New class azaphilone produced by a marine fish-derived Chaetomium globosum. the stereochemistry and biological activities

Article abstract of DOI:10.1016/j.bmc.2011.05.008

Four new metabolites, chaetomugilins P-R and 11-epi-chaetomugilin I, were isolated from a strain of Chaetomium globosum originally obtained from the marine fish Mugil cephalus, and their absolute stereostructures were elucidated on the basis of spectrosco

Full text of DOI:10.1016/j.bmc.2011.05.008

Bioorganic & Medicinal Chemistry 19 (2011) 4106–4113
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New class azaphilone produced by a marine fish-derived Chaetomium
globosum. The stereochemistry and biological activities
⇑
Takeshi Yamada , Yasuhide Muroga, Masaaki Jinno, Tetsuya Kajimoto, Yoshihide Usami,
Atsushi Numata, Reiko Tanaka
Osaka University of Pharmaceutical Sciences, 4-20-1, Nasahara, Takatsuki, Osaka 569-1094, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Four new metabolites, chaetomugilins P–R and 11-epi-chaetomugilin I, were isolated from a strain of
Chaetomium globosum originally obtained from the marine fish Mugil cephalus, and their absolute stereo-
structures were elucidated on the basis of spectroscopic analyses, including 1D and 2D NMR techniques
and various chemical transformations. Particularly, the skeleton of chaetomugilin P is different from that
of other azaphilones isolated from this fungal strain to date. In addition, these compounds significantly
inhibited the growth of cultured P388, HL-60, L1210 and KB cell lines.
Received 17 March 2011
Revised 6 May 2011
Accepted 6 May 2011
Available online 15 May 2011
Keywords:
Ó 2011 Elsevier Ltd. All rights reserved.
Chaetomium globosum
Chaetomugilins
Cytotoxicity
Azaphilones
Marine fish
1. Introduction
carcinoma cell line. We describe herein the absolute configuration
of the stereogenic centers and biological activities of these
Marine products include a number of compounds with unique
structures, some of which may exhibit unusual bioactivities. We
have focused on potential new antitumor materials from marine-
derived microorganisms and found a number of antitumour and/
or cytotoxic compounds.1–3 We have already reported the absolute
stereostructures and cytotoxic activities of chaetomugilins A–O,
azaphilones isolated as cytostatic metabolites from the fungus
Chaetomium globosum OUPS-T106B-6 originally obtained from
the marine fish Mugil cephalus.4–9 Our continuing search for cyto-
toxic metabolites from this fungal strain led to the isolation of four
new azaphilones designated as chaetomugilins P–R (1–3) and
11-epi-chaetomugilin I (4). Azaphilones have various bioactivities
such as antimicrobial activity, nitric oxide inhibition (cohaerins),10
gp120-CD4 binding inhibition (isochromophiliones, ochrephilone,
screotiorin and rubrorotiorin),11 monoamine oxidase inhibition
(luteusins, TL-1, TL-2 and chaetoviridins),12,13 platelet-derived
growth factor binding inhibition (RP-1551s)14 and antimalarial
activity (cochliodones).15 These metabolites have a chlorine atom
at C-5 and a methyl group at C-7, however, chaetomugiline P (1)
has a methyl group at C-5 and no substituent at C-7. Compounds
1 and 4 exhibited significant cytotoxic activity against the murine
P388 leukemia cell line, the human HL-60 leukemia cell line, the
murine L1210 leukemia cell line, and the human KB epidermoid
compounds.
2. Results and discussion
2.1. Identification and structure determination
The microorganism from M. cephalus fish was cultured at 27 °C
for six weeks in a medium (50 L) containing 1% soluble starch and
0.1% casein in 50% artificial seawater adjusted to pH 7.4 as reported
previously.4–9 The EtOAc extract of the culture filtrate was purified
by bioassay-directed fractionation (cytotoxicity against the P388
cell line) employing Sephadex LH-20, silica gel column chromatog-
raphy and the reverse phased HPLC to afford chaetomugilins P–R
(1–3) and 11-epi-chaetomugilin I (4) (Fig. 1).
Chaetomugilin
P (1) was assigned the molecular formula
C
22H27ClO5 based on the [M+H]+ peak in HRFABMS and the ratio
of the intensity of isotope peaks(MH+/[MH+2]+). Its IR spectrum
exhibited bands at 3440, 1678 and 1666 cmꢀ1, characteristic of hy-
droxyl and a, b-unsaturated carbonyl groups. A close inspection of
the 1H and 13C NMR spectra (Table 1) of 1 in DEPT and HMQC exper-
iments revealed the presence of two secondary methyls (11-CH3
and C-13), one tertiary methyl (10-CH3), one secondary olefin
methyl (C-50), one tertiary olefin methyl (5-CH3), one sp3-hybrid-
ized methylene (C-7), three sp3-methines (C-8, C-11 and C-12)
including an oxygen-bearing carbon (C-12), five sp2-methines
(C-1, C-4, C-9, C-10 and C-40) including an oxygen-bearing carbon
⇑
Corresponding author.
E-mail address: yamada@gly.oups.ac.jp (T. Yamada).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.05.008

T. Yamada et al. / Bioorg. Med. Chem. 19 (2011) 4106–4113
4107
Figure 1. The structures of metabolites from A. fumigatus and the derived compound.
Table 1
NMR spectroscopic data (CDCl3) for chaetomugilin P (1)
a
Position
dH
J/Hz
NOE
dc
HMBC (C)b
3, 4a, 8, 8a
1
3
4
4a
5
6
7 A
7 B
8
8a
9
10
11
12
13
6.71
5.98
s
s
8, 40, 10-CH3, 10-OH
142.3
153.2
105.5
139.7
116.7
194.6
35.9
(d)
(s)
(d)
(s)
(s)
(s)
(t)
5-CH3
3, 8a, 9
2.65
2.83
3.44
dd
dd
dd
16.1 (7B), 7.0 (8)
16.1 (7A), 2.1 (8)
7.0 (7A), 2.1 (7B)
7B, 8, 10-CH3
5, 6, 8, 8a
5, 6, 8, 8a
1, 4a, 6, 7, 8a, 10
7A, 8, 10-CH3
1, 7A, 7B, 40, 10-CH3
42.0
(d)
(s)
(d)
(d)
(d)
(d)
(q)
(q)
(q)
(s)
(s)
(s)
(d)
(q)
(q)
116.4
122.9
138.3
44.0
71.0
20.3
5.98
6.32
2.38
3.77
1.17
1.76
1.10
d
15.6 (10)
10, 11, 13, 11-CH3
9, 11, 12, 13, 11-CH3
9, 10, 12, 13, 11-CH3
10, 11, 13, 11-CH3
9, 10, 11, 12, 11-CH3
4
3, 4, 10, 11
3, 11, 12, 11-CH3
9, 10, 12, 13, 11-CH3
10, 11-CH3
11, 12
dd
dqd
Quint
d
s
d
15.6 (9), 7.8 (11)
7.8 (10), 6.9 (11-CH3), 6.0 (12)
6.0 (11,13)
6.0 (12)
5-CH3
11-CH3
10
9.3
4a, 5, 6
10, 11, 12
6.9 (11)
9, 10, 11, 12, 13
14.9
83.8
197.2
131.1
141.0
15.7
23.8
20
30
40
7.38
2.00
1.56
3.33
q
d
s
6.9 (50)
6.9 (40)
1, 8, 50, 10-CH3, 10-OH
20, 30, 50
50
40
30, 40
10-CH3
10-OH
1, 7A, 7B, 8, 40
1, 40
8, 10, 20
s
8, 10, 20, 10-CH3
a
1H chemical shift value (d ppm) followed by multiplicity and then the coupling constants (J/Hz). Figures in parentheses indicate the proton coupling with that position.
HMBC correlations are from proton stated to the indicated carbon.
b
(C-1), one quarternary oxygen-bearing sp3-carbon (C-10), five quar-
ternary sp2-carbons (C-3, C-4a, C-5, C-8a and C-30) including an
oxygen-bearing carbon (C-3), and two carbonyls (C-6 and C-20).
The 1H–1H COSY analysis of 1 led to three partial structural units
as shown by bold-faced lines in Figure 2. The geometrical configu-
ration of the double bond moiety (C-9–C-10) was deduced as the E
configuration from the coupling constant of the olefinic protons
(J9,10 = 15.6 Hz). The connection of these units and the remaining
functional groups was determined on the basis of the key HMBC
correlations summarized in Figure 2, and this fact suggested that
a chlorine atom connected to C-30, a quarternary olefin carbon. In
addition, the geometry for the double bond moiety (C-30–C-40)
was deduced as the Z configuration from NOE correlations (H-40/
H-8 and H-40/10-CH3). Thus the planar structure of 1 was elucidated
as shown in Figure 2. To determine the absolute configurations for
C-8, C-11, C-12 and C-10, 1 was transformed to chaetomugilin I (5),
whose absolute stereostructure had been determined.8 The treat-
ment of 1 in MeOH with p-TsOH gave the product 5 (yield 21.4%)
(Scheme 1), which was confirmed to be identical to the natural sub-
stance 5 in IR, UV, NMR spectra and optical rotation. As shown in
Scheme 1, the plausible mechanism for this reaction is unusual,
with both Diels-Alder and retro-Diels–Alder reactions occuring.

4108
T. Yamada et al. / Bioorg. Med. Chem. 19 (2011) 4106–4113
exciton spilt Cotton effects, each sign of which agreed with that of
the screw sense between the side chain enone chromophore (C-20
and C-30) and the conjugated chromophore (C-4–C-6) of the azaphi-
lone moiety (Fig. 3).
Chaetomugilin Q (2) was assigned the molecular formula
C
22H29ClO6 deduced from HRFABMS. Its general spectral features
closely resembled those of 5 except for 1H and 13C NMR signals
at C-20–C-50 (Table 2). The planar structure of 2 was confirmed
1
by analyzing H–1H COSY and HMBC correlations (from 30-CH3 to
C-20, from H-40 to C-20, and from H-50 to C-30). The above evidence
and the 13C NMR chemical shift of C-40 (dC 69.74), together with the
molecular formula of 2, suggested the addition of H2O to the dou-
ble bond (C-30–C-40) in 5. The absolute configurations at C-7, C-8,
C-11 and C-12 in 2 were determined by the transformation from
2 to 5. The treatment of 2 in MeOH with p-TsOH produced 5 with
the elimination of H2O (yield 20.9%), which was confirmed to be
identical with the natural 5 in all spectral data and optical rotation.
Figure 2. 1H–1H COSY and key HMBC correlations in 1.
This conversion from 1 to 5 revealed the absolute stereostructure of
1 (8S, 11R, 12R and 10S). The CD spectra of 1 and 5 exhibited typical
Scheme 1. Plausible mechanism for transformation from 1 to 5.
Figure 3. CD spectra of 1, 4 and 5.

T. Yamada et al. / Bioorg. Med. Chem. 19 (2011) 4106–4113
4109
Table 2
NMR spectroscopic data (CDCl3) for chaetomugilin Q (2) and acetonide 6
Chaetomugilin Q (2)
Acetinide 6
a
a
Position
dH (J/Hz)
dc, multi.
HMBC (C)b
3, 4a, 8, 8a
dH (J/Hz)
dc
1
3
4
4a
5
6
7
8
8a
9
10
11
12
13
7-CH3
7-OH
11-CH3
7.42
s
s
145.2
156.7
104.9
141.4
107.0
191.7
74.1
(d)
(s)
(d)
(s)
(s)
(s)
(s)
(d)
(s)
(d)
(d)
(d)
(d)
(q)
(q)
7.21
6.48
s
s
143.5 (d)
156.3 (s)
105.1 (d)
139.2 (s)
110.9 (s)
188.8 (s)
85.1 (s)
42.7 (d)
116.6 (s)
122.4 (d)
141.2 (d)
44.1 (d)
70.9 (d)
20.4 (q)
25.0 (q)
6.47
3, 5, 8a, 9
3.46
dd (9.5, 3.5)
40.6
1, 4a, 6, 7, 8a, 7-CH3, 10, 20
3.45
dd (12.5, 7.2)
119.5
122.2
142.0
44.1
70.8
20.4
6.11
6.59
2.44
3.81
1.19
1.31
4.03
1.12
2.39
3.2
d (15.5)
3, 4, 10, 11
3, 11, 12, 11-CH3
9, 10, 12, 11-CH3
10, 11, 13, 11-CH3
11, 12
6.12
6.57
2.44
3.81
1.19
1.44
d (15.5)
dd (15.5, 8.0)
dqd (8.0, 6.5, 5.8)
qd (6.5, 5.8)
d (6.5)
s
br s
d (6.5)
dd (18.0, 9.5)
dd (18.0, 3.5)
dd (15.5, 8.0)
dqd (8.0, 6.5, 6.0)
Quint (6.0)
d (6.0)
26.6
6, 7, 8
6, 7, 8
s
14.7
40.9
(q)
(t)
10, 11, 12
1.13
2.20
1.85
d (6.5)
t (12.5)
dd (12.5, 7.2)
14.8 (q)
42.7 (t)
10
10
20
30
40
50
A
B
7, 8, 8a, 20
7, 8, 8a, 20
213.7
54.0
69.7
20.9
13.7
(s)
(d)
(d)
(q)
(q)
106.4 (s)
40.8 (d)
67.3 (d)
19.9 (q)
11.3 (q)
98.8 (s)
25.0 (q)
31.2 (q)
2.41
3.86
1.13
1.04
dq (7.5, 7.0)
dq (7.5, 6.1)
d (6.1)
20, 40, 50, 30-CH3
20, 30, 50, 30-CH3
30, 40
1.56
4.09
1.17
0.75
dq (10.5, 7.0)
dq (10.5, 6.0)
d (6.0)
30-CH3
d (7.0)
20, 30, 40
d (7.0)
60
70
80
1.63
1.35
s
s
a
As in Table 1.
As in Table 1.
b
The above findings revealed the absolute configuration of the chiral
centers as 7S, 8S, 11R and 12R except for C-30 and C-40. To deter-
mine the absolute configurations of C-30 and C-40 in 2, we designed
an acetonide, in which an isopropyridene group is interposed be-
tween 7-OH and 40-OH. The treatment of 2 in CHCl3 with 2, 2-
dimethoxypropane gave the acetonide 6 (yield 33.1%), however,
13
the C NMR data for 6 suggested that the carbonyl group at C-20
(dC 213.7) in 2 was replaced by an acetal (dC 106.4) in 6 (Table
2). HMBC correlations (from H-40 to C-60) and HRFABMS ([M+H]+
m/z 465.2020) in 6 revealed that ether linkages were formed be-
tween C-20and C-7, and an acetonide was bridged between C-20and
C-40. In the NOESY experiment of 6, observed NOEs (H-10/30-CH3,
30-CH3/7-CH3 and H-40/H-70) implied that 30-CH3 is oriented trans
to 40-CH3, and the C-20 – C-30 bond is cis to 7-CH3 (Fig. 4). In addi-
tion, NOE correlation from H-70 to H-4 and J value (J3 ,4 = 10.5 Hz)
implied the six members ring including an isopropyridene group
to exist in a chair conformation. These evidences revealed the
absolute configuration for C-30 and C-40 in 2 to be S and S,
respectively.
0
0
Figure 4. Key NOEs for 6.
Chaetomugilin R (3) was assigned the molecular formula
was identified with 2E-5-hydroxy-4-methylhex-2-enoic acid ob-
tained from 5 by a similar manner in IR, UV and NMR spectra except
the specific rotations. The carboxylic acid obtained from 3 showed
C
16H21ClO5 which contained six carbons atom less than that of 5.
13
In the 1H and C NMR spectra (Table 3), the signals for C-10–C-50
in 5 disappeared, and a hydroxyl methine (dH 3.73, dC 73.89), a
methylene (dH 3.81, 4.84, dC 68.43) and a sp3-methine (dH 2.88, dC
38.27) newly appeared in 3. The geometrical configuration of the
double bond moiety (C-9–C-10) was deduced as trans from the cou-
pling constant of the olefinic protons (J9,10 = 15.5 Hz). The 1H–1H
COSY correlations (H-1/H-8a and H-8a/H-8) and the HMBC correla-
tions (from 7-CH3 to C-8, H-1 to C-3, H-4 to C-8a, and H-8a to C-5)
revealed the planar structure of 3. In NOESY experiments for 3, the
the [a]D value opposite that obtained from 5. This evidence revealed
that the absolute configurations for C-11 and C-12 of 3 were estab-
lished as S and S, respectively. In addition, the modified Mosher’s
method16 was used to determine the absolute configuration of
C-8 in 3, and the 1H chemical-shift difference between (R)- and
(S)-MTPA esters (3a and 3b) implied an S configuration at C-8a,
and supported an S configuration at C-12 (Fig. 6). These evidences
allowed assignment of the absolute configuration of all the asym-
metric centers (7R, 8S, 8aS, 11S and 12S).
11-Epi-chaetomugilin I (4) had the same molecular formula
(C22H27ClO5) as 1 and 5 as deduced from HRFABMS. The general
features of its UV, IR and NMR spectra (Table 4) closely resembled
those of chaetomugilin I (5) except that the NMR signals for the
NOE correlations (H-8a/7-CH3 H-8a/H-1b and H-1a/H-8) implied
that 70-CH3 is oriented trans to H-8, and cis to H-8a (Fig. 5). In order
to determine the absolute configurations at C-11 and C-12 in 3, the
alkaline degradation of 3 was carried out.8 The degradation of 3
with 5% potassium hydroxide afforded a carboxylic acid, which

4110
T. Yamada et al. / Bioorg. Med. Chem. 19 (2011) 4106–4113
Table 3
NMR spectroscopic data (CDCl3) for chaetomugilin R (3)
a
Position
dH
J/Hz
13.1 (8a), 11.0 (1b)
11.0 (1 ), 5.0 (8a)
NOE
dc
HMBC (C)b
1
a
3.81
4.84
dd
dd
1b, 8
68.4
(t)
3, 4a, 8a
3, 4a, 8a
1 b
a
1a
, 8a
3
4
4a
5
6
7
8
8a
9
10
11
12
13
7-CH3
11-CH3
162.4
100.9
146.6
117.5
193.2
77.9
73.9
38.3
124.6
142.0
44.2
(s)
(d)
(s)
(s)
(s)
(s)
(d)
(d)
(d)
(d)
(d)
(d)
(q)
(q)
(q)
6.02
s
3, 5, 8a, 9
3.73
2.88
6.04
6.55
2.42
3.79
1.19
1.34
1.11
d
ddd
d
dd
dqd
Quint
d
10.0 (8a)
13.1 (1 ), 10.0 (8), 5.0 (1b)
15.5 (10)
15.5 (9), 7.5 (11)
7.5 (10), 6.3 (11-CH3), 6.0 (12)
6.0 (11, 13)
1
a
1, 7, 8a, 7-CH3
1, 4a, 5, 8
a
1b, 7-CH3
10, 11, 11-CH3
9, 11, 12, 13, 11-CH3
9, 10, 12, 13, 11-CH3
10, 11, 13, 11-CH3
10, 11, 12
3, 4, 10, 11
3, 11, 12, 11-CH3
9, 10, 12, 13, 11-CH3
10, 11-CH3
11, 12
71.0
20.4
19.1
14.8
6.0 (12)
s
d
8a
6, 7, 8
10, 11, 12
6.3 (11)
9, 10, 11, 12
a
As in Table 1.
As in Table 1.
b
observed. In comparison between 4 and 5 in NMR spectra, the
difference was the same as the above feature for 7 and chaeto-
mugilin A. Based on this evidence, we guessed that 4 was a epimer
of 5 at C-11. In future, we will carry out the modified Mosher’s
method to confirm the absolute configuration at C-12.
2.2. Cytotoxic activities
As a primary screen for antitumor activity, cancer cell growth
inhibitory properties of chaetomugilins P–R (1–3) and 11-epi-
chaetomugilin I (4) were examined using the murine P388 leuke-
mia cell line, the human HL-60 leukemia cell line, the murine
L1210 leukemia cell line, and the human KB epidermoid carcinoma
cell line. As shown in Table 5, 1 and 4 exhibited significant cyto-
toxic activity against the cancer cell lines, more than 5-FU used
as a positive control. This result suggests that the cytotoxicity re-
quires an enone moiety at C-20–C-40, because the activities of 1
and 4 were nearly equal to that of 5. Henceforth, compounds 1
and 2 will be examined using a disease-oriented panel of 39 hu-
man cell lines7,8 to reveal their selective cytotoxic activity and
mode of action.
Figure 5. Key NOEs for 3.
3. Conclusion
The marine fish-derived fungus C. globosum OUPS-T106B-6 pro-
duced chaetomugilins P–R (1–3) and 11-epi-chaetomugilin I (4) as
metabolites with growth inhibitory effects on cancer cells. Inter-
estingly, chaetomugilin P (1) had a unique skeleton and is a new
class of azaphilones. 1 and 4 have exhibited significant potent cyto-
toxicity, and their activities are equal to that of 5-fluorouracil in
IC50 value.
Figure 6. 1H chemical-shift difference (
esters (3a and 3b).
D
d = dS ꢀ dR) between the (R)- and (S)-MTPA
side chain moiety (C-9–C-13) [the proton signals; H-11 (dH 2.35,
dqd), H-12 (dH 3.74, quint), the carbon signal; 11-CH3 (dC 16.11)]
revealed a chemical shift difference. The 1H–1H COSY and HMBC
correlations (vide info) led to elucidation of the planar structure
of 4. In terms of the stereochemistry, the CD spectra of 4 and 5
showed similar Cotton curves, allowing for the assignment of the
absolute configuration of the asymmetric centers, except C-11
and C-12 (7S and 8S) (Fig. 3). This suggested that 4 was the stereo-
isomer of 5 at C-11 or C-12. Most chaetomugilins produced by this
fungal strain have the R configuration at C-11 and R configuration
at C-12, however, we had isolated 11-epi-chaetomugilin A (7),
which had the opposite absolute configuration at C-11 to chaeto-
mugilin A.9 The 1H and 13C NMR signals for the core part of 7 clo-
sely resembled those of chaetomugilin A. In the side chain moiety,
the chemical shift difference of the proton signals for H- 11 and
H-12 and the carbon signal for 11-CH3 (Table 4) had been
4. Experimental
4.1. General procedures
Mps were determined on a Yanagimoto micro-melting point
apparatus and are uncorrected. UV spectra were recorded on a Hit-
achi U-2000 spectrophotometer and IR spectra on a JASCO FT/IR-
680 plus. NMR spectra were recorded at 27 °C on Varian UNITY
INOVA-500 and MERCURY spectrometers with tetramethylsilane
(TMS) as an internal reference. FABMS was determined using a
JOEL JMS-700 (Ver. 2) mass spectrometer. Optical rotations were
recorded on a JASCO J-820 polarimeter. Liquid chromatography

T. Yamada et al. / Bioorg. Med. Chem. 19 (2011) 4106–4113
4111
Table 4
differential refractometer (R 401) and Shim-pack PREP-ODS
(25 cm ꢁ 20 mm i.d.). Analytical TLC was performed on precoated
Merck aluminium sheets (DC-Alufolien Kieselgel 60 F254,
0.2 mm) with the solvent system CH2Cl2–MeOH (19:1), and
compounds were viewed under a UV lamp, and sprayed with
10% H2SO4, and then heated.
(a) NMR spectroscopic data (CDCl3) for 11-epi-chaetomugilin I (4) and chaetomugilin
I
(5). (b) NMR spectroscopic data (CDCl3) for 11-epi-chaetomugilin
A
(7) and
chaetomugilin A
11-Epi-chaetomugilin I (4)
Chaetomugilin I (5)
a
a
Position
dH
dc
145.7
dH
dc
1
3
4
4a
5
6
7
8
8a
9
10
11
12
13
7-CH3
11-CH3
7.47
6.47
s
(d)
(s)
(d)
(s)
(s)
(s)
(s)
(d)
(s)
(d)
(d)
(d)
(d)
(q)
(q)
(q)
(t)
7.48
6.47
s
s
145.7
156.7
105.0
141.6
106.8
191.9
74.1
(d)
(s)
(d)
(s)
(s)
(s)
(s)
(d)
(s)
(d)
(d)
(d)
(d)
(q)
(q)
(q)
(t)
4.2. Culturing and isolation of metabolites
156.5
105.1
141.6
106.9
191.9
74.1
s
A strain of C. globosum was initially isolated from the marine
fish M. cephalus, collected in Katsuura bay, Japan in October
2000. The marine fish was wiped with EtOH and its gastrointesti-
nal tract applied to the surface of nutrient agar layered in a Petri
dish. Serial transfers of one of the resulting colonies provided a
pure strain of C. globosum. The fungal strain was cultured at
27 °C for six weeks in a liquid medium (50 L) containing 1% soluble
starch and 0.1% casein in 50% artificial seawater adjusted to pH 7.4.
The culture filtrate was extracted thrice with EtOAc. The combined
extracts were evaporated in vacuo to afford a mixture of crude
3.48
dd
40.5
3.48
dd
40.5
119.5
122.8
141.8
44.2
119.5
122.3
141.9
44.2
6.12
6.56
2.35
3.74
1.21
1.33
1.11
2.62
3.27
d
6.12
6.58
2.44
3.80
1.19
1.33
1.12
2.62
3.27
d
dd
dqd
Quint
d
s
d
dd
sex
Quint
d
s
d
71.1
70.8
20.4
26.7
14.8
21.0,
26.8,
16.1
metabolites (20.3 g), which exhibited cytotoxicity (ED50 35.8 lg/
10
10
20
30
40
50
A
B
dd
dd
34.8
dd
dd
34.8
ml). The EtOAc extract was passed through Sephadex LH-20, using
CHCl3/MeOH (1:1) as the eluent. The second fraction (7.2 g), in
which the activity was concentrated, was chromatographed on a
silica gel column with a CHCl3–MeOH gradient as the eluent. The
MeOH/CHCl3 (1:99) eluate (489.3 mg) was purified by HPLC using
MeCN/H2O (50:50) as the eluent to afford 1 (4.1 mg), 4 (2.9 mg)
and 5 (15.7 mg). The MeOH–CHCl3 (1:99) eluate (1.8 g) was puri-
fied by HPLC using MeCN/H2O (35:65) as the eluent, following
HPLC using MeCN/H2O (50:50) as the eluent, to afford 2 (7.4 mg)
and 7 (6.2 mg). The MeOH/CHCl3 (2:98) eluate (220.7 mg) was
purified by HPLC using MeCN/H2O (35:65) as the eluent to afford
3 (3.3 mg).
199.4
138.0
138.0
14.7
(s)
(s)
(d)
(q)
(q)
199.4
138.0
138.0
14.7
(s)
(s)
(d)
(q)
(q)
6.66
1.81
1.73
q
d
s
6.66
1.81
1.73
q
d
s
30-CH3
11.0
10.5
11-Epi-chaetomugilin A (7)
Chaetomugilin A
a
a
Position
dH
dc, multi.
dH
dc
1
3
4
4a
5
6
7
8
8a
9
10
11
12
13
7.29
6.57
s
s
145.5
157.1
105.4
140.2
110.3
189.2
83.8
(d)
7.27
s
s
145.7
(d)
(s)
(d)
(s)
(s)
(s)
(s)
(d)
(s)
(d)
(d)
(d)
(d)
(q)
(q)
(q)
(s)
(d)
(s)
(d)
(d)
(q)
(q)
(s)
(d)
(s)
(s)
(s)
(s)
(d)
(s)
(d)
(d)
(d)
(d)
(q)
(q)
(q)
(s)
(d)
(s)
(d)
(d)
(q)
(q)
157.1
105.5
140.1
110.3
189.3
84.0
6.57
Chaetomugilin P (1): yellow powder; mp 103–105 °C (CHCl3–
MeOH); ½a 2D2
ꢂ
ꢀ49.7 (c 0.06, EtOH); UV kmax (EtOH) nm (log
e) 283
3.00
d
50.4
2.98
d
50.6
(3.54), 384 (3.68), 415 (3.24); IR mmax (KBr) cmꢀ1 3440, 1678,
1666, 1563, 1522; HRFABMS m/z, found 407.1618 [M+H]+ (calcd
for C22H2835ClO5, 407.1626). 1H and 13C NMR data are listed in
Table 1.
114.4
122.5
142.5
44.8
71.0
20.9
114.3
122.1
142.5
44.3
6.16
6.62
2.39
3.77
1.22
1.40
1.12
d
6.15
6.61
2.45
3.81
1.20
1.40
1.13
d
dd
sex
Quint
d
s
d
dd
sex
br s
d
s
d
70.9
20.5
23.2
14.8
Chaetomugilin Q (2): yellow powder; mp 110–112 °C (CHCl3–
MeOH); ½a 2D2
ꢂ
ꢀ18.3 (c 0.14, EtOH); UV kmax (EtOH) nm (log
e) 291
7-CH3
11-CH3
10
23.3
16.1
(3.77), 373 (3.75), 412 (3.83); IR mmax (KBr) cmꢀ1 3407, 1642,
1560, 1522; HRFABMS m/z, found 425.1728 [M+H]+ (calcd for
170.7
58.2
170.7
58.2
104.2
44.9
20
3.05
d
3.06
d
C
22H3035ClO6, 425.1731). 1H and 13C NMR data are listed in Table
30
104.0
44.9
77.2
18.7
8.8
2 and S1 (Supplementary data).
40
1.90
4.31
1.41
1.13
dq
dq
d
1.90
4.30
1.41
1.13
dq
dq
d
50
76.9
18.7
8.8
Chaetomugilin R (3): yellow powder; mp 104–106 °C (CHCl3–
60
MeOH); ½a 2D2
ꢂ
ꢀ130.2 (c 0.11, EtOH); UV kmax (EtOH) nm (log
e)
40-CH3
d
d
331 (3.73), 371 (3.73), 393 (3.78); IR mmax (KBr) cmꢀ1 3424,
1666, 1564, 1547; HRFABMS m/z, found 329.1149 [M+H]+ (calcd
for C16H2235ClO5, 329.1156). 1H and 13C NMR data are listed in
Table 3.
a
As in Table 1
Table 5
Cytotoxity of the metabolites against P388, HL-60, L1210 and KB cells
11-Epi-chaetomugilin
I
(4) yellow powder; mp 85–87 °C
(CHCl3–MeOH); ½a D22
ꢂ
77.5 (c 0.12, EtOH); UV kmax (EtOH) nm (log e)
290 (3.87), 373 (3.84), 410 (3.84); IR mmax (KBr) cmꢀ1 3448, 1645,
1617, 1560, 1522; HRFABMS m/z, found 407.1618 [M+H]+ (calcd
for C22H2835ClO5: 391.1626). 1H and 13C NMR data are listed in
Table 4 and S3 (Supplementary data).
Compounds
Cell line
P388
Cell line
HL-60
Cell line
L1210
Cell line
KB
IC50 (pM)a IC50 (pM)a IC50 (pM)a IC50 (pM)a
Chaetomugilin I (5)
1.1
0.7
49.5
32.0
1.1
1.2
47.2
51.8
1.0
1.9
1.5
80.2
67.1
1.6
2.3
1.8
>100
67.1
1.2
P (1)
Q (2)
R (3)
4.3. Transformation of 1 and 2–5
11-Epichaetomugilin I (4) 0.7
5-FUb
1.7
2.7
1.1
7.7
p-TsOH (0.5 mg) was added to a MeOH solution (1 mL) of chae-
tomugilin P (1) (5.6 mg), and the reaction mixture was left at room
temperature for 3 h. The solvent was evaporated off under reduced
pressure, and the residue was purified by HPLC using MeCN/H2O
(50:50) as the eluent to afford 5 (1.2 mg).8
a
DMSO was used for vehicle.
Positive control.
b
Using the same proceudure as above with 1, chaetomugilin Q
(2) (10.5 mg) was treated with p-TsOH (1.9 mg) in MeOH (1 mL)
over silica gel (mesh 230–400) was performed at medium pressure.
HPLC was run on a Waters ALC-200 instrument equipped with a

4112
T. Yamada et al. / Bioorg. Med. Chem. 19 (2011) 4106–4113
and the products were purified by HPLC using MeCN/H2O (50:50)
761.1952); 1H NMR
d ppm (CDCl3): 1.10 (3H, d, J = 6.5 Hz,
as the eluent to afford 5 (2.1 mg).8
11-CH3), 1.26 (3H, d, J = 6.0 Hz, H-13), 1.35 (3H, s, 7-CH3), 2.61
(1H, dqd, J = 7.5, 6.5, 6.0 Hz, H-11), 3.02 (1H, ddd, J = 13.1, 11.5,
5.0 Hz, H-8a), 3.51 (3H, s, MTPA–OCH3), 3.59 (3H, s, MTPA–
OCH3), 3.86 (1H, dd, J = 13.1, 11.0 Hz, H-10a), 4.28 (1H, dd,
J = 11.0, 5.0 Hz, H-10b), 5.11 (1H, quint, J = 6.0 Hz, H-12), 5.37
(1H, d, J = 11.5 Hz, H-8), 5.95 (1H, d, J = 15.5 Hz, H-9), 6.00 (1H,
s, H-4), 6.48 (1H, dd, J = 15.5, 7.5 Hz, H-10), 7.37–7.42 (3H, m,
Ar–H), 7.42–7.47 (3H, m, Ar–H), 7.49–7.52 (2H, m, Ar–H),
7.67–7.70 (2H, m, Ar–H).
4.4. Formation of the acetonide 6 from 2
2,2-Dimethoxypropane (1 mL) and p-toluenesulfonate (3.6 mg)
were added to a CH2Cl2 solution (1 mL) of chaetomugilin Q (2)
(17.9 mg), and the reaction mixture was left at room temperature
for 2 h. The solvent was evaporated off under reduced pressure,
and the residue was purified by HPLC using MeCN/H2O (70:30)
as the eluent to afford the acetonide 6 (6.5 mg) as a yellow powder.
Acetonide 6: ½a 2D2
ꢂ
ꢀ105.0 (c 0.16, EtOH); FABMS m/z (rel. int.)
4.7. Cytotoxic assay
465 ([M+H]+, 100%); HRFABMS m/z found 465.2050 [M+H]+ (calcd
for C25H3435ClO6, 465.2054). 1H and 13C NMR data are listed in
Table 2 and S2 (Supplementary data).
Cytotoxic activities of chaetomugilins P–R (1–3) and 11-epi-
chaetomugilin I (4) were examined by the 3-(4, 5-dimethyl-2-
thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) method.
P388, HL-60, L1210 and KB cells were cultured in the Eagle’s
Minimum Essential Medium (10% fetal carf serum) at 37 °C in
5% CO2. The test material was dissolved in dimethyl sulfoxide
(DMSO) to give a concentration of 10 mm, and the solution
was diluted with the Essential Medium to give concentrations
4.5. Degradation by potassium hydroxide of 3
Chaetomugilin R (3) (18.6 mg) was dissolved in 10 mL of 5% aq
potassium hydroxide and stirred for 3 h at 100 °C. The reaction
mixture was extracted with 10 mL of CHCl3. The water layer was
adjusted to pH 3.0 with 9% sulfuric acid and reextracted with
10 mL of AcOEt. The organic extract was concentrated to dryness
in vacuo. The residue was purified by HPLC using a MeCN/H2O gra-
dient (0:100)–(60:40) as the eluent to afford (4S, 5S)-2E-5-hydro-
xy-4-methylhex-2-enoic acid (0.6 mg) as a colorless oil. Using the
same procedure, chaetomugilin I (5) (16.8 mg), whose absolute
stereostructure had been established, was treated with 5% aq
potassium hydroxide (10 mL), and purified by HPLC to afford (4R,
5R)-2E-5-hydroxy-4-methylhex-2-enoic acid (0.5 mg) as a color-
less oil.
of 200, 20 and 2 lM, respectively. Each solution was combined
with each cell suspension (1 ꢁ 105 cells/ml) in the medium,
respectively. After incubation at 37 °C for 72 h in 5% CO2, the
grown cells were labeled with 5 mg/ml MTT in phosphate-buf-
fered saline (PBS), and the absorbance of formazan dissolved
with 20% sodium dodecyl sulfate (SDS) in 0.1 N HCl was mea-
sured at 540 nm using microplate reader (Model 450, BIO-
RAD). Each value was expressed as a percentage, relative to a
control cell suspension prepared without the test substance. All
assays were performed three times, semilogarithmic plots were
constructed from the averaged data, and the effective dose of
the substance required to inhibit cell growth by 50% (IC50) was
determined.
(4R, 5R)-2E-5-hydroxy-4-methylhex-2-enoic acid: ½a D22
ꢂ
90.0 (c
0.05, EtOH); HRFABMS m/z: 145.0867 found [M+H]+ (calcd for
C7H13O3,145.0865); 1H NMR
d ppm (CDCl3): 1.12 (3H, d,
J = 6.5 Hz, 4-CH3), 1.19 (3H, d, J = 6.2 Hz, H-6), 2.44 (1H, dqd,
J = 7.5, 6.5, 6.2 Hz, H-4), 3.80 (1H, quint, J = 6.2 Hz, H-5), 5.90 (1H,
d, J = 15.5 Hz, H-2), 7.06 (1H, dd, J = 15.5, 7.5 Hz, H-3).
Acknowledgments
(4S, 5S)-2E-5-hydroxy-4-methylhex-2-enoic acid: ½a D22
ꢂ
–89.8 (c
We thank Dr. T. Ito (National Institute of Technology and
Evaluation, Biological Resource Center) and Dr. Endo (Kanazawa
University) for identification of the fungal strain, and for supply
of the cancer cells, respectively. We are grateful to Ms. M. Fujitake
and Dr. K. Minoura of this university for MS and NMR measure-
ments, respectively. This study was supported by a Grant-in-aid
for High Technology from the Ministry of Education, Culture,
Sports, Science and Technology, Japan.
0.06, EtOH); HRFABMS m/z: 145.0868 found [M+H]+ (calcd for
C7H13O3, 145.0865).
4.6. Formation of the (R)- and (S)-MTPA esters 3a and 3b from 3
(R)-MTPA (4.0 mg), dicyclohexylcarbodiimide (DCC) (4.0 mg)
and 4-(dimethylamino) pyridine (DMAP) (1.0 mg) were added to
a CH2Cl2 solution (0.5 mL) of chaetomugilin R (3) (2.0 mg), and
the reaction mixture was left at room temperature for 3 h. The sol-
vent was evaporated off under reduced pressure, and the residue
was purified by HPLC using MeCN/H2O (85:15) as the eluent to af-
ford (R)-MTPA ester 3a (1.8 mg) as a yellow powder. The same
reaction with 3 (2.3 mg) using (S)-MTPA (4.8 mg) gave the ester
3b (1.7 mg).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.05.008.
Compuond 3a: FABMS m/z (rel. int.) 761 ([M+H]+, 23.3%).
HRFABMS m/z found 761.1943 [M+H]+ (calcd for C36H3635ClF6O9,
761.1952); 1H NMR d ppm (CDCl3): 1.03 (3H, d, J = 6.8 Hz, 11-
CH3), 1.31 (3H, d, J = 6.5 Hz, H-13), 1.33 (3H, s, 7-CH3), 2.57
(1H, dqd, J = 8.0, 6.8, 6.5 Hz, H-11), 3.10 (1H, ddd, J = 13.1, 12.0,
5.0 Hz, H-8a), 3.55 (3H, s, MTPA–OCH3), 3.57 (3H, s, MTPA–
OCH3), 3.87 (1H, dd, J = 13.1, 11.0 Hz, H-10a), 4.38 (1H, dd,
J = 11.0, 5.0 Hz, H-10b), 5.13 (1H, quint, J = 6.5 Hz, H-12), 5.35
(1H, d, J = 12.0 Hz, H-8), 5.81 (1H, d, J = 15.5 Hz, H-9), 6.00 (1H,
s, H-4), 6.32 (1H, dd, J = 15.5, 8.0 Hz, H-10), 7.38-7.43 (3H, m,
Ar–H), 7.43–7.47 (3H, m, Ar–H), 7.52–7.56 (2H, m, Ar–H),
7.60–7.64 (2H, m, Ar–H).
References and notes
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Perkin Trans. 1 2001, 3046.
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Compuond 3b: FABMS m/z (rel. int.): 761 ([M+H]+, 14.9%).
HRFABMS m/z found 761.1940 [M+H]+ (calcd for C36H3635ClF6O9,

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