 1.45 (m, 1H, H-10b), 1.30 (m, 2H, H-11a,
H-11b), 1.50 (m, 2H, H-12a, H-12b), 4.87 (m, 1H, H-13), 1.19 (t, J =
6.6, 3H, H-14), 1.18 (t, J = 6.6, 3H, H-15), 2.14 (s, 3H, 3-OAc), 2.02
(s, 3H, 13-OAc); 13C NMR (150 MHz, CDCl3): δ = 174.4 (s,
C-1), 45.4 (d, C-2), 73.3 (d, C-3), 129.1 (d, C-4), 129.2 (d, C-5), 32.7
(t, C-6), 28.2 (t, C-7), 33.1 (t, C-8), 75.9 (d, C-9), 35.7 (t, C-10), 21.0
Corresponding Author
*E-mail: (L.L.) lilingty@126.com; (W.Z.) wenzhang1968@
163.com.
ACKNOWLEDGMENTS
■
This research was financially supported by the Natural Science
Foundation of China (Nos. 30873200, 41076082, 81172979),
the Shanghai Pujiang Program (PJ2008), the Scientific &
Technological Major Project (2009ZX09301-011). S.A. and
T.K. thank the Hungarian Scientific Research Fund (OTKA,
́
K-81701), TAMOP 4.2.1./B-09/1/KONV-2010-0007, and
National Information Infrastructure Development Institute (NIIFI
10038). We are grateful to Prof. L. Ernst and Mrs. P. H. Schulz
(Technische Universitat Braunschweig, Germany) and Prof.
̈
E. Hans (University of Paderborn, Germany) for the assistance
with the NOEDIFF experiments. We are also indebted to
9709
dx.doi.org/10.1021/jo201755v|J. Org. Chem. 2011, 76, 9699−9710

The Journal of Organic Chemistry
Article
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