Novel Open-Chain Cytochalsins from the Marine-Derived Fungus Spicaria elegans

Article abstract of DOI:10.1021/np070539b

Six novel open-chain cytochalasins (1-6) and one known [12]-cytochalasin (7) have been isolated from the fermentation broth of a marine-derived fungus, Spicaria elegans. Cytochalasins Z₁₀-Z₁₅(1-6) are the first reported cytochalasins that contain an open chain to date. The structures of these new compounds were elucidated by spectroscopic methods. The cytotoxic effects on P388, A-549, HL-60, and BEL-7402 cell lines of all compounds were evaluated by the MTT method.

Full text of DOI:10.1021/np070539b

J. Nat. Prod. 2008, 71, 1127–1132
1127
Novel Open-Chain Cytochalsins from the Marine-Derived Fungus Spicaria elegans
Rui Liu,§ Zhenjian Lin,§ Tianjiao Zhu, Yuchun Fang, Qianqun Gu,* and Weiming Zhu*
Key Laboratory of Marine Drugs, Chinese Ministry of Education, Institute of Marine Drugs and Food, Ocean UniVersity of China,
Qingdao 266003, People’s Republic of China
ReceiVed September 30, 2007
Six novel open-chain cytochalasins (1-6) and one known [12]-cytochalasin (7) have been isolated from the fermentation
broth of a marine-derived fungus, Spicaria elegans. Cytochalasins Z10-Z15 (1-6) are the first reported cytochalasins
that contain an open chain to date. The structures of these new compounds were elucidated by spectroscopic methods.
The cytotoxic effects on P388, A-549, HL-60, and BEL-7402 cell lines of all compounds were evaluated by the MTT
method.
In our effort to search for anticancer compounds,1–3 a fungal
strain identified as Spicaria elegans exhibited cytotoxic activity.
We have previously reported on the isolation and structural
elucidation of three [12]-cytochalasins, Z7, Z8, and Z9, and two [13]-
cytochalasins, E and K,4 from S. elegans. Our ongoing investiga-
tions of the mass reculture of this species in the same media have
led to the isolation of a further six new cytochalasins, Z10-Z15
(1-6), and one known [12]-cytochalasin (7). Compounds 1-6
(cytochalasins Z10-Z15) are the first ones in a new class of
cytochalasins that possess a highly substituted perhydroisoindol-
1-one core usually fused with an 11-,5–8 13-,9 12-,4,10 or 14-
membered11 macrocyclic ring. These compounds display a wide
range of biological activities9,12,13 and, thus, have great potential
in cell biology and medicine. In addition to a highly substituted
perhydroisoindol-1-one skeleton, the novel cytochalasins (1-6)
were found to have an open 8-carbon chain system rather than a
macrocyclic ring, which was strikingly different from previously
reported cytochalasins. Therefore, precise information on the
structure and conformation of these novel cytochalasins was
important in order to understand their chemical and biological
activities. In this paper, we report the isolation, structural elucida-
tion, and cytotoxic activities of six novel open-chain cytochalasins,
Z10-Z15 (1-6).
(167.7, qC)] that is present in the 13C NMR spectrum of cytochalasin
Z7 was missing, and chemical shifts of C-9 (δC 79.4 qC) and C-18
(δC 68.8 CH) in 1 were upfield and downfield, respectively, which
suggested that two hydroxyl groups were located on C-9 and C-18
and the 12-membered macrocyclic ring in cytochalasin Z7 was
changed to an open chain in 1, which was consistent with the
molecular formula. The positions of the substituents in the open
chain were confirmed as occurring at C-16 (methyl) and C-17 and
C-18 (two hydroxyls) using the HMBC spectrum, which showed
correlations from CH3-19 (1.19 ppm, d) to the two oxygenated
methine sp3 carbons C-17 (78.4 ppm, CH) and C-18 (68.8 ppm,
CH) and correlations from CH3-20 (0.86 ppm, d) to C-15 (38.5
ppm, CH2), C-16 (35.1 ppm, CH), and C-17. Therefore the structure
of the new compound 1 was elucidated.
Cytochalasin Z11 (2) was an amorphous solid like 1. Its molecular
formula was established as C25H33NO5 by the [M + H]+ ion peak
observed at m/z 428.2434 (calcd for [M + H]+ 428.2437) in its
HRESIMS. Analysis of the 1D NMR spectra of 2 indicated that it
had a structure very similar to 1. The only difference was that the
13C NMR spectrum (Table 1) of 2 contained one more carbonyl
carbon at 216.8 ppm (C-17, qC) in place of an oxygenated carbon
at 78.4 ppm (C-17, CH) in 1. This change could be further
confirmed by the HMBC correlations from CH3-19 (1.21 ppm, d)
and CH3-20 (1.01 ppm, d) to the carbonyl carbon at 216.8 ppm
(C-17, qC). Thus the structure of 2 was established.
Results and Discussion
The bioactive EtOAc extract of S. elegans was chromatographed
over a Si gel column and by preparative HPLC to give six new
cytochalasins (1-6) and [12]-cytochalasin (7).10 Their structures
were established by detailed analysis of NMR spectra, which
included 1H-1H COSY, HMQC, HMBC, and NOESY experiments,
and also by comparison with the NMR data for the known
cytochalasins Z7, Z8, Z9, E, and K we had reported previously.4
Cytochalasin Z10 (1) was a white, amorphous solid. Its HRESIMS
gave a [M + H]+ ion peak at m/z 430.2596 (calcd for [M + H]+
430.2593), in agreement with the molecular formula C25H35NO5,
36 mass units smaller than that of cytochalasin Z7.4 The IR spectrum
showed the presence of hydroxyl and carbonyl groups. Analysis
of the 1D NMR data for 1 revealed one carbonyl, three quaternary
carbons, 15 methines, three methylenes, and three methyls.
Comparison of the 1H and 13C NMR data of 1 with those of
cytochalasin Z7 showed the presence of the same 10-phenyl-
substituted perhydroisoindol-1-one skeleton. Compound 1 differed
from cytochalasin Z7 only in the macrocyclic ring, where the R,ꢀ-
unsaturated ester system [C-19 (157.7, CH), C-20 (123.3, CH), C-21
Another metabolite isolated from the same extract is cytochalasin
Z12 (3), obtained as a yellow, amorphous solid. The molecular
formula of 3 was also C28H35NO5, as determined by HRESIMS
([M + Na]+ at m/z 452.2411, calcd for 452.2413). This compound
was isomeric with 1. Comparison of their 1H and 13C NMR spectra
suggested the two exocyclic double-bond resonances at 150.9 ppm
(C-6, qC) and 112.8 ppm (C-12, CH2) in 1 had been replaced by
two carbon resonances at 125.0 ppm (C-5, qC) and 129.6 ppm (C-
6, qC) attributable to a tetrasubstituted double bond, which were
also similar to those observed in the 13C spectrum of cytochalasin
1
K. Furthermore, in the H NMR spectrum of 3, the proton signal
at 1.83 ppm due to the C-11 methyl appeared as a singlet, not a
doublet. Therefore, as an isomer of 1, compound 3 was only
different in having a tetrasubstituted bond of the perhydroisoindol-
1-one core in 1. This change was also confirmed by 2D NMR
correlations (1H-1H COSY correlation of H-8 to H-7).
Cytochalasin Z13 (4), a colorless oil, was isomeric with 2, with
the same molecular formula C25H33NO5. HRESIMS showed a [M
+ H]+ ion peak at m/z 428.2399 (calcd for 428.2437). Comparison
of the 1D NMR spectra of compounds 4 and 2 showed the only
difference was also that the exocyclic double bond in 2 [148.1 ppm
(C-6, qC,), 112.3 ppm (C-12, CH2)] was replaced by a tetrasub-
stituted double bond in 4 [124.9 ppm (C-5, qC), 129.4 ppm (C-6,
* To whom correspondence should be addressed. Tel: 0086-532-
82032065. Fax: 0086-532-82033054. E-mail: guqianq@ouc.edu.cn and
weimingzhu@ouc.edu.cn.
§ These authors made equal contributions to this paper.
10.1021/np070539b CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 05/29/2008

1128 Journal of Natural Products, 2008, Vol. 71, No. 7
Liu et al.
qC)]. Further analysis of the 2D NMR (COSY, HMQC, and
HMBC) experiments confirmed the structure as 4.
Cytochalasin Z14 (5) was a yellow oil. Its HRESIMS (m/z
410.2337 [M - H2O+ H]+ (calcd for C25H32NO4, 410.2331)
suggested that it was an isomer of compounds 2 and 4, with a
molecular formula of C25H33NO5. Comparison of 5 and 4 showed
5 contained the same tetrasubstituted double bond at 124.9 ppm
(C-5, qC) and 129.9 ppm (C-6, qC). The only difference between
the two compounds was the exchange of the position of the carbonyl
and the hydroxyl carbons. This change could be explained by the
downfield shift of CH3-19 in 5 at δH 2.06 ppm, δC 25.1 ppm,
compared to CH3-19 in 4 at δH 1.20 ppm, δC 18.9 ppm. This
structure was also in agreement with the observed significant HMBC
correlations from CH3-20 (0.60 ppm, d) to C-15 (37.3 ppm, CH2),
C-16 (35.5 ppm, CH), and C-17 (77.8 ppm, CH). Thus the structure
of 5 was established.
Cytochalasin Z15 (6) was a yellow oil. Its HRESIMS ([M + H]+
m/z 428.2451, calcd for C25H34NO5 428.2437) suggested that it was
an isomer of compounds 2, 4, and 5, also with a molecular formula
of C25H33NO5. Comparison of 6 and 5, combined with the HMBC
correlations from CH3-20 (0.93 ppm, s) to C-15 (33.6 ppm, CH2),
C-16 (35.8 ppm, CH), and C-17 (80.8 ppm, CH), showed these
two compounds had the same planar structure. The only difference
of 6 from 5 was the downshift of C-17 (δH 3.99 ppm, δC 80.8 ppm
compared to δH 3.97 ppm, δC 77.8 ppm in 5) and the downshift of
CH3-20 in 6 (δH 0.93 ppm, δC 17.1 ppm compared to δH 0.60 ppm,
δC 12.7 ppm in 5), suggesting that the configuration of C-17 and
CH3-20 in 6 was different from that in 5.
Previous studies have suggested that the essential elements of
most cytochalasins’ skeleton have the same stereochemistry, viz.,
cis-stereochemistry across the 5/6 ring junction and the trans-
stereochemistry of the macrocyclic ring.8 A series of NOESY and
NOE experiments on these new cytochalasins (1-6) suggested the
same relative stereochemistry of the 10-phenyl-substituted perhy-
droisoindol-1-one skeleton, as correlations were observed between
protons H-3/CH3-11, H-4/H-8 and 2H-10, H-5/H-8, and H-8/H-
14. Correlations between CH3-11/H-12E and H-12Z/H-7 were also
observed in compounds 1 and 2. The relative configurations of the
three consecutive stereogenic centers (C-16, C-17, and C-18) in
the open 8-carbon chain in 1 were first proposed on the basis of
the comparison of the 13C NMR chemical shifts of 1 with the
published values for synthetic diastereomers (a-d) sharing common
partial structures from C-16 to C-20 with C-17 substitution (Figure
3).14 Detailed analysis of 13C NMR data of a-d indicated that the
chemical shifts of C-19 and C-20 methyl groups were clearly
dependent on the relative configurations of the methyl-hydroxy-
hydroxy-substituted stereogenic centers of these compounds. The
similarities between the chemical shifts of C-19 and C-20 in
compounds 1 and 3 (Table 2) and those in compound c suggested
that C-19 and C-20 in 1 and 3 had the same relative configuration
as that of c. Thus, the 8-carbon chain’s relative configurations of 1
and 3 were assigned as 16S*, 17R*, 18R*.
Comparison of the 1D NMR spectra (Tables 1 and 2) of 2 with
those of 4 showed almost identical 13C NMR chemical shifts for
the 8-carbon chains in the two compounds. As a result, the relative
configurations of the side chain in 2 should be the same as those
in 4. Comparison of the 1D NMR spectra (Tables 1 and 2) of 5
with those of 6 found that the differences were at C-17 and C-20
(3.97, H-17, 77.8, CH, C-17; 0.60, CH3-20, 12.7, qC, C-20 in 5;
3.98, H-17, 80.8, CH, C-17; 0.93, CH3-20, 17.1, qC, C-20 in 6).
These data implied that the relative orientations of the methyl at
C-16 and the hydroxyl group at C-17 in compounds 5 and 6 were
different (Figure 1) in these two compounds.
The absolute configuration of 1 was established by a convenient
Mosher ester method using the (S)- and (R)-MTPA esters. Tri-
Mosher ester derivatives (1a and 1b) of 1 were prepared (Figure

Open-Chain Cytochalsins from Spicaria elegans
Journal of Natural Products, 2008, Vol. 71, No. 7 1129
Figure 1. Structures of compounds 1-7.
Figure 2. ∆δ value [∆δ (in ppm) ) δS - δR] obtained for (S)- and (R)-MTPA esters of compounds 1, 4, and 6.
2), and the positive value of δS-R for H-11 and H-12 and negative
value of δS-R for H-14 and H-1515established the 7S configuration,
which was consistent with that in cytochalasins Z7-Z9.4 It had been
confirmed that the modified Mosher’s method was useful for acyclic
1,2-glycols possessing simple alkyl groups,16a-c and most recently
Riguera’s group16d has predicted four general patterns for 1,2-diols.
The absolute configuration of the vicinal carbinol carbons in 1 was
uration in 4, and the 17R configuration in 6 were also established
by the Mosher ester method (Figure 2).
Finally, it is noteworthy that in all cytochalasins isolated thus
far, the S configuration of C-16 in the macrocyclic ring moieties is
maintained throughout the series. Open-chain cytochalsins are
presumably biosynthesized from those with a 12-membered mac-
rocyclic ring. Thus, we tentatively concluded that all the open-
chain cytochalasins have an S configuration at C-16. This is also
consistent with the 16S configuration deduced above in compound
1. Therefore, the absolute configurations of all six new compounds
have been assigned as shown in Figure 1.
1
determined by the analysis of the H NMR spectrum of the di-
MTPA ester of 1 in accordance with method of Riguera. The
negative values of δS-R for H-17 and H-18 indicated 17R, 18R
configurations in 1.16 Taken together, these data established the
d
absolute configurations of compound 1 as 7S, 16S, 17R, 18R.
Similarly, the 7S configurations in both 4 and 6, the 18R config-
Compounds 1-7 were evaluated for their cytotoxicities against
the P388, A-549, HL-60, and BEL-7402 cell lines by the MTT

1130 Journal of Natural Products, 2008, Vol. 71, No. 7
Liu et al.
transferred into a clean reaction bottle and dried completely under
vacuum. Deuterated pyridine (0.5 mL) and (S)-(+)-R-methoxy-R-
(trifluoromethyl)phenylacetyl chloride (1 equiv) were added into the
reaction bottle quickly under a N2 gas stream and then stirred for 24 h
at room temperature. The organic layer was then washed with water,
HCl (1 M), water, NaHCO3 (sat), and water, dried (Na2SO4), and
concentrated under reduced pressure to obtain the ester. Final purifica-
tion was achieved by HPLC (90% MeOH, 4.0 mL/min): 1a and 1b, tR
) 11.7 min; 4a and 4b, tR ) 6.9 min; 4c and 4d, tR ) 11.4 min; 6a
and 6b, tR ) 6.0 min. 1H NMR data of the (S)-MTPA ester derivative
(1a) of 1 (600 MHz, CDCl3): δ 5.63 (1H, brs, 2-NH), 3.33 (1H, m,
H-3), 2.39 (1H, dd, J ) 3.6, 5.4 Hz, H-4), 3.04 (1H, m, H-5), 5.47
(1H, d, J ) 8.8 Hz, H-7), 2.90 (1H, dd, J ) 8.8, 9.2 Hz, H-8), 2.93
(1H, dd, J ) 4.7, 13.6 Hz, H-10a), 2.74 (1H, dd, J ) 8.8, 13.6 Hz,
H-10b), 1.05 (3H, d, J ) 6.6 Hz, H-11), 5.38 (1H, brs, H-12a), 5.17
(1H, brs, H-12b), 5.73 (1H, dd, J ) 9.9, 5.4 Hz, H-13), 5.36 (1H, ddd,
J ) 7.0, 7.3, 15.0 Hz, H-14), 1.84 (1H, m, H-15a), 1.74 (1H, m, H-15b),
1.73 (1H, m, H-16), 5.15 (1H, dd, J ) 4.4, 4.4 Hz, H-17), 5.25 (1H,
m, H-18), 1.11 (3H, d, J ) 6.2 Hz, H-19), 0.79 (3H, d, J ) 6.2 Hz,
Figure 3. Comparison of 13C NMR chemical shifts (in CD3OD-
d4) of compound 1 with four synthetic model compounds (a-d) to
establish the relative configurations at C-16, C-17, and C-18 in 1.
1
H-20), 7.12-7.53 (20H, m, Ph-H). H NMR data of the (R)-MTPA
ester derivative (1b) of 1 (600 MHz, CDCl3): δ 5.61 (1H, brs, 2-NH),
3.31 (1H, m, H-3), 2.34 (1H, dd, J ) 4.4, 4.4 Hz, H-4), 2.87 (1H, m,
H-5), 5.46 (1H, d, J ) 7.2 Hz, H-7), 2.89 dd, J ) 8.8, 9.2 Hz), 2.94
(1H, dd, J ) 4.7, 13.6 Hz, H-10a), 2.70 (1H, dd, J ) 8.8, 13.6 Hz,
H-10b), 0.99 (3H, d, J ) 6.6 Hz, H-11), 5.32 (1H, brs, H-12a), 5.11
(1H, brs, H-12b), 5.72 (1H, dd, J ) 9.9, 15.4 Hz, H-13), 5.39 (1H,
ddd, J ) 7.0, 7.3, 15.0 Hz, H-14), 1.91 (1H, m, H-15a), 1.78 (1H, m,
H-15b), 1.60 (1H, m, H-16), 5.15 (1H, dd, J ) 4.4, 4.4 Hz, H-17),
5.29 (1H, m, H-18), 1.27 (3H, d, J ) 6.2 Hz, H-19), 0.70 (3H, d, J )
method.17 Compounds 1 and 2 showed moderate cytotoxicities
against the A-549 cell line with IC50 values of 9.6 and 4.3 µM,
respectively (Table 3).
Experimental Section
General Experimental Procedures. Optical rotations were obtained
on a JASCO P-1020 digital polarimeter. UV spectra were recorded on
a Beckman DU 640 spectrophotometer. IR spectra were taken on a
Nicolet Nexus 470 spectrophotometer in KBr disks. 1H, 13C NMR and
DEPT spectra and 2D NMR were recorded on a JEOL JNM-ECP 600
spectrometer using TMS as internal standard, and chemical shifts were
recorded as δ values. NOESY experiments were carried out using a
mixing time of 0.5 s. 1D NOE spectra were obtained on a Varian
INOVA-400 spectrometer. ESIMS was measured on a Q-TOF Ultima
Global GAA076 LC mass spectrometer. Semiprepartive HPLC was
performed using an ODS column [Shin-pak ODS (H), 10 × 250 mm,
5 µm, 4 mL/min].
Fungal Material. The fungus Spicaria elegans was isolated from
the marine sediments collected in Jiaozhou Bay, China. It was preserved
in the China Center for Type Culture Collection (patent depository
number: KLA03 CCTCC M 205049). Working stocks were prepared
on potato dextrose agar slants stored at 4 °C.
Fermentation and Extraction. The fungus was grown under static
conditions at 24 °C for 25 days in 80 × 1000 mL conical flasks
containing liquid medium (300 mL/flask) composed of glucose (20 g/L),
peptone (5 g/L), malt extract (3 g/L), yeast extract (3 g/L), and seawater
after adjusting its pH to 7.0. The fermented whole broth (24 L) was
filtered through cheesecloth to separate it into supernatant and mycelia.
The former was concentrated under reduced pressure to about a quarter
of the original volume and then extracted three times with EtOAc to
give an EtOAc solution, while the latter was extracted three times with
acetone. The acetone solution was concentrated under reduced pressure
to afford an aqueous solution. The aqueous solution was extracted three
times with EtOAc to give another EtOAc solution. Both EtOAc
solutions were combined and concentrated under reduced pressure to
give a crude extract (23.0 g).
Purification. The crude extract (23.0 g) was separated into 15
fractions on a silica gel column using gradient elution of petroleum
ether/acetone and TLC monitoring (on silica gel plates with CHCl3/
MeOH, 95:5, as eluent). Fraction 12, eluted with petroleum ether/
acetone, 5:5 (2.7 g), was purified into eight subfractions by another
silica gel column using stable elution of chloroform/MeOH, 9:1.
Subfraction 12-1 was further purified by extensive HPLC (60% MeOH,
4.0 mL/min), giving compound 1 (20 mg, tR 16 min). Subfraction 12-3
was further purified by extensive HPLC (60% MeOH, 4.0 mL/min) to
yield compounds 7 (24 mg, tR 22min) and 4 (30 mg, tR 30 min).
Subfraction 12-4 was further purified by HPLC (60% MeOH, 4.0 mL/
min) to yield compounds 2 (40 mg, tR 11 min), 3 (8 mg, tR 15 min),
and 5 (3 mg, tR 25 min). Subfraction 12-5 was further purified by HPLC
(60% MeOH, 4.0 mL/min) to yield compound 6 (25 mg, tR 24 min).
Preparation of the (S)- and (R)-MTPA Ester Derivatives of 1, 4,
and 6 and Determination of the Absolute Stereochemistry. Com-
pounds 1 (5.0 mg), 4 (1.0 mg), and 6 (1.0 mg) were separately
1
6.2 Hz, H-20), 7.16-7.54 (20H, m, Ph-H). H NMR data of the (S)-
MTPA ester derivative (4a) of 4 (600 MHz, CDCl3): δ 5.69 (1H, brs,
2-NH), 3.43 (1H, m, H-3), 2.64 (1H, brs, H-4), 5.55 (1H, d, J ) 7.3
Hz, H-7), 2.67 (1H, dd, J ) 7.0, 9.5 Hz, H-8), 3.18 (1H, dd, J ) 5.5,
13.6 Hz, H-10a), 2.87 (1H, dd, J ) 8.8, 13.6 Hz, H-10b), 1.61 (3H, s,
H-11), 1.66 (3H, s, H-12), 5.69 (1H, dd, J ) 9.9, 15.4 Hz, H-13), 5.54
(1H, ddd, J ) 7.0, 7.3, 15.0 Hz, H-14), 2.37 (1H, m, H-15a), 2.05
(1H, m, H-15b), 2.71 (1H, m, H-16), 5.25 (1H, q, J ) 7.0 Hz, H-18),
1.41 (3H, d, J ) 7.3 Hz, H-19), 1.06 (3H, d, J ) 6.6 Hz, H-20),
7.18-7.64 (15H, m, Ph-H). 1H NMR data of the (R)-MTPA ester
derivative (4b) of 4 (600 MHz, CDCl3): δ 5.82 (1H, brs, 2-NH), 3.44
(1H, m, H-3), 2.65 (1H, brs, H-4), 5.56 (1H, d, J ) 7.3 Hz, H-7), 2.73
(1H, m, H-8), 3.16 (1H, dd, J ) 5.9, 13.2 Hz, H-10a), 2.92 (1H, dd, J
) 8.5, 13.2 Hz, H-10b), 1.41 (1H, s, H-11), 1.58 (1H, s, H-12), 5.79
(1H, dd, J ) 9.9, 15.4 Hz, H-13), 5.52 (1H, ddd, J ) 7.0, 7.3, 15.0
Hz, H-14), 2.33 (1H, m, H-15a), 2.13 (1H, m, H-15b), 2.72 (1H, m,
H-16), 5.34 (1H, q, J ) 7.3 Hz, H-18), 1.49 (3H, d, J ) 7.0 Hz, H-19),
1.03 (3H, d, J ) 6.9 Hz, H-20), 7.18-7.60 (15H, m, Ph-H). 1H NMR
data of the (S)-MTPA ester derivative (4c) of 4 (600 MHz, CDCl3): δ
5.74 (1H, brs, 2-NH), 3.41 (1H, m, H-3), 3.54 (1H, brs, H-4), 3.61
(1H, brs, H-7), 3.00 (1H, brd, J ) 9.1 Hz, H-8), 3.36 (1H, dd, J ) 2.5,
13.6 Hz, H-10a), 2.88 (1H, dd, J ) 10.6, 13.6 Hz, H-10b), 1.74 (3H,
s, H-11), 1.85 (3H, s, H-12), 5.07 (1H, dd, J ) 9.5, 15.0 Hz, H-13),
5.56 (1H, ddd, J ) 7.7, 7.3, 15.0 Hz, H-14), 2.35 (1H, m, H-15a),
1.93 (1H, m, H-15b), 2.61 (1H, m, H-16), 5.21 (1H, q, J ) 7.0 Hz,
H-18), 1.35 (3H, d, J ) 7.0 Hz, H-19), 0.94 (3H, d, J ) 7.0 Hz, H-20),
7.24-7.60 (15H, m, Ph-H). 1H NMR data of the (R)-MTPA ester
derivative (4d) of 4 (600 MHz, CDCl3): δ 5.73 (1H, brs, 2-NH), 3.44
(1H, m, H-3), 3.45 (1H, brs, H-4), 3.50 (1H, brs, H-7), 3.04 (1H, brd,
J ) 7.3 Hz, H-8), 3.32 (1H, dd, J ) 2.9, 13.6 Hz, H-10a), 2.81 (1H,
dd, J ) 10.3, 13.6 Hz, H-10b), 1.90 (3H, s, H-11), 1.90 (3H, s, H-12),
5.12 (1H, dd, J ) 9.9, 15.4 Hz, H-13), 5.49 (1H, ddd, J ) 7.0, 7.3,
15.0 Hz, H-14), 2.65 (1H, m, H-15a), 2.01 (1H, m, H-15b), 2.58 (1H,
m, H-16), 5.27 (1H, q, J ) 7.0 Hz, H-18), 1.41 (3H, d, J ) 7.0 Hz,
H-19), 0.88 (3H, d, J ) 7.0 Hz, H-20), 7.20-7.61 (15H, m, Ph-H). 1H
NMR data of the (S)-MTPA ester derivative (6a) of 6 (600 MHz,
CDCl3): δ 5.55 (1H, brs, 2-NH), 3.40 (1H, m, H-3), 2.64 (1H, brs,
H-4), 5.44 (1H, brs, H-7), 2.67 (1H, dd, J ) 5.5, 9.2 Hz, H-8), 3.24
(1H, dd, J ) 1.5, 13.6 Hz, H-10a), 2.77 (1H, dd, J ) 9.1, 13.6 Hz,
H-10b), 1.67 (3H, s, H-11), 1.75 (3H, s, H-12), 5.52 (1H, m, H-13),
5.44 (1H, m, H-14), 1.98 (1H, m, H-15a), 1.81 (1H, m, H-15b), 2.16
(1H, m, H-16), 5.04 (1H, d, J ) 3.6 Hz, H-17), 2.11 (3H, s, H-19),
0.89 (3H, d, J ) 7.0 Hz, H-20), 7.15-7.58 (15H, m, Ph-H). 1H NMR
data of the (R)-MTPA ester derivative (6b) of 6 (600 MHz, CDCl3): δ
5.57 (1H, brs, 2-NH), 3.41 (1H, m, H-3), 2.65 (1H, brs, H-4), 5.44
(1H, brs, H-7), 2.78 (1H, brd, J ) 5.3, 9.3 Hz, H-8), 3.22 (1H, dd, J

Open-Chain Cytochalsins from Spicaria elegans
Journal of Natural Products, 2008, Vol. 71, No. 7 1131
Table 2. 13C NMR Data for Compounds 1-6a
C
1
2
3 (ppm)
4
5
6
1
3
4
5
6
7
8
9
177.6 qC
54.3 CH
53.5 CH
31.0 CH
150.9 qC
74.4 CH
54.0 CH
79.4 qC
175.0 qC
52.9 CH
53.6 CH
27.8 CH
148.1 qC
76.1 CH
53.2 CH
79.4 qC
176.2 qC (177.9)
57.2 CH (58.8)
52.5 CH (53.8)
125.0 qC (127.1)
129.6 qC (131.4)
72.8 CH (73.5)
50.6 CH (52.7)
78.2 qC (78.8)
175.4 qC
57.1 CH
52.6 CH
124.9 qC
129.4 qC
73.0 CH
50.5 CH
78.6 qC
175.4 qC
56.9 CH
52.9 CH
124.9 qC
129.9 qC
73.0 CH
51.4 CH
78.2 qC
175.7 qC
57.3 CH
52.9 CH
124.8 qC
130.3 qC
72.4 CH
51.1 CH
77.7 qC
10
11
12
13
14
15
16
17
18
19
20
1′
44.1 CH2
15.1 CH3
112.8 CH2
128.6 CH
135.7 CH
38.5 CH2
35.1 CH
78.4 CH
68.8 CH
20.2 CH3
13.5 CH3
138.5 qC
130.7 CH
129.6 CH
127.8 CH
43.7 CH2
15.9 CH3
112.3 CH2
128.1 CH
132.1 CH
38.4 CH2
40.9 CH
216.8 qC
73.0 CH
18.8 CH3
17.6 CH3
137.3 qC
129.1 CH
128.8 CH
127.0 CH
43.6 CH2 (44.0)
18.4 CH3 (17.0)
17.5 CH3 (18.3)
126.5 CH (127.8)
134.0 CH (135.8)
36.7 CH2 (38.6)
34.0 CH (35.4)
76.2 CH (78.8)
68.1 CH (68.9)
17.1 CH3 (20.2)
14.6 CH3 (13.5)
137.0 qC (138.7)
128.8 CH (129.7)
129.0 CH (130.8)
126.9 CH (128.1)
44.3 CH2
18.7 CH3
18.0 CH3
127.8 CH
132.1 CH
38.6 CH2
40.8 CH
216.7 qC
73.2 CH
18.9 CH3
17.8 CH3
137.3 qC
129.3 CH
128.8 CH
127.0 CH
44.1 CH2
18.6 CH3
17.6 CH3
127.4 CH
134.1 CH
37.3 CH2
35.5 CH
77.8 CH
210.0 qC
25.1 CH3
12.7 CH3
137.0 qC
128.9 CH
128.9 CH
127.2 CH
43.9 CH2
18.3 CH3
17.3 CH3
127.2 CH
133.9 CH
33.6 CH2
35.8 CH
80.8 CH
210.1 qC
25.8 CH3
17.1 CH3
137.2 qC
129.0 CH
128.9 CH
127.0 CH
2′, 6′
3′, 5′
4′
a Spectra were recorded at 150 MHz for 13C using TMS as internal standard.
Biological Assays. Active fractions were assayed using the MTT
method15 with the mouse temperature-sensitive p34cdc2 mutant cell line
tsFT210. Cytotoxic activity was evaluated by the MTT method using
P388, A-549, HL60, and BEL-7402 cell lines. The cell lines were grown
in RPMI-1640 supplemented with 10% FBS under a humidified
atmosphere of 5% CO2 and 95% air at 37 °C (tsFT210 cell line at 32
°C). An aliquot (200 µL) of these cell suspensions at a density of 5 ×
104 cell mL-1 was plated in 96-well microtiter plates and incubated
for 24 h at the above conditions. Then 2 µL of the test compound
solutions (in DMSO) at different concentrations was added to each
well and further incubated for 72 h in the same conditions. MTT
solution (20 µL of 5 mg/mL in IPMI-1640 medium) was added to each
well and incubated for 4 h. Old medium containing MTT (150 µL)
was then gently replaced by DMSO and pipetted to dissolve any
formazan crystals that had formed. Absorbance was then determined
on a Spectra Max Plus plate reader at 570 nm.
Table 3. Cytotoxicities of Compounds 1-7 in Four Cancer Cell
Lines
cytotoxicity (IC50, µM)
compd
P388
A-549
HL-6
BEL-7402
1
2
3
4
5
6
7
>100
69
9.6
4.3
92
69
45
89
>100
>100
>100
88
>100
>100
94
>100
67
76
80
>100
>100
79
>100
>100
96
>100
>100
66
) 1.5, 13.6 Hz, H-10a), 2.80 (1H, dd, J ) 9.1, 13.6 Hz, H-10b), 1.47
(3H, s, H-11), 1.69 (3H, s, H-12), 5.54 (1H, m, H-13), 5.53 (1H, m,
H-14), 2.01 (1H, m, H-15a), 1.80 (1H, m, H-15b), 2.14 (1H, m, H-16),
5.05 (1H, q, J ) 3.6 Hz, H-17), 2.20 (3H, s, H-19), 0.82 (3H, d, J )
7.0 Hz, H-20), 7.15-7.68 (15H, m, Ph-H).
Acknowledgment. This work was financially supported by the
Chinese National Natural Science Fund (No.30772640), the Chinese
Ocean Mineral Resource R & D Association (DY105-2-04), and
Shandong Province (No. Z2006C13). The antitumor assay was per-
formed at the Shanghai Institute of Materia Medica, Chinese Academy
of Sciences.
Cytochalasin Z10 (1): colorless oil (MeOH); [R]25D +58.0 (c 0.080,
MeOH); UV (MeOH) λmax (log ε) 225 (3.40), 258 (1.03) nm; IR (KBr)
ν
max 3344, 2924, 1696 1458, 1374, 1072, 974 cm-1; 1H and 13C NMR
(see Tables 1 and 2); HRESIMS m/z 430.2596 [M + H]+ (calcd for
C25H36NO5, 430.2593).
Cytochalasin Z11 (2): colorless oil; [R]25D +114.8 (c 0.080, MeOH);
UV (MeOH) λmax (log ε) 222 (2.613), 258 (0.698) nm; IR (KBr) νmax
3368, 2969, 1694 1454, 1015, 697 cm-1; 1H and 13C NMR (see Tables
1 and 2); HRESIMS m/z 428.2434 [M + H]+ (calcd for C25H34NO5,
428.2434).
References and Notes
(1) Liu, R.; Cui, C. B.; Duan, L.; Gu, Q. Q.; Zhu, W. M. Arch. Pharm.
Res. 2005, 28, 1341–1344.
(2) Liu, R.; Zhu, W. M.; Zhang, Y. P.; Zhu, T. J.; Liu, H. B.; Fang, Y. C.;
Gu, Q. Q. J. Antibiot. 2006, 59, 362–365.
(3) Liu, R.; Gu, Q. Q.; Zhu, W. M.; Cui, C. B.; Fan, G. T. Arch. Pharm.
Res. 2005, 28, 1042–1046.
(4) Liu, R.; Gu, Q. Q.; Zhu, W. M.; Cui, C. B.; Fan, G. T.; Fang, Y. C.;
Zhu, T. J.; Liu, H. B. J. Nat. Prod. 2006, 69, 871–875.
(5) Buchanan, M. S.; Hashimoto, T.; Asakawa, Y. Phytochemistry 1995,
40, 135–140.
(6) Buchanan, M. S.; Hashimoto, T.; Asakawa, Y. Phytochemistry 1996,
41, 821–828.
(7) Buchanan, M. S.; Hashimoto, T.; Asakawa, Y. Phytochemistry 1996,
42, 173–176.
(8) Feng, Y. J.; Blunt, J. W.; Cole, A. L. J.; Munro, M. H. G. J. Nat.
Prod. 2002, 65, 1274–1277.
(9) Edwards, R. L.; Maitland, D. J.; Whalley, A. J. S. J. Chem. Soc., Perkin
Trans. 1 1989, 1, 57–65.
Cytochalasin Z12 (3): colorless oil: [R]25D +43.2 (c 0.080, MeOH);
UV (MeOH) λmax (log ε) 222 (2.052), 258 (0.555) nm; IR (KBr) νmax
3368, 2965, 1688, 1447, 1082, 983, 758 cm-1; H and 13C NMR (see
1
Tables 1 and 2); HRESIMS m/z 412.2477 [M - H2O + H]+ (calcd for
C25H34NO4, 412.2488).
Cytochalasin Z13 (4): colorless oil; [R]25D +80.8 (c 0.160, MeOH);
UV (MeOH) λmax (log ε) 223 (2.879), 258 (1.097) nm; IR (KBr) νmax
3323, 2965, 1712, 1454, 1348, 1155, 976, 764 cm-1; 1H and 13C NMR
(see Tables 1 and 2); HRESIMS m/z 428.2399 [M + H]+ (calcd for
C25H34NO5, 428.2437).
Cytochalasin Z14 (5): colorless oil; [R]25D +8.87 (c 0.080, MeOH);
UV (MeOH) λmax (log ε) 223 (2.134), 258 (1.003) nm; IR (KBr) νmax
3367, 2988, 1690 1452, 1000, 697 cm-1; 1H and 13C NMR (see Tables
1 and 2); HRESIMS m/z 410.2337 [M - H2O + H]+ (calcd for
C25H32NO4, 410.2331).
Cytochalasin Z15 (6): colorless oil; [R]25D +98.0 (c 0.086, MeOH);
UV (MeOH) λmax (log ε) 222 (2.022), 258 (0.550) nm; IR (KBr) νmax
3367, 2989, 1690 1453, 1005, 697 cm-1; 1H and 13C NMR (see Tables
1 and 2); HRESIMS m/z 428.2451 [M + H]+ (calcd for C25H34NO5
428.2437).
(10) Sharma, S. V.; Oneyama, C.; Nakano, H.; Agatsuma, T. Kanda, Y.;
Tsumagari, N.; Ando, K. PCT Int. Appl. 2003.
(11) Evidente, A.; Andolfi, A.; Vurro, M.; Zonno, M. C.; Motta, A.
Phytochemistry 2002, 60, 45–53.
(12) Carter, S. B. Nature 1967, 213, 261–264.

1132 Journal of Natural Products, 2008, Vol. 71, No. 7
Liu et al.
(13) Mookerijee, B. K.; Cuppoletti, J.; Rampa, A.; Jung, C. Y. J. Biol.
Chem. 1981, 256, 1290–1300.
(14) Martin, S. F.; Li, W. J. Org. Chem. 1989, 54, 6129–6133.
(15) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem.
Soc. 1991, 113, 4092–4096.
(16) (a) Ichikawa, A. Enantiomer 1997, 2, 327–331. (b) Ichikawa, A.
Enantiomer 1998, 3, 255–261. (c) Ivanchina, N. V.; Kicha, A. A.;
Kalinovsky, A. I.; Dmitrenok, P. S.; Dmitrenok, A. S.; Chaikina, E. L.;
Stonik, V. A.; Gavagnin, M.; Cimino, G. J. Nat. Prod. 2006, 69, 224–
228. (d) Freire, F.; Seco, J. M.; Quinoa, E.; Riguera, R. J. Org. Chem.
2005, 70, 3778–3790.
(17) Mosmann, T. J. Immunol. Methods 1983, 65, 55–63.
NP070539B