Trichodermates A-F, New Cytotoxic Trichothecenes from the Plant Pathogenic Fungus Trichoderma sp.

Article abstract of DOI:10.1002/hlca.201500182

Trichodermates A - F (1-6, resp.), six new trichothecene polyunsaturated octadioic acid esters, and (-)-harzianum B (7) were isolated from the fermentation extract of Trichoderma sp., a plant pathogenic fungus isolated from stem rot of an unidentified tree in Thailand. The structures of 1-7 were elucidated by NMR experiments. The absolute configuration at C(12) in 3 was assigned by in situ dimolybdenum circular dichroism method, whereas that in 7 was deduced after hydrolysis of 7 to 8 via modified Mosher's method. Compounds 1 and 2 showed modest cytotoxic activities against the K562 (human myelogenous leukemia) cell line with IC₅₀ values of 12.12 and 13.08 μM, respectively.

Full text of DOI:10.1002/hlca.201500182

Helv. Chim. Acta 2016, 99, 63 – 69
63
FULL PAPER
Trichodermates A – F, New Cytotoxic Trichothecenes from the Plant Pathogenic Fungus
Trichoderma sp.
a
b
b
b
a
b
b
b
b
by Jiaying Li ) ), Jinwei Ren ), Li Bao ), Tao Jin* ), Wenzhao Wang ), Yunfei Pei ), Xingzhong Liu ), and Erwei Li* )
a) Laboratory of Microbiology, College of Life Science, Heilongjiang University, Harbin 150080, P. R. China
b) State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, P. R. China
(
phone: þ 86-10-64806154; fax: þ 86-10-64807515; e-mail: liew@im.ac.cn)
Trichodermates A – F (1 – 6, resp.), six new trichothecene polyunsaturated octadioic acid esters, and (À)-harzianum B (7)
were isolated from the fermentation extract of Trichoderma sp., a plant pathogenic fungus isolated from stem rot of an
unidentified tree in Thailand. The structures of 1 – 7 were elucidated by NMR experiments. The absolute configuration at C(12)
in 3 was assigned by in situ dimolybdenum circular dichroism method, whereas that in 7 was deduced after hydrolysis of 7 to 8 via
modified Mosherꢀs method. Compounds 1 and 2 showed modest cytotoxic activities against the K562 (human myelogenous
leukemia) cell line with IC50 values of 12.12 and 13.08 mm, respectively.
Introduction. – Mycotoxins are toxic secondary metab-
Results and Discussion. – The AcOEt extract of solid-
olites produced by different types of fungi, belonging substrate fermentation of Trichoderma sp. was subjected to
mainly to the genera Aspergillus, Penicillium, Fusarium, chromatographic separation using silica gel, ODS, and
Cladosporium, and Alternaria [1]. Currently, more than 400 Sephadex LH-20, and finally purified by preparative HPLC
mycotoxins are known. The six major classes of frequently to yield six new trichothecene derivatives, trichodermates
occuring mycotoxins are aflatoxins, trichothecenes, fumo- A – F (1 – 6, resp.), and (À)-harzianum B (7). Compound 7
nisins, zearalenones, ochratoxins, and ergot alkaloids [1]. was assigned the same constitutional formula and relative
Trichothecenes, produced by several species of Fusarium, configuration as harzianum B by comparison of its
Myrothecium, Stachybotrys, Trichoderma, and Trichothe- spectroscopic data with the literature data [6]. However,
2
5
cium, are the largest group of mycotoxins known to date, the negative value of the optical rotation of 7 ([a] ¼
D
2
5
consisting of more than 180 structurally-related toxic À29.0 (c ¼ 0.10, CHCl ) as opposed to [a] ¼ þ27.94
3
D
compounds, but only a few are significant to human health (c ¼ 0.3, CHCl ) in [6]), indicated that 7 was the enan-
3
[
2]. Trichothecenes show various biological activities, such tiomer of (þ)-harzianum B. The absolute configuration
as cytotoxic and antitumor activities [3 – 6], and are of 7 was determined after hydrolysis of 7 to 8 by modified
extremely potent inhibitors of protein and/or DNA syn- Mosherꢀs method [12]. Compound 8 was treated with
thesis [7]. The 12,13-epoxide group of trichothecenes is (À)-(R)- and (þ)-(S)-a-methoxy-a-(trifluoromethyl)phe-
essential for the inhibition of protein synthesis, and nylacetyl chloride (MTPACl), respectively, to give the
reduction of the C(9)¼C(10) bond reduces toxicity [8]. corresponding (S)- and (R)-Mosher monoesters 8s and 8r.
1
Trichoderma is a genus of fungi that is present in all soils, Interpretation of the H-NMR chemical shift differences
where they are the most prevalent culturable fungi. Many (Dd ¼ d À d ) between 8s and 8r, established the absolute
S
R
species of this genus can be characterized as opportunistic configuration at C(4) as (R) (Fig. 2). Related to the
avirulent plant symbionts [9]. Some Trichoderma sp. absolute configuration at C(4), the absolute configuration
produce enzymes and antibiotics and were used as bio- of 7 was assigned as (2R,4R,5S,6R,11R,12S).
control agents against plant diseases [10].
Trichodermate A (1) was assigned the molecular
During an ongoing search for biologically active natural formula of C H O (nine degrees of unsaturation) on
2
4
32
6
1
13
products from plant endophytic or pathogenic fungi [11], the basis of HR-ESI-MS. Analysis of the H- and C-NMR
we initiated chemical investigation on Trichoderma sp. data (Table 1) revealed the presence of four Me (including
3
which was isolated from stem rot of an unidentified tree in one MeO), six CH , and three CHÀO groups, three sp
2
Thailand. Bioassay-directed fractionation of the AcOEt C -atoms (one O-bearing), six olefinic C-atoms (five H-
q
extract prepared from the solid-substrate fermentation bearing), and two carboxylate C-atoms (d(C) 173.3 and
products resulted in the isolation of six new trichothecene 166.4, resp.). These data accounted for all NMR resonan-
polyunsaturated octadioic acid esters, which we named ces of 1 and five of the nine degrees of unsaturation,
trichodermates A –F (1–6, resp.), and (À)-harzianum B (7; suggesting that 1 was a tetracyclic compound. Interpreta-
1
1
Fig. 1). Details of the isolation, structure elucidation, and tion of the H, H-COSY data of 1 revealed the presence
cytotoxic activities of these metabolites are reported herein. of three isolated spin systems, which were CH(2)À
DOI: 10.1002/hlca.201500182
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Helv. Chim. Acta 2016, 99, 63 – 69
Fig. 1. Structures of 1 – 8
due to an epoxide ring at C(12) and C(13). Therefore, the
constitutional formula of trichodermate A (1) was pro-
posed.
1
1
The relative configuration of 1 was deduced by H, H
coupling constants and NOESY data. The C(2’)¼C(3’)
bond was assigned (Z) geometry on the basis of the
coupling constant (J ¼ 11.4) observed between HÀC(2’)
and HÀC(3’), whereas the C(4’)¼C(5’) bond was assigned
(E) geometry due to the large coupling constant (J ¼ 15.2)
observed between HÀC(4’) and HÀC(5’). The NOESY
correlations HÀC(4)/HÀC(11), Me(15) and HÀC(11)/
Fig. 2. Dd (¼d À d ) Values obtained from MTPA esters 8r and 8s
S
R
CH (3)ÀCH(4), CH (7)ÀCH (8)ÀC(9)¼CH(10)ÀCH(11), H ÀC(3), Me(15) indicated that these H-atoms were
2
2
2
a
and CH(2’)¼CH(3’)ÀCH(4’)¼CH(5’)ÀCH (6’)ÀCH (7’). located at the same side of the 2-oxabicyclo[3.2.1]octane
2
2
In the HMBC spectrum of 1, the correlations Me(14)/ ring. In turn, the correlations HÀC(2)/H ÀC(13) and
a
C(5,6,12), Me(15)/C(5,6,7), C(11) (d(C) 70.7), HÀC(11) H ÀC(13)/H ÀC(7,8), Me(14) showed that these H-atoms
b
b
(
d(H) 3.63)/C(2) (d(C) 79.3), C(5,6,7), C(9) (d(C) 140.4), were located at the opposite side, thereby establishing the
C(10) (d(C) 118.8), C(15), and HÀC(2) (d(H) 3.83)/ relative configuration of trichodermate A (1). Considering
C(5,11,12) established the C(6)/C(11)-fused hexahydro- the identical relative configurations and similar optical
2
H-1-benzopyran moiety, with Me(14) and Me(15) attach- rotation values of 1 and 7, the absolute configuration of 1
ed to C(5) and C(6), respectively. The additional HMB was assigned as (2R,4R,5S,6R,11R,12S).
correlations Me(16)/C(8,9,10) and CH (8), HÀC(10)/
The elemental composition of trichodermate B (2) was
2
C(16) indicated that the third Me group was attached to determined to be C H O (ten degrees of unsaturation) by
24
30
6
C(9). The HMB correlations CH (3)/C(2,5,12), HÀC(4)/ HR-ESI-MS, which means that M is two mass units lower
2
r
C(2,5,6,12), and Me(14)/C(4), indicated that the fragment than for 1. Analysis of the NMR data (Table 1) revealed
C(3)ÀC(4) was connected to C(2) and C(5), respectively, that 2 possesses a similar structure to 1, except that two CH2
establishing the third ring fused via C(2)ÀC(12)ÀC(5). The groups (d(H) 2.53 – 2.52, d(C) 28.1 and d(H) 2.48 – 2.47,
HMBC cross peaks HÀC(4,3’)/C(1’) and CH (6’,7’)/C(8’) d(C) 33.2, resp.) in 1 were replaced by an olefin unit (d(H)
2
revealed that the 1’-O-1’,8’-dicarboxylate unit was attached 7.32, d(C) 143.2 and d(H) 6.07, d(C) 124.7, resp.) in 2. These
1
1
to C(4). In addition, the MeO group was located at C(8’) observations were supported by H, H-COSY and HMB
1
1
which was supported by the correlation Me(1’’)/C(8’). The correlations. By comparison of the H, H coupling con-
HMB correlations CH (13)/C(2,5,12) confirmed that C(13) stants and NOESY data of 2 with those of 1, the relative
2
was connected to C(12). Based on the chemical shifts of configuration of 2 was deduced to be the same as that of 1,
C(12) (d(C) 65.7) and C(13) (d(C) 48.0), the degrees of except for the configurations of the 1’,8’-dicarboxylate side
unsaturation, and the number of O-atoms in the molecular chain. The large coupling constants (J ¼ 15.4) observed
formula of 1, the remaining one degree of unsaturation was between HÀC(2’) and HÀC(3’), HÀC(6’) and HÀC(7’)
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65
Table 1. 1H- and 13C-NMR Data (500 and 125 MHz, resp.) for 1 – 3. d in ppm, J in Hz.
Posi- 1a)
2a)
3 )
b
tion
d(H)
d(C) HMBC
d(H)
d(C) d(H)
d(C)
2
3
3.83 (d, J ¼ 5.3)
79.3 4, 5, 11, 12
37.0 2, 4, 5, 12
3.84 (d, J ¼ 5.2)
79.3 4.10 (dd, J ¼ 11.5, 6.3)
36.8 2.34 (dd, J ¼ 11.4, 5.6),
1.75 (dd, J ¼ 11.4, 11.4)
75.5 5.13 (dd, J ¼ 11.6, 5.6)
49.4
77.9
37.9
2.55 (dd, J ¼ 15.5, 7.8),
2.57 (dd, J ¼ 15.5, 7.8),
2.03 (ddd, J ¼ 15.5, 5.3, 3.6)
5.65 (dd, J ¼ 7.8, 3.6)
2
.02 (ddd, J ¼ 15.5, 5.3, 3.6)
4
5
6
7
8
9
0
1
2
3
5.63 (dd, J ¼ 7.8, 3.6)
74.8 2, 5, 6, 12, 1’
49.2
40.6
24.6 6, 8, 9, 11
28.1 6, 7, 9, 10, 16
140.4
72.9
57.8
44.1
26.1
28.4
139.9
119.5
77.4
90.9
65.2
40.6
1.96 – 1.92 (m), 1.43 – 1.40 (m)
1.99 – 1.97 (m)
1.96 – 1.92 (m), 1.43 – 1.40 (m)
1.99 – 1.97 (m)
24.6 1.67 – 1.63 (m), 1.28 – 1.26 (m)
28.1 2.01 – 1.99 (m), 1.90 – 1.86 (m)
140.4
118.7 5.48 (d, J ¼ 5.5)
70.7 3.75 (d, J ¼ 5.5)
65.7
1
1
1
1
5.41 (d, J ¼ 5.4)
3.63 (d, J ¼ 5.4)
118.8 6, 8, 11, 16
5.41 (d, J ¼ 5.8)
70.7 2, 5 – 7, 9, 10, 15 3.63 (d, J ¼ 5.8)
65.7
3.12 (d, J ¼ 4.1),
48.0 2, 5, 12
3.13 (d, J ¼ 4.0),
2.83 (d, J ¼ 4.0)
0.72 (s)
0.95 (s)
1.71 (s)
48.0 3.79 (d, J ¼ 11.7),
2
.83 (d, J ¼ 4.1)
3.74 (d, J ¼ 11.7)
1
1
1
4
5
6
1
2
3
4
5
6
7
8
1
0.72 (s)
0.95 (s)
1.71 (s)
6.1 4 – 6, 12
16.2 5 – 7, 11
23.4 8 – 10
166.4
116.6 3’, 4’, 6’
145.2 1’, 4’, 5’
128.0 2’, 3’, 6’
142.9 3’, 4’, 6’, 7’
28.1 4’, 5’, 7’, 8’
33.2 5’, 6’, 8’
173.3
6.1 1.20 (s)
10.7
13.6
23.4
16.2 0.70 (s)
23.4 1.68 (s)
166.4
124.4 6.13 (d, J ¼ 15.4)
143.1 7.35 (dd, J ¼ 15.4, 10.3)
137.1 6.89 (dd, J ¼ 10.3, 10.3)
137.3 6.89 (dd, J ¼ 10.3, 10.3)
143.2 7.39 (dd, J ¼ 15.4, 10.3)
124.7 6.14 (d, J ¼ 15.4)
167.0
’
’
’
’
’
’
’
’
165.9
125.2
143.9
137.8
138.5
144.0
125.2
165.9
5.62 (d, J ¼ 11.4)
6.54 (t, J ¼ 11.4)
6.03 (d, J ¼ 15.4)
7.32 (dd, J ¼ 15.4, 10.2)
6.61 (dd, J ¼ 10.2, 10.2)
6.64 (dd, J ¼ 10.2, 10.2)
7.32 (dd, J ¼ 15.4, 10.2)
6.07 (d, J ¼ 15.4)
7.43 (dd, J ¼ 15.2, 11.4)
6.06 (dt, J ¼ 15.2, 6.6)
2.53 – 2.52 (m)
2.48 – 2.47 (m)
’’
3.68 (s)
51.8 8’
3.77 (s)
51.9
a) Recorded in CDCl3. b) Recorded in (D )acetone.
6
indicated that the C(2’)¼C(3’) and C(6’)¼C(7’) bonds solution of 3 in DMSO, a metal complex was generated as
possess (E) geometry, whereas the C(4’)¼C(5’) bond was an auxiliary chromophore. Since the contribution from the
assigned (Z) geometry due to the coupling constant (J ¼
inherent CD resulting from the C(1’)¼O group was
1
0.2) observed between HÀC(4’) and HÀC(5’). The subtracted to give the induced CD of the complex, the
absolute configuration of the tetracyclic structure skeleton observed sign of the Cotton effect in the induced spectrum
in 2 was determined to be the same as in 7 based on the originates solely from the chirality of the vic-diol moiety
identical relative configurations and similar optical rota- expressed by the sign of the OÀCÀCÀO torsion angle. The
tion values.
positive Cotton effect observed at 310 nm in the induced
The molecular formula of trichodermate C (3) was CD spectrum (Fig. 3) permitted assignment of the config-
established as C H O (nine degrees of unsaturation) on uration (12S) on the basis of the empirical rule proposed by
23
30
7
the basis of HR-ESI-MS data, which means that M is 18 Snatzke [15]. In agreement with the relative configuration
r
mass units higher than for 7. Analysis of the NMR data established by NOESY data, the configuration (2R,4R,
(
Table 1) revealed that 3 possesses a similar structure to 7, 5S,6R,11R,12S) was assigned for 3.
except that the chemical shifts of C(12) (d(C) 65.5) and The molecular formula of trichodermate D (4) was
CH (13) (d(H) 3.14 and 2.85, d(C) 47.8) in 7 were assigned as C H O (nine degrees of unsaturation) on the
2
24 32
5
obviously downfield shifted in 3 (d(C) 90.9 and d(H) 3.79 basis of HR-ESI-MS data. Analysis of the NMR data
and 3.74, d(C) 65.2, resp.). Considering the additional 18 (Table 2) revealed that 4 possesses a similar structure to 1,
mass units compared to 7, the epoxide ring at C(12) and except that the epoxide ring at C(12) (d(C) 65.7) and C(13)
C(13) was opened to a 1,2-diol moiety in 3. These (d(H) 3.12 and 2.83, d(C) 48.0) in 1 was replaced by a C¼C
observations were supported by the HMBC cross-peaks bond in 4 (d(C) 152.8 and d(H) 5.12 and 4.70, d(C) 105.1,
HÀC(2)/C(12,13), CH (3)/C(12), CH (13)/C(2,5,12), and resp.). These observations were supported by the HMBC
2
2
1
1
Me(14)/C(12). By comparison of the H, H coupling con- cross-peaks HÀC(2)/C(12,13), CH (3)/C(12), HÀC(4)/
2
stants and NOESY data of 3 with those of 2, the relative C(12), CH (13)/C(2,5,12), and Me(14)/C(12). The relative
2
configuration of 3 was deduced to be the same as that of 2. configuration of 4 was deduced to be the same as that of 1
1
1
The absolute configuration at C(12) of the acyclic by comparing their H, H coupling constants and NOESY
12,13-diol unit in 3 was assigned using the in situ data. The absolute configurations at C(2), C(4) – C(6), and
dimolybdenum circular dichroism (CD) method developed C(11) in 4 were the same as in 1, in view of their identical
by Frelek [13 – 15]. Upon addition of (AcO) Mo to a relative configurations and similar optical rotation values.
4
2
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Helv. Chim. Acta 2016, 99, 63 – 69
Fig. 3. CD Spectrum of 3 in DMSO containing (AcO) Mo with inherent CD spectrum subtracted
4
2
Table 2. 1H- and 13C-NMR Data (500 and 125 MHz, resp.; in CDCl ) for 4 – 6. d in ppm, J in Hz.
3
Position
4
5
6
d(H)
d(C)
d(H)
d(C)
d(H)
d(C)
2
3
4.43 (d, J ¼ 5.2)
78.9
38.1
3.84 (d, J ¼ 5.1)
79.5
36.9
3.87 (d, J ¼ 5.0)
79.5
37.0
2.59 (dd, J ¼ 15.4, 7.7),
2.51 (dd, J ¼ 15.4, 7.8),
2.01 (ddd, J ¼ 15.4, 5.0, 3.6)
5.55 (dd, J ¼ 7.8, 3.6)
2.53 (dd, J ¼ 15.4, 7.7),
2.02 (ddd, J ¼ 15.4, 5.0, 3.6)
5.57 (dd, J ¼ 7.7, 3.6)
1
.74 (ddd, J ¼ 15.4, 5.2, 3.5)
4
5
6
7
5.61 (dd, J ¼ 7.7, 3.5)
74.9
52.2
40.4
23.7
75.4
49.8
41.5
28.1
75.2
49.7
41.4
28.1
1.86 – 1.80 (m),
1.88 (dt, J ¼ 13.0, 3.4),
1.41 (br. d, J ¼ 13.0)
1.65 (br. d, J ¼ 13.2),
1.89 (dt, J ¼ 13.3, 3.7),
1.43 (br. d, J ¼ 13.3)
1.65 (br. d, J ¼ 13.0),
1.18, (dd, J ¼ 13.0, 2.8)
1.81 – 1.77 (m)
1.73 (br. d, J ¼ 14.5),
1.49 (dt, J ¼ 14.1, 2.8)
3.57 – 3.55 (m)
1.30 – 1.26 (m)
8
9
1.98 – 1.95 (m)
28.2
24.7
24.7
1
.17 (dd, J ¼ 13.0, 2.7)
140.0
119.0
1.81 – 1.77 (m)
34.7
30.3
34.7
30.3
1
0
5.37 (d, J ¼ 5.2)
3.66 (d, J ¼ 5.2)
1.72 (br. d, J ¼ 15.2),
1
.48 (dt, J ¼ 15.4, 2.9)
1
1
1
1
1
1
1
2
3
4
5
6
7
8
1
1
2
3
4
5
6
’
’
’
’
’
70.6
152.8
105.1
10.4
16.4
23.4
166.2
116.5
145.2
127.9
142.8
28.2
33.2
173.2
51.9
3.56 – 3.54 (m)
72.4
65.8
48.7
5.8
17.8
72.4
65.8
48.7
6.0
17.9
5.12 (s), 4.70 (s)
0.95 (s)
0.95 (s)
3.17 (d, J ¼ 4.0), 2.87 (d, J ¼ 4.0)
0.69 (s)
1.03 (s)
3.19 (d, J ¼ 4.0), 2.89 (d, J ¼ 4.0)
0.70 (s)
1.04 (s)
1.68 (s)
3.45 – 3.43 (m), 3.48 – 3.46 (m)
68.2
3.45 – 3.43 (m), 3.50 – 3.48 (m)
68.2
166.3
125.2
143.0
137.9
136.9
144.9
123.8
170.7
166.8
121.6
142.8
136.7
137.5
145.8
123.6
171.1
5.57 (d, J ¼ 11.4)
6.53 (t, J ¼ 11.4)
6.09 (d, J ¼ 15.3)
5.86 (d, J ¼ 11.4)
7.33 (dd, J ¼ 15.3, 10.2)
6.65 (dd, J ¼ 10.2, 10.2)
6.66 (dd, J ¼ 10.2, 10.2)
7.38 (dd, J ¼ 15.3, 10.2)
6.02 (d, J ¼ 15.3)
6.65 (dd, J ¼ 11.4, 11.4)
7.93 (dd, J ¼ 14.8, 11.4)
6.56 (dd, J ¼ 14.8, 11.4)
7.50 (dd, J ¼ 15.3, 11.4)
6.02 (d, J ¼ 15.3)
7.38 (dd, J ¼ 15.2, 11.4)
6.06 (dt, J ¼ 15.2, 6.6)
2.53 – 2.51 (m)
’
’
’
’’
2.47 – 2.46 (m)
3.69 (s)
The molecular formula of trichodermate E (5) was C(9,10). Furthermore, Me(16) in 7 was oxidized to a
assigned as C H O (nine degrees of unsaturation) on the HOCH group in 5 (d(H) 3.48 – 3.46 and 3.45 – 3.43, d(C)
23
30
7
2
basis of HR-ESI-MS data. Analysis of the NMR data 68.2), which was confirmed by the HMB correlations
(
Table 2) revealed that 5 possesses a similar structure to 7, HÀC(9)/C(16) and CH (16)/C(8,9,10). HÀC(9) in 5 pos-
2
except that the C(9)¼C(10) bond in 7 was hydrogenated to sesses the same orientation as HÀC(4), HÀC(11), and
one CH (d(H) 1.81 – 1.77, d(C) 34.7) and one CH group Me(15) as supported by the NOESY correlations HÀC(4)/
2
(
d(H) 1.72 and 1.48, d(C) 30.3). These observations were HÀC(11), Me(15), and HÀC(11)/HÀC(9), Me(15), and
supported by the HMBC cross-peaks CH (8)/C(9,10), CH (16)/H ÀC(13). The relative configurations at the
2
2
a
HÀC(9)/C(7,8), CH (10)/C(6,8,9,11), and HÀC(11)/ other C-atoms in 5 were deduced to be the same as in 7
2
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Helv. Chim. Acta 2016, 99, 63 – 69
67
1
1
by comparing their H, H coupling constants and NOESY
data. Considering the similar optical rotation values, and
the biosynthesis homology of 5 and 7, and in agreement
with the relative configuration determined by NOESY
data, the absolute configuration of 5 was assigned as
UV detector. Optical rotations: Anton Paar MCP 200 automatic
polarimeter. UV Spectra: Thermo Genesys-10S UV/VIS spectropho-
tometer; lmax (log e) in nm. CD Spectra: Applied Photophysics
Chirascan spectropolarimeter; l in nm. IR Spectra: Nicolet IS5 FT-
max
À1
1
13
IR spectrophotometer; n˜in cm . H- and C-NMR spectra: Bruker
Avance-500 spectrometer (500 and 125 MHz, resp.; Bruker, Rheinstet-
(2R,4R,5S,6R,9R,11R,12S).
ten, Germany); d in ppm rel. to residual solvent peaks (CDCl : d(H)
3
The molecular formula of trichodermate F (6) was
7.26, d(C) 77.7; (D )acetone: d(H) 2.05, d(C) 29.8 and 206.1), J in Hz.
HMQC and HMBC experiments were optimized for 145.0 and 8.0 Hz,
resp. ESI-MS and HR-ESI-MS: Agilent Accurate-Mass-Q-TOF LC/MS
6
assigned as C H O (nine degrees of unsaturation) on the
23
30
7
basis of HR-ESI-MS data, which is same as of 5.
Comparison of the NMR data (Table 2) with those of 5
suggested 6 to possess a similar structure to 5; they were
only differing in the chemical shifts and coupling constants
of the C(1’) – C(8’) side chain. The (2’Z,4’E,6’E) geometry
in 6, instead of (2’E,4’Z,6’E) geometry occurring in 5, was
deduced by the coupling constants observed between
HÀC(2’) and HÀC(3’) (J ¼ 11.4), HÀC(4’) and HÀC(5’)
6
520 instrument; in m/z. The fragmentor and capillary voltages were
kept at 125 and 3500 V, resp. N was supplied as nebulizing and drying
2
gas. The temp. of the drying gas was set at 3008. The flow rate of the
À1
drying gas and the pressure of the nebulizer were 10 l min and 10 psi,
resp. All MS experiments were performed in positive-ion mode. Full-
scan spectra were acquired at 1.03 spectra/s.
Fungal Material. The culture of Trichoderma sp. was isolated from
the stem rot of an unidentified tree collected in Thailand in August
2
012. The isolate was identified by X. L. based on morphology and
(
J ¼ 14.8), and HÀC(6’) and HÀC(7’) (J ¼ 15.3). The
absolute configuration of 6 was assigned to be (2R,4R,
S,6R,9R,11R,12S) as for 5, based on the same relative
sequence (Genbank Accession No. KP317835) analysis of the ITS
region of the rDNA. The fungal strain was cultured on slants of potato
dextrose agar (PDA) at 258 for 5 d. Agar plugs were cut into small
pieces (ca. 0.5 Â 0.5 Â 0.5 cm) under aseptic conditions, 15 pieces were
used to inoculate three Erlenmeyer flasks (250 ml), each containing
5
configuration and the obvious biogenetic homology.
Compounds 1 – 7 and hydroxylation product 8 were
tested for their cytotoxic activities against K562 (human
myelogenous leukemia), HCT116 (human colon carcino-
ma), and HepG2 (human hepatocellular liver carcinoma)
cell lines by a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-
methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)
assay. Compounds 1, 2, and 7 showed modest cytotoxic
effects against K562 cells with IC50 values of 12.12 Æ 1.03,
5
0 ml of media (0.4% glucose, 1% malt extract, and 0.4% yeast extract;
the final pH of the media was adjusted to 6.5, and the medium was
sterilized by autoclave). Three flasks of the inoculated media were
incubated at 258 on a rotary shaker at 170 rpm for 5 d to prepare the
seed culture. Fermentation was carried out in 60 Fernbach flasks
(500 ml), each containing 80 g of rice. Dist. H O (120 ml) was added to
2
each flask, and the contents were soaked overnight before autoclaving
at 15 psi for 30 min. After cooling to r.t., each flask was inoculated with
5
.0 ml of the spore inoculum and incubated at 258 for 40 d.
1
3.08 Æ 1.12, and 25.17 Æ 3.09 mm, respectively (the positive
Extraction and Isolation. The fermented material was extracted
control taxol showed an IC value of 0.34 Æ 0.08 mm). In
5
0
repeatedly with AcOEt (4 Â 9.0 l), and the org. solvent was evaporated
contrast, 1 – 8 did not show detectable cytotoxic activities
against HCT116 and HepG2 cell lines at 100 mm.
to dryness in vacuo to afford the crude extract (35.0 g), which was
fractionated by reversed-phase (RP) CC (ODS; H O/MeOH, gra-
2
dient) to obtain 20 fractions. The fraction (1.5 g) eluted with 65%
MeOH was separated by CC (SiO ; CH Cl /MeOH, gradient) to give
Trichodermates A – F (1 – 6, resp.) represent further
examples of trichothecene sesquiterpenoid polyunsaturat-
ed octadioic acid esters [6]. Compounds 1 and 2 are
structurally closely related to harzianum B (7) [6], except
that the terminal COOH group is methyl-esterified and the
geometry of the C(2’)¼C(3’) and C(4’)¼C(5’) bonds differs.
In addition, the C(6’)¼C(7’) bond is hydrogenated in 1.
Compounds 3 and 4 are new trichothecene sesquiterpenes
with the 12,13-expoxide moiety opened to form a vic-diol
and an olefin unit, respectively. Compounds 5 and 6 differ
2
2
2
1
5 subfractions, Frs. 1 – 15. Fr. 2 (25 mg) was separated by semi-prep.
À1
RP-HPLC (MeOH/H O 45 :55 over 45 min; 2 ml min ) to afford 4 (tR
2
2
(
7.0 min; 2.3 mg). Using the same conditions as described above, Fr. 4
40 mg) was separated to afford 1 (tR 18.3 min; 9.6 mg) and 2 (tR
20.5 min; 3.5 mg), Fr. 6 (50 mg) was separated to afford 7 (tR
34.5 min; 18.0 mg), and Fr. 13 (40 mg) was separated to afford 3 (t
R
2
5.0 min; 3.4 mg). Fr. 2 (650 mg) was separated by CC (Sephadex LH-
2
0; MeOH) and the resulting subfractions were further purified by
À1
semi-prep. RP-HPLC (MeOH/H O 55 :45 over 50 min; 2 ml min ) to
2
afford 5 (tR 29.0 min; 6.5 mg) and 6 (tR 40.0 min; 6.7 mg).
from harzianum B (7) in having a HOCH group instead of
2
Trichodermate A (¼ 8-Methyl 1-[(2R,4R,5S,5aR,9aR)-2,3,4,5,
5a,6,7,9a-Octahydro-5,5a,8-trimethylspiro[2,5-methano-1-benzoxepine-
10,2’-oxiran]-4-yl] (2Z,4E)-Octa-2,4-dienedioate; 1). Colorless oil.
a Me group linked to C(9), and in the hydrogenation of the
3
C(9)¼C(10) bond to sp CH and CH C-atoms.
2
2
5
[
a] ¼ À20.0 (c ¼ 0.10, MeOH). UV (MeOH): 302 (3.88). IR (neat):
D
1
13
We gratefully acknowledge financial support from the National
Program of Drug Research and Development (2012ZX09301002-003),
National Natural Science Foundation of China (21302216), and Open
Project of Key Laboratory of Microbiology in Helongjiang Province
3673 (br.), 2972, 1654, 1032. H- and C-NMR, and HMBC: see
Table 1. Key NOESY correlations (500 MHz, CDCl
): HÀC(2)/
3
H
ÀC(13); H ÀC(3)/HÀC(11); HÀC(4)/HÀC(11), Me(15); HÀC(11)/
a
a
Me(15); H
ÀC(13)/H
ÀC(7,8), Me(14). HR-ESI-MS: 417.2272 ([M þ
b
b
þ
þ
(
2012MOI-4).
H] , C24
H
33
O
6
; calc. 417.2272).
Trichodermate B (¼ Methyl (2R,4R,5S,5aR,9aR)-2,3,4,5,5a,6,7,9a-
Octahydro-5,5a,8-trimethylspiro[2,5-methano-1-benzoxepine-10,2’-ox-
2
5
iran]-4-yl (2E,4Z,6E)-Octa-2,4,6-trienedioate; 2). Colorless oil. [a]
D
¼
Experimental Part
À25.0 (c ¼ 0.14, MeOH). UV (MeOH): 302 (4.44). IR (neat): 3689
1
13
General. Column chromatography (CC): silica gel (SiO ; 300 –
(br.), 2889, 1653, 1037. H- and C-NMR: see Table 1. HMB
Correlations (500 MHz, CDCl (3)/
): HÀC(2)/C(4,5,11,12); CH
C(2,4,5,12); HÀC(4)/C(1’,2,6,12); CH (7)/C(6,8,9,11); CH (8)/
C(6,7,9,10);
HÀC(11)/C(2,6,7,9,10,15);
CH
2
4
5
00 mesh; Qingdao Haiyang Chemical Co., Ltd.), ODS (12 nm; S-
0 mm, YMC Co., Ltd.), and Sephadex LH-20 (GE Healthcare Bio-
3
2
2
2
Sciences AB). HPLC: Waters 1525 instrument (Waters, USA); Waters
HÀC(10)/C(6,8,11,16);
ꢂ
Symmetry Prep C18 column (7.8 Â 300 mm, 7 mm); variable wavelength
2
(13)/C(2,5,12); Me(14)/C(4,5,6,12); Me(15)/C(5,6,7,11); Me(16)/
www.helv.wiley.com
ꢁ
2016 Verlag Helvetica Chimica Acta AG, Zürich

6
8
Helv. Chim. Acta 2016, 99, 63 – 69
C(8,9,10); HÀC(2’)/C(1’,3’,4’); HÀC(3’)/C(1’,2’,5’); HÀC(4’)/C(2’,3’,6’);
HÀC(5’)/C(3’,4’,7’); HÀC(6’)/C(4’,7’,8’); HÀC(7’)/C(5’,6’,8’); Me(1’’)/
pine-10,2’-oxiran]-4-yl]oxy}-8-oxoocta-2,4,6-trienoic Acid; 7). Color-
less oil. [a]2
5
¼ À29.0 (c ¼ 0.10, CHCl ).
D
3
C(8’). Key NOESY correlations (500 MHz, CDCl ): HÀC(2)/
Hydrolysis of 7. To a soln. (2.0 ml) of 7 (6.0 mg, 15 mmol) in
CH Cl /MeOH 9 :1, MeONa (3.3 mg, 60 mmol) was added, and the
3
H ÀC(13); H ÀC(3)/HÀC(11); HÀC(4)/HÀC(11), Me(15); HÀC(11)/
a
a
2
2
Me(15); H ÀC(13)/H ÀC(7,8), Me(14). HR-ESI-MS: 415.2115 ([M þ
mixture was stirred for 1 h. The solvents were removed in vacuo, the
residue was extracted repeatedly with CH Cl (4 Â 1.0 ml), and the org.
b
b
þ
þ
6
H] , C H O ; calc. 415.2115).
24
31
2
2
Trichodermate C (¼(2E,4Z,6E)-8-{[(2R,4R,5S,5aR,9aR)-2,3,4,5,
a,6,7,9a-Octahydro-10-hydroxy-10-(hydroxymethyl)-5,5a,8-trimethyl-
,5-methano-1-benzoxepin-4-yl]oxy}-8-oxoocta-2,4,6-trienoic Acid; 3).
solvent was evaporated to dryness in vacuo. The product was purified
À1
5
2
by semi-prep. RP-HPLC (MeOH/H O 45: 55 over 45 min; 2 ml min
;
2
detected at 195 nm [16]) to afford 8 (t 31.0 min; 2.6 mg). Colorless
R
25
25
1
Colorless oil. [a]D ¼ À3.6 (c ¼ 0.14, MeOH). UV (MeOH): 302 (4.32).
powder. [a]D ¼ À12.0 (c ¼ 0.16, MeOH). H-NMR (500 MHz, CDCl ):
3
1
13
IR (neat): 3675 (br.), 2981, 1685, 1032. H- and C-NMR: see Table 1.
5.39 (d, J ¼ 5.5, HÀC(10)); 4.33 (dd, J ¼ 7.5, 2.9, HÀC(4)); 3.82 (d, J ¼
HMB Correlations (500 MHz, (D )acetone): HÀC(2)/C(3,12,13);
5.4, HÀC(2)); 3.51 (d, J ¼ 5.5, HÀC(11)); 3.10 (d, J ¼ 3.9, H ÀC(13));
6
a
CH (3)/C(2,4,5,12); HÀC(4)/C(1’,3,5,6,14); CH (7)/C(6,8,9,11,15);
2.81 (d, J ¼ 3.9, H ÀC(13)); 2.61 (dd, J ¼ 15.7, 7.5, H ÀC(3)); 1.99 – 1.97
2
2
b
a
CH (8)/C(6,7,9,10); HÀC(10)/C(6,8,11,16); HÀC(11)/C(2,6,7,9,10,15);
(m, H ÀC(3), CH (8)); 1.92 – 1.90 (m, H ÀC(7)); 1.70 (s, Me(16));
2
b
2
a
CH (13)/C(2,5,12); Me(14)/C(4,5,6,12); Me(15)/C(5,6,7,11); Me(16)/
1.46 – 1.42 (m, H ÀC(7)); 0.85 (s, Me(15)); 0.80 (s, Me(14)).
2
b
C(8,9,10); HÀC(2’)/C(1’,3’,4’); HÀC(3’)/C(1’,2’,5’); HÀC(4’)/C(2’,3’,6’);
HÀC(5’)/C(3’,4’,7’); HÀC(6’)/C(4’,7’,8’); HÀC(7’)/C(5’,6’,8’). Key NO-
ESY correlations (500 MHz, (D )acetone): HÀC(2)/H ÀC(13);
Preparation of (R)- and (S)-MTPA Esters (¼ 8r and 8s). A sample
of
8 (1.1 mg, 4.4 mmol), (S)-MTPACl (5.0 ml, 26 mmol), and
(D )pyridine (0.5 ml) were quickly added to a clean NMR tube at
6
a
5
HÀC(4)/HÀC(11), Me(15); HÀC(11)/Me(15); H ÀC(13)/Me(14).
ambient temp., and all compounds were mixed thoroughly by shaking
b
þ
þ
1
HR-ESI-MS: 419.2064 ([M þ H] , C H O
7
; calc. 419.2064).
the NMR tube carefully. The H-NMR spectrum of the (R)-MTPA
23
31
Trichodermate D (¼ 8-Methyl 1-[(2R,4R,5S,5aR,9aR)-2,3,4,5,
tbcrules=bot5a,6,7,9a-Octahydro-5,5a,8-trimethyl-10-methylidene-2,5-
methano-1-benzoxepin-4-yl] (2Z,4E)-Octa-2,4-dienedioate; 4). Color-
ester derivative was recorded directly every 4 h, and the reaction was
1
found to be complete after 12 h. H-NMR (500 MHz, (D )pyridine) for
5
8r: 5.96 (dd, J ¼ 7.7, 3.5, HÀC(4)); 5.52 (d, J ¼ 5.5, HÀC(10)); 3.92 (d,
J ¼ 5.2, HÀC(2)); 3.68 (d, J ¼ 5.5, HÀC(11)); 3.10 (d, J ¼ 4.2,
H ÀC(13)); 2.82 (d, J ¼ 4.2, H ÀC(13)); 2.68 (dd, J ¼ 15.4, 7.7,
less oil. [a]2 ¼ À26.5 (c ¼ 0.16, MeOH). UV (MeOH): 302 (4.26). IR
5
D
1
13
(
neat): 3670 (br.), 2732, 1655, 1037. H- and C-NMR: see Table 2.
a
b
HMB Correlations (500 MHz, CDCl
3
): HÀC(2)/C(4,5,11,13); CH
2
(3)/
H
a
ÀC(3)); 2.08 (ddd, J ¼ 15.4, 5.2, 3.5, H ÀC(3)); 1.93 – 1.91 (m,
b
C(2,5,12); HÀC(4)/C(1’,2,6,12); CH (7)/C(6,8,9,11); CH (8)/
H ÀC(8)); 1.90 – 1.88 (m, H ÀC(8)); 1.85 – 1.83 (m, H ÀC(7)); 1.63 (s,
2
2
a
b
a
C(6,7,9,10); HÀC(10)/C(6,8,11,16); HÀC(11)/C(7,9,10,15); CH (13)/
Me(16)); 1.30 – 1.27 (m, H ÀC(7)); 0.93 (s, Me(15)); 0.91 (s, Me(14)).
2
b
C(2,5,12); Me(14)/C(4,5,6,12); Me(15)/C(5,6,7,11); Me(16)/C(8,9,10);
HÀC(2’)/C(1’,4’); HÀC(3’)/C(1’,5’); HÀC(4’)/C(2’,3’,6’); HÀC(5’)/
C(3’,6’,7’); HÀC(6’)/C(3’,4’,7’,8’); HÀC(7’)/C(5’,6’,8’); Me(1’’)/C(8’).
Key NOESY correlations (500 MHz, CDCl ): HÀC(2)/H ÀC(13);
Similarly, a mixture of 8 (1.1 mg, 4.4 mmol), (R)-MTPACl (5.0 ml,
26 mmol), and (D )pyridine (0.5 ml) was processed as described above
5
1
for 8r to afford 8s. H-NMR (500 MHz, (D )pyridine) for 8s: 5.89 (dd,
5
J ¼ 7.6, 3.3, HÀC(4)); 5.52 (d, J ¼ 5.8, HÀC(10)); 3.96 (d, J ¼ 5.2,
3
a
H ÀC(3)/HÀC(11); HÀC(4)/HÀC(11), Me(15); HÀC(11)/Me(15);
HÀC(2)); 3.68 (d, J ¼ 5.5, HÀC(11)); 3.11 (d, J ¼ 4.2, H ÀC(13)); 2.79
a
a
þ
þ
H ÀC(13)/Me(14). HR-ESI-MS: 401.2325 ([M þ H] , C H O ; calc.
(d, J ¼ 4.2, H ÀC(13)); 2.75 (dd, J ¼ 15.4, 7.7, H ÀC(3)); 2.20 (ddd, J ¼
b
24 33
5
b
a
4
01.2323).
15.4, 5.2, 3.5, H ÀC(3)); 1.93 – 1.91 (m, H ÀC(8)); 1.90 – 1.88 (m,
b
a
Trichodermate E (¼(2E,4Z,6E)-8-{[(2R,4R,5S,5aR,8R,9aR)-Deca-
H ÀC(8)); 1.85 – 1.83 (m, H ÀC(7)); 1.63 (s, Me(16)); 1.30 – 1.27 (m,
b
a
hydro-8-(hydroxymethyl)-5,5a-dimethylspiro[2,5-methano-1-benzoxe-
H ÀC(7)); 0.90 (s, Me(15)); 0.75 (s, Me(14)).
b
pine-10,2’-oxiran]-4-yl]oxy}-8-oxoocta-2,4,6-trienoic Acid; 5). Color-
Absolute Configuration at C(12) of the 12,13-Diol Moiety in 3 [13].
HPLC Grade DMSO was dried (4 Š molecular sieves). According to
the published procedure, a 1:1.5 mixture of diol 3/(AcO) Mo was
less oil. [a]2 ¼ À27.0 (c ¼ 0.10, MeOH). UV (MeOH): 302 (4.80). IR
5
D
1
13
(
neat): 3687 (br.), 1699, 1081. H- and C-NMR: see Table 2. HMB
4
2
À1
Correlations (500 MHz, CDCl ): HÀC(2)/C(4,5,11,13); CH (3)/
subjected to CD measurements at a concentration of 0.2 mg ml . The
first CD spectrum was recorded immediately after mixing, and its time
evolution was monitored until stationary (ca. 10 min after mixing). The
inherent CD was subtracted. The observed sign of the diagnostic band
at 310 nm in the induced CD spectrum correlated with the absolute
configuration at C(12) in the 12,13-diol moiety.
3
2
C(2,5,12); HÀC(4)/C(1’,2,6,12); CH (7)/C(6,8,9,11,15); CH (8)/
2
2
C(6,7,9,10); HÀC(9)/C(7,8,16); CH (10)/C(6,8,9,11); HÀC(11)/
2
C(7,9,10,15); CH (13)/C(2,5,12); Me(14)/C(4,5,6,12); Me(15)/
2
C(5,6,7,11); CH (16)/C(8,9,10); HÀC(2’)/C(1’,3’,4’); HÀC(3’)/C(1’,5’);
2
HÀC(4’)/C(2’,3’,6’); HÀC(5’)/C(3’,6’,7’); HÀC(6’)/C(4’,7’,8’); HÀC(7’)/
C(5’,6’,8’). Key NOESY correlations (500 MHz, CDCl ): HÀC(2)/
MTS Assay [17]. In a 96-well plate, each well was plated with (2 –
5) · 103 cells (depending on the cell multiplication rate). After cell
attachment overnight, the medium was removed, and each well was
treated with 100 ml of medium containing 0.1% DMSO, or appropriate
concentrations of the test compounds and the positive control cisplatin
(100 mm stock soln. in DMSO and serial dilutions). The plate was
incubated for 48 h at 378 in a humidified 5%-CO2 atmosphere.
Proliferation was assessed by adding 20 ml of MTS (Promega) to each
well in the dark, followed by 90 min incubation at 378. The assay plate
was read at 490 nm using a microplate reader. The assay was run in
triplicate.
3
H ÀC(13); H ÀC(3)/HÀC(11); HÀC(4)/HÀC(11), Me(15); HÀC(11)/
a
a
HÀC(9), Me(15); H ÀC(13)/CH (16); H ÀC(13)/Me(14). HR-ESI-
a
2
b
þ
þ
7
MS: 419.2069 ([M þ H] , C H O , calc. 419.2064).
23
31
Trichodermate F (¼(2E,4E,6Z)-8-{[(2R,4R,5S,5aR,8R,9aR)-Deca-
hydro-8-(hydroxymethyl)-5,5a-dimethylspiro[2,5-methano-1-benzoxe-
pine-10,2’-oxiran]-4-yl]oxy}-8-oxoocta-2,4,6-trienoic Acid; 6). Color-
less oil. [a]2 ¼ À6.3 (c ¼ 0.14, MeOH). UV (MeOH): 302 (4.23). IR
5
D
1
13
(
neat): 3687 (br.), 2889, 1708, 1037. H- and C-NMR: see Table 2.
HMB Correlations (500 MHz, CDCl ): HÀC(2)/C(4,5,11,12); CH (3)/
3
2
C(2,5,12); HÀC(4)/C(1’,2,5,6,12); CH (7)/C(5,6,8,9,15); CH (8)/
2
2
C(6,7,9); HÀC(9)/C(8,10,16); CH (10)/C(6,8,9,11,16); HÀC(11)/
2
C(2,7,9,10,15); CH (13)/C(2,5,12); Me(14)/C(4,5,6,12); Me(15)/
2
C(5,6,7,11); CH (16)/C(8,9,10); HÀC(2’)/C(1’,3’,4’); HÀC(3’)/C(1’,5’);
2
HÀC(4’)/C(2’,3’,6’); HÀC(5’)/C(3’,6’,7’); HÀC(6’)/C(4’,7’,8’); HÀC(7’)/
REFERENCES
C(5’,6’,8’). Key NOESY correlations (500 MHz, CDCl ): HÀC(2)/
3
H ÀC(13); H ÀC(3)/HÀC(11); HÀC(4)/HÀC(11), Me(15); HÀC(11)/
[1] J. W. Bennett, M. Klich, Clin. Microbiol. Rev. 2003, 16, 497.
[2] P. A. Murphy, S. Hendrich, C. Landgren, C. M. Bryant, J. Food Sci.
2006, 71, R51.
[3] K. Konishi, A. Iida, M. Kaneko, K. Tomioka, H. Tokuda, H.
Nishino, Y. Kumeda, Bioorg. Med. Chem. 2003, 11, 2511.
a
a
HÀC(9), Me(15); H ÀC(13)/CH (16); H ÀC(13)/Me(14). HR-ESI-
a
2
b
þ
þ
7
MS: 419.2069 ([M þ H] , C H O ; calc. 419.2064).
23
31
(
À)-Harzianum B (¼(2E,4Z,6E)-8-{[(2R,4R,5S,5aR,9aR)-2,3,4,
5
,5a,6,7,9a-Octahydro-5,5a,8-trimethylspiro[2,5-methano-1-benzoxe-
www.helv.wiley.com
ꢁ 2016 Verlag Helvetica Chimica Acta AG, Zürich

Helv. Chim. Acta 2016, 99, 63 – 69
69
[
[
[
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Received March 23, 2015
Accepted August 12, 2015
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