Synthesis and biological evaluation of novel trichodermin derivatives as antifungal agents

Article abstract of DOI:10.1016/j.bmcl.2014.05.047

To discover more potential antifungal agents, 17 novel trichodermin derivatives were designed and synthesized by modification of 3 and 4a. The structures of all the synthesized compounds were confirmed by ¹H NMR, ESI-MS and HRMS. Their antifungal activities against Ustilaginoidea oryzae and Pyricularia oryzae were evaluated. Most of the target compounds showed potent inhibitory activity, in which 4g showed superior inhibitory effects than 4a and commercial fungicide prochloraz. Furthermore, 4h demonstrated comparable inhibitory activity to 4a. Moreover, 4i and 4l exhibited excellent inhibitory activity for Pyricularia oryzae. Additionally, compound 9 was found to be more active against all tested fungal strains than 3, with EC₅₀ values of 0.47 and 3.71 mg L^-1, respectively.

Full text of DOI:10.1016/j.bmcl.2014.05.047

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of novel trichodermin derivatives
as antifungal agents
⇑
Min Zheng, Ting-Ting Yao, Xiao-Jun Xu, Jing-Li Cheng, Jin-Hao Zhao , Guo-Nian Zhu
College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310029, China
a r t i c l e i n f o
a b s t r a c t
Article history:
To discover more potential antifungal agents, 17 novel trichodermin derivatives were designed and
synthesized by modification of 3 and 4a. The structures of all the synthesized compounds were confirmed
by 1H NMR, ESI-MS and HRMS. Their antifungal activities against Ustilaginoidea oryzae and Pyricularia
oryzae were evaluated. Most of the target compounds showed potent inhibitory activity, in which 4g
showed superior inhibitory effects than 4a and commercial fungicide prochloraz. Furthermore, 4h
demonstrated comparable inhibitory activity to 4a. Moreover, 4i and 4l exhibited excellent inhibitory
activity for Pyricularia oryzae. Additionally, compound 9 was found to be more active against all tested
fungal strains than 3, with EC50 values of 0.47 and 3.71 mg LÀ1, respectively.
Received 10 March 2014
Revised 13 May 2014
Accepted 15 May 2014
Available online xxxx
Keywords:
Trichodermin derivatives
Synthesis
Ó 2014 Elsevier Ltd. All rights reserved.
Antifungal activities
Fungal diseases have long been one of the major causes of crop
losses, and to control the epidemic of these diseases has a vital
importance.1 To date, the commonly treatment method for the
epidemic spread of fungal diseases is the application of chemical
fungicide. The excessive use of these synthetic chemicals over
the years, however, has led to the rapid development of fungicide
resistance, which in turn makes the management of fungal dis-
eases more precarious.2–4 Therefore, there is an urgent need for
the development of novel, effective and environmentally-friendly
agents to replace the conventional chemical fungicides.
Natural products have received a considerable attention as
shown by the numerous studies published on their broad-spec-
trum biological activities, such as insecticidal, anti-inflammatory,
antifungal, antitumor and antibacterial activities.5–11 Trichodermin
(1, Fig. 1), a naturally occurring sesquiterpene antibiotic, was iso-
lated from metabolites of fungi, which had been found to possess
excellent antifungal activity.12,13 Trichodermin was considered as
one of the ideal lead compounds for new fungicides because of
its unique mechanism of action that could inhibit protein synthesis
in eukaryotes.14–16
bioactivity.17,18 Among these compounds, 3 and 4a, which all
contained conjugated structure, exhibited relatively stronger
antifungal activities against Ustilaginoidea oryzae (U. ory.) and
Pyricularia oryzae (P. ory.) than trichodermin (Fig. 1). Therefore,
inspired by the above results, we designed and synthesized a series
of novel trichodermin derivatives based on the general structures of
3 and 4a to better understand the structure–activity relationship.
The synthesis of the key intermediates 2 and 3 were readily
obtained in one step from starting material trichodermin. The
synthetic pathway was depicted in Scheme 1. In our previous
manuscript, we disclosed a method to obtain 2 and 3 by reacting
1 with stannic oxide in 1,4-dioxane.18 Through this method, 2
could be obtained as expected. However, intermediate 3 as by-
product resulted in a relatively low yield. To optimize this reaction,
several different solvent systems were investigated (Table 1).
According to the screening of solvent systems, acetonitrile/H2O
(10:1, v/v) (Table 1, entry 3) presented a relatively higher yield
of both 2 and 3, which was then introduced to produce these
two key intermediates.
In an attempt to explore the electronic effect of the substituent
in the benzene ring on the investigated biological activity, the
desired compounds 4a–4m were prepared through treating 2 with
substituted cinnamoyl chloride in the presence of triethylamine as
acid acceptor and N,N-dimethylaminopyridine as catalyst, as
shown in Scheme 2. In addition, considering that the chirality of
drug isomer sometimes has significant effects on their biological
activity,19,20 we designed 4n (cis-conformer of 4a) via the route
outlined in Scheme 2. On the other hand, we also did some proper
structure modification on 3 with instability of aldehyde group
taken into account. As outlined in Scheme 3, thiazole amine, which
As far as structure-activity relationship is concerned, in our
previous studies, series of structure modifications on trichodermin
mother nucleus have been carried out, indicating that appropriate
substituents introduced to the C-4 and C-8 position could help
improve the antifungal activities of the compounds, and the conju-
gated structure in molecular could contribute to enhancing their
⇑
Corresponding author. Tel.: +86 (0)571 86971220; fax: +86 (0)571 6430193.
E-mail address: jinhaozhao@zju.edu.cn (J.-H. Zhao).
http://dx.doi.org/10.1016/j.bmcl.2014.05.047
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Zheng, M.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.05.047

2
M. Zheng et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Figure 1. Chemical structures of trichodermin (1), compounds 3 and 4a.
Scheme 1. Synthetic route to compounds 2 and 3.
of 3, we were successful in preparing X-ray quality single crystal
Table 1
of intermediate 3 obtained by slow evaporation of its 95% ethanol
solution (CCDC 934209). As shown in Figure 2, the crystal
was monoclinic with space group P212121, and the cell parameters
were a = 7.1994(3) Å, b = 12.9564(5) Å, c = 16.9369(7) Å, V =
1579.85(11) Å3, Z = 4. The final R = 0.0398, wR = 0.0987, S = 1.000,
the difference density were <0.001 e/Å3 (max) and À0.145 e/Å3
(min).
The in vitro antifungal activities of all the target compounds
have been evaluated against two representative pathogenic fungi
including U. ory. (ACCC 36443) and P. ory. (ACCC 37631) by the
mycelium growth rate method.21,22 Commercial fungicide prochlo-
raz was used as a comparative control. Each treatment was
The optimization of compounds 2 and 3
Entry
Solvent
Yield (%)
2
3
1
2
3
4
5
6
7
8
9
1,4-Dioxane/H2O (10:1, v/v)
1,4-Dioxane /TBHPa (10:1, v/v)
Acetonitrile/H2O (10:1, v/v)
Acetonitrile/TBHP (10:1, v/v)
Dicloromethane/TBHP (10:1, v/v)
Ethanol (95%)
50
32
50
45
36
26
70
56
30
25
15
35
20
22
12
5
THFb
Xylene
Acetone
2
1
a
TBHP: tert-butyl hydroperoxide.
THF: tetrahydrofuran.
performed three times, and the effective concentration 50 (EC50
)
b
defined as the concentration required to inhibit 50% of the fungal
growth, was used to describe the inhibitory activity of the
compounds. The antifungal activities of the target compounds
along with the standard drug for comparison are summarized in
Tables 2 and 3.
Initially, we aimed to investigate the influence of substituent in
the benzene ring on the antifungal activity. From the data
presented in Table 2, we can see that when 4a was modified by
varying the substituents on the benzene ring, the antifungal activ-
ities of these target compounds were changed obviously. When
para-position of the benzene ring of 4a was substituted with a
is usually used as antifungal pharmacophore, was reacted with 3 in
dry methanol to give compounds 7a and 7b. Compound 8 was pre-
pared by treatment of 3 with potassium carbonate, which reacted
further with acetyl chloride to yield 9 (to improve its lipophilicity).
The structures of all the synthesized compounds were confirmed
by 1H NMR, ESI-MS and HRMS analyses.
In our previous study, the 3D structural information of
compound 4a was confirmed by single-crystal X-ray diffraction
analysis.18 In this Letter, in order to identify the stereostructure
Scheme 2. Reagents and conditions: (a) Substituted cinnamoyl chloride, Et3N, DMAP, CH2Cl2, rt, 75–85%; (b) CrO3, pyridine, 80 °C, 3 h, 68%; (c) KBH4, NH4Cl, H2O, CH3OH,
24 h, rt, 45%; (c) Cinnamoyl chloride, Et3N, DMAP, CH2Cl2, rt, 12 h, 84%.
Please cite this article in press as: Zheng, M.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.05.047

M. Zheng et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
Scheme 3. Reagents and conditions: (a) H2NR2, CH3OH, 45 °C, 12 h, 70–76%; (b) NH2OHÁHCl, K2CO3, CH3OH, reflux, 5 h, 75%; (c) CH3COCl, DMAP, CH2Cl2, rt, 1 h, 71%.
Table 3
Antifungal activity of 7a, 7b, 8, 9, and 3 against U. ory. and P. ory.
Compd
Antifungal activity (EC50, mg LÀ1
)
U. ory.
P. ory.
7a
7b
8
9
2.54
1.52
3.67
0.47
0.80
0.82
13.27
12.48
8.91
3.71
11.82
0.96
3
Prochloraz
These results demonstrated that the volume of the substituent was
very important for the inhibitory activity of U. ory. and P. ory. Nota-
bly, the in vitro antifungal profile of 4b–4m is shown to be
increased when groups such as –OCH3, –F and –Br are present at
ortho position as is also inferred by Table 2. For example, with
the comparisons of compounds 4j (o-Br), 4k (m-Br) and 4l (p-Br),
it can be seen that their inhibitory capacities in decreasing order
were: 4l, 4k and 4j. Changing the configuration of the 4a also led
to a remarkable destruction of inhibitory activity in both U. ory.
and P. ory. The result indicated that the trans-isomer at the C-8
position was crucial for the antifungal activity.
On the other side, some preliminary structure–activity relation-
ships on the 3 derivatives were also investigated. As shown in
Table 3, compound 9 showed better inhibitory activity against all
two tested fungi than 3. Notably, compound 9 also exhibited more
promising antifungal activity than prochloraz (an EC50 value: 0.47
vs 0.82 mg LÀ1). However, the loss of the antifungal activities were
observed for compounds 7a and 7b containing a thiazole amine
group, revealing that thiazole amine group was not favorable for
the antifungal activity. In addition, compound 8 by replacement
aldehyde group with oxime group obviously decreased the anti-
fungal activity against U. ory. (an EC50 value: 3.67 vs 0.80 mg LÀ1).
These above results suggested that improving the lipophilicity of
trichodermin derivatives was more beneficial for the bioactivity.
In summary, we have developed a series of trichodermin deriv-
atives based on the general structures of 3 and 4a. Antifungal activ-
ities of these compounds were evaluated. The SARs revealed that
the nature of the substituent, the lipophilicity and the chirality of
the compounds were very important for the antifungal activity.
The biological test results showed that these compounds displayed
moderate to high antifungal activity against U. ory. and P. ory.
Among them, 4g showed better antifungal activity than the lead
compound 4a and prochloraz. Furthermore, 4h exhibited excellent
inhibitory activity compared with 4a and prochloraz. Moreover, 4i
and 4l demonstrated competitive inhibitory activity against P. ory.
to 4a. Additionally, compound 9 displayed superior activity against
all two tested fungi compared with 3, with EC50 values of 0.47 and
Figure 2. The single-crystal X-ray crystallography of 3.
Table 2
Antifungal activity of 4a–4n against U. ory. and P. ory.
Compd
R1
(R) or (S)
Antifungal activity (EC50, mg LÀ1
)
U. ory.
P. ory.
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
H
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(S)
—
1.48
6.21
4.23
6.86
11.04
10.31
0.56
1.47
3.12
3.17
6.20
11.28
2.45
>20
0.74
5.99
6.18
6.68
11.70
12.47
0.53
1.09
1.31
2.70
3.82
12.62
1.47
>20
4-CH3
2-OCH3
4-OCH3
3,4,5-(OCH3)3
3-CF3
2-F
4-F
2-Cl
2-Br
3-Br
4-Br
2-Cl-4-F
H
4m
4n
Prochloraz
—
0.82
0.96
methyl (compound 4b) or methoxy (compound 4d) group, their
fungicidal activity generally decreased. However, introducing hal-
ogen atoms into the benzene ring of 4a led to more than one half
compounds showed remarkable antifungal activity. To our delight,
both of the compounds 4g and 4h containing a fluorine atom
showed competitive inhibitory activities to 4a. Especially com-
pound 4g (2-fluorine) exhibited the better inhibitory activity
against all two tested fungi than prochloraz (EC50 values: 0.56 vs
0.82 mg LÀ1; 0.53 vs 0.96 mg LÀ1). Altering the substitution groups
at the ortho-position of the benzene ring with F, Cl, Br systemati-
cally (4g, 4i, 4j), we found the antifungal activity against U. ory.
decreased in that order, and the same trend was observed in the
activity against P. ory. Using 3,4,5-triethoxy group (4e) instead of
a methoxy to determine its importance, the results showed that
there was remarkable activity decrease against all two tested fungi.
Please cite this article in press as: Zheng, M.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.05.047

4
M. Zheng et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
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Supplementary data
Supplementary data associated with this article can be found, in
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