Synthesis and biological evaluations of a series of calycanthaceous analogues as antifungal agents

Article abstract of DOI:10.1080/14786419.2019.1644635

Starting from indole-3-acetonitrile, a total of 66 new calycanthaceous alkaloid analogues were synthesised in excellent yields. The prepared compounds were evaluated for their biological activities against a broad range of plant pathogen fungi. The results of bioassays indicated that the majority of tested compounds displayed comparable or better in vitro bioactivities than the positive control. Notably, Compound a1 displayed a significant activities against B. cereus, Escherichia sp and R. solanacearum, even better than the positive control streptomycin and Penicillin, with the same MIC value of 15.63 μg mL^?1. Compound a1 displayed a broad spectrum and remarkably activities among the tested calycanthaceous analogues and might be a novel potential leading compound for further development of antifungal agents. The results obtained in the study will be very helpful for further design and structural optimisation of calycanthaceous alkaloids as potential agrochemical lead for plant disease control.

Full text of DOI:10.1080/14786419.2019.1644635

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Synthesis and biological evaluations of a series of
calycanthaceous analogues as antifungal agents
Shaojun Zheng, Rui Zhu, Bing Tang, Lizhuang Chen, Hongjin Bai & Jiwen
Zhang
To cite this article: Shaojun Zheng, Rui Zhu, Bing Tang, Lizhuang Chen, Hongjin Bai & Jiwen
Zhang (2019): Synthesis and biological evaluations of a series of calycanthaceous analogues as
antifungal agents, Natural Product Research, DOI: 10.1080/14786419.2019.1644635
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2019.1644635
Synthesis and biological evaluations of a series of
calycanthaceous analogues as antifungal agents
Shaojun Zhenga, Rui Zhua, Bing Tanga, Lizhuang Chena, Hongjin Baib and
Jiwen Zhangc
aSchool of Environmental and Chemical Engineering, Jiangsu University of Science and Technology,
b
Zhenjiang Jiangsu, China; Key Laboratory of Protection & Utilization of Biological Resources in
Tarim Basin of Xinjiang Production and Construction Corps/College of Life Sciences, Tarim University,
c
Alar, Xinjiang, China; Key Laboratory of Botanical Pesticide R & D in Shaanxi Province, Yangling,
Shaanxi, China
ABSTRACT
ARTICLE HISTORY
Received 5 May 2019
Accepted 13 July 2019
Starting from indole-3-acetonitrile, a total of 66 new calycantha-
ceous alkaloid analogues were synthesised in excellent yields. The
prepared compounds were evaluated for their biological activities
against a broad range of plant pathogen fungi. The results of bio-
assays indicated that the majority of tested compounds displayed
comparable or better in vitro bioactivities than the positive
control. Notably, Compound a1 displayed a significant activities
against B. cereus, Escherichia sp and R. solanacearum, even better
than the positive control streptomycin and Penicillin, with
the same MIC value of 15.63 mg mLꢀ1. Compound a1 displayed
a broad spectrum and remarkably activities among the tested
calycanthaceous analogues and might be a novel potential
leading compound for further development of antifungal agents.
The results obtained in the study will be very helpful for further
design and structural optimisation of calycanthaceous alkaloids
as potential agrochemical lead for plant disease control.
KEYWORDS
Calycanthaceous alkaloids;
synthesis; plant pathogen
fungi; biological
activity; SAR
1. Introduction
Pesticides play a key role in our life, not only for crop protection in agriculture, but
also for human health. The exploitation of new pesticides, especially the pesticides
with high efficacy and selectivity against target species, has become an increasing
CONTACT Shaojun Zheng
sz281cam@just.edu.cn; Hongjin Bai
bhj67@163.com; Jiwen Zhang
nwzjw@
nwsuaf.edu.cn
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1644635x.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
S. ZHENG ET AL.
Figure 1. Structures of calycanthaceous alkaloids.
focus in agricultural chemistry. Imitating the chemistry of biologically active natural
products is one approach for developing such pesticides (Du 2003; Huang et al. 2007).
The Calycanthaceae plants (Figure 1), mainly distributed in China, North America
and Australia, are an important class of alkaloids that can be isolated from the roots,
leaves, flowers, and fruits of Chimonanthus praecox (Bowman et al. 2005; De Fusco
et al. 2017). Calycanthaceous alkaloids, which contain hexahydropyrroloindole skele-
tons, have been used as traditional chinese medicines for the treatment of fungal
infection (Bowman et al. 2005), hypertension, tumour, inflammatory, and melanogene-
sis (Hino and Yamada 1963; Hall et al. 1967; Fang et al. 1994; Movassaghi and Schmidt
2007; Ruiz-Sanchis et al. 2011; Li et al. 2012; Araki et al. 2013; Peng et al. 2013; Kim
and Movassaghi 2015; Xu and Cheng 2015).
Our group has recently reported the preparation and biological evaluations of a ser-
ies of calycanthaceous analogues. We discovered that different substituents at N pos-
ition could remarkably affect the biological activities. (Zheng et al. 2012, 2016a, 2016b,
2017, 2018a, 2018b). In addition, introducing Fluorine Substitutes into the natural
product derivatives could enhance their biological activity (Jia et al. 2018). These find-
ings inspired us to further modify the structure of calycanthaceous alkaloids with func-
tional motifs so as to acquire potential agrochemical leads for plant disease control.
As a continuation of the development of new natural-product-based antifungal
agents, a series of N-substituted calycanthaceous alkaloid analogues were designed
1
and prepared, and their structures were characterised by H NMR, 13C NMR and MS.
To the best of our knowledge, the biological activities of the prepared analogues
are reported for the first time.
2. Results and discussion
2.1. Design and synthesis of calycanthaceous alkaloids analogues
The synthetic route of title compounds is given in Scheme 1. The calycanthaceous
alkaloid analogues were prepared according to a previously reported procedure in our
group, and their spectral data were consistent with reported values (Zheng et al.
2016a, 2016b, 2017, 2018a, 2018b). The derivatives of calycanthaceous alkaloids were
prepared from indole-3-acetonitrile via acylation at the N position. A total of 66 caly-
1
canthaceous analogues were prepared and characterised by H-NMR, 13C-NMR spec-
troscopy and ESI-MS.

NATURAL PRODUCT RESEARCH
3
Scheme 1. Synthetic route to the title compounds a1–a22, b1–b22 and c1–c22.
2.2. Antimicrobial activity
Inhibitory effects of calycanthaceous alkaloid analogues against a wide range of plant
pathogen fungi are listed in Table 1. MIC were examined with Carbendazim,
Amphotericin B, Chlorothalonil, Gentamicin, Streptomycin, Penicillin, and Fluconazole
as the positive control, to evaluate the activities of the synthesised calycanthaceous
alkaloid analogues against Verticillium dahliae, Fusarium oxysperium sp. vasinfectum,
Cytospora juglandis, Aspergillu sflavu, Penicillium citrinum, Fusarium oxysporum,
Colletotrichum orbiculare, Aspergillus niger, B. cinerea Pers, Curvularia lunaia, Escherichia
sp, Pseudomonas aeruginosa, Ralstonia solanacearum, Bacillus cereus, Staphylococcus
aureus, Candida krusei, Crytococcus Neofonmans and Candida tropicalis.
Antibacterial results are shown in Table 1. It is manifested that these series of analogues
generally exhibit more effective antimicrobial activity than the positive control.
Compounda1 displayed better activity against Escherichia sp than that of streptomycin,
with MIC value of 15.63 mg mLꢀ1. Compounds b13 and c13 illustrated much more effect-
ive activities against V. dahliae than the positive control Chlorothalonil, with MIC value of
31.25 mg mLꢀ1. Compound c13 showed improved activity compared with the positive con-
trol Chlorothalonil against V. dahliae, with MIC value of 31.25mg mLꢀ1. The activity of
Compound a17 is more potent than carbendazol, amphotericin B and Chlorothalonil
against F. oxysporum f. sp., with MIC value of 31.25mg mLꢀ1. Compound c2 illustrated bet-
ter activity against C. juglandis than the positive control carbendazol, with MIC value of
31.25 mg mLꢀ1. Compound c2 illustrated much more effective activity against C. juglandis
than that of amphotericin B and Chlorothalonil, with the same MIC value of
31.25 mg mLꢀ1. Compound b8 revealed better activity against C. orbiculare than the posi-
tive control amphotericin B and Chlorothalonil, with the same MIC value of 31.25mg mLꢀ1
.
Compounds a8 and a13 manifested improved activity compared with the positive control
carbendazol, amphotericin B and Chlorothalonil against F. oxysporum f. sp., with the same
MIC value of 62.5 mg mLꢀ1. Compound a2 illustrated much more active against A. niger
than that of carbendazol, with MIC value of 62.5 mg mLꢀ1. Compounds a3, a8, b1, b7, b17,
b18, c3 and c8 showed better activities against C. juglandis than the positive control
amphotericin B, with the same MIC value of 62.50 mg mLꢀ1. Compounds b3 and b4 illus-
trated better activity than the control Chlorothalonil against F. oxysporum f. sp., with the

4
S. ZHENG ET AL.

NATURAL PRODUCT RESEARCH
5

6
S. ZHENG ET AL.
same MIC value of 125mg mLꢀ1. Compounds a1, a2, a4, a17, a19, a21, a22, b2, b3, b5,
b6, b12, b13, b15, b16, c4, c7, c17 and c20 revealed better activities against C. juglandis
than that of amphotericin B, with the same MIC value of 125mg mLꢀ1. Compounds a1, a3,
a10, b12 and b13 illustrated much more activities against A. niger than that of carbenda-
zol, with the same MIC value of 125 mg mLꢀ1. Compounds a1, a8, c2 and c8 manifested
better activities against C. hmaia than that of carbendazol, with the same MIC value of
125 mg mLꢀ1
Compounds a1, a2, a3 and c3 illustrated better activities against B. cereus, with
MIC values of 15.63 mg mLꢀ1, 31.25 mg mLꢀ1, 31.25 mg mLꢀ1 and 31.25 mg mLꢀ1
.
,
respectively. Compound b8 manifested much more effective activity against R. solana-
cearum than the positive control gentamicin and streptomycin, with MIC value of
31.25 mg mLꢀ1. Compound b7 illustrated better activity against R. solanacearum than
the positive control streptomycin, with MIC value of 62.5 mg mLꢀ1. Compounds a8,
a15, b1, b6 and b9 displayed improved activity compared with the positive control
streptomycin against R. solanacearum, with MIC value of 125 mg mLꢀ1
.
Compound c12 illustrated better activity against C. krolimus than that of
Fluconazole, with MIC value of 62.5 mg mLꢀ1. Compounds a4 and a14 manifested
much more effective activities against C. krolimus than that of carbendazol and
amphotericin B, with MIC value of 125 mg mLꢀ1. Compounds a5, a6, a14 and a15
revealed better activities against C. tropicalis, with MIC value of 125 mg mLꢀ1
.
Compounds b2 and b13 showed moderate activities against F. oxysperium sp. vasin-
fectum to that of carbendazol, with MIC value of 62.5 mg mLꢀ1. Compounds a3, a8,
b1, b7, b17, b18, c3 and c8 manifested comparable control efficacy against C. juglan-
dis than that of Chlorothalonil, with MIC value of 62.5 mg mLꢀ1. Compounds b2 and
b13 showed moderate activities against F. oxysperium sp. vasinfectum to that of car-
bendazol, with MIC value of 62.5 mg mLꢀ1. Compounds a1, a3, a4, a5, a6, a7, a10,
a20, b2, b7 and b8 displayed comparable control efficacy against F. oxysporum than
that of carbendazol, with MIC value of 125 mg mLꢀ1. Compound b8 illustrated compar-
able control efficacy against C. orbiculare than that of carbendazol, with MIC value of
125 mg mLꢀ1. Compounds a1, a8, c2 and c8 displayed comparable control efficacy
against C. lunaia than that of Chlorothalonil, with MIC value of 125 mg mLꢀ1
.
Compound a1 revealed comparable control efficacy against R. solanacearum than that
of Penicillin, with MIC value of 15.63 mg mLꢀ1. Compound b8 illustrated comparable control
efficacy against R. solanacearum than that of gentamicin, with MIC value of 62.5 mg mLꢀ1
.
3. Experimental
3.1. Instruments and chemicals
All reagents and solvents were reagent grade or purified according to standard methods
before use. Analytical thin-layer chromatography (TLC) was performed with silica gel
plates using silica gel 60 GF254 (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China).
Melting points were measured on an Electrothermal digital apparatus (Beijing, China) and
1
were uncorrected. The Hꢀ NMR (500 MHz), and 13Cꢀ NMR (125 MHz) were obtained on
an AM ꢀ 500 FT ꢀ NMR spectrometer (Bruker Corporation, Switzerland) with CDCl3 as the
solvent and TMS as the internal standard. MS were recorded under ESI conditions using a

NATURAL PRODUCT RESEARCH
7
LCQ Fleet instrument (Thermo Fisher, Waltham, MA, USA). Yields were not optimised. The
title compounds were synthesised under a nitrogen atmosphere.
3.2. Synthesis
3.2.1. Synthesis of intermediates 1-3
Intermediates 1-3 were synthesised according to our previously reported procedure
(Zheng et al. 2016b).
3.2.2. Synthesis of compounds a1–a22, b1–b22 and c1–c22
Compound 3 was dissolved in pyridine (10 mL), then, the corresponding desired reagent
was added at 0 ꢁC. After refluxing for 2 h. The mixture was warmed to room temperature.
Then, the resulting mixture was reacted for 1.5 h. At last, the resulting mixture was
quenched with methanol (2 mL), and extracted with ethyl acetate. The organic extracts
were combined, washed with brine, dried over Na2SO4, and concentrated. Purification by
flash chromatography on silica gel afforded the compounds a1–a21 in yields from 80%
to 96% (Characterization data see Supplementary materials).
3.3. Biological activity
The antimicrobial activity of calycanthaceous alkaloids analogues were measured
according to the previously reported method (Zhang et al. 2009, 2013).
The tested compounds dissolved in 5% dimethyl sulfoxide (DMSO), to a concentration
of 1.02 mg/mL, 100 lL of the solutions were added to the first well and serially diluted
from first well by taking 100 lL into second. This two-fold dilution was continued down
the plate and 100 lL from the 8th column of the plated discarded. The 9th column of the
plate was reserved for negative control wells (without inocula) and the 10th column, for
the positive growth control wells (without antibacterial agent). The antibacterial concen-
trations were 256, 128, 64, 32, 16, 8, 4 and 2 lg/mL, respectively. The antibacterial test
plates were incubated aerobically at 37 ꢁC for 24 h, the antifungal test plates were incu-
bated aerobically at 28 ꢁC for 48 h. The MICs, MBC and MFC were examined. MBC and
MFC were determined by plating 10 lL from each negative well and from the positive
growth control on LB Agar and Sabouraud Dextrose Agar. MBC and MFC were defined as
the lowest concentration yielding negative subcultures or only on colony. All tests were
performed in triplicate and repeated if the results differed.
4. Conclusions
A total of 66 novel tetrahydropyrroloindole-based calycanthaceous alkaloid analogues
were prepared using indole-3-acetonitrile as the starting material via acylation at the
N3 position, and their activities against a wide range of plant pathogen fungi were
screened. The results of bioassays revealed that most of the title compounds mani-
fested potent activities against a broad variety of plant pathogen fungi, which were
more effective than the positive controls. Notably, Compound a1 displayed a signifi-
cant activities against B. cereus, Escherichia sp and R. solanacearum, even better than

8
S. ZHENG ET AL.
the positive control streptomycin and Penicillin, with the same MIC value of 15.63 mg
mLꢀ1. Compound a1 displayed a broad spectrum and remarkably high activities
among the tested calycanthaceous analogues and might be a novel potential leading
compound for further development of antifungal agents. The results obtained in the
study will be very helpful for further design, structural optimisation, and development
of calycanthaceous alkaloids as antimicrobial agents.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the National Natural Science Foundation of China (21502073 and
31360079), the Natural Science Foundation of Jiangsu Province (Grant nos. BK 20150465 and
BK20180978) and the Key Research and Development Program (Modern Agriculture) of
Zhenjiang City (NY2018002).
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