Synthesis, antifungal activity, and QSAR study of novel trichodermin derivatives

Article abstract of DOI:10.1080/10286020.2014.962522

In an attempt to discover more potential antifungal agents, in this study, 21 novel trichodermin derivatives containing conjugated oxime ester (5a-5u) were designed and synthesized and were screened for in vitro antifungal activity. The bioassay tests sho

Full text of DOI:10.1080/10286020.2014.962522

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Synthesis, antifungal activity, and QSAR
study of novel trichodermin derivatives
Jing-Li Chenga, Min Zhenga, Ting-Ting Yaoa, Xiao-Liang Lib, Jin-Hao
Zhaoa, Min Xiab & Guo-Nian Zhua
a Ministry of Agriculture Key Laboratory of Agricultural
Entomology, Institute of Pesticide and Environmental Toxicology,
Zhejiang University, Hangzhou 310029, China
b Institute of Sciences, Zhejiang Sci-Tech University, Hangzhou
310018, China
Published online: 07 Oct 2014.
To cite this article: Jing-Li Cheng, Min Zheng, Ting-Ting Yao, Xiao-Liang Li, Jin-Hao Zhao, Min
Xia & Guo-Nian Zhu (2014): Synthesis, antifungal activity, and QSAR study of novel trichodermin
derivatives, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2014.962522
To link to this article: http://dx.doi.org/10.1080/10286020.2014.962522
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Journal of Asian Natural Products Research, 2014
http://dx.doi.org/10.1080/10286020.2014.962522
Synthesis, antifungal activity, and QSAR study of novel trichodermin
derivatives
Jing-Li Chenga, Min Zhenga, Ting-Ting Yaoa, Xiao-Liang Lib, Jin-Hao Zhaoa*, Min Xiab
and Guo-Nian Zhua
aMinistry of Agriculture Key Laboratory of Agricultural Entomology, Institute of Pesticide and
Environmental Toxicology, Zhejiang University, Hangzhou 310029, China; Institute of Sciences,
Zhejiang Sci-Tech University, Hangzhou 310018, China
b
(Received 4 March 2014; final version received 2 September 2014)
In an attempt to discover more potential antifungal agents, in this study, 21 novel
trichodermin derivatives containing conjugated oxime ester (5a–5u) were designed
and synthesized and were screened for in vitro antifungal activity. The bioassay tests
showed that some of them exhibited good inhibitory activity against the tested
pathogenic fungi. Compound 5a exhibited better activity against Pyricularia oryzae
and Sclerotonia sclerotiorum than trichodermin, and compound 5j showed particular
activity against P. oryzae and Botrytis cinerea. The quantitative structure–activity
relationship (QSAR) indicated that log P and hardness were two critical parameters for
the biological activities. The result suggested that these would be potential lead
compounds for the development of fungicides with further structure modification.
Keywords: trichodermin derivatives; conjugated oxime ester; antifungal activity;
QSAR
1. Introduction
for further synthesis and structure
optimization.
The extensive application of traditional
fungicides for crop disease control has led
to the rapid development of fungicide
resistance and severe environmental pro-
blems [1]. This situation has prompted an
increased demand for more eco-friendly
agents to replace conventional chemical
fungicides. For decades, natural products
have attracted widespread attentions due to
their broad-spectrum biological activities,
such as insecticidal [2], anti-inflammatory
[3], antifungal [4], antitumor [5], and
antibacterial activities [6]. The develop-
ment and commercial success of some
natural products, such as validamycin and
streptomycin, make them become ideal
lead compounds for novel fungicide
discovery. Therefore, it is worth searching
for new antifungal agents based on natural
products as the bioactive prototypes
Trichodermin (1, Figure 1), a sesqui-
terpene antibiotic originally isolated from
metabolites of fungi, has been found to
exhibit excellent antifungal activity
against a broad spectrum of pathogenic
fungi [7]. It has already been discovered
that the antifungal activity of trichodermin
is mainly owing to its novel mechanism of
action by inhibiting protein synthesis in
eukaryotes [8]. Thus, compounds derived
from trichodermin are considered worthy
of exploring.
In our previous research, we success-
fully isolated a new fungus Trichoderma
taxi sp. nov. (ZJUF0986) which could
produce trichodermin in a fairly high yield.
Moreover, several series of structure
modifications on trichodermin were car-
ried out in order to study the structure–
*Corresponding author. Email: jinhaozhao@zju.edu.cn
q 2014 Taylor & Francis

2
J.-L. Cheng et al.
oxime ester have been designed and
synthesized as an important part of our
program to discovery novel compounds
with superior performance [10–12]. Fur-
thermore, to further explore the structural
requirements of the derivatives for the
activity, the quantitative structure–
activity relationship (QSAR) were also
studied.
Figure 1. Chemical structure of trichodermin
(1).
activity relationship (SAR) [9–11].
As indicated by the SAR analysis, appro-
priate substituents introduced to the C-4
and C-8 positions could help improving
the inhibitory activities of the compounds,
and the conjugated structure could pro-
mote intensive efforts to enhance their
bioactivity [12]. On the other hand, since
the first commercial oxime ether fungi-
cide, cymoxanil, was first marketed in the
mid-1970s, there have been numerous
researches on oxime ether compounds
[13], and some agents by incorporating
conjugated oxime ether into mother
nucleus have been commercialized, such
as trifloxystrobin, metominostrobin, and
dimoxystrobin (Figure 2). Thus, conju-
gated oxime ether can be considered as
potential pharmacophore.
2. Results and discussion
A series of novel trichodermin derivatives
containing conjugated oxime ester (5a–
5u) were synthesized by a four-step
procedure as shown in Scheme 1. The
key intermediate 2, obtained according to
our previously described procedure [11],
reacted with hydroxylamine hydrochloride
in the presence of K2CO3 to form
compound 3 in good yield. With 3 in
hand, conversion of 3 to 4 was then carried
out with iodoalkane in the anhydrous N,N-
dimethylformamide using sodium hydride
as a base. Finally, different carboxylic
acids were treated with 4 in the presence of
N,N-dimethylpyridin-4-amine (DMAP)
and
N,N-dicyclohexylcarbodiimide
(DCC) to afford the desired compounds
5a–5u. All of the target compounds were
obtained in moderate to good yields
(47.76–85.32%). However, it was worth
noting that the volume of the groups at C-4
position affected the yields to an extent.
That is, a bulk group generally made the
reaction run much slower with a relatively
lower yield. Of course, all spectral and
analytical data were in good agreement
Inspired by the above result, we first
introduced conjugated oxime ester groups
with different lengths of carbon chain into
position C-8. In addition, with appropriate
lipophilicity of derivatives taken into
account, we also incorporated different
ester moieties into position C-4 (Figure 3).
Therefore, in this paper, 21 novel tricho-
dermin derivatives containing conjugated
Figure 2. Commercial fungicides with conjugated oxime ester groups.

Journal of Asian Natural Products Research
3
Figure 3. Design strategy of the target molecules.
Scheme 1. Synthesis route of target compounds. Reagents and conditions: (a) CrO3·Py2, CH2Cl2,
reflux, 12 h; (b) NH2OH·HCl, K2CO3, CH3OH, reflux, 5 h; (c) NaH, R1I, DMF, Rt, 4 h; (d) R2COOH,
DMAP, DCC, CH2Cl2, Rt, 12 h.
Figure 4. Structures of the target compounds.
with the proposed structures of the
compounds.
derivatives (5a–5u) were evaluated on
three representative plant pathogenic fungi
including Pyricularia oryzae, Botrytis
cinerea, and Sclerotonia sclerotiorum by
using the mycelium growth rate method
With the above synthesized com-
pounds in hand (Figure 4), the in vitro
antifungal activities of these trichodermin

4
J.-L. Cheng et al.
[14]. Trichodermin, prochloraz, pyri-
methanil, and chlorothalonil were used as
comparative controls. Each treatment was
performed three times, and the effective
concentration 50 (EC50) defined by the
concentration corresponding to 50%
growth inhibition in vitro was used to
describe the inhibitory activity of the
compounds.
values of 2.40–30.51 mg L21. Among
these derivatives, compounds 5b, 5d, and
5j showed excellent antifungal activity,
with EC50 values of 4.83, 5.71, and
5.31 mg L21, respectively, comparable
with the lead compound trichodermin
(EC50: 4.25 mg L21). Compounds 5a
(EC50: 2.40 mg L21
) and 5i (EC50:
3.61 mg L21) displayed better inhibitory
activity against P. oryzae than that of
trichodermin. Moreover, compound 5a
(EC50: 1.59 mg L21) exhibited the highest
antifungal activity against Sclerotonia.
Sclerotiorum, which is higher than that of
trichodermin (EC50: 3.20 mg L21) and
commercial fungicide chlorothalonil
(EC50: 7.21 mg L21). In addition, com-
pounds 5i (EC50: 7.49 mg L21) and 5j
(EC50: 6.34 mg L21) were found to exhibit
the nearest approximation of inhibitory
activity against B. cinerea to pyrimethanil
(EC50: 6.98 mg L21).
The data presented in Table 1 demon-
strated that the biological activities of
these molecules were obviously dependent
on not only the length of carbon chain at
the C-8 position but also the nature of the
substituents at the C-4 position. All of
these assayed trichodermin derivatives
possessed moderate to good inhibitory
activity against P. oryzae, which is one of
the most serious threats against rice
productivity worldwide, with the EC50
Table 1. Antifungal activities of compounds
5a–5u expressed as EC50 (mg L21).
From a deep analysis of the data in
Table 1, some interesting phenomena
could be found. For example, with the
comparisons of compounds 5a–5h, 5i–
5p, and 5q–5u, it can be seen that, when
R1 was replaced by substituents with
different lengths of carbon chain, the
antifungal activities of the corresponding
compounds were changed obviously.
Generally, compounds with a short carbon
chain at C-8 position seemed to be
more active than long ones. The substi-
tuent at C-4 position also has great effect
on its antifungal activity, and aliphatic
acyloxy group is the most preferred. For
instance, compounds 5a and 5b with an
aliphatic acyloxy group at C-4 position
were generally more active than com-
pounds 5c–5h with an aromatic acyloxy
group. Therefore, the volume of the groups
at C-4 position may be an important factor
for activity. Introducing a phenylacetyl
group at C-4 position gives higher
bioactivities than that of the benzoyl
group. For example, compound 5d (R1 ¼
methyl; R2 ¼ 4-benzyl) showed higher
fungicidal activities than compound 5c
Compd
P. ory.
B. cin.
S. scl.
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
5t
5u
1
2.40
4.83
8.32
5.71
8.83
12.90
16.92
12.78
9.36
26.31
.30
20.12
7.49
1.59
7.52
14.31
10.34
7.91
21.28
.30
.30
3.17
5.95
11.28
16.59
7.85
3.61
5.31
9.77
6.79
9.46
14.33
20.45
11.29
10.96
14.02
26.69
30.51
21.32
4.25
6.34
4.34
18.26
10.37
17.38
14.29
.30
10.25
21.74
28.93
.30
.30
.30
0.45
16.51
11.27
14.05
20.41
.30
26.28
.30
.30
.30
.30
.30
3.20
Prochloraz
Pyrimethanil
Chlorothalonil
0.96
ND
ND
ND
6.98
ND
ND
ND
7.21
Note: Pyricularia oryzae (P. ory.), Botrytis cinerea
(B. cin.), and Sclerotonia sclerotiorum (S. scl.).
ND: Not determined.

Journal of Asian Natural Products Research
5
(R1 ¼ methyl; R2 ¼ 4-phenyl). There may
be a flexible methylene bridge in the
phenylacetyl group, which favors bioac-
tivity [15].
Table 2. Values of selected descriptors with
correlation (r . 0.5).
Compd LUMO Hardness Log P
MR
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
20.047 20.186
20.044 20.186
20.053 20.178
20.042 20.188
20.043 20.186
20.069 20.159
20.063 20.154
20.074 20.155
20.030 20.184
20.028 20.182
20.040 20.173
20.029 20.184
20.029 20.182
20.075 20.137
20.069 20.141
20.080 20.143
20.042 20.186
20.041 20.185
20.068 20.158
20.063 20.154
20.074 20.154
0.84
1.49
87.31
92.06
Because of the good antifungal effects
against P. oryzae of the target compounds,
our further studies in this work focused to
determine the relationships between
chemical structures and biological activity
data for P. oryzae. Herein, a quantitative
structure–activity relationships (QSAR)
study was undertaken.
2.73 107.44
2.68 111.76
3.17 117.65
3.08 118.21
2.95 125.46
3.23 118.61
1.18
1.83
92.11
96.86
EC50 values are first transformed to log
(1/EC50) and used as dependent variables
to get the linear relationship in the QSAR
model [16]. These were correlated with
different descriptors such as the highest
occupied molecular orbital (HOMO)
energy, the lowest unoccupied molecular
orbital (LUMO) energy, hardness
(HOMO–LUMO energy gap), n-octanol/
water partition coefficient (log P), dipole
moment (DM), and molar refractivity
(MR) [17]. To obtain these parameters,
the three-dimensional structures of the
studied compounds were first sketched in
the ChemBio3D Ultra 12.0 software [18],
and then pre-optimized using molecular
mechanics (MM) force field procedure,
and the resulting geometric conclusions
conformations were further refined by a
more precise optimization using the semi-
empirical AM1 method [19]. Finally, the
geometry with the lowest energy for each
derivative was then exported to Gaussian
03W (version 6.0) [20] to generate a set of
molecular. The descriptor used in the
present study was selected for its individ-
ual correlation with corresponding bio-
logical activity of compounds (r . 0.5),
and the key descriptors correlating with
the final equation are summarized in
Table 2. Cross-correlation between these
selected parameters is given in Table 3.
Figure 5 displays the correlation relation-
ship between experimental and predicted
activity for the 21 compounds.
3.07 112.24
3.02 116.56
3.50 122.45
3.41 123.01
3.29 130.26
3.57 123.41
2.50 105.91
3.15 110.66
4.73 136.81
4.61 144.06
4.89 137.21
5t
5u
correlated with hardness (0.890) and
log P was also strongly related to MR
(0.955). Therefore, we must choose one
between LUMO and hardness, as well as
between log P and MR [21].
The quality of each of the regression
models was determined by the correlation
coefficient (r), squared correlation coeffi-
cient (r 2), standard deviation (SE), and
Fisher ratio (F) [22]. Of the combination
of the parameters described before as
independent variables, the best model for
P. oryzae is Equation (1) as shown in
Table 4.
As illustrated in Table 4, the inhibitory
potential of each compound is highly
correlated with log P and hardness. Log P
is a measure of the lipophilicity of a
chemical substance. A low log P indicates
that the compound is lipophilic, while a
high log P suggests a hydrophilic nature
[23]. Lipophilicity is a very important
molecular descriptor that often correlates
well with the bioactivity of chemicals.
In this study, the model shows that the
Correlation of the four descriptors
demonstrated that LUMO was highly

6
J.-L. Cheng et al.
Table 3. Matrix of correlation between the selected variables used in equations.
LUMO
Hardness
Log P
MR
Activity
1.000
LUMO
Hardness
Log P
MR
Activity
1.000
20.890
20.561
20.617
0.599
1.000
20.593
20.702
20.688
1.000
0.955
20.879
1.000
20.896
Figure 5. Plots of experiment versus predicted activity of compounds 5a–5u screened against
Pyricularia oryzae (Equation 1).
value of log P negatively correlates to
antifungal activity. That is, a lower log P
value will result in higher inhibitory
activity, and high lipophilicity may be
beneficial for derivatives to penetrate into
the body of fungus.
accept electron, respectively [15]. Hard-
ness is defined as the energy difference
between HOMO and LUMO, which is
widely used as a measure of chemical
reactivity with bigger gaps implying larger
excitation energies and higher stability
[24]. It is indicated from Equation (1) that
hardness places negative contribution
towards the bioactivity.
It is worth noting that HOMO and
LUMO are two most important factors
which represent the ability to donate and
Table 4. QSAR models for activity against Pyricularia oryzae.
QSAR models
n
r
r 2
F
SE
Log (1/EC50) ¼ 21.102–0.197 log P 2 4.168 Hardness (1) 21 0.903 0.815 39.710 0.129
Log (1/EC50) ¼ 0.446–0.015 MR–1.903 Hardness (2)
Log (1/EC50) ¼ 20.209–0.215 log P þ 2.535 LUMO (3)
Log (1/EC50) ¼ 0.911–0.016 MR þ 1.215 LUMO (4)
21 0.900 0.809 38.161 0.131
21 0.888 0.789 33.592 0.138
21 0.898 0.806 37.287 0.133

Journal of Asian Natural Products Research
7
In conclusion, a series of novel
trichodermin derivatives with antifungal
activity were designed and synthesized by
incorporating the pharmacophore of con-
jugated oxime ether within the scaffold of
trichodermin. Generally, most of the new
compounds displayed excellent fungicidal
activity, in which compounds 5a and 5i
could be used as new lead compounds for
the development of antifungal agent with
further structure modification. In addition,
the QSAR model indicated that log P and
hardness were very important descriptors
for the biological activities of the
compounds.
mycelium growth rate method [14]. Each
of the test compounds was first dissolved in
DMF, and a proper dilution was aseptically
added to the medium at 458C to generate a
solution of corresponding concentration. P.
oryzae, B. cinerea, and S. sclerotiorum were
cultivated in potato dextrose agar (PDA) at
25 ^ 18C for 4 days to make a new
mycelium for the identification of bioactiv-
ity. Then, mycelia dishes of approximately
4 mm diameter were cut from the culture.
A mycelium was obtained using a germ-free
inoculation needle and inoculated in the
middle of the PDA plate aseptically. The
inoculated plates were incubated at
25 ^ 18C for 4 days. The growth rate was
conducted by measuring daily colony
diameter for 4 days after the transport of
the fungus onto dishes containing the
substance to be tested. Each treatment had
threerepetitions. The relativeinhibitory rates
(I %) of the test compounds were calculated
as follows:
3. Experimental
3.1 General experimental procedures
All reagents were commercially available
and all anhydrous solvents were dried by
standard techniques before use. All fungal
materials were obtained from the National
Engineering Research Center. Analytical
thin-layer chromatography (TLC) was
performed with silica gel plates using
silica gel 60 GF254 (Qingdao Haiyang
Chemical Co., Qingdao, China). Melting
points were determined on a X-4 digital
melting-point apparatus (Beijing Tech
Instruments Co., Ltd., Beijing, China)
CK 2 PT
Ið%Þ ¼
£ 100;
CK 2 0:4
where CK represents the diameter of fungal
growth on untreated PDA, PT represents the
diameter of fungi on treated PDA, and I
represents the inhibition rate. Finally, the
EC50 values were calculated to describe the
inhibitory activity of the compounds.
1
and were uncorrected. H and 13C NMR
(400 and 100 MHz, respectively) spectra
were recorded on a Bruker AVANCE III
instrument (Bruker CO., Bremen,
Germany) in CDCl3 using tetramethylsi-
lane (TMS) as the internal standard. Mass
spectra were recorded with a Finnigan
Trace Mass 2000 spectra (Thermo Finni-
gan, California, USA) using the electro-
spray ionization (ESI) method. HRMS was
determined with an APEX III 7.0 FT-ICR
mass spectrometer (Bruker Daltonic Inc.,
New York, USA).
3.3 Synthesis of title compounds
3.3.1 Synthesis of (4b)-4-acetoxy-8-
oximido-12,13-epoxytrichothec-9-ene (3)
To a solution of compound 2 (0.92 g,
3 mmol) and hydroxylamine hydrochlo-
ride (0.63 g, 9 mmol) in anhydrous CH3OH
(10 ml) was added dropwise a solution of
K2CO3 (1.24 g, 9 mmol) in water (10 ml) at
658C. The reaction was stirred for 5 h at
this temperature and monitored by TLC.
After completion, the mixture was evap-
orated and extracted with ethyl acetate (3
£ 5 ml). The combined extracts were
washed with saturated brine, dried (anhy-
3.2 Bioassay
The antifungal activities of the target
compounds were evaluated by the

8
J.-L. Cheng et al.
drous Na2SO4), and filtered. The filtrate was
evaporated, and the crude product was
purified via silica gel column chromatog-
raphy, using a 1:3 (v/v) mixture of ethyl
acetate and petroleum ether (boiling point
range: 60–908C) as the eluting solution to
afford compound 3 as a colorless crystal;
12.6 Hz, 1H, H-7), 2.92 (d, J ¼ 3.2 Hz, 2H,
H-13), 2.65 (dd, J1 ¼ 6.0 Hz, J2 ¼
12.6 Hz, 1H, H-3), 2.30 (d, J ¼ 12.6 Hz,
1H, H-7), 1.93–1.98 (m, 1H, H-3), 1.89 (s,
3H, H-16), 0.87 (s, 3H, H-14), 0.86 (s, 3H,
H-15); 13C NMR (100 MHz, CDCl3): d
5.88, 17.33, 18.31, 26.09, 30.90, 40.22,
47.55, 49.15, 61.97, 65.71, 69.80, 73.53,
78.92, 126.17, 135.17, 153.73; ESI-MS
(m/z): 294 [M þ H]þ.
1
yield 89.83%; m.p.: 209–2118C; H NMR
(400MHz, CDCl3, d ppm): d 8.57 (s, 1H,
NOH), 5.86–5.84 (m, 1H, H-10), 4.31 (s,
1H, H-4), 3.89 (d, J ¼ 4.4Hz, 1H, H-11),
3.71 (d, J ¼ 4.8 Hz, 1H, H-2), 3.15 (d, J ¼
3.2 Hz, 1H, H-13), 3.12 (d, J ¼ 12.4 Hz, 1H,
H-7), 2.94 (d, J ¼ 3.2Hz, 1H, H-13), 2.66
(dd, J1 ¼ 6.0 Hz, J2 ¼ 12.8 Hz, 1H,
H-3), 2.38 (d, J ¼ 12.4 Hz, 1H, H-7),
1.98–1.94 (m, 1H, H-3), 1.90 (s, 3H,
O ¼ CCH3), 1.65 (s, 3H, H-16), 0.90 (s,
3H, H-14), 0.89 (s, 3H, H-15); 13C NMR
(100MHz, CDCl3): d 4.88, 16.44, 17.46,
20.08, 24.60, 35.82, 40.24, 46.85, 47.97,
64.56, 69.11, 73.42, 78.35, 125.92, 133.71,
153.77, 170.01; ESI-MS (m/z): 322
[M þ H]þ. HR-ESI-MS: m/z 322.1649
[M þ H]þ (calcd for C17H24NO5,
322.1655).
Similar procedures as for the prep-
aration of compound 4a were used for the
synthesis of compounds 4b and 4c.
3.3.3 Synthesis of (4b)-8-methoxime-12,
13-epoxytrichothec-9-en-4-yl acetate (5a)
Compound 4a (0.59 g, 2 mmol), ethanoic
acid (0.18 g, 3 mmol), DMAP (0.24 g,
2 mmol), and DCC (0.82 g, 4 mmol) were
dissolved in dried dichloromethane
(10 ml). The reaction was stirred at room
temperature for 12 h. The process of the
reaction was detected by TLC. After
completion, the mixture was poured into
water and extracted with dichloromethane
(3 £ 5 ml). The organic layer was washed
with saturated brine (15 ml), dried over
anhydrous Na2SO4, and filtered. The
solvent was removed under reduced
pressure, and the residue was purified by
silica gel column chromatography to give
compound 5a as a colorless oil; yield
85.32%; 1H NMR (400 MHz, CDCl3,
d ppm): d 5.80–5.82 (m, 1H, H-10), 5.51
(dd, J1 ¼ 2.8, J2 ¼ 6.4 Hz, 1H, H-4), 3.93
(s, 3H, NOCH3), 3.87 (d, J ¼ 4.3 Hz, 1H,
H-11), 3.78 (d, J ¼ 4.8 Hz, 1H, H-2), 3.15
(d, J ¼ 3.2 Hz, 1H, H-13), 3.00 (d,
J ¼ 12.4 Hz, 1H, H-7), 2.94 (d,
J ¼ 3.2 Hz, 1H, H-13), 2.58 (dd,
J1 ¼ 6.2 Hz, J2 ¼ 12.5 Hz, 1H, H-3), 2.33
(d, J ¼ 12.4 Hz, 1H, H-7), 2.10 (s, 3H,
O ¼ CCH3), 2.04–2.00 (m, 1H, H-3), 1.90
(s, 3H, H-16), 0.94 (s, 3H, H-14), 0.79 (s,
3H, H-15); ESI-MS (m/z): 336 [M þ H] þ.
The target compounds 5b–5u were
prepared by following the same pro-
cedures as for 5a.
3.3.2 Synthesis of (4b)-8-methoxime-12,
13-epoxytrichothec-9-ene-4-ol (4a)
To a stirred solution of 3 (0.96 g, 3 mmol)
and 60% sodium hydride (0.24 g, 6 mmol)
in N,N-dimethylformamide (10 ml) was
slowly added iodomethane (0.57 g,
4 mmol). The reaction mixture was stirred
at room temperature for 4 h. The mixture
was poured into water (20 ml) and
extracted with ethyl acetate (3 £ 10 ml).
The combined organic phase was washed
with saturated brine (30 ml), dried over
anhydrous Na2SO4, concentrated in vacuo,
and purified via silica gel column chroma-
tography to obtain compound 4a as a
colorless oil; yield 56.21%; 1H NMR
(400 MHz, CDCl3, d ppm): d 5.80–5.78
(m, 1H, H-10), 4.29 (s, 1H, H-4), 3.92
(s, 3H, NOCH3), 3.87 (d, J ¼ 4.3 Hz, 1H,
H-11), 3.68 (d, J ¼ 4.7 Hz, 1H, H-2), 3.12
(d, J ¼ 3.0 Hz, 1H, H-13), 3.01 (d, J ¼

Journal of Asian Natural Products Research
9
G.N. Zhu, Chem. Biodivers. 10, 600
(2013).
Acknowledgment
This work was financially supported by the
National Key Technology R&D Program of
China under Grant [2011BAE06B04-10].
[13] Y.S. Xiao, X.J. Yan, Y.J. Xu, J.X. Huang,
H.Z. Yuan, X.M. Liang, J.J. Zhang, and
D.Q. Wang, Pest Manag. Sci. 69, 814
(2013).
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