Novel S-Thiazol-2-yl-furan-2-carbothioate Derivatives as Potential T3SS Inhibitors against Xanthomonas oryzae on Rice

Article abstract of DOI:10.1021/acs.jafc.9b04085

Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv oryzae (Xoo) is considered as the most destructive disease of rice. The use of bactericides is among the most widely used traditional methods to control this destructive disease. The excessive an

Full text of DOI:10.1021/acs.jafc.9b04085

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Agricultural and Environmental Chemistry
Novel S-thiazol-2-yl-furan-2-carbothioate derivatives as
potential T3SS inhibitors against Xanthomonas oryzae on rice
Shan Jiang, Min He, Xuwen Xiang, Muhammad Adnan, and Zi-Ning Cui
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b04085 • Publication Date (Web): 04 Oct 2019
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1 Novel S-thiazol-2-yl-furan-2-carbothioate derivatives as
2 potential T3SS inhibitors against Xanthomonas oryzae on
3 rice
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Shan Jiang†,#, Min He†,#, Xu-Wen Xiang†, Muhammad Adnan†, Zi-Ning Cui†,*
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†State Key Laboratory for Conservation and Utilization of Subtropical
Agro-bioresources, Integrative Microbiology Research Centre, Guangdong Province
Key Laboratory of Microbial Signals and Disease Control, South China Agricultural
University, Guangzhou 510642, China
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#Both authors contributed equally to this paper.
*Corresponding author. Tel.: +86-20-85288229; Fax: +86-20-85288229
E-mail address: ziningcui@scau.edu.cn (Z. C.)
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ABSTRACT: Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae
(Xoo) is considered as the most destructive disease of rice. Using bactericides is
among the widely used traditional methods to control this destructive disease. The
excessive and repeated use of the same bactericides is also becoming the reason
behind bactericide resistance development. The widely used method for finding the
new anti-microbial agents often involves the bacterial virulence factors as a target
without affecting bacterial growth. Type III secretion system (T3SS) is a protein
appendage and considered as essential virulence factors in most gram-negative
bacteria. Due to the conserved construct, T3SS has been regarded as an important
mark for the blooming of novel anti-microbial drugs. Towards the search of new
T3SS inhibitors, an alternative series of 1,3-thiazole derivatives were designed and
1
synthesized. Their structures were characterized and confirmed by H NMR, 13C
NMR, MS, and elemental analysis. All the title compounds inhibited the promoter
activity of hpa1 gene, significantly. Eight of them showed better inhibition than our
previous T3SS inhibitor TS006 (o-coumaric acid, OCA). The treatment of Xoo with
eight compounds significantly attenuated HR without affecting bacterial growth. The
mRNA levels of some representative genes (hrp/hrc genes) were reduced up to
different extents. In vivo bioassay results showed that eight T3SS inhibitors could
reduce bacterial leaf blight and bacterial leaf streak symptoms on rice, significantly.
KEYWORDS: thiazole; synthesis; type III secretion system (T3SS); inhibitors
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INTRODUCTION
Xanthomonas oryzae pv. oryzae (Xoo) is the most serious rice bacterial disease that
causes bacterial leaf blight worldwide.1 It devastates rice growth and then affects rice
production, and ultimately results in huge crop yield and economic losses.2 Various
traditional antibiotics have been used for many years and now they are reported to be
restricted in many countries because of their environmental impact and development
of disease resistance.3,4 Therefore, there is always a need for the development of new
compounds targeting bacterial virulence factors without affecting their growth.5 Such
compounds which do not affect the bacterial growth little selective pressure, hence,
preventing the development of bacterial disease resistance.6 Xoo and many other
gram-negative bacteria invade host plant cells by using type three secretion system.7,8
T3SS is conserved in different species,7 and the T3SS is encoded by the hrp, hrc and
hpa genes.9,10 The hrp genes play a critical role in hypersensitive response (HR) and
plant pathogenicity.7,11 In Xanthomonas oryzae, HrpG-HrpX regulatory cascade
activates the most hrp genes expression.12 HrpG belongs to the OmpR family, which
plays an important role in responding to the environmental stimulation.13 The
expression of hrpX was regulated by hrpG. HrpX is a member of the AraC-family
regulator.14,15 It is essential for the expression of five operons (hrpB, hrpC, hrpD,
hrpE and hrpF) in the hrp gene cluster, while these operons encode the protein
products required by the T3SS.14,15
Various natural and synthetic compounds have been identified and reported, which
are capable of inhibiting T3SS in human or plant pathogens like Pseudomonas,16
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Yersinia,17 Salmonella,18 and Xanthomonas5, etc. These compounds with different
structures (salicylidene acylhydrazide, p-coumaric acid, o-coumaric acid,
N-hydroxybenzimidazole, etc.) have shown the great activity in vitro or in vivo
experiments. The thiazole is a special five-membered heterocyclic ring containing
nitrogen and sulfur atoms. Its aromatic structure enables thiazole to be modified into
compounds with various biological activities. It has been reported that thiazole
derivatives are not only used widely in medical fields (antiviral and anticancer
drugs),19,20 but also plays an indispensable role in agrochemicals (insecticide,
herbicides, fungicides).21-23 Such broad-spectrum biological activities make thiazole
derivatives become a hotspot in the design and synthesis of compounds. In our
previous work, we have synthesized and screened some thiazole and thiadiazole
derivatives as anti-virulence agents through suppressing the T3SS (Scheme 1). It can
be concluded that thiazole is a framework with good biological activity. The
compounds obtained by substituting thiazole on its rings have great worth in the
pharmaceutical and agrochemical industries. Therefore, it is very important to
continue the research on the design and synthesis of thiazole-related derivatives.
In this study, we extended our previous work24-26 to synthesis a new series of
1,3-thiazole derivatives. We have screened all compounds by using flow cytometry
analysis. Positive candidate compounds exhibited an excellent ability to control
bacterial leaf blight on rice by suppressing T3SS functionality without affecting
bacterial growth.
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MATERIALS AND METHODS
Instrumental analysis.
Mass spectrum analysis were done by using a Bruker APEX IV spectrometer
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(Bruker, Fallanden, Switzerland). H NMR and C NMR spectra were recorded on
Bruker DPX400 and Bruker AV600 (Bruker, Fallanden, Switzerland), while
tetramethylsilane was used as an internal standard. Chemical shifts δ are given in ppm
and coupling constant J are in Hz. signal patterns are indicated as follows: s, singlet; d,
doublet; dd, doublet of doublets; dt, doublet of triplets; ddd, doublet of doublets of
doublets; t, triplet; m, multiplet; bs, broad singlet, quin., quintet. Melting points were
recorded with a Cole-Parmer melting point apparatus (ColeParmer, Vernon Hills,
Illinois, USA). Elemental analyses were operated on a Vario EL elemental analyzer.
Analytical thin-layer chromatography was carried out on silica gel 60 F254 plates.
Technical grade ethyl acetate and hexane used for column chromatography were
distilled prior to use. The promoter activity of hpa1 was checked by a FACS-Caliber
flow cytometer (CytoFLEX USA). The growth rates were recorded using a Bioscreen
(Bioscreen, Finland). RNA concentration and purity were monitored using the
Nanovue UV-Vs spectrophotometer (GE Healthcare Bio-Science, Sweden). The
cDNA levels were quantified by real-time PCR using a SYBR Green Master Mix
(Thermo, USA).
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Synthesis methods of key intermediate I
According to the previous reports,27,28 by using the method of Meerwein arylation
reaction, a series of 5-substituted phenyl-2-furoic acid I were synthesized with
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substituted aniline and furoic acid as starting reagents.
Synthesis methods of key intermediate II
At room temperature, concentrated hydrochloric acid (150 mmol) was added to a
round-bottomed flask containing chloracetaldehyde solution (100 mmol). The
reaction lasted for two hours. And then the reaction solution was dissolved in 12.12 g
water. Under the condition of the water bath, ammonium dithiocarbamate (100 mmol)
was added. The mixture was stirred at room temperature for 60 minutes, then the
temperature of the reaction system was increased to 74 oC, and the reaction continued
for 3 hours. Upon completion, the reaction was cooled to room temperature. The
mixture was extracted three times with ethyl acetate, and the organic phase was dried
with anhydrous magnesium sulfate. Concentration and recrystallization of the filtrate
were done to obtain thiazole-2-thiol (compounds II).
Synthesis methods of title compound III
Compound I (2 mmol) and dichlorosulfoxide (5 mL) were stirred and heated in a
round bottom flask. Two hours later, the excess dichlorosulfoxide was removed by
decompression and distillation. Acetonitrile, triethylamine, 4-dimethylaminopyridine
and compound II were added to the round bottom flask and stirred overnight at room
temperature. When the reaction was completed, the mixture was extracted three times
with water and dichloromethane. Later, the organic phase was washed successively
with 10% hydrochloric acid, 10% sodium bicarbonate and water, dried with
anhydrous magnesium sulfate. Concentration and recrystallization of the filtrate were
done to obtain compounds III.
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S-(thiazol-2-yl) 5-(2-chlorophenyl) furan-2-carbothioate (III-1). Pink solid: yield
74%. m.p. 137.8-138.7 oC. 1H NMR (400 MHz, Chloroform-d) δ 7.98 (d, J = 3.4 Hz,
1H, ThiaH), 7.79 (t, J = 1.5 Hz, 1H, PhH), 7.70 (dt, J = 7.1, 1.7 Hz, 1H, PhH), 7.61 (d,
J = 3.4 Hz, 1H, ThiaH), 7.45 – 7.36 (m, 3H, PhH + FuH), 6.88 (d, J = 3.8 Hz, 1H,
FuH). 13C NMR (101 MHz, DMSO-d6) 13C NMR (101 MHz, CDCl3) δ 175.18 (C=O),
157.16 (C-2, Thia), 153.64 (C-5, Fu), 148.48 (C-2, Fu), 143.04 (C-4, Thia), 142.89
(C-2, Ph), 135.14 (C-1, Ph), 130.37 (C-4, Ph), 129.68 (C-3, Ph), 125.06 (C-6, Ph),
123.35 (C-5, Ph), 123.15 (C-3, Fu), 119.68 (C-5, Thia), 108.78 (C-4, Fu).
ESI-MS:m/e 322.5 [M+H]+. Anal. Calcd. (%) for C14H8ClNO2S2: C,52.26; H, 2.51; N,
4.35. Found: C, 52.44;H, 2.30 ; N, 4.60.
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S-(thiazol-2-yl) 5-(3-chlorophenyl) furan-2-carbothioate (III-2). Pink solid: yield
o
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78%. m.p. 136.8-137.7 C. H NMR (600 MHz, Chloroform-d) δ 8.00 (dd, J = 7.9,
1.6 Hz, 1H, PhH), 7.97 (d, J = 3.3 Hz, 1H, ThiaH), 7.60 (d, J = 3.3 Hz, 1H, ThiaH),
7.50 (dd, J = 8.0, 1.2 Hz, 1H, PhH), 7.44 (d, J = 3.8 Hz, 1H, FuH), 7.41 (td, J = 7.7,
1.3 Hz, 1H, PhH), 7.35 (dd, J = 7.5, 1.7 Hz, 1H, , PhH), 7.33 (d, J = 3.8 Hz, 1H, FuH).
13C NMR (151 MHz, CDCl3) δ 175.33 (C=O), 155.02 (C-2, Thia), 153.80 (C-5, Fu),
147.97 (C-2, Fu), 143.04 (C-4, Thia), 131.56 (C-3, Ph), 131.06 (C-1, Ph), 130.26 (C-5,
Ph), 129.00 (C-4, Ph), 127.48 (C-2, Ph), 127.31 (C-6, Ph), 123.27 (C-3, Fu), 119.25
(C-5, Thia), 113.42 (C-4, Fu). ESI-MS(m/e): 322.6 [M+H]+. Anal. Calcd. (%) for
C14H8ClNO2S2: C, 52.26; H, 2.51; N, 4.35. Found: C, 52.09; H, 2.75; N, 4.27.
S-(thiazol-2-yl) 5-(4-chlorophenyl) furan-2-carbothioate (III-3). Pink solid: yield
73%. m.p. 130.4-134.0 oC. 1H NMR (600 MHz, Chloroform-d) δ 7.97 (d, J = 3.3 Hz,
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1H, ThiaH), 7.77 – 7.73 (m, 2H, PhH), 7.60 (d, J = 3.4 Hz, 1H, ThiaH), 7.46 – 7.43
(m, 2H, PhH), 7.42 (d, J = 3.8 Hz, 1H, FuH), 6.84 (d, J = 3.8 Hz, 1H, FuH). 13C NMR
(151 MHz, CDCl3) δ 175.04 (C=O), 157.75 (C-2, Thia), 153.77 (C-5, Fu), 148.38
(C-2, Fu), 143.05 (C-4, Thia), 135.79 (C-4, Ph), 129.38 (C-1, Ph), 127.31 (C-2, C-6,
Ph), 126.36 (C-3, C-5, Ph), 123.31 (C-3, Fu), 119.83 (C-5, Thia), 108.24 (C-4, Fu).
ESI-MS(m/e): 322.5 [M+H]+. Anal. Calcd. (%) for C14H8ClNO2S2: C, 52.26; H, 2.51;
N, 4.35. Found: C, 52.38; H, 2.42; N, 4.21.
S-(thiazol-2-yl) 5-(2-fluorophenyl) furan-2-carbothioate (III-4). White solid: yield
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80%. m.p. 63.7-65.0 C. H NMR (600 MHz, Chloroform-d) δ 8.00 (td, J = 7.7, 1.7
Hz, 1H, PhH), 7.97 (d, J = 3.4 Hz, 1H, ThiaH), 7.60 (d, J = 3.4 Hz, 1H, ThiaH), 7.45
(d, J = 3.8 Hz, 1H, FuH), 7.39 (dddd, J = 8.3, 7.0, 5.2, 1.8 Hz, 1H, PhH), 7.29 (td, J =
7.6, 1.1 Hz, 1H, PhH), 7.18 (ddd, J = 11.3, 8.3, 1.1 Hz, 1H, PhH), 7.04 (t, J = 3.6 Hz,
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1H, FuH). C NMR (151 MHz, Chloroform-d) δ 175.18 (C=O), 159.71 (d, JC-F
=
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253.2 Hz, C-2, Ph), 153.83 (C-2, Thia), 153.08 (d, JC-F = 3.0 Hz, C-4, Fu), 147.88
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(C-2, Fu), 143.04( C-5, Thia), 130.97 (d, JC-F = 8.7 Hz, C-4, Ph), 127.07 (d, JC-F
=
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2.1 Hz, C-5, Ph), 124.79 (d, JC-F = 3.5 Hz, C-6, Ph), 123.27 (C-3, Fu), 119.83 (C-5,
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Thia), 117.31 (d, JC-F = 11.5 Hz, C-1, Ph), 116.30 (d, JC-F = 21.3 Hz, C-3, Ph),
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112.70 (d, JC-F = 12.3 Hz, C-5, Fu). ESI-MS(m/e): 306.1 [M+H]+. Anal. Calcd. (%)
for C14H8FNO2S2: C, 55.07; H, 2.64; N, 4.59. Found: C, 55.22; H, 2.71; N, 4.41.
S-(thiazol-2-yl) 5-(3-fluorophenyl) furan-2-carbothioate (III-5). White solid: yield
72%. m.p. 108.6-109.6 oC. 1H NMR (400 MHz, Chloroform-d) δ 7.98 (d, J = 3.4 Hz,
1H, ThiaH), 7.64 – 7.57 (m, 2H, PhH + ThiaH), 7.54 – 7.49 (m, 1H, PhH), 7.48 –
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7.41 (m, 2H, PhH + FuH), 7.12 (tdd, J = 8.4, 2.6, 0.9 Hz, 1H, PhH), 6.88 (d, J = 3.8
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Hz, 1H, FuH). C NMR (101 MHz, Chloroform-d) δ 175.22 (C=O), 163.07 (d, JC-F
= 246.8 Hz, C-3, Ph), 157.38 (d, 4JC-F = 3.1 Hz, C-5, Fu), 153.65 (C-2, Thia), 148.43
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(C-2, Fu), 143.06 (C-4, Thia), 130.79 (d, JC-F = 8.1 Hz, C-5, Ph), 130.67 (d, JC-F
=
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8.1 Hz, C-1, Ph), 123.37 (C-3, Fu), 120.81 (d, JC-F = 3.1 Hz, C-6, Ph), 119.68 (C-5,
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Thia), 116.67 (d, JC-F = 21.3 Hz, C-4, Ph), 112.02 (d, JC-F = 23.8 Hz, C-2, Ph),
108.77 (C-4, Fu). ESI-MS(m/e): 306.3 [M+H]+. Anal. Calcd. (%) for C14H8FNO2S2: C,
55.07; H, 2.64; N, 4.59. Found: C, 55.31; H, 2.45; N, 4.70.
S-(thiazol-2-yl) 5-(4-fluorophenyl) furan-2-carbothioate (III-6). Gray solid: yield
89%. m.p. 129.7-131.3 oC. 1H NMR (600 MHz, Chloroform-d) δ 7.96 (d, J = 3.3 Hz,
1H, ThiaH), 7.83 – 7.77 (m, 2H, PhH), 7.59 (d, J = 3.4 Hz, 1H, ThiaH), 7.41 (d, J =
3.8 Hz, 1H, FuH), 7.19 – 7.12 (m, 2H, PhH), 6.79 (d, J = 3.8 Hz, 1H, FuH). 13C NMR
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(151 MHz, Chloroform-d) δ 175.06 (C=O), 163.66 (d, JC-F = 250.9 Hz, C-4, Ph),
158.11 (C-2, Thia), 153.94 (C-5, Fu), 148.30 (C-2, Fu), 143.13 (C-4, Thia), 127.28 (d,
3JC-F = 8.4 Hz, C-2, C-6, Ph), 125.31 (d, JC-F = 3.3 Hz, C-1, Ph), 123.39 (C-3, Fu),
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120.06 (C-5, Thia), 116.40 (d, JC-F = 22.2 Hz, C-3, C-5, Ph), 107.75 (C-4, Fu).
ESI-MS(m/e): 306.2 [M+H]+. Anal. Calcd. (%) for C14H8FNO2S2: C, 55.07; H, 2.64;
N, 4.59. Found: C, 54.89; H, 2.84; N, 4.36.
S-(thiazol-2-yl)5-(2,4-difluorophenyl) furan-2-carbothioate (III-7). Yellow solid:
o
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yield 81%. m.p. 135.9-138.6 C. H NMR (400 MHz, Chloroform-d) δ 7.98 (q, J =
7.1, 6.5 Hz, 2H, ThiaH + PhH), 7.61 (d, J = 2.8 Hz, 1H, FuH), 7.44 (d, J = 3.3 Hz, 1H,
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ThiaH), 7.04 (t, J = 8.2 Hz, 1H, PhH), 7.00 – 6.87 (m, 2H, FuH + PhH). C NMR
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(101 MHz, Chloroform-d) δ 175.14 (C=O), 163.36 (dd, JC-F= 253.7, JC-F =12.1 Hz,
C-4, Ph), 159.93 (dd, 1J = 255.8, 3JC-F =11.9 Hz, C-2, Ph), 153.67 (C-2, Thia), 152.25
( C-5, Fu), 147.81 (C-2, Fu), 143.04 (C-4, Thia), 128.21 (dd, 3JC-F= 9.8, 3JC-F= 3.9 Hz,
C-6, Ph), 123.39(C-3, Fu), 119.94(C-5, Thia), 113.93 (dd, 2JC-F = 11.9, JC-F=4.0 Hz,
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C-1, Ph), 112.42 (dd, JC-F= 21.8, JC-F=3.6 Hz, C-5, Ph), 112.10 (d, JC-F= 12.2 Hz,
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C-4, Fu), 104.91 (dd, JC-F= 25.5 Hz, JC-F= 25.5 Hz, C-3, Ph). ESI-MS(m/e): 324.1
[M+H]+. Anal. Calcd. (%) for C14H8F2NO2S2: C, 52.01; H, 2.18; N, 4.33. Found: C,
52.25; H, 2.36; N, 4.18.
S-(thiazol-2-yl) 5-(2,6-difluorophenyl) furan-2-carbothioate (III-8). Yellow solid:
o
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yield 70%. m.p. 86.0-86.8 C. H NMR (600 MHz, Chloroform-d) δ 7.97 (d, J = 3.3
Hz, 1H, ThiaH), 7.59 (d, J = 3.3 Hz, 1H, ThiaH), 7.44 (d, J = 3.8 Hz, 1H, FuH), 7.37
(tt, J = 8.4, 6.1 Hz, 1H,, PhH), 7.07 – 7.02 (m, 3H, FuH + PhH). 13C NMR (151 MHz,
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Chloroform-d) δ 175.66 (C=O), 159.91 (dd, JC-F = 256.5, JC-F =6.1 Hz, C-2, C-6,
Ph), 153.87 (C-2, Thia), 148.83 (dd, 4JC-F = 2.3 Hz, 4JC-F = 2.3 Hz，C-4, Fu), 148.70
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(C-2, Fu), 143.05 (C-4, Thia), 130.84 (t, JC-F = 10.7 Hz, C-4, Ph), 123.26 (C-3, Fu),
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118.39 (C-5, Thia), 114.82 (dd, JC-F = 6.3 Hz, JC-F = 6.3 Hz, C-5, Fu), 112.35 (dd,
2JC-F = 21.8, 4JC-F =4.1 Hz, C-3, C-5, Ph), 107.67 ( dd, 2JC-F= 15.5 Hz, 2JC-F= 15.6 Hz,
C-1, Ph). ESI-MS(m/e): 324.2 [M+H]+. Anal. Calcd. (%) for C14H8F2NO2S2: C, 52.01;
H, 2.18; N, 4.33. Found: C, 51.96; H,1.96; N, 4.52.
S-(thiazol-2-yl) 5-(2-nitrophenyl) furan-2-carbothioate (III-9). Yellow solid: yield
77%. m.p. 110.8-111.9 oC. 1H NMR (400 MHz, Chloroform-d) δ 7.97 (d, J = 3.3 Hz,
1H, ThiaH), 7.83 (ddd, J = 7.8, 4.1, 1.3 Hz, 2H, PhH), 7.70 (td, J = 7.7, 1.3 Hz, 1H,
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PhH), 7.63 – 7.56 (m, 2H, ThiaH + PhH), 7.40 (d, J = 3.8 Hz, 1H, FuH), 6.83 (d, J =
3.8 Hz, 1H, FuH). 13C NMR (101 MHz, CDCl3) δ 175.54 (C=O), 153.26 (C-2, Thia),
152.93 (C-2, Ph), 149.32 (C-5, Fu), 147.95 (C-2, Fu), 143.13 (C-4, Thia), 132.50 (C-5,
Ph), 130.46 (C-4, Ph), 129.90 (C-6, Ph), 124.40 (C-1, Ph), 123.54 (C-3, Ph), 122.58
(C-3, Fu), 118.89 (C-5, Thia), 112.27 (C-4, Fu). ESI-MS(m/e): 333.2 [M+H]+. Anal.
Calcd. (%) for C14H8N2O4S2: C, 50.60; H, 2.43; N, 8.43. Found: C, 50.82; H, 2.66; N,
8.28.
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S-(thiazol-2-yl) 5-(3-nitrophenyl) furan-2-carbothioate (III-10). Yellow solid: yield
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70%. m.p. 236.4-237.9 C. H NMR (600 MHz, Chloroform-d) δ 8.62 (t, J = 1.8 Hz,
1H, PhH), 8.26 (ddd, J = 8.2, 2.2, 0.9 Hz, 1H, PhH), 8.15 (dt, J = 7.8, 1.3 Hz, 1H,
PhH), 7.99 (d, J = 3.3 Hz, 1H, ThiaH), 7.68 (t, J = 8.0 Hz, 1H, PhH), 7.62 (d, J = 3.4
13
Hz, 1H, ThiaH), 7.46 (d, J = 3.8 Hz, 1H, FuH), 7.03 (d, J = 3.8 Hz, 1H, FuH). C
NMR (151 MHz, CDCl3) δ 175.31 (C=O), 155.84 (C-2, Thia), 153.21 (C-5, Fu),
149.10 (C-3, Ph), 148.83 (C-2, Fu), 143.19 (C-4, Thia), 130.42 (C-6, Ph), 130.40 (C-5,
Ph), 130.28 (C-1, Ph), 123.97 (C-4, Ph), 123.52 (C-3, Fu), 119.91 (C-2, Ph), 119.49
(C-5, Thia), 109.71 (C-4, Fu). ESI-MS(m/e): 333.3 [M+H]+. Anal. Calcd. (%) for
C14H8N2O4S2: C, 50.60; H, 2.43; N, 8.43. Found: C, 50.51; H, 2.37; N, 8.62.
S-(thiazol-2-yl) 5-(4-nitrophenyl) furan-2-carbothioate (III-11). Yellow solid: yield
75%. m.p. 229.1-232.4 oC. 1H NMR (400 MHz, DMSO-d6) δ 8.39 (d, J = 8.7 Hz, 2H,
PhH), 8.17 (d, J = 8.8 Hz, 2H, PhH), 8.10 (dd, J = 3.3 Hz, 2H, Thia), 7.86 (d, J = 3.9
13
Hz, 1H, FuH), 7.68 (d, J = 3.8 Hz, 1H, FuH). C NMR (151 MHz, DMF) δ 175.07
(C=O), 156.40 (C-2, Thia), 152.47 (C-5, Fu), 149.14 (C-4, Ph), 148.09 (C-2, Fu),
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143.57 (C-4, Thia), 134.45 (C-1, Ph), 126.18 (C-2, C-6, Ph), 125.33 (C-3, C-5, Ph),
124.65 (C-3, Fu), 120.91 (C-5, Thia), 112.54 (C-4, Fu). ESI-MS(m/e): 331.1 [M+H]+.
Anal. Calcd. (%) for C14H8N2O4S2: C, 50.60; H, 2.43; N, 8.43. Found: C, 50.81; H,
2.22; N, 8.31.
S-(thiazol-2-yl) 5-(4-bromophenyl) furan-2-carbothioate (III-12). Gray solid: yield
83%. m.p. 150.1-152.0 oC. 1H NMR (400 MHz, Chloroform-d) δ 7.97 (d, J = 3.4 Hz,
1H, Thia), 7.67 (dt, J = 8.5, 1.7 Hz, 2H, PhH), 7.62 – 7.55 (m, 3H, PhH + Thia), 7.40
13
(dd, J = 3.8, 1.2 Hz, 1H, FuH), 6.85 (dd, J = 3.8, 1.1 Hz, 1H, FuH). C NMR (101
MHz, CDCl3) δ 175.04 (C=O), 157.69 (C-2, Thia), 153.70 (C-5, Fu), 148.27 (C-2, Fu),
142.99 (C-4, Thia), 132.26 (C-3, C-5, Ph), 127.61 (C-1, Ph), 126.48 (C-2, C-6, Ph),
123.99 (C-4, Ph), 123.32 (C-3, Fu), 119.85 (C-5, Thia), 108.34 (C-4, Fu).
ESI-MS(m/e): 367.2 [M+H]+. Anal. Calcd. (%) for C14H8BrNO2S2: C, 45.91; H, 2.20;
N, 3.82. Found: C, 46.12; H, 2.44; N, 3.65.
S-(thiazol-2-yl) 5-(p-tolyl) furan-2-carbothioate (III-13). Yellow solid: yield 79%.
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m.p. 122.9-124.0 C. H NMR (400 MHz, Chloroform-d) δ 7.95 (d, J = 3.4 Hz, 1H,
Thia), 7.68 (d, J = 8.2 Hz, 2H, PhH), 7.57 (d, J = 3.4 Hz, 1H, Thia), 7.39 (d, J = 3.8
Hz, 1H, FuH), 7.24 (d, J = 8.0 Hz, 2H, PhH), 6.78 (d, J = 3.8 Hz, 1H, FuH), 2.38 (s,
13
3H, CH3). C NMR (101 MHz, CDCl3) δ 174.77 (C=O), 159.26 (C-2, Thia), 154.12
(C-5, Fu), 147.71 (C-2, Fu), 142.85 (C-4, Thia), 140.08 (C-4, Ph), 129.70 (C-3, C-5,
Ph), 126.00 (C-1, Ph), 125.05 (C-2, C-6, Ph), 123.13 (C-3, Fu), 120.09 (C-5, Thia),
107.33 (C-4, Fu), 21.48 (CH3). ESI-MS(m/e): 302.1 [M+H]+. Anal. Calcd. (%) for
C15H11NO2S2: C, 59.78; H, 3.68; N, 4.65. Found: C, 59.92; H, 3.88; N, 4.45.
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S-(thiazol-2-yl) 5-(4-methoxyphenyl) furan-2-carbothioate (III-14). Yellow solid:
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yield 84%. m.p. 129.7-130.4 C. H NMR (400 MHz, Chloroform-d) δ 7.96 (d, J =
3.4 Hz, 1H, Thia), 7.78 – 7.74 (m, 2H, PhH), 7.59 (d, J = 3.4 Hz, 1H, Thia), 7.42 (d, J
= 3.8 Hz, 1H, FuH), 7.00 – 6.97 (m, 2H, PhH), 6.73 (d, J = 3.8 Hz, 1H, FuH), 3.87 (s,
13
3H, OCH3). C NMR (101 MHz, CDCl3) δ 174.64 (C=O), 160.88 (C-4, Ph), 159.28
(C-2, Thia), 154.27 (C-5, Fu), 147.53 (C-2, Fu), 142.85 (C-4, Thia), 126.79 (C-1, Ph),
123.10 (C-2, C-6, Ph), 121.59 (C-3, Fu), 120.39 (C-5, Thia), 114.47 (C-3, C-5, Ph),
106.57 (C-4, Fu), 55.43 (OCH3). ESI-MS(m/e): 318.2 [M+H]+. Anal. Calcd. (%) for
C15H11NO3S2: C, 56.77; H, 3.49; N, 4.41. Found: C, 56.59; H, 3.31; N, 4.62.
S-(thiazol-2-yl) 5-phenylfuran-2-carbothioate (III-15). Yellow solid: yield 77%.
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m.p. 68.3-70.3 C. H NMR (600 MHz, Chloroform-d) δ 7.97 (d, J = 3.3 Hz, 1H,
Thia), 7.85 – 7.80 (m, 2H, PhH), 7.59 (d, J = 3.3 Hz, 1H, Thia), 7.49 – 7.45 (m, 2H,
PhH), 7.44 – 7.39 (m, 2H, PhH + FuH), 6.86 (d, J = 3.8 Hz, 1H, FuH). 13C NMR (151
MHz, CDCl3) δ 175.05 (C=O), 158.99 (C-2, Thia), 154.02 (C-5, Fu), 148.20 (C-2, Fu),
142.99 (C-4, Thia), 129.79 (C-4, Ph), 129.06 (C-1, Ph), 128.83 (C-3, C-5, Ph), 125.16
(C-2, C-6, Ph), 123.21 (C-3, Fu), 119.88 (C-5, Thia), 107.93 (C-4, Fu). ESI-MS(m/e):
288.3 [M+H]+. Anal. Calcd. (%) for C14H9NO2S2: C, 58.52; H, 3.16; N, 4.87. Found:
C, 58.36 ;H, 2.98 ; N, 4.99.
Bioassays
Bacterial strains, plasmids and growth conditions
The bacterial strains and plasmids used in this study are listed in Table S1.
PXO99A strain (Xoo wild-type strain) and other derived strains were grown in rich
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medium (M210) or on PSA plates at 28 oC.5,29 XOM2 medium was a culture medium
that used to induce the expressions of hrp genes. And XOM2 medium was prepared
as previous reported.29 Escherichia coli was grown in Luria Bertani (LB) medium at
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37 C. Ampicillin (Ap) and cephalexin (Cp) were used at the final concentrations of
100 μg/mL and 25 μg/mL. All title compounds were dissolved in dimethyl sulfoxide
(DMSO) at a final concentration of 10 μg/mL.
Flow cytometry analysis
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pPhpa1 strain (Figure S31) was grown in M210 to OD600 ≈ 2.0 at 28 C and
transferred to XOM2 along with the tested compounds. The promoter activity of hpa1
was tested by a FACS-Caliber flow cytometer (CytoFLEX USA).16 DMSO was used
as a negative control. Three independent experiments were performed with three
replications each. The similar method was used to analyze the promoter activities of
hrpG, hrpX and hrcT.
Measurement of the growth rate
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Xoo PXO99A strain was grown in M210 to OD600 ≈ 1.0 at 28 C. The bacterial
suspension was transferred to M210 or XOM2 (plus 0.5% sucrose) medium
containing 10 μg/mL of tested compounds or DMSO, which started at an OD600 of 0.1.
The growth rates were recorded every 3 h during the 48 h period using a Bioscreen
(Bioscreen, Finland). Three replicates were used each time, and three independent
experiments were performed.
HR assay
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suspended in sterile distilled water to OD600 ≈ 0.5. Nicotiana benthamiana plants were
used for HR assays. Each flag leaf was inoculated with bacterial suspensions with
15–20 independent individuals, respectively.The HR symptoms were observed at 24
hours after inoculation.
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RNA extraction and qPCR analysis
Xoo PXO99A strain was grown in M210 to OD600 ≈ 2.0 at 28 oC and subcultured to
XOM2 at OD600 ≈ 0.6. The total RNA was extracted from the collected cells using
RNAprep Pure Bacteria Kit (Promega, USA). cDNA was synthesized using an
HiScriptII Q RT SuperMix Kit (Tiangen, Beijing, China). The cDNA levels were
quantified by real-time PCR using a SYBR Green Master Mix (Thermo Scientific,
MA, USA). The relative levels of genes expressions were analyzed by the 2−∆∆Ct
method.30 Expression values are the means of three biological repeats in each
experiment. The Student’s t-test was used for statistical analysis.
Pathogenicity assays
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Xoo PXO99A strain was grown in M210 to OD600 ≈ 2.0 at 28 C and then
suspended in sterile distilled water to OD600 ≈ 0.8. The rice cultivar IR24 (Oryza
sativa ssp. indica) was used for pathogenicity assays. Rice seedlings (2-week-old,
using a needleless syringe) and adult plants (2-month-old, by the leaf clipping
method)were inoculated with Xoo. Plants were scored and the symptoms were
observed at three days post-inoculation (dpi) in seedlings, and at 14 dpi for lesion
lengths (the length from the tip to the leading edge of the grayish symptom) in adult
rice plants. And X. oryzae pv. oryzicola (Xoc) used similar experimental methods with
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Xoo.
In vivo protection activity test
The protection activity of title compounds against rice bacterial leaf blight in potted
plants was conducted under greenhouse conditions. The experiment was conducted
following a previous study.31
Other virulence factors assay
For analyzing extracellular cellulase activity, PSA plates containing 0.5%
carboxymethyl cellulose were used. Xoo was grown in M210 to OD600 ≈ 0.5 at 28 oC,
and were inoculated into the PSA plates. After incubating at 28 oC for 24 h, the plates
were dyed with 0.1% Congo red for 20 min and washed twice with 1.0 M NaCl;
cellulase-positive colonies are supposed to show pale-yellow clear zones against a red
background.32
For analyzing the production of extracellular xylanase activity, PSA plates
containing 0.2% RBB-xylan were used to analyze xylanase activity. Xoo was grown
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in M210 to OD600≈0.5 at 28 C, and they were inoculated on the PSA plates. After
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incubating at 28 C for 48 h, the plates appeared as the white clear zones among a
blue background.32
For analyzing exopolysaccharides (EPS) production, Xoo was grown in M210 to
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OD600 ≈ 2.0 at 28 C. 100 mL bacterial culture was centrifuged at 12,000 rpm for 10
min, then the supernatants of bacterial culture were collected. The supernatants were
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added two volumes of absolute ethanol and kept at –20 C for at least 15 h. The
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before determination of dry weight.33,34
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Biofilm formation was performed according to reported studies.35 Bacterial
suspension (2 mL) was placed in each glass tube with 10 µg/mL III-15 compounds
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and incubated at 28 C for 72 h. The culture medium was poured out, and attached
bacterial cells were gently washed three times with distilled water. The cells were
then stained with 0.1% crystal violet (2 mL) for 15 min. Unbound crystal violet was
poured out, and the glass tubes were washed three times with distilled water. The
crystal violet-stained cells were solubilized in DMSO (2 mL).
RESULTS AND DISCUSSION
Synthesis
Figure 1 demonstrated the synthetic route to compound III. Following the reported
procedure via Meerwein arylation, key intermediate I was synthesized using
substituted aniline and furoic acid as starting substrate.27,28 This 5-substituted
phenyl-2-furancarboxylic acid I and thionyl chloride were refluxed in anhydrous
toluene for 3h to produce 5-phenyl-2-furancarbonyl chloride, which was reacted with
thiazole-2-thiol, triethylamine and 4-dimethylaminopyridine in acetonitrile to afford
compounds III in moderate to good yields. Title compounds were characterized and
confirmed by 1H NMR, 13C NMR, and elemental analyses.
Screening compounds that suppress hpa1 transcription
To detect potential T3SS inhibitors, we used a strain containing a gene named hpa1
promoter into the pPROBE-AT vector, which exhibited a green fluorescence protein
(GFP) reporter gene without a promoter. The hpa1 gene in Xoo encodes a harpin
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protein that could cause HR on non-host plants like tobacco, and its expression is
regulated by HrpX protein. The strain was grown in an induced medium called XOM2
that could induce hpa1 gene expression. Hpa1 promoter activity was measured 15
hours after incubation with each of the compounds at 10 μg/mL by
fluorescence-activated cell sorting (FACS) system. The mean fluorescence intensity
(MFI) was listed in Table 1, which showed the promoter activity of hpa1. %DMSO
was calculated by the ratio of MFI after incubation with each compound to that of the
DMSO (dimethyl sulfoxide) control. All compounds showed significant inhibition of
hap1 promoter activity, and the inhibition rate of III-1, III-2, III-3, III-4, III-6, III-8,
III-9 and III-15 were higher than our previous published T3SS inhibitor called TS006
(Table 1). Among all the compounds, eight were selected as positive candidates for
further experiments.
Effects of positive candidates on bacterial growth
T3SS inhibitors should target T3SS without affecting bacterial growth. Thus, we
tested bacterial growth treated with compounds III-1, III-2, III-3, III-4, III-6, III-8,
III-9 and III-15 compounds in 48 hours at different stages. It was measured in M210
(rich medium) and XOM2 (hrp-inducing medium and supplement 0.5% sucrose to
sustain bacterial growth). In comparison with the wild type and the DMSO solvent
control, Xoo treated with eight different compounds showed no significant difference.
As these compounds met the requirements of potential T3SS inhibitors (Figure 2), we
continued further study using these eight compounds.
Hypersensitive response inhibition by positive candidates
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In Xoo, Hpa1 could trigger HR on tobacco and secrete by T3SS. Therefore, we
checked the HR-inducing ability of Xoo on tobacco to know whether eight
compounds influence the T3SS in Xoo. All of the eight compounds inhibited HR,
significantly (Figure 3).
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Positive candidates inhibit T3SS expression at hrpG and hrpX level
HrpG and HrpX are two key regulatory proteins in Xanthomonas, and their hrp
genes expressions are mainly regulated by HrpX-HrpG pathway. Thus, it is important
to verify whether positive candidates affect the expression of hrpG/hrpX. We also
used a strain that contained hrpG promoter into the pPROBE-AT vector, and the hrpG
promoter activity was measured by FACS system. As shown in Figure 4A, the mRNA
level of hrpG was reduced by eight compounds in comparison with the solvent
control DMSO. We also used the same way to construct an expression vector to test
hrpX promoter activity, the results showed that the mRNA level of hrpX was also
reduced at different extent (Figure 4B). III-2, III-4, III-8, III-9, III-15 reduced the
mRNA level of hrpX more significantly than the other three compounds (Figure 4B).
Meanwhile, HrpX activates the transcription of hrp genes (hrpB to hrpF) and T3SS
effector genes. The promoter region regulated by HrpX usually has a conserved
sequence containing PIP-box (TTCGC-N15-TTCGC), like that of hpa1. Therefore,
we selected hrpT as a representative gene containing a complete PIP-box in its
promoter region to identify the eight positive candidates could affect the transcription
of T3SS genes in Xoo (Figure S32). The activity of PhrcT was observed by FACS
assays, and the results showed that all of the eight compounds truly affected the hrcT
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in mRNA level (Figure 4C). These results along with statistical analysis of the hpa1
promoter may infer that these potential T3SS inhibitors target the promoters with a
PIP-box. And it also revealed that HrpX has played important role in regulating the
inhibitory function.
Positive candidates inhibit representative hrp/hrc genes expression
The above results showed that eight compounds inhibited the T3SS genes of Xoo
without affecting bacterial growth. Indicating that these compounds may work on the
regulatory pathway to affect the expression of T3SS genes. We selected some special
hrp/hrc gene to check the effect of eight compounds for influencing the expression of
T3SS genes by doing qRT-PCR experiment. HrpE gene is used to encode the hrp
pilus protein, and the other hrp gene named hrpF encodes a putative translocon
protein. HrpC encodes the outer-membrane secretin, while hrcU is export apparatus
gene like hrcT.36 The hrp/hrc genes in mRNA levels were reduced significantly as
compared to DMSO control (Figure 5).
Our results showed that eight potential T3SS inhibitors reduced the expression of
hrp/hrc gene probably by HrpX-HrpG pathway. In this study, six genes were used
(hpa1, hrpE, hrpF, hrcC, hrcT and hrcU) to identify the expression of T3SS in Xoo.
Three of the six genes (hpa1, hrcT, and hrcU) contained the PIP-box in their promoter
region. Interestingly, except the hrcC gene, five genes were reduced significantly by
our compounds in mRNA levels. So, more works still need to be done in the future to
find out the exact mechanism involved in making these eight T3SS inhibitors as
potential candidates.
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Positive candidates reduce bacterial leaf blight symptoms on rice
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This study aimed to find out the most potential T3SS inhibitors, which could
control the virulence phenotypes of Xoo in planta. In Xanthomonads, water soaking is
induced by the TAL (transcription activator-like) effectors.37 There are several TAL
effectors in Xoo strain PXO99A, which confer gene-specific resistant to host plants.38
We used the seedlings of susceptible rice (cultivar IR24) to estimate the symptoms of
water-soaked lesions induced by Xoo. After treatment of Xoo with the compounds, the
water soaked symptoms on seedlings IR24 plants (Figure 6A) and the yellowish
disease symptoms on adult (Figure 6B) were reduced significantly as compared to
control. The effect of III-2, III-4, III-6, III-9, and III-15 compounds on rice was
better than our previous T3SS inhibitors TS006 (Figure 6B).
Xanthomonas oryzae pv. oryzae (Xoo) and X. oryzae pv. oryzicola (Xoc) belong to
Xanthomonas oryzae. They are closely related that have similar physiological
properties and T3SS regulatory pathways.5 So, we wanted to check the compounds
whether had the same effect to control the Xoc. The results showed that some
compounds could reduce the disease symptoms caused by Xoc in different extent
(Figure 6C). Those helped to enhance the broad spectrum of the compounds.
As mentioned earlier, T3SS invades plant cells by secreting TAL effectors. So we
selected compound III-15 which showed the most significant control effect on rice to
test the transcription levels of two TAL effectors named avrXa10 and pthXo6.
AvrXa10 could activate the Xa10 (the host resistance gene) to confer the immune or
HR in IRRB10.39,40 PthXo6 could induce the virulence of PXO99A and ectopic the
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bZIP transcription factor (OsTFX1) expression that conduces to host susceptibility.41
The compound III-15 suppressed the expression of the avrXa10 and pthXo6 (Figure
6D), so we guessed the compound not only inhibited the T3SS but reduced the
effectors’ transcription to control the virulence of the bacteria.
In vivo, protection activity was evaluated and the results showed that title
compound III-15 exhibited the best protective activity of 63.77% (Table 2). In
greenhouse test, compounds III-2, III-4, III-6, and III-9 gave better protective activity
(54.51%, 54.63%, 54.63%, and 54.74%) than that of compounds III-1, III-3, III-8 and
TS006 (45.71%, 45.49%, 45.60%, and 45.60%) against rice bacterial leaf blight as
compared with commercial drugs bismerthiazol (49.66%) and thiodiazole copper
(39.39%). Meanwhile, the tested compounds were also found safe to the plants.
Other virulence factors assay
In search of finding the best T3SS inhibitors without affecting other virulence
factors, we investigated whether our T3SS inhibitors affect some other representative
virulence factors, such as exopolysaccharides (EPS), extracellular cellulase,
extracellular xylanase, and biofilm.23,35 We also selected compound III-15 to do the
phenotypic experiments. As shown in Figure 7A and 7B, no significant difference was
observed as compared to the negative control indicating that III-15 may not affect the
expression of extracellular cellulase and extracellular xylanase. Similarly, III-15
caused no significant effect on the production of EPS and surface morphology (Figure
7C). Crystal violet assay was used to monitor the biofilm formation by Xoo. The
crystal violet stained adherent biofilms retained on glass test tubes (Figure 7D). As
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expected, III-15 compound was not found to restrict the production of biofilms by
Xoo.
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CONCLUSION
A series of structurally novel S-thiazol-2-yl-furan-2-carbothioate derivatives were
designed and synthesized. Bioassay results demonstrated that eight of title compounds
showed better bioactivities than our previous published T3SS inhibitor named TS006.
Compounds III-2, III-4, III-6, III-9, and III-15 could control bacterial leaf blight on
rice, significantly. These findings provide a new series of compounds, which may
help to increase rice production by effectively controlling Xoo.
ACKNOWLEDGEMENTS
We acknowledge the financial supports from the National Natural Science
Foundation of China (31570122), the National Key Research and Development
Program of China (2017YFD0200504), the National Key Project for Basic Research
(973 Program, 2015CB150600). We give our thanks to Professor Chenyang He
(Institute of Plant Protection, Chinese Academy of Agricultural Sciences) for
providing some strains and plasmids used in this study.
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