Novel plant activators with thieno[2,3-d]-1,2,3-thiadiazole-6-carboxylate scaffold: Synthesis and bioactivity

Article abstract of DOI:10.1016/j.cclet.2013.07.003

The 1,2,3-thiadiazole-carboxylate moiety was reported to be an important pharmacophore of plant activators. In this study, a series of novel plant activators based on thieno[2,3-d]-1,2,3-thiadiazole-6-carboxylate were designed and synthesized and their biological activity as plant activators was studied. The structures of the novel compounds were identified by ¹H NMR, ¹⁹F NMR and HRMS. The in vivo bioassay showed that these novel compounds had good efficacy against seven plant diseases. Especially, compounds 1a and 1c were more potent than the commercialized plant activator BTH. Almost no fungicidal activity was observed for the active compounds in the in vitro assay, which matched the requirements as plant activators.

Full text of DOI:10.1016/j.cclet.2013.07.003

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Chinese Chemical Letters xxx (2013) xxx–xxx
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
Novel plant activators with thieno[2,3-d]-1,2,3-thiadiazole-6-
carboxylate scaffold: Synthesis and bioactivity
Qing-Shan Du a, Yan-Xia Shi b, Peng-Fei Li a, Zhen-Jiang Zhao a, Wei-Ping Zhu a,
a,
Xu-Hong Qian a, Bao-Ju Li b, , Yu-Fang Xu
*
*
a Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
b Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing100081, China
A R T I C L E I N F O
A B S T R A C T
Article history:
The 1,2,3-thiadiazole-carboxylate moiety was reported to be an important pharmacophore of plant
activators. In this study, a series of novel plant activators based on thieno[2,3-d]-1,2,3-thiadiazole-6-
carboxylate were designed and synthesized and their biological activity as plant activators was studied.
The structures of the novel compounds were identified by 1H NMR, 19F NMR and HRMS. The in vivo
bioassay showed that these novel compounds had good efficacy against seven plant diseases. Especially,
compounds 1a and 1c were more potent than the commercialized plant activator BTH. Almost no
fungicidal activity was observed for the active compounds in the in vitro assay, which matched the
requirements as plant activators.
Received 17 April 2013
Received in revised form 5 June 2013
Accepted 9 June 2013
Available online xxx
Keywords:
Thieno[2,3-d]-1,2,3-thiadiazole-6-
carboxylate derivatives
Plant activator
ß 2013 Bao-Ju Li and Yu-Fang Xu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All
rights reserved.
Systemic acquired resistance
1. Introduction
tobacco [14,15]. Recently, a novel plant activator, 3,4-dichloro-20-
cyano-1,2-thiazole-5-carboxanilide (Isotianil) was developed by
Plant diseases can cause lethal consequences to crops and lead to
great losses in agriculture [1]. As a result, agrochemicals play
important roles in plant disease control by directly killing the
pathogens. However, the increasing applications of agrochemicals
have caused serious environmental problems and induced severe
drug resistances of pathogens. Therefore, it is of great importance to
exploit novel environmentally friendly methods for plant protec-
tion. Plant activators that can stimulate the defensive system of the
plant have attracted much attention [2–4]. By activating SAR, a plant
can be conferred with long-lasting resistance to a broad-spectrum of
pathogens, including viruses, bacteria, fungi, and oomycetes [5,6].
Several chemicals have been reported to induce SAR efficiently in
plants. Among them, S-methyl-benzo[1,2,3]thiadiazole-7-car-
bothioate (BTH), 2,6-dichloroisonicotinic acid (INA), and N-cyano-
methyl-2-choloisonicotiamide (NCI) are analog of SA and showed
better SAR-inducing activity [7,8]. As a result, the most successful
compound, BTH, has been well exploited for agricultural applica-
tions [9–13]. Similarly, N-(3-chloro-4-methylphenyl)-4-methyl-
1,2,3-thidiazole-5-carboxamide (TDL) and its derivatives are
another class of plant activators for the control of rice diseases,
which could also induce the expression of SAR marker genes in
Bayer CropScience AG for the control of rice blast. Isotianil showed
no antimicrobial activity against various fungi and bacteria, but
remarkably displayed high efficacies against these diseases in field
tests [16,17]. Until now, most of the commercialized plantactivators
bore similar 1,2,3-thiadiazole, or
a thiazole moiety in their
structures; hence 1,2,3-thiadiazole was the promising pharmaco-
phore for the development of novel plant activators.
Currently, an increasing effort is being put into the study of
novel plant activators. However, only a few promising chemicals
have been reported to date, and the agricultural applications of
plant activators are far from developed [7,14,18–20]. The efficacy
of 1,2,3-thidaizoles, as plant activators, has been fully proved,
which found that 1,2,3-thidiazole was essential for activity [7,21].
While in our previous study, 7-carboxylate derivatives of BTH have
been developed as plant secondary metabolites elicitors and plant
activators [22,23]. The results showed that introduction of
fluorine-containing 7-carboxylate esters significantly enhanced
the activity of target compounds. Till now, however, much effort
has been given to the active study of 1,2,3-thidiazole, and there are
only few structure–activity studies on the heterocyclic-fused 1,2,3-
thidiazole. Stanetty and colleagues have reported the synthesis of
methyl-thieno[2,3-d]-1,2,3-thiadiazole-6-carboxylate and sug-
gested the potential applications as plant activator [24,25], while
no biological activity was investigated. In this study, a series of
novel thieno[2,3-d]-1,2,3-thiadiazole-6-carboxylate derivatives
*
Corresponding authors.
E-mail addresses: libj@mail.caas.net.cn (B.-J. Li), yfxu@ecust.edu.cn (Y.-F. Xu).
1001-8417/$ – see front matter ß 2013 Bao-Ju Li and Yu-Fang Xu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
http://dx.doi.org/10.1016/j.cclet.2013.07.003
Please cite this article in press as: Q.-S. Du, et al., Novel plant activators with thieno[2,3-d]-1,2,3-thiadiazole-6-carboxylate scaffold:
Synthesis and bioactivity, Chin. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.cclet.2013.07.003

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ROOC
HOOC
H COOC
were designed and synthesized as novel plant activators. Various
ester groups, including fluorine-containing groups, were intro-
duced and their activity as plant activators was evaluated both in
vivo and in vitro against several plant diseases.
3
1. NaOH(aq)
2. HCl
1. (COCl)
S
S
S
2
N
3
N
N
2. ROH
S
N
S
N
S
N
2
1a
1b-1m
1j: R=CH CH CH CH CH
1b: R=CH CH
1f: R=CH CF CF
2 2 3
2. Experimental
2
3
2
2
2
2
1k: R=CH CF CF CF CHF
1c: R=CH CF
1g: R=CH(CF )
2
2
2
2
2
2
3
3 2
1l: R=CH CH(OH)CH OH
1h: R=CH CH CH CH
2
2
1d: R=CH CH CH
3
2.1. Synthesis of the new compounds
2
2
2
3
2
2
1m: R=CH C F
6 5
1i: R=CH CF CF CF
2
1e: R=CH(CH )
2
2
2
3
3 2
The starting compound 1a was synthesized according to
literature report [25]. Compounds 1b–1m were synthesized from
1a via two steps including hydrolization and esterification
(Scheme 1). Synthetic procedures of the novel compounds are
described in Supporting information, and data of the novel
compounds are shown in Tables S1 and S2 (see Supporting
information).
Scheme 1. General route for the synthesis of target compounds.
and physical properties of the novel compounds are shown in Table
S1. All compounds were characterized by 1H NMR, HRMS, and
compounds containing fluorine atoms were also identified by 19F
NMR, as shown in Table S2.
3.2. Biological activity
2.2. SAR-inducing activity evaluation
Plant activators are a class of chemicals that could activate the
defense response of plants being invaded by pathogens and usually
have no in vitro fungicidal activity [26]. Therefore, firstly we
evaluated the in vivo efficacy of the novel compounds, then in vitro
test of the high efficacy compounds was conducted to illustrate the
potential as plant activator. Methods of inoculation are described
in Table S3 (see Supporting information). The in vivo efficacy of the
novel compounds against seven diseases is shown in Table 1. In
order to make a better assessment and comparison of the efficacy
of the novel compounds, we defined herein the efficacy above 40%
as demonstrating active efficacy against the disease, due partly to
the fact that positive control, 50% procymidone (WP), only showed
efficacy of 42% toward Botrytis cinerea. With this standard, we can
establish from Table 1 that the positive control BTH was active
against four of the seven tested diseases, including Mycosphaerella
melonis (88%), Pseudomonas syringae pv. Lachrymans (76%),
Phytophthora infestans (65%) and B. cinerea (47%). The free acid
of compound 2 only showed efficacy against two of the seven test
diseases, including M. melonis (74%) and P. infestans (57%), and that
In order to screen plant activators efficiently, the assay was
conducted in two steps: firstly, all compounds were tested in vivo
toward seven pathogens including four types of fungi, two types of
bacteria and a type of oomycete. After that, compounds with high
activities were selected for the in vitro anti-microbial activity
assay. As plant activator, it should protect plants by activating the
SAR in vivo and show no direct activity in vitro. Test methods are
described in Supporting information.
3. Results and discussion
3.1. Preparation of thieno[2,3-d]-1,2,3-thiadiazole-6-carboxylate
derivatives
Compound methyl thieno[2,3-d]-1,2,3-thiadiazole-6-carboxyl-
ate (1a) was synthesized according to the literature [25].
Compound 2 was synthesized from 1a via the hydrolysis with
good yield, followed by esterification with various alcohols to
obtain novel compounds 1b–1m, as shown in Scheme 1. The yields
Table 1
In vivo induction activity of the target compounds.
Compd.a
Efficacy (%)b
MM
CC
-9 ꢀ 2
PL
PI
57 ꢀ 5
RS
FO
BC
2
74 ꢀ 3
90 ꢀ 3
30 ꢀ 4
69 ꢀ 6
55 ꢀ 5
41 ꢀ 3
51 ꢀ 4
62 ꢀ 2
76 ꢀ 9
45 ꢀ 5
90 ꢀ 2
88 ꢀ 6
88 ꢀ 7
89 ꢀ 5
88 ꢀ 3
98 ꢀ 1
18 ꢀ 2
42 ꢀ 4
45 ꢀ 3
42 ꢀ 4
44 ꢀ 4
10 ꢀ 3
48 ꢀ 5
41 ꢀ 2
45 ꢀ 3
32 ꢀ 4
24 ꢀ 2
41 ꢀ 1
36 ꢀ 5
27 ꢀ 4
76 ꢀ 8
ꢁ6 ꢀ 2
ꢁ6 ꢀ 1
ꢁ6 ꢀ 4
ꢁ6 ꢀ 3
ꢁ6 ꢀ 5
0
3
5
9
7
ꢀ 3
ꢀ 4
ꢀ 4
ꢀ 3
14 ꢀ 3
37 ꢀ 4
50 ꢀ 6
41 ꢀ 5
1a
77 ꢀ 3
79 ꢀ 4
52 ꢀ 5
66 ꢀ 7
70 ꢀ 2
58 ꢀ 3
59 ꢀ 7
34 ꢀ 5
35 ꢀ 4
81 ꢀ 5
47 ꢀ 7
67 ꢀ 6
35 ꢀ 2
35 ꢀ 4
74 ꢀ 4
40 ꢀ 3
1b
1c
1d
33 ꢀ 5
2
ꢀ 3
1e
2
9
ꢀ 1
ꢀ 1
39 ꢀ 6
32 ꢀ 3
10 ꢀ 1
23 ꢀ 4
18 ꢀ 3
30 ꢀ 5
35 ꢀ 4
11 ꢀ 8
17 ꢀ 3
47 ꢀ 6
1f
0
1g
ꢁ1 ꢀ 2
64 ꢀ 5
31 ꢀ 4
46 ꢀ 6
36 ꢀ 4
48 ꢀ 5
47 ꢀ 3
16 ꢀ 4
14 ꢀ 4
1h
3
8
ꢀ 2
ꢀ 7
7
ꢀ 3
1i
ꢁ5 ꢀ 3
ꢁ6 ꢀ 4
1j
7
ꢀ 2
ꢁ9 ꢀ 4
1k
80 ꢀ 4
41 ꢀ 5
23 ꢀ 2
ꢁ8 ꢀ 4
1
9
9
ꢀ 1
ꢀ 1
1l
5
ꢀ 3
1m
ꢁ18 ꢀ 4
65 ꢀ 5
ꢁ8 ꢀ 5
19 ꢀ 2
BTH
50% kresoxim-methyl (WG)
75% chlorothalonil (WP)
20% bismerthlazol (WP)
50% dimethomorph (WP)
5% validamycin A (WP)
70% Mildothane (WP)
50% procymidone (WP)
90 ꢀ 3
68 ꢀ 5
97 ꢀ 2
83 ꢀ 6
75 ꢀ 5
42 ꢀ 5
a
BTH represents S-methyl benzo[1,2,3]thiadiazole-7-carbothioate and was synthesized in our lab.
b
Micro-organisms used: Mycosphaerella melonis (MM), Corynespora cassiicola (CC), Fusarium oxysporum (FO), Botrytis cinerea (BC), Pseudomonas syringae pv. Lachrymans
(PL), Phytophthora infestans (PI) and Rhizoctonia solani (RS); efficacy is the average of all repeated tests; MM, CC, BC, FO and PL were tested on cucumber seedlings; PI was
tested on tomato seedlings; RS was tested on rice seedlings.
Please cite this article in press as: Q.-S. Du, et al., Novel plant activators with thieno[2,3-d]-1,2,3-thiadiazole-6-carboxylate scaffold:
Synthesis and bioactivity, Chin. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.cclet.2013.07.003

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Q.-S. Du et al. / Chinese Chemical Letters xxx (2013) xxx–xxx
3
the introduction of various ester groups significantly enhanced the
activity, among which, compounds 1d, 1k and 1l showed efficacy
against three diseases. Compounds 1a, 1b, 1f and 1g showed
efficacy against four diseases; and especially compound 2,2,2-
trifluoroethyl-6-ester (1c) showed efficacy against five of the seven
tested diseases, including M. melonis (69%), Corynespora cassiicola
(52%), P. syringae pv. Lachrymans (42%), P. infestans (67%), and B.
cinerea (41%). In addition, compounds 1a, 1b, 1c, 1f and 1g showed
efficacy against various types of pathogens including fungi,
oomycetes, and bacteria. Therefore, we concluded that the
introduction of the proper ester groups, such as small alkyl groups
like methyl (1a) and ethyl (1b), increased the efficacy, while larger
alkyl groups, such as propyl (1d) and isopropyl (1e), decreased the
activity, as these two compounds only showed activity toward
three and two tested diseases, respectively. Furthermore, intro-
duction of fluorine atoms in these alkyl groups could further
enhance their activities, as compounds 1c (2,2,2-trifluoroethyl), 1f
(2,2,3,3,3-pentafluropropyl) and 1g (1,1,1,3,3,3-hexafluoro-2-pro-
pyl) all showed better efficacy than 1b (ethyl), 1d (propyl) and 1e
(isopropyl), respectively.
the National High Technology Research and Development Program
of China (863 Program, No. 2011AA10A207), the China 111 Project
(No. B07023), and the Fundamental Research Funds for the Central
Universities.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.cclet.2013.07.003.
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Acknowledgments
This work is financially supported by the National Basic
Research Program of China (973 Program, No. 2010CB126100),
Please cite this article in press as: Q.-S. Du, et al., Novel plant activators with thieno[2,3-d]-1,2,3-thiadiazole-6-carboxylate scaffold:
Synthesis and bioactivity, Chin. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.cclet.2013.07.003