Design, Synthesis, and Evaluation of New Sulfone Derivatives Containing a 1,3,4-Oxadiazole Moiety as Active Antibacterial Agents

Article abstract of DOI:10.1021/acs.jafc.7b06061

This study aimed to synthesize some new sulfone derivatives containing a 1,3,4-oxadiazole moiety and investigate their in vitro antibacterial activities against Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas axonopodis pv. citri (Xac), the pathogens

Full text of DOI:10.1021/acs.jafc.7b06061

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Agricultural and Environmental Chemistry
Design, Synthesis, and Evaluation of New Sulfone Derivatives
Containing a 1,3,4-Oxadiazole Moiety as Active Antibacterial Agents
Pei Li, De-Yu Hu, Dandan Xie, Jixiang Chen, Linhong Jin, and Song Baoan
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b06061 • Publication Date (Web): 05 Mar 2018
Downloaded from http://pubs.acs.org on March 5, 2018
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Design, Synthesis, and Evaluation of New Sulfone Derivatives Containing a
1,3,4-Oxadiazole Moiety as Active Antibacterial Agents
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Pei Li,†,‡ Deyu Hu,† Dandan Xie,† Jixiang Chen,† Linhong Jin,† Baoan Song†,*
†State Key Laboratory Breeding Base of Green Pesticide and Agricultural
Bioengineering; Key Laboratory of Green Pesticide and Agricultural Bioengineering,
Ministry of Education; Research and Development Center for Fine Chemicals,
Guizhou University, Guiyang 550025, P.R. China
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‡Qiandongnan Engineering and Technology Research Center for Comprehensive
Utilization of National Medicine, Kaili University, Kaili 556011, P.R. China
*Corresponding author: Tel.: +86(0851)362ꢀ0521. Fax: +86(0851)362ꢀ2211. Eꢀmail:
songbaoan22@yahoo.com
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ABSTRACT: This study aimed to synthesize some new sulfone derivatives
containing a 1,3,4ꢀoxadiazole moiety and investigate their in vitro antibacterial
activities against Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas axonopodis
pv. citri (Xac), the pathogens of rice bacterial leaf blight and citrus canker,
respectively, by performing turbidimeter tests. Antibacterialꢀbioassay results showed
that compound 6d revealed excellent bioactivities against Xoo and Xac, with the 50%
effective concentration (EC50) values of 0.17 and 1.98 µg/mL, respectively, than
thiodiazole copper (121.82 and 77.04 µg/mL, respectively) and bismerthiazol (92.61
and 58.21 µg/mL, respectively). Meanwhile, greenhouseꢀcondition trials indicated
that, compared with thiodiazole copper and bismerthiazol, compound 6d more
effectively reduced rice bacterial leaf blight.
KEYWORDS: synthesis, antibacterial activity, sulfone derivative, 1,3,4ꢀoxadiazole
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INTRODUCTION
Rice bacterial leaf blight, a prevalent rice bacterial disease and caused by
Xanthomonas oryzae pv. oryzae (Xoo), can affect rice plants at each growth stage and
could result in leaf blight at tillering stage and up to 80% yield loss.1,2 Meanwhile,
citrus canker, a devastating citrus disease and caused by Xanthomonas axonopodis pv.
citri (Xac), could significantly affect the citrus production.3,4 The longꢀterm use of
available traditional bactericides, such as thiodiazole copper, bismerthiazol,
zhongshengmycin, Zn thiazole, etc., not only leads to the drug resistance of bacterial
populations but also results in a harmful influence on the safety of environment and
plants.5 Therefore, new active antibacterial agents that possess a novel mechanism of
action must be discovered and developed.
Recent works have highlighted that the sulfone group, which has an ideal structure
for developing agrochemicals and is found in many biologically active compounds,
also exhibits various biological activities including antibacterial,6–15 antifungal,16–20
antiviral,21,22 insecticidal,23 and herbicidal24 activities. Meanwhile, 1,3,4ꢀoxadiazole
ring, a common feature of medicinal agents, exhibits various biological activities,
such as antibacterial,6–15,25 antifungal,16–21 antiviral,21,22,26,27 insecticidal,28–31
herbicidal,32,33 and nematocidal25 activities. In our previous work, we investigated the
biological properties of some series of sulfone derivatives containing a
1,3,4ꢀoxadiazole moiety, which, compared with commercial bactericides, showed
better antibacterial activities against Ralstonia solanacearum (R. solanacearum), Xoo,
Xac, and Xanthomonas oryzae pv. oryzicola (Xoc), however, the control efficiencies
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of the greenhouse conditions and field trials were unsatisfactory compared with
commercial bactericides.7–9
To extend our previous study and develop new lead compounds with highly
bioactivity, in this study, we aimed to introduce a sulfone group into the 5ꢀposition of
the 1,3,4ꢀoxadiazole ring or introduce two sulfone groups into the 2,5ꢀpositions of the
1,3,4ꢀoxadiazole ring to build some new sulfone derivatives containing a
1,3,4ꢀoxadiazole moiety as active antibacterial agents.
MATERIALS AND METHODS
Instruments and Chemicals
Melting points (uncorrected) were measured on an XTꢀ4 microꢀmelting point
apparatus (Beijing Tech. Instrument Co., China). Using TMS as an internal standard,
1H and 13C NMR spectra were taken in a JEOL ECXꢀ500 NMR spectrometer (JEOL,
Tokyo, Japan) in DMSOꢀd6 or CDCl3. HRMS was conducted using a Q Exactive
(Thermo Scientific, MO). TLC was used to monitor the chemical reactions and
visualized under UV light at 254 nm. All reagents products from the Chinese
Chemical Reagent Company were analytical or chemical pure.
General Procedures for Preparing Intermediates 2–4
Using different substituted phenthiols as the starting materials, as shown in Figure
1, intermediates 2–4 were prepared in four steps, namely, substitution, oxidation,
hydrazidation, and cyclization reactions, according to previously reported methods.8,34
The substitution reaction proceeded at a reflux temperature in the presence of
different substituted phenthiols (0.01 mol) in acetonitrile (10 mL), ethyl chloroacetate
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(0.012 mol), and potassium carbonate (0.012 mol) within 6–8 h to obtain intermediate
1. Using ethanol (10 mL) as the solvent, intermediate 1 (0.01 mol) was treated with 30%
H2O2 (0.03 mol) and ammonium molybdate (0.5 mmol) to produce intermediate 2.
Then, 80% hydrazine hydrate (0.015 mol) was added dropwise to intermediate 2
dissolved in methanol. After refluxing for 6–8 h, the reaction was cooled down to
room temperature to obtain intermediate 3. Then, to a 250 mL round bottom flask,
intermediate 3 (0.01 mol) dissolved in ethanol (10 mL), KOH (0.02 mol) in distilled
water (5 mL), and carbon disulfide (0.03 mol) were added and refluxed for 6–8 h.
After that, the mixture was acidified with dilute HCl to pH 1–2. Finally, the crude
products were filtered and recrystallized from ethanol to give intermediate 4.
General Procedures for Preparing the Target Compounds 5a–5m
To a 25 mL round bottom flask, intermediate 4 (0.01 mol), K2CO3 (0.012 mol),
DMF (10 mL), and R2X (0.012 mol) were added. After reacting for 2–4 h at room
temperature, the mixture was decanted into distilled water. The crude products were
filtered or extracted using CH2Cl2, then recrystallized from ethanol or purified
through column chromatography to obtain the pure target compounds 5a–5m with
yields of 49–82%.
General Procedures for Preparing the Target Compounds 6a–6h
To a 25 mL round bottom flask, compound 5 (0.01 mol), acetic acid (10 mL), and
ammonium molybdate (0.5 mmol) dissolved in 30% H2O2 (0.05 mol) were mixed and
reacted for 1–3 h at room temperature. The mixture was decanted into distilled water
and then the crude products were filtered or extracted using CH2Cl2. The crude
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products were recrystallized from ethanol or purified through column chromatography
to obtain the pure target compounds 6a–6h with yields of 43–66%. The representative
data for compound 6d are presented in the following.
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Data for 2ꢀ(methylsulfonyl)ꢀ5ꢀ(((4ꢀfluorophenyl)sulfonyl)methyl)ꢀ1,3,4ꢀoxadiazole
(6d). White soil; m.p. 135–136 °C; yield, 46%; 1H NMR (500 MHz, DMSOꢀd6, ppm)
δ: 7.88–7.86 (m, 2H, PhꢀH), 7.76–7.73 (m, 2H, PhꢀH), 5.59 (s, 2H, ꢀCH2ꢀ), 3.63 (s,
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3H, ꢀCH3); C NMR (125 MHz, DMSOꢀd6, ppm) δ: 163.92, 160.18, 140.57, 136.96,
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130.88, 130.27, 51.87, 43.21; HRMS (ESI) [M+H]+ calcd for C10H9ClN2O5S2:
336.9714, found: 336.9713.
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In Vitro Antibacterial Activity Test
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In our present study, their in vitro antibacterial activities of the title compounds
against Xoo and Xac were determined using the turbidimeter tests.35 To a 15 mL tube,
4 mL of nutrient broth (NB) media, 1 mL of the test compounds or the commercial
bactericides solution (final concentration: 200 and 100 ꢁg/mL), and 40 ꢁL Xoo or Xac
bacterium solution were added. Then, the test tubes were incubated in a constant
temperature shaker for 24–48 h at 180 rpm and 28 ± 1 °C. The optical density (OD595)
of the NB media in each test tube was measured on a microplate reader (Model 680,
BIOꢀRAD, Hercules, CA) until the bacteria in untreated NB media were at
logarithmic growth. The in vitro inhibition rates I (%) are calculated as in the
following formula, where C represents the corrected absorbance value (OD595) of the
untreated NB media, T represents the corrected absorbance value (OD595) of the
treated NB media.
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Inhibition rate I (%) = (C − T)/C × 100
Based on the preliminary biological activity, the EC50 values were also determined
and calculated via software SPSS 17.0 (SPSS Inc, Chicago, USA). Three times were
repeated for each experiment.
In Vivo Antibacterial Activity Test
The curative and protection activities in potted plants of compound 6d against rice
bacterial leaf blight were determined by Schaad’s method with some slight
modifications.36 Bismerthiazol (20% wettable powder) and thiodiazole copper (20%
suspending agent), the bactericides registered for rice bacterial leaf blight and
purchased from the market, served as the positive controls.
The curative activity in potted plants for reducing rice bacterial leaf blight of
compound 6d was determined under controlled conditions in a greenhouse. After
sowing the rice seeds of variety ‘Fengyouxiangzhan’ approximately 5 weeks, rice
leaves were inoculated with Xoo, which was incubated at logarithmic growth, using
sterilized scissors. One day after inoculation, compound 6d solution at 200 ꢁg/mL
was uniform sprayed onto the rice leaves until dripping down, whereas distilled water
was uniform sprayed onto the negative control plants. Then, all the inoculated rice
plants were placed in a plant growth chamber (28 °C and 90% RH). At 14 days after
spraying, the disease index of the inoculated rice leaves was measured.
Similarly, the protection activity in potted plants for reducing rice bacterial leaf
blight of compound 6d was also conducted under controlled conditions in a
greenhouse. After sowing the rice seeds of variety ‘Fengyouxiangzhan’ approximately
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5 weeks, compound 6d solution at 200 ꢁg/mL was uniform sprayed onto the rice
leaves until dripping down, whereas distilled water was uniform sprayed onto the
negative control plants. One day after spraying, Xoo, which was incubated at
logarithmic growth, was inoculated on the rice leaves using sterilized scissors. All the
inoculated rice plants were placed in a growth chamber (28 °C and 90% RH). At 14
days after inoculation, the disease index of the inoculated rice leaves was measured.
The control efficiencies I (%) for the curative and protection activities are
calculated as in the following equation. In the equation, C is the disease index of the
negative control, and T is the disease index of the treatment group.
Control efficiency I (%) = (C − T)/C × 100
Statistical analysis was conducted by ANOVA with software SPSS 17.0 (SPSS Inc,
Chicago, USA). Different uppercase letters following the control efficiency values
indicate that there is significant difference (P < 0.05) among different treatment
groups.
RESULTS AND DISCUSSION
Chemistry
Using different substituted phenthiols as the starting materials, the target
compounds 5a–5m were synthesized in five steps (Figure 1), namely, substitution,
oxidation, hydrazidation, cyclization, and thioetherification reactions. Then, the target
compounds 6a–6h were prepared through the oxidation reactions of the corresponding
compound 5.
In Vitro Antibacterial Activity
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The in vitro bioactivities of the target compounds 5a–5m and 6a–6h against Xoo
and Xoc were determined by the turbidimeter tests, and the bioassay results are listed
in Tables 1 and 2. As shown, all test compounds displayed moderate to good
antibacterial activities against Xoo and Xac. Table 1 shows that compounds 5a, 5f, 5g,
5h, 5j, 5k, 5l, 5m, 6a, 6b, 6c, 6d, 6e, 6f, 6g, and 6h exhibited excellent in vitro
antibacterial activity (100%) against Xoo at concentrations of 200 and 100 µg/mL,
which was superior to thiodiazole copper (64% and 43%, respectively) and
bismerthiazol (72% and 54%, respectively). Table 2 shows that, at 200 and 100
µg/mL, compounds 5a, 5f, 5g, 6a, 6b, 6c, 6d, 6e, 6f, 6g, and 6h displayed better in
vitro antibacterial activity (100%) against Xac than bismerthiazol (89% and 72%).
The EC50 values of the synthesized compounds were also determined based on the
preliminary bioassay results and are presented in Tables 1 and 2. Table 1 shows that
the target compounds displayed excellent in vitro antibacterial activities against Xoo,
with EC50 values in the range of 0.17–45.78 µg/mL, which were superior to
thiodiazole copper (121.82 µg/mL) and bismerthiazol (92.61 µg/mL). Meanwhile,
Table 2 shows that the target compounds expressed moderate to good bioactivities
(EC50 = 1.98–76.10 µg/mL) against Xac. In particular, compound 6d exhibited the
best in vitro antibacterial activities against Xoo (EC50 = 0.17 µg/mL) and Xac (EC50 =
1.98 µg/mL).
In Vivo Antibacterial Activity
Table 3 and Figure 2 indicate that compound 6d exerted a better in vivo curative
activity (45.05%) for reducing rice bacterial leaf blight than bismerthiazol (37.80%)
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and thiodiazole copper (39.89%). Table 4 and Figure 3 show that, compared with
bismerthiazol (43.31%) and thiodiazole copper (44.70%), compound 6d demonstrated
excellent in vivo protection activity (51.77%) against rice bacterial leaf blight.
Structure-Activity Relationship Analysis
The structure–activity relationships (SAR) analysis was deduced based on the
antibacterial activity in Tables 1 and 2. First, with the presence of the –Cl group at the
4ꢀposition of the R1 substituent group on phenyl, the corresponding compounds
presented better in vitro bioactivities against Xoo and Xac following the order of 5f
(R1 = 4ꢀCl, R2 = ꢀCH3) > 5a (R1 = 4ꢀF, R2 = ꢀCH3) and 6d (R1 = 4ꢀCl, R2 = ꢀCH3) >
6a (R1 = 4ꢀF, R2 = ꢀCH3). Second, compared with the same substituent on the R1
substituent group, smaller substituent groups, such as methyl, on the R2 substituent
group can increase the antibacterial activities against Xoo and Xac following the order
of 5a (R1 = 4ꢀF, R2 = ꢀCH3) > 5b (R1 = 4ꢀF, R2 = ꢀC2H5), 5f (R1 = 4ꢀCl, R2 = ꢀCH3) >
5g (R1 = 4ꢀCl, R2 = ꢀC2H5), and 6d (R1 = 4ꢀCl, R2 = ꢀCH3) > 6e (R1 = 4ꢀCl, R2 =
ꢀC2H5). Third, compared with the same substituent on the R1 and R2 substituent
groups, the corresponding compound 6 presented better in vitro bioactivities against
Xoo and Xac than compound 5 following the order of 6a (R1 = 4ꢀF, R2 = ꢀCH3) > 5a
(R1 = 4ꢀF, R2 = ꢀCH3) and 6d (R1 = 4ꢀCl, R2 = ꢀCH3) > 5f (R1 = 4ꢀCl, R2 = ꢀCH3).
Meanwhile, it worth to note that when two sulfone groups at the 2,5ꢀpositions of the
1,3,4ꢀoxadiazole ring, the corresponding target compounds exhibited excellent
bioactivities against Xoo and Xac compared with all the compounds that were
reported by our previously work.
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This study demonstrated that this series of sulfone derivatives containing a
1,3,4ꢀoxadiazole moiety can be used to develop the potential agrochemicals. In
accordance with the pesticide registration requirements in China, further field and
toxicities studies of compound 6d will be undertaken in future studies.
ASSOCIATED CONTENT
Supporting Information
1
The physical characteristics, H NMR, 13C NMR, and HRMS data for the target
compounds are provided in the Supporting Information. This material is available free
of charge on the Internet at http://pubs.acs.org.
Funding
The authors gratefully acknowledge the Natural Sciences Foundation of China (No.
21672044).
Notes
The authors declare no competing financial interest.
ABRREVIATIONS USED
Xoo, Xanthomonas oryzae pv. oryzae; Xac, Xanthomonas axonopodis pv. citri; Xoc,
Xanthomonas oryzae pv. oryzicola; R. solanacearum, Ralstonia solanacearum; EC50,
50% effective concentration; NB, nutrient broth; SAR, structure–activity
relationships.
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Figure Captions
Figure 1. Synthetic route of the target compounds.
Figure 2. Curative activity of compound 6d against rice bacterial leaf blight under
greenhouse conditions at 200 µg/mL.
Figure 3. Protection activity of compound 6d against rice bacterial leaf blight under
greenhouse conditions at 200 µg/mL.
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Table 1. In Vitro Antibacterial Activity of the Target Compounds against Xoo.
Inhibition rate
EC50
Compounds
(ꢁg/mL)a
R1
R2
200 (ꢁg/mL) 100 (ꢁg/mL)
4ꢀF
4ꢀF
ꢀCH3
ꢀC2H5
100±0.62
100±0.54
90±3.06
100±0.49
80±2.21
19.44±2.06
27.66±2.89
33.95±2.32
45.78±2.65
38.49±1.21
10.44±2.30
12.22±3.34
16.90±2.38
24.27±2.53
28.46±3.03
31.44±2.94
23.00±3.86
17.09±2.77
0.22±0.14
6.77±2.01
5.92±1.14
0.17±1.02
0.64±0.24
1.11±0.42
5a
5b
5c
5d
5e
5f
4ꢀF
Benzyl
82±1.74
4ꢀF
4ꢀClꢀBenzyl
4ꢀFꢀBenzyl
ꢀCH3
82±5.01
71±4.95
4ꢀF
84±2.34
74±3.34
4ꢀCl
4ꢀCl
4ꢀCl
4ꢀCl
4ꢀCl
4ꢀCl
4ꢀCl
4ꢀCl
4ꢀF
100±0.12
100±0.58
100±0.51
100±0.97
100±0.65
100±0.18
100±0.32
100±0.22
100±0.43
100±0.42
100±0.41
100±0.67
100±0.32
100±0.16
100±0.54
100±0.51
100±0.23
83±2.72
ꢀC2H5
5g
5h
5i
ꢀCH2CH2CH3
ꢀCH2CH2CH2CH3
ꢀCH2CH2CH2CH2CH3
Benzyl
100±0.52
100±0.32
100±0.52
100±0.14
100±0.36
100±0.48
100±0.23
100±0.41
100±0.12
100±0.40
5j
5k
5l
4ꢀClꢀBenzyl
4ꢀFꢀBenzyl
ꢀCH3
5m
6a
6b
6c
6d
6e
6f
4ꢀF
Benzyl
4ꢀF
ꢀOH
4ꢀCl
4ꢀCl
4ꢀCl
ꢀCH3
ꢀC2H5
Benzyl
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4ꢀCl
4ꢀClꢀBenzyl
4ꢀFꢀBenzyl
100±0.12
100±0.43
72±0.65
64±2.76
100±0.54
100±0.51
54±1.23
43±3.15
2.25±0.98
1.66±0.82
6g
6h
4ꢀCl
Bismerthiazol
92.61±2.15
121.82±3.59
Thiodiazole copper
a The experiments were repeated three times.
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Table 2. In Vitro Antibacterial Activity of the Target Compounds against Xac.
Inhibition rate
EC50
Compounds
(ꢁg/mL)a
R1
R2
200 (ꢁg/mL) 100 (ꢁg/mL)
4ꢀF
4ꢀF
ꢀCH3
ꢀC2H5
100±0.86
100±0.56
100±0.22
80±1.87
100±0.13
90±1.32
82±2.85
50±2.02
60±2.87
100±0.13
100±0.23
87±2.01
75±2.84
77±2.07
87±1.67
76±2.67
79±2.38
100±0.24
100±0.37
100±0.67
100±0.12
100±0.53
100±0.84
25.61±2.29
29.50±3.62
34.82±2.90
76.10±4.29
61.88±1.96
21.11±2.06
24.90±2.89
29.25±4.40
45.27±3.39
37.43±3.30
28.00±3.35
49.31±4.04
43.72±3.35
3.88±1.92
5a
5b
5c
5d
5e
5f
4ꢀF
Benzyl
4ꢀF
4ꢀClꢀBenzyl
4ꢀFꢀBenzyl
ꢀCH3
4ꢀF
85±1.93
4ꢀCl
4ꢀCl
4ꢀCl
4ꢀCl
4ꢀCl
4ꢀCl
4ꢀCl
4ꢀCl
4ꢀF
100±0.86
100±0.43
100±0.22
90±1.18
ꢀC2H5
5g
5h
5i
ꢀCH2CH2CH3
ꢀCH2CH2CH2CH3
ꢀCH2CH2CH2CH2CH3
Benzyl
95±1.92
5j
100±0.34
84±1.56
5k
5l
4ꢀClꢀBenzyl
4ꢀFꢀBenzyl
ꢀCH3
89±2.10
5m
6a
6b
6c
6d
6e
6f
100±0.43
100±0.49
100±0.65
100±0.09
100±0.84
100±0.33
4ꢀF
Benzyl
15.27±1.83
12.39±1.92
1.98±0.86
4ꢀF
ꢀOH
4ꢀCl
4ꢀCl
4ꢀCl
ꢀCH3
ꢀC2H5
8.09±1.06
Benzyl
23.17±2.95
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4ꢀCl
4ꢀClꢀBenzyl
4ꢀFꢀBenzyl
100±0.98
100±0.33
89±2.74
100±0.65
100±0.76
72±4.12
67±2.19
49.25±4.21
27.60±1.86
58.21±2.77
77.04±1.96
6g
6h
4ꢀCl
Bismerthiazol
Thiodiazole copper
a The experiments were repeated three times.
100±1.11
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351
Table 3. Curative Activity of Compound 6d against Rice Bacterial Leaf Blight under Greenhouse
Conditions at 200 µg/mL.
14 Days after spraying
Treatment
Morbidity (%)
Disease index (%)
Control efficiency (%)b
100
100
100
100
44.07
49.88
48.21
80.20
45.05A
37.80B
39.89B
/
6d
Bismerthiazol
Thiodiazole copper
CKa
a Negative control; b Statistical analysis was conducted via the ANOVA method at a condition of
equal variances assumed (P > 0.05) and equal variances not assumed (P < 0.05). Different uppercase
letters indicate the values of curative activity with significant difference among different treatment
groups at P < 0.05.
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Table 4. Protection Activity of Compound 6d against Rice Bacterial Leaf Blight under Greenhouse
Conditions at 200 µg/mL.
14 Days after inoculation
Treatment
Morbidity (%)
Disease index (%)
Control efficiency (%)b
100
100
100
100
38.68
45.46
45.05
80.20
51.77A
43.31B
44.70B
/
6d
Bismerthiazol
Thiodiazole copper
CKa
a
b
Negative control; The statistical analysis was conducted by ANOVA method at the condition of
equal variances assumed (P > 0.05) and equal variances not assumed (P < 0.05). Different uppercase
letters indicate the values of protection activity with significantly difference among different
treatment groups at P < 0.05.
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Figure 1. Synthetic route of the target compounds.
119x78mm (300 x 300 DPI)
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Figure 2. Curative activity of compound 6d against rice bacterial leaf blight under greenhouse conditions at
200 µg/mL.
170x124mm (300 x 300 DPI)
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Journal of Agricultural and Food Chemistry
Figure 3. Protection activity of compound 6d against rice bacterial leaf blight under greenhouse conditions at
200 µg/mL.
170x124mm (300 x 300 DPI)
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Top Graphic
84x47mm (300 x 300 DPI)
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