Tandyukisin, a novel ketoaldehyde decalin derivative, produced by a marine sponge-derived Trichoderma harzianum

Article abstract of DOI:10.1016/j.tetlet.2013.11.107

Tandyukisin (1), a novel decalin derivative with an enolic β-ketoaldehyde, has been isolated from a strain of Trichoderma harzianum OUPS-111D-4 originally derived from the marine sponge Halichondria okadai, and its structure has been elucidated on the basis of spectroscopic analyses using 1D and 2D NMR techniques. In addition, the absolute configuration for 1 was established by the application of CD spectrum to the tribenzoate derivative. This compounds exhibited moderate cytotoxicity against human cancer cell lines.

Full text of DOI:10.1016/j.tetlet.2013.11.107

Tetrahedron Letters 55 (2014) 662–664
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Tandyukisin, a novel ketoaldehyde decalin derivative, produced
by a marine sponge-derived Trichoderma harzianum
⇑
Takeshi Yamada , Yuki Mizutani, Yoshihide Umebayashi, Naoko Inno, Maiko Kawashima,
Takashi Kikuchi, 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:
Tandyukisin (1), a novel decalin derivative with an enolic b-ketoaldehyde, has been isolated from a strain
of Trichoderma harzianum OUPS-111D-4 originally derived from the marine sponge Halichondria okadai,
and its structure has been elucidated on the basis of spectroscopic analyses using 1D and 2D NMR tech-
niques. In addition, the absolute configuration for 1 was established by the application of CD spectrum to
the tribenzoate derivative. This compounds exhibited moderate cytotoxicity against human cancer cell
lines.
Received 18 September 2013
Revised 26 November 2013
Accepted 29 November 2013
Available online 4 December 2013
Keywords:
Decalin
Ó 2013 Elsevier Ltd. All rights reserved.
Trichoderma harzianum
Marine microorganism
Halichondria okadai
Cytotoxicity
Tandyukisin
Marine microorganisms are potentially prolific sources of
highly bioactive secondary metabolites that might serve as useful
leads in the development of new pharmaceutical agents. We have
focused on potential new antitumor materials from marine-
derived microorganisms that produce a number of compounds
with unique structures.1–3 As a part of this study, we have exam-
ined metabolites from the fungus Trichoderma harzianum
OUPS-T111D-4 originally obtained from the marine sponge Hali-
chondria okadai, and isolated a new compound together with two
known compounds, which have a decalin skeleton. The new com-
pound was designated tandyukisin (1).4 On the other hand, the
known compounds were identified with trichoharzin (2)5 and euj-
avanicol A (3).6 Trichoharzin5 isolated from the same class of fun-
gus, eujavanicols6 from Eupenicillium javanicum, betaenones7 from
Phoma betae, stemphyloxin I8 from Stemphylium botryosum, fusar-
ielins9 from Fusarium sp., pannomycin10 from Geomyces pannorum,
and australifungin from Spopormiella australis11 had been reported
previously as metabolites having a similar alkylated decalin skele-
ton. They have various bioactivities such as antifungal, phytotoxic,
and antibacterial activities, however cytotoxicities of these
compounds were not reported to date. We describe herein the elu-
cidation of the absolute stereostructure for 1, and the cytotoxic
activity of metabolites 1–3 (Fig. 1).
T. harzianum, the microorganism from H. okadai, was cultured at
27 °C for 6 weeks in a medium (70 L) containing 1% glucose, 1%
malt extract, and 0.05% pepton in artificial seawater adjusted to
pH 7.5. After incubation, the EtOAc extract of the culture filtrate
was purified by bioassay-directed fractionation (cytotoxicity
against P388 cells) employing a stepwise combination of silica
gel column chromatography and reverse phased HPLC to afford
tandyukisin (1) (1, 14.3 mg), trichoharzin (2, 5.4 mg),5 and eujava-
nicol A (3, 15.5 mg)6 as pale yellow oil, respectively (vide info.).
Tandyukisin (1) had the molecular formula C26H38O7 as estab-
lished from the [M]+ peak in HRFABMS. The UV spectrum of 1
exhibited absorption bands characteristic of a conjugated carbonyl
group. In addition, the IR spectrum exhibited absorption bands at
3424, 1718, and 1649 cm–1 characteristic of hydroxyl groups and
carbonyls. A close inspection of the 1H and 13C NMR spectra of 1
(Table 1) using DEPT and 1H–13C correlation spectroscopy (HMQC)
revealed the presence of three secondary methyls (C-16–C-18), a
tertiary methyl (C-19), an olefin methyl (C-60), a methoxy group
(50-OCH3), three sp3-hybridized methylenes (C-7, C-14 and C-40),
seven sp3-methines (C-5, C-6, C-8 – C-10, C-13 and C-15) including
two oxymethines (C-8 and C-9), a quaternary sp3-carbon (C-4), five
sp2-methines (C-1, C-2, C-11, C-12 and C-20) including an oxygen-
bearing carbon (C-1), a quaternary sp2-carbon (C-30), and three car-
bonyls (C-3, C-10and C-50). The 1H–1H COSY analysis of 1 led to four
partial structures including isobutyl groups (H-14/H-15, H-15/
H-16, and H-15/H-17), as shown by bold-faced lines in Figure 2.
The connection of these four units and the remaining functional
⇑
Corresponding author.
E-mail address: yamada@gly.oups.ac.jp (T. Yamada).
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.11.107

T. Yamada et al. / Tetrahedron Letters 55 (2014) 662–664
663
OH
OH
1
2
O
O
18
6
3
19
14
16
H
H
H
6'
3'
4
13
7
8
5
15
17
O
O
O
O
H
H
10
12
9
5'
1'
O
O
HO
O
11
4'
H
H
H
2'
OH
OH
1
2
OR
OH
O
O
H
H
H
H
RO
HO
H
H
4
5
R = H
OR
OH
R = p-Br-Bz
3
Figure 1.
Table 1
NMR spectral data for 1 in CDCl3
a
b
a
b
Position
dH
J/Hz
dC
Position
dH
J/Hz
dC
1
2
3
4
5
6
7.95
5.82
br s
d
175.8 (d)
99.8 (d)
205.5 (s)
49.0 (s)
43.2 (d)
31.0 (d)
39.0 (t)
14A
14B
15
16
17
18
19
10
0.78
m
m
m
d
d
d
25.8 (t)
4.3 (1)
1.54
1.32
0.91
0.79
0.74
1.20
36.8 (d)
19.2 (q)
12.1 (q)
21.0 (q)
19.2 (q)
166.2 (s)
119.3 (d)
152.1 (s)
45.9 (t)
6.8 (15)
6.8 (15)
7.2 (6)
1.97
1.67
1.88
1.55
5.26
3.57
2.14
6.05
5.74
1.83
t
m
dt
tq
10.5 (6,10)
7a
12.3 (7
12.3 (6, 7b), 2.5 (8)
2.5 (7 , 7b, 9)
10.5 (10), 2.5 (8)
a), 2.5 (6, 8)
s
7b
8
9
10
11
12
13
q
a
72.1 (d)
74.0 (d)
40.0 (d)
126.1 (d)
124.3 (d)
54.0 (d)
20
5.86
3.17
2.25
s
s
dd
tdd
ddd
ddd
m
30
10.5 (5, 9), 4.5 (11), 2.5 (12)
10.0 (12), 4.5 (10), 2.5 (13)
10.0 (11), 4.5 (13), 2.5 (10)
40
50
170.2 (s)
19.0 (q)
52.0 (q)
60
s
s
5-OCH3
3.722
a
Measured at 600 MHz in CDCl3.
Measured at 150 MHz in CDCl3.
b
groups was determined on the basis of the key HMBC correlations
summarized in Figure 2, and the planar structure of 1 was eluci-
dated. For the compound having the enol moiety in the side chain,
betaenones,7 stemphyloxin I,8 and australifungin11 had been re-
ported up to now. The geometrical configuration of two olefin moi-
eties (C-1–C-2 and C-20–C-30) in the side chains was deduced as cis
and trans from NOEs (H-1/H-2 and H-20/H-40), respectively.
The relative stereochemistry of 1 was deduced from NOESY
experiments (Fig. 3). NOE correlations (H-7b/H-5, H-7b/H-9 and
H-5/H-9) implied that the A ring existed in a chair conformation
with H-7b, H-9, and H-5 in coaxial arrangements. This evidence
OH
1
2
O
3
18
19
12
14
16
4
6'
6
9
7
13
5
15
17
O
5'
O
1'
10
8
3'
O
O
11
2'
4'
OH
and the vicinal proton coupling constants (J7 ,8 = J7b,8 = J9,8 = 2.5
a
1H-1H COSY
HMBC
Hz) showed that the esterified side chain at C-8 was oriented cis
to 9-OH in an axial arrangement. On the other hand, in the B ring,
observed NOEs (H-10/H-19, H-13/H-19 and H-5/H-14) demon-
strated a half boat conformation, and showed that an isobutyl
Figure 2. Typical 2D NMR correlations in 1.

664
T. Yamada et al. / Tetrahedron Letters 55 (2014) 662–664
the human HL-60 leukemia, and the murine L1210 leukemia cell
lines. 1 and 2 exhibited moderate cytotoxic activity against these
cancer cells, and 3 showed no activity. In this time, we could not
find the compound exhibiting potent activity. We continue to rese
the related compound from this fungal metabolite, and study the
structure-activity relationship (Table 2).
18
1
17
2
6
3
7
6'
4
8
5
15
2'
16
14
1'
Acknowledgements
9
19
11
10
3'
13
4'
We thank Dr. Endo (Kanazawa University) for supply of the
cancer cells. We are grateful to Ms. M. Fujitake and Dr. K. Minoura
of this university for MS and NMR measurements, respectively.
This study was supported by a Grant-in-aid for Scientific Research
from the Japan Society for the Promotion of Science.
5'
12
NOEs
Supplementary data
Figure 3. Observed NOEs for 1.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2013.11.
107.
Table 2
Cytotoxicity of 1–3 against three cancer cell lines
Compounds
Cell line P388
IC50 (mM)a
Cell line HL-60
IC50 (mM)a
Cell line LI210
IC50 (mM)a
References and notes
Tandyukisin (1)
Trichoharzin (2)
Eujavanicol A (3)
5-Fluorouracilb
54.5
22.5
N.Ac
2.5
42.2
14.8
N.A
2.2
41.3
23.4
N.A
2.1
1. Yamada, T.; Iritani, M.; Ohishi, H.; Tanaka, K.; Minoura, K.; Doi, M.; Numata, A.
Org. Biomol. Chem. 2007, 5, 3979–3986. and references cited therein.
2. Yamada, T.; Kitada, H.; Kajimoto, T.; Numata, A.; Tanaka, R. J. Org. Chem. 2010,
7512, 4146–4153.
3. Yamada, T.; Kikuchi, T.; Tanaka, R.; Numata, A. Tetrahedron Lett. 2012, 53,
2842–2846.
a
b
c
DMSO was used as vehicle.
Positive control.
No activity
4. (1): pale yellow oil, [a]D + 30.3 (c 0.11 in MeOH); kmax (EtOH)/nm 226 (log e
3.56), 272 (3.17); mmax (liquid)/cm–1 2958, 1718 and 1649; HRFABMS m/z
463.2697 [M+H]+ (calcd for C26H39O7: 463.2677). 1H and 13C NMR data are
listed in Table 1 and Table S1 (Supplementary Data).
5. Kobayashi, M.; Uehara, H.; Matsunami, K.; Aoki, S.; Kitagawa, I. Tetrahedron
Lett. 1993, 34, 7925–7928.
6. Nakadate, S.; Nozawa, K.; Horie, H.; Fujii, Y.; Nagai, M.; Hosoe, T.; Kawai, K.;
Yaguchi, T.; Fukushima, K. J. Nat. Prod. 2007, 70, 1510–1512.
7. Ichihara, A.; Oikawa, H.; Hayashi, K.; Sakamura, S. J. Am. Chem. Soc. 1983, 105,
2907–2908.
8. Barash, I.; Pupkin, G.; Netzer, D.; Kashman, Y. Plant Physiol. 1982, 69, 23–27.
9. Kobayashi, H.; Sunaga, R.; Furihara, K.; Morisaki, N.; Iwasaki, S. J. Antibiot. 1995,
48, 42–52.
10. Parish, C. A.; Cruz, M.; Smith, S. K.; Zink, D.; Baxter, J.; Tucker-Samaras, S.;
Collado, J.; Platas, G.; Bills, G.; Diez, M. T.; Vicente, F.; Pelaez, F.; Wilson, K. J.
Nat. Prod. 2009, 72, 59–62.
group was oriented cis to the ketoaldehyde chain in an axial
arrangement, allowing for the relative stereostructure of 1.
The absolute stereostructure of 1 was elucidated by the manner
shown as below. The treatment of 1 with NaOH in aqueous MeOH
gave the hydrolysis product 4, and then the acylation with
p-bromobenzoyl chloride led to the tribenzoate 5 (vide info.).5 In
the CD spectrum of 5, a negative Cotton effect (
D
e253 ꢀ18.3) and
a positive Cotton effect ( 234 +4.5) indicated that the screw sense
D
e
of two benzoate chromophores at C-8 and C-9 was counterclock-
wise, therefore, the absolute configurations for C-8 and C-9 were
revealed as R and S, respectively.
11. Otto, D. H.; Helms, Gregory L.; Tracy, T. J.; Guy, H. H. J. Org. Chem. 1995, 60,
1772–1776.
The cancer cell growth inhibitory properties of the decalin
derivatives 1–3 were examined using the murine P388 leukemia,