Deoxy-cytochalasins from a marine-derived fungus Spicaria elegans

Article abstract of DOI:10.1139/V09-006

Treatment of Spicaria elegans with cytochrome P-450 inhibitor resulted in two new deoxy-cytochalasins, 7-deoxy-Z7 (1) and 7-deoxy-cytochalasin Z9 (2), which were recognized as plausible precursors of cytochalasins 7 and Z9, respectively. Their structures

Full text of DOI:10.1139/V09-006

486
Deoxy-cytochalasins from a marine-derived
fungus Spicaria elegans
Zhen-Jian Lin, Tian-Jiao Zhu, Guo-Jian Zhang, Hong-Juan Wei, and Qian-Qun Gu
Abstract: Treatment of Spicaria elegans with cytochrome P-450 inhibitor resulted in two new deoxy-cytochalasins, 7-de-
oxy-cytochalasin Z7 (1) and 7-deoxy-cytochalasin Z9 (2), which were recognized as plausible precursors of cytochalasins
Z7 and Z9, respectively. Their structures were elucidated by spectroscopic methods and the absolute configuration of 1 was
determined by the conventional Mosher ester method. Their cytotoxicities against two cancer cell lines were evaluated.
Key words: cytochalasin, Spicaria elegans, cytochrome P-450 inhibitor, cytotoxicity.
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Resume : Le traitement du Spicaria elegans par l’inhibiteur du cytochrome P-450 conduit a la formation de deux nouvel-
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les desoxycytochalasines, le 7-desoxycytochalasine Z7 (1) et 7-desoxycytochalasine Z9 (2) qui ont ete reconnues comme
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des precurseurs plausibles respectivement pour les cytochalasines Z7 et cytochalsaines Z9. Les structures des composes 1
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et 2 ont ete elucidees par des methodes spectroscopiques et la configuration absolue du produit 1 a ete determinee par la
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methode conventionnelle de l’ester de Mosher. On a evalue leurs cytotoxicites contre deux series de cellules cancereuses.
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Mots-cles : cytochalasine, Spicaria elegans, inhibiteur de cytochrome P-450, cytotoxicite.
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[Traduit par la Redaction]
(1 and 2) were recognized by HPLC-MS analysis and iso-
lated by semiprepared HPLC.
Introduction
In our ongoing search for new bioactive secondary metabo-
lites from marine fungi, a series of cytochalasins has been iso-
lated from the marine-derived fungus Spicaria elegans, some
of which exhibited interesting cytotoxicities against the A-
549 cell line,1,2 which attracted our continuous attentions.
Given that P450 mono-oxygenases are believed to catalyze
the oxidative polyketide tailoring reactions of cytochalasins,3
it is possible to use the inhibitors to obtain various biosyn-
thetic precursor products. The achievement of this was real-
ized by the discovery of 7-deoxy-cytochalasin Z7 (1) and 7-
deoxy-cytochalasin Z9 (2) by the addition of metyrapone, an
inhibitor of P-450 dependent monooxygenases, in the culture
of fungus S. elegans. We describe herein the isolation, struc-
ture elucidation, and biological activities of 1 and 2.
7-Deoxy-cytochalasin Z7 (1) was isolated as a white pow-
der. Its molecular formula was established as C28H35NO4 by
HR-ESI-MS (m/z 450.2652 [M + H]+, anal. calcd. for
C28H36NO4: 450.2644) and indicated twelve degrees of
unsaturation. The IR spectrum had bands due to NH and
(or) OH (3392 cm–1), an a,b-unsaturated ester/lactone car-
bonyl (1698 cm–1), and an amide carbonyl (1680 cm–1). The
1H NMR spectrum (Table 1) showed the presence of an
amide NH (dH: 5.79), three methyl doublets (dH: 0.97, 1.05,
and 1.17), a methyl singlet (dH: 1.72), and 11 methine and
(or) methylene protons. Ten olefinic protons (dH: 5.10, 5.34,
5.66, 5.96, and 7.16–7.33), suggesting a monosubstituted
benzene ring, were also observed. The 13C NMR spectrum
of 1 displayed signals for 28 carbon atoms and confirmed
the presence of an a,b-unsaturated ester/lactone carbonyl
(dC: 167.4) and an amide carbonyl (dC: 172.5) (Table 1).
Analysis of the DEPT spectrum revealed the presence of
four methyl, two methylene, 17 methine, and three quater-
nary carbons, in addition to the two carbonyls. All of these
1D NMR features of 1 suggested it was closely related to an
analog of cytochalasins. Accurate inspection of its 1D NMR
spectra revealed, with respect to that of cytochalasin Z7, the
absence of exocyclic olefinic methylene (12-CH2). Whereas
a trisubstituted double bond (dH: 5.34, dC: 123.9, 140.0) and
a methyl goup (12-Me, dH: 1.72, dC: 19.8) was present.
These findings indicated that the 7-OH and the exocyclic
olefinic methylene were missing, instead, a trisubstituted
double bond was located between C-6 and C-7. The struc-
ture of 1 was further supported by its molecular formula
(C28H35NO4), with one fewer oxygen than cytochalasin Z7.
Compound 1 possessed a 12-membered macrocyclic ring,
the same as that in cytochalasin Z7. Accurate inspection of
Results and discussion
Previous study had shown that the strain S. elegans
produced 10 phenyl-cytochalasins (Z7–Z15)1,2 as the major
components, which were all oxidized at the 7-position.
Metyrapone (1 mmol/L), a cytochrome P-450 inhibitor, was
added on the sixth day after inoculation to the culture of
S. elegans. After an additional 14 days of static fermentation,
the metabolites were extracted from the mycelium. Compared
with nontreated samples, two new 7-deoxy-cytochalasins
Received 14 November 2008. Accepted 10 December 2008.
Published on the NRC Research Press Web site at
canjchem.nrc.ca on 11 February 2009.
Z. Lin, T. Zhu, G. Zhang, H. Wei, and Q. Gu.1 Key
Laboratory of Marine Drugs, Chinese Ministry of Education,
Institute of Marine Drugs and Food, Ocean University of China,
Qingdao 266003, PR China.
1Corresponding author (e-mail: guqianq@ouc.edu.cn).
1
1
the H and 13C NMR data, including H NMR coupling con-
Can. J. Chem. 87: 486–489 (2009)
doi:10.1139/V09-006
Published by NRC Research Press

Lin et al.
487
1
Table 1. H (600 MHz) and 13C (150 MHz) NMR spectroscopic data for 1 and 2 in CDCl3
Compound
position
1
2
dH (J in Hz)
dC
dH (J in Hz)
dC
1
172.5 (s)
172.1 (s)
2-NH
3
4
5
5.79 (brs)
3.22 (m)
3.04 (m)
3.04 (m)
5.89 (brs)
3.20 (m)
2.91 (m)
2.91 (m)
55.9 (d)
50.8 (d)
33.8 (d)
140.0 (s)
55.9 (d)
51.5 (d)
34.1 (d)
139.9 (s)
123.8 (d)
46.7 (d)
87.7 (s)
44.8 (t)
13.8 (q)
19.8 (q)
126.3 (d)
137.1 (d)
6
7
8
9
5.34 (brs)
3.47 (m)
123.9 (d) 5.36 (brs)
45.4 (d)
88.0 (s)
45.1 (t)
13.8 (q)
19.8 (q)
3.38 (m)
10
11
12
13
14
15
16
16-CH3
17
18
18-CH3
19
20
21
1’
2.89 (dd, J = 13.2, 4.1), 2.70 (d, J = 13.7)
1.17 (d, J = 6.8)
1.72 (brs)
5.96 (ddd, J = 15.1, 10.0, 2.0)
5.10 (ddd, J = 15.1, 10.1, 4.6)
2.08 (m)
1.62 (m)
0.97 (d, J = 7.3)
3.87 (dd, J = 3.2, 4.1)
2.72 (m)
1.05 (d, J = 6.4)
2.89 (m), 2.85 (m)
1.14 (d, J = 6.6)
1.74 (brs)
125.9 (d) 5.91 (ddd, J = 15.1, 10.0, 2.7)
137.2 (d) 5.28 (ddd, J = 15.1, 10.1, 2.7)
42.0 (t)
32.5 (d)
18.4 (q)
77.3 (d)
42.8 (d)
8.7 (q)
2.10 (brd, J = 14.3), 1.97 (ddd, J = 14.3, 10.4, 10.4) 43.5 (t)
1.39 (m)
1.05 (d, J = 7.1)
1.77 (dd, J = 13.6, 5.5), 1.64 (dd, J = 13.6, 2.2)
32.5 (d)
22.2 (q)
53.2 (d)
72.8 (s)
1.34 (s)
27.1 (q)
159.5 (d)
118.4 (d)
167.4 (s)
137.7 (s)
128.9 (d)
129.0 (d)
126.9 (d)
129.0 (d)
128.9 (d)
7.16–7.33 (m)
5.66 (dd, J = 16.0, 1.9)
157.5 (d) 7.45 (d, J = 16.0)
120.5 (d) 5.75 (d, J = 16.0)
167.4 (s)
137.4 (s)
2’
3’
4’
5’
7.16–7.33 (m)
7.16–7.33 (m)
7.16–7.33 (m)
7.16–7.33 (m)
7.16–7.33 (m)
128.8 (d) 7.18–7.33 (m)
129.0 (d) 7.18–7.33 (m)
127.0 (d) 7.18–7.33 (m)
129.0 (d) 7.18–7.33 (m)
128.8 (d) 7.18–7.33 (m)
6’
Fig. 1. Structures of compounds 1 and 2.
Fig. 2. The key Dd values [Dd (in ppm) = dS – dR] obtained for (S)-
and (R)-MTPA esters of compound 1.
stants, for compound 1 and cytochalasin Z7 should have the
most coincident relative configuration (structure from C-15
to C-19). The absolute configuration of the 17 position in 1
was established by a conventional Mosher ester method using
the (S)- and (R)-a-methoxy-a-trifluoromethyl-a-phenylacetic
acid (MTPA) esters (1a and 1b, respectively) (Fig. 1), where
the positive values of DdS–R for H-16 and 16-CH3 and neg-
ative values of DdS–R for H-18 and 18-CH3 (Fig. 2) sug-
gested a 17-(R) configuration in 1, which was the same as
that of cytochalasin Z7. Therefore, the absolute configura-
tions of 1 could be deduced as shown in Fig. 1.
of C28H35NO4 showing one fewer oxygen atom than cyto-
chalasin Z9. This assumption could be confirmed by its 1D
NMR spectra. Analysis of its 1D NMR spectra, with respect
to that of cytochalasin Z9, revealed that the singlet of 11-
CH3 in cytochalasis Z9 was upfiled shifted to dH: 1.14 and
changed to a doublet, which suggested the double bond at
C-5,6 was missing in 2. Most similarly to 1, the 1D NMR
spectra of 2 (Table 1) also showed the absence of the oxy-
genated methine group at C-7, whereas there was the ap-
pearance of a trisubstituted double bond (dH: 5.36, dC:
123.8, 139.9) assigned between C-6 and C-7. On the basis
of these 1D NMR spectra features, the structure of 2 was
elucidated as 7-deoxy-cytochalasis Z9.
7-Deoxy-cytochalasis Z9 (2) was isolated as a white pow-
der. Its HR-ESI-MS (m/z 450.2638 [M + H]+, anal. calcd.
for C28H36NO4: 450.2644) suggested that it might be a de-
oxy-derivative of cytochalasin Z9, with a molecular formula
Compounds 1and 2 were evaluated for their cytotoxic-
ities against the A-549 and P-388 cell lines by the MTT [3-
Published by NRC Research Press

488
Can. J. Chem. Vol. 87, 2009
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
method. Compound 1 showed moderate cytotoxicity against
the A-549 cell line with an IC50 value of 15.0 mmol/L,
while 2 was inactive. In comparison to that of cytochala-
sins Z7 and Z9, the double bond (C-6,7) in 1 and 2 re-
sulted in a much weaker cytotoxicity.
extracted three times with EtOAc to give an EtOAc solution,
which was concentrated under reduced pressure to give a
crude extract (5.5 g).
Purification
The crude extract (5.5 g) was separated into four fractions
(Fr. 1–4) on a Si gel column using a gradient elution of
CHCl3:MeOH. Fr. 2 was eluted with CHCl3:MeOH (100:1,
0.4 g) and was purified into 8 subfractions (Fr. 2-1 ~ Fr. 2-8)
by Si gel column using stable elution of CHCl3:MeOH
(200:1). Subfraction Fr. 2-6 was further purified by reverse-
phase column using stable elution of MeOH:H2O (7: 3) to
give 4 subfractions (Fr. 2-6-1 ~ Fr. 2-6-4), respectively.
Further purification of subfraction Fr. 2-6-4 by extensive
HPLC (75% MeOH, 4.0 mL/min) gave compounds 1 (3 mg)
and 2 (2 mg).
A new generation of plausible precursors of chaetoglobo-
sin A4 and epoxycytochalasin H5 had been obtained by treat-
ment fungi with cytochrome P-450 inhibitors. Those results,
as well as the experiments in this paper, indicated that the
biosynthetic oxidation at the 7 position of cytochalasins was
exactly due to the cytochrome P-450. More frequently, the
cytochalasins, likewise cytochalasins Z7 and Z9, have an ox-
idation at the C-9 position, which was believed to be formed
from the acetate-derived polyketide chain by an enzymatic
‘Baeyer–Villager’-like oxidation.6 Even though a cyto-
chrome P-450 monooxygenase CYP85A2 had been reported
to have a Baeyer–Villiger oxidation activity,7 it was still
unknown whether the Baeyer–Villager-like oxidation in cyto-
chalasins was related to the cytochrome P-450. In our ex-
periments, no more deoxy-cytochalasin was detected except
for 1 and 2 when S. elegans was treated with metyrapone,
therefore, the P-450 enzyme inhibitor can’t block the
Baeyer–Villager-like oxidation in this strain. Maybe it is
not the P-450 enzyme that catalyzed the Baeyer–Villager-
like oxidation in the biosynthetic pathway of cytochalasins.
This warrants further investigations.
7-Deoxy-cytochalasin Z7 (1)
White powder. [a]2D5 +94.8 (c 0.1, MeOH). UV (MeOH)
lmax (nm) (log 3): 233 (2.43). IR (KBr, cm–1) nmax: 3368,
1
2937, 1700, 1444, 1214, 1076, 990. H and 13C NMR (see
Table 1). HR-ESI-MS m/z: 450.2652 [M + H]+. Anal. calcd.
for C28H36NO4: 450.2644.
7-Deoxy-cytochalasin Z9 (2)
White powder. [a]2D5 +12.8 (c 0.1, MeOH). UV (MeOH)
lmax (nm) (log 3): 230 (2.00). IR (KBr, cm–1) nmax: 3298,
2900, 1694, 1321 1201, 999. 1H and 13C NMR (see Table 1).
HR-ESI-MS m/z: 450.2638 [M + H]+. Anal. calcd. for
C28H36NO4: 450.2644.
Experimental section
General
Optical rotations were obtained on a JASCO P-1020
digital polarimeter. UV spectra were recorded on Beckman
DU1 640 spectrophotometer. IR spectra were taken on a
Preparation of the (S)- and (R)-MTPA ester derivatives
of 1
Compound 1 (2.0 mg) was divided into two aliquots and
transferred into two clean reaction bottles and was dried
completely under vacuum. Each solubilized in 0.5 mL of
pyridine. The two samples were treated with (R)- and (S)-a-
methoxy-a-trifluoromethylphenylacetyl chloride (1 equiv.)
under a N2 gas stream, and then stirred for 24 h at room tem-
perature. The organic layer was then washed with water, HCl
(1 mol/L), water, NaHCO3(satd.), and water, then dried
(Na2SO4) and concentrated under reduced pressure to obtain
the ester. Final purification achieved by HPLC gave 1a and
1b, respectively. 1H NMR data for the (S)-MTPA ester
derivative (1a) of 1 (600 MHz, CDCl3) d: 5.76 (1H, brs, 2-NH),
3.24 (1H, m, H-3), 3.05 (1H, m, H-4), 3.05 (1H, m, H-5),
5.35 (1H, brs, H-7), 3.48 (1H, m, H-8), 2.90 (1H, dd, J =
4.1, 13.8 Hz, H-10a), 2.69 (1H, dd, J = 9.6, 13.8 Hz, H-10b),
1.20 (3H, d, J = 6.9 Hz, H-11), 1.73 (3H, brs, H-12), 6.04
(1H, ddd, J = 13.0, 10.6, 1.8 Hz, H-13), 5.07 (1H, m, H-14),
2.30 (1H, ddd, m, H-15a), 2.09 (1H, brd, J = 14.6 Hz, H-15b),
1.80 (1H, m, H-16), 0.88 (3H, d, J = 6.4 Hz, 16-CH3),
5.09 (1H, m, H-17), 2.80 (1H, m, H-18), 0.97 (3H, d, J =
7.3 Hz, 18-CH3), 5.68 (1H, dd, J = 15.5, 2.3 Hz, H-19),
1
NICOLET NEXUS 470 spectrophotometer in KBr discs. H,
13C NMR and DEPT, and 2D NMR spectra were recorded on
a JEOL JNM-ECP 600 spectrometer using TMS as an inter-
nal standard and chemical shifts were recorded as d values.
NOESY experiments were carried out using a mixing time of
0.5 s. ESI-MS 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 mm  250 mm, 5 mm, 4 mL/min).
Fungal material
The fungus Spicaria elegans was isolated from the marine
sediments collected in Jiaozhou Bay, China. It was pre-
served in the China Center for Type Culture Collection
(patent depositary number: KLA03 CCTCC M 205049).
Working stocks were prepared on potato dextrose agar slants
stored at 4 8C.
Fermentation and extraction
The fungus was incubated at 28 8C under static condi-
tions in 30 1000 mL conical flasks containing the liquid
medium (300 mL/flask) composed of glucose (20 g/L), pep-
tone (5 g/L), malt extract (3 g/L), yeast extract (3 g/L), and
sea-water after adjusting its pH to 7.0. The inhibitor metyr-
apone (1 mmol/L) was added on the sixth day after inocula-
tion to the culture of S. elegans. After an additional 14 days
of static fermentation, the fermented whole broth (9 L) was
1
7.15*7.56 (11H, m, H-20 and Ph-H). H NMR data for
the (R)-MTPA ester derivative (1b) of 1 (600 MHz,
CDCl3) d: 5.82 (1H, brs, 2-NH), 3.24 (1H, m, H-3), 3.05
(1H, m, H-4), 3.05 (1H, m, H-5), 5.35 (1H, brs, H-7),
3.48 (1H, m, H-8), 2.90 (1H, dd, J = 4.1, 13.8 Hz, H-10a),
2.69 (1H, dd, J = 9.6, 13.8 Hz, H-10b), 1.20 (3H, d, J =
6.9 Hz, H-11), 1.73 (3H, brs, H-12), 6.04 (1H, ddd, J =
Published by NRC Research Press

Lin et al.
489
13.0, 10.6, 1.8 Hz, H-13), 5.07 (1H, m, H-14), 2.34 (1H,
ddd, m, H-15a), 2.05 (1H, brd, J = 14.6 Hz, H-15b), 1.78
(1H, m, H-16), 0.81 (3H, d, J = 6.4 Hz, 16-CH3), 5.12
(1H, m, H-17), 2.83 (1H, m, H-18), 1.07 (3H, d, J =
7.3 Hz, 18-CH3), 5.71 (1H, dd, J = 15.5, 2.3 Hz, H-19),
7.15*7.56 (11H, m, H-20 and Ph-H).
Acknowledgements
This work was financially supported by the Chinese Na-
tional Natural Science Fund (No. 30772640) and the Shan-
dong Province (No. Z2006C13). The antitumor assay was
performed at the Shanghai Institute of Materia Medica, Chi-
nese Academy of Sciences.
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