Synthesis and antibacterial evaluation of new sulfone derivatives containing 2-aroxymethyl-1,3,4-oxadiazole/thiadiazole moiety

Article abstract of DOI:10.3390/molecules22010064

Sulfones are one of the most important classes of agricultural fungicides. To discover new lead compounds with high antibacterial activity, a series of new sulfone derivatives were designed and synthesized by introducing the aroxymethyl moiety into the scaffold of 1,3,4-oxadiazole/thiadiazole sulfones. Antibacterial activities against three phytopathogens (Xanthomonas oryzae pv. oryzae, Ralstonia solanacearum, Xanthomonas axonopodis pv. citri.) were assayed in vitro. As compared to the control of commercial fungicides and some reported sulfone fungicides, seven compounds 5I-1-5I-7 exerted remarkably higher activities with EC₅₀ values ranging from 0.45-1.86 μg/mL against X. oryzae and 1.97-20.15 μg/mL against R. solanacearum. Exhilaratingly, 5I-1, 5I-2 and 5I-4 displayed significant in vivo activity against X. oryzae with protective effect of 90.4%, 77.7%, and 81.1% at 200 μg/mL, respectively, much higher than that exhibited by Bismerthiazol (25.6%) and Thiadiazole-copper (32.0%). And the differential phytotoxicity of active derivatives was preliminarily checked. The results demonstrated that derivative of 2-aroxymethyl-1,3,4-oxadiazole/thiadiazole sulfone can serve as potential alternative bactericides for the management of plant bacterial diseases.

Full text of DOI:10.3390/molecules22010064

molecules
Article
Synthesis and Antibacterial Evaluation of New
Sulfone Derivatives Containing
2-Aroxymethyl-1,3,4-Oxadiazole/Thiadiazole Moiety
Shihu Su 1,2, Xia Zhou 1,*, Guoping Liao 1, Puying Qi 1 and Linhong Jin 1,
*
1
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, China; happytt365@foxmail.com (S.S.);
lgp899017@163.com (G.L.); qpuying000@163.com (P.Q.)
Zunyi Agricultural Products Quality and Safety Inspection and Testing Center, Room 698, Shanghai Road,
Huichuan District, Zunyi 563000, Guizhou, China
2
*
Correspondence: zhouxia_j@126.com (X.Z.); linhong_j@126.com (L.J.); Tel.: +86-851-362-0521 (X.Z. & L.J.);
Fax: +86-851-362-2211 (X.Z. & L.J.)
Academic Editor: Panayiotis A. Koutentis
Received: 21 November 2016; Accepted: 27 December 2016; Published: 31 December 2016
Abstract: Sulfones are one of the most important classes of agricultural fungicides. To discover new
lead compounds with high antibacterial activity, a series of new sulfone derivatives were designed and
synthesized by introducing the aroxymethyl moiety into the scaffold of 1,3,4-oxadiazole/thiadiazole
sulfones. Antibacterial activities against three phytopathogens (Xanthomonas oryzae pv. oryzae,
Ralstonia solanacearum, Xanthomonas axonopodis pv. citri.) were assayed in vitro. As compared to the
control of commercial fungicides and some reported sulfone fungicides, seven compounds 5I-1
exerted remarkably higher activities with EC50 values ranging from 0.45–1.86 g/mL against X. oryzae
and 1.97–20.15 g/mL against R. solanacearum. Exhilaratingly, 5I-1 5I-2 and 5I-4 displayed significant
in vivo activity against X. oryzae with protective effect of 90.4%, 77.7%, and 81.1% at 200 g/mL,
–5I-7
µ
µ
,
µ
respectively, much higher than that exhibited by Bismerthiazol (25.6%) and Thiadiazole-copper
(32.0%). And the differential phytotoxicity of active derivatives was preliminarily checked.
The results demonstrated that derivative of 2-aroxymethyl-1,3,4-oxadiazole/thiadiazole sulfone
can serve as potential alternative bactericides for the management of plant bacterial diseases.
Keywords: synthesis; 1,3,4-oxadiazole/thiadiazole sulfone; aryloxymethyl; antibacterial activity;
structure-activity relationship
1. Introduction
Xanthomonas, a large genus of gram-negative plant-associated bacteria, causes important diseases
such as bacterial blight of rice by Xanthomonas oryzae pv. Oryzae (X. oryzae) and citrus canker by
Xanthomonas axonopodis pv. citri. (X. axonopodis) leading to serious quality and yield losses in the host
crop [
wilt on more than 200 plant species including important crops such as potato, tomato, tobacco and
banana [ ]. Controlling those pathogens with bactericides such as copper based bactericides serves as
the most effective way to cure plant disease [ ]. However, the application of many currently available
antibacterial agents is compromised by their low efficiency, phytotoxic effect and high accumulation in
the soil [ ]. For example, Bismerthiazol, one of the major bactericides for the control of rice bacterial
leaf blight, only shows the control efficiency of 25.49% at a high dosage of 200 g/mL [ ]. Furthermore,
the emergence of bactericide resistance [ 10] coupled with above drawbacks has led to the rising need
for development of high-efficient bactericides to effectively control those agricultural diseases.
1]. Ralstonia solanacearum (R. solanacearum), a soil-borne bacterium, is responsible for bacterial
2,3
4,5
6,7
µ
8
9,
Molecules 2017, 22, 64; doi:10.3390/molecules22010064
www.mdpi.com/journal/molecules

Molecules 2017, 22, 64
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Extensive biochemical studies on sulfone derivatives bearing suitably substituted heterocyclic
units have confirmed that those molecular building blocks, as a common pharmacophore model are
effective against various phytopathogens in agriculture [11
agents based on heterocyclic sulfones have been patented and employed for crop protection [
For example, 1,3,4-oxadiazole/thiadiazole bearing sulfone derivatives showed potent antibacterial
activities against X. oryzae and R. solanacearum [ ]. We have sought new synthetic compounds related
to phenyl/benzyl substituted 1,3,4-oxadiazole sulfone that have similar antibacterial action [ 15 16].
–
13]. In this regard, many bactericidal
4,
12–16].
4
4, ,
Quite interestingly, it was demonstrated that replacement of phenyl group with benzyl fragment at the
2-position of the 1,3,4-oxadiazole/thiadiazole unit (Figure 1) could significantly enhance the flexibility
of the molecular backbone to combine with the receptor protein of pathogenic bacteria [14,15]. Another
evidence indicated that the incorporation of an aroxymethyl group to the heterocyclic component
can serve as an effective approach for enhancing antibacterial activity [17]. Intriguingly, in the course
of structure optimization, the modifying of the oxime ether pharmacophore (strobilurins) with a
phenoxymethyl side chain lead to the successful landing of Kresoxim-methy [18]. Fascinated by these
investigations, herein we explored to build the heterocyclically substituted sulfones incorporating
an aroxymethyl fragment with -O-attached to the heterocycle through linker (-CH2-). The design
strategy and synthetic route are schematically depicted in Figures 1 and 2 respectively. In our synthetic
design, the aroxymethyl group involved is typical and expected to play an important role in increasing
bioactivity. The inhibitory activity of target compounds on growth of various important pathogenic
bacteria in vitro and in infected rice were investigated and their structure-activity relationships (SARs)
were discussed. Phytotoxicity in tobacco leaves and rice seed were also determined.
Figure 1. Strategic Design for the Target Compounds.
2. Results and Discussion
2.1. Chemistry
Compounds 5I and 5II were prepared by analogous processes [17
Figure 2. Starting from phenol or substituted phenol, the key intermediate 2-aroxymethyl-1,3,4-
oxadiazole-2-thiol was synthesized in four steps involving esterification [17], hydrazidation,
salt formation, and cyclization [19]. Subsequently, thiol 3 was converted into its corresponding
thioether derivative by thioetherification with dimethyl/diethyl sulfate or benzyl bromide. Finally,
the target sulfonyl oxadiazole/thiadiazole 5I/II was obtained through oxidation of the thioether
,19,20], as presented in
3
4
4
with potassium permanganate [20]. The structures of all the target compounds were characterized by
elemental analyses, and 1H, 13C NMR and MS(ESI).

Molecules 2017, 22, 64
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Figure 2. Synthetic Route to the Target Compounds 5I-1–5I-14 and 5II-1–5II-1.
2.2. In Vitro Antibacterial Activity
The antibacterial activities of all the compounds were evaluated in vitro against X. oryzae,
R. solanacearum and X. axonopodis via the turbidimeter test [21]. To make a judgment on the bactericidal
potency of the synthesized compounds, three commercial antibacterials, Bismerthiazol, Kocide 3000
and Thiodiazole-copper were selected as the positive controls. As shown in Table 1, most of the
products 5I exhibited better or equivalent antibacterial activities compared to positive controls against
the tested plant bacteria. Among them, compounds 5I-1
R. solanacearum with an observed control efficacy of 100% both at 200 and 100
5I-1 5I-7 exhibited inhibition rates of 65%–100% at 200 g/mL and 48%–90% at 100 µ
–
5I-7 appeared highly active for X. oryzae and
g/mL. Interestingly,
g/mL against
µ
–
µ
X. axonopodis which were also superior to that of positive controls. On the other hand, the present
synthesis and bioactive tests were parallel conducted on 5II. While no anticipated bioactivity was
observed, except 5II-1, 5II-2 showed better activities than positive controls against X. oryzae with

Molecules 2017, 22, 64
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inhibition rates of 84%–99% at 200
µg/mL. It was thus confirmed that compounds 5I bearing
1,3,4-oxidiazole moiety were more potent in combating tested bacteria and displayed significantly
higher activity as compared to compounds 5II and positive controls.
Table 1. Inhibition rate of Compounds 5I and 5II against Pathogenic Bacteria X. oryzae, R. solanacearum
and X. axonopodis a.
X. oryzae
R. solanacearum
X. axonopodis
Compd.
R1/R2
200 µg/mL
100 µg/mL
200 µg/mL
100 µg/mL
200 µg/mL
100 µg/mL
5I-1
5I-2
4-F/CH3
100
100
100
100
100
100
100
100
100
90
100
100
100
100
100
100
100
87
100
48
38
70
13
78
92
73
28
18
16
55
13
8
100
100
100
100
100
100
100
97
33
24
18
33
15
69
50
56
26
18
9
100
100
100
100
100
100
99
43
25
19
13
20
8
37
49
45
7
15
8
12
0
80
88
91
69
100
88
65
62
72
74
11
26
7
76
40
27
25
23
25
15
32
24
14
5
72
76
90
48
54
57
53
36
50
62
16
23
0
53
36
16
18
20
24
18
6
4-Cl/CH3
5I-3
4-Br/CH3
5I-4
2,4-diCl/CH3
2-Me-4-Cl/CH3
H/CH3
5I-5
5I-6
5I-7
4-F/CH2CH3
2,4-diCl/CH2CH3
4-F/CH2Ph
5I-8
5I-9
5I-10
5I-11
5I-12
5I-13
5I-14
5II-1
5II-2
5II-3
5II-4
5II-5
5II-6
5II-7
5II-8
5II-9
5II-10
5II-11
5II-12
4-Cl/CH2Ph
4-Br/CH2Ph
2,4-diCl/CH2Ph
2-Me-4-Cl/CH2Ph
H/CH2Ph
56
98
33
95
4-F/CH3
99
4-Cl/CH3
84
4-Br/CH3
30
2,4-diCl/CH3
2-Me-4-Cl/CH3
4-F/CH2CH3
2,4-diCl/CH2CH3
4-F/CH2Ph
16
42
65
7
20
0
24
30
17
11
27
0
10
18
1
26
0
99
100
35
21
23
8
32
33
45
25
47
4-Cl/CH2Ph
4-Br/CH2Ph
2,4-diCl/CH2Ph
2-Me-4-Cl/CH2Ph
19
14
0
10
37
14
0
54
52
35
38
10
67
39
65
5
Bismerthiazol b
Kocide 3000 b
72
100
100
50
69
69
Thiodiazole-copper b
a
Average of three replicates; b The commercial agricultural antibacterial agents Bismerthiazol, Kocide 3000,
and Thiodiazole-copper were used as positive control.
The compounds acting better than positive controls (Table 1) were further performed for
dose response with lower gradient concentration for their EC50 values (Table 2). Compounds
5I-1
1.67, 1.86, 0.52
displayed by Bismerthiazol. Meanwhile their EC50 (1.97–20.51
were shown to be much lower compared to the figure 45.91
Additionally, 5I-1 5I-7 inhibited the growth of X. axonopodis with EC50 values ranging from 24.89 to
96.83 g/mL. Although they were not as effective to the other two type of bacteria, but still superior to
–
5I-7 revealed outstanding activity against X. oryzae with EC50 values of 0.45, 1.44, 1.67, 0.72,
g/mL respectively, which are about 1/50–1/200 fraction lower than 96.2 g/mL
g/mL) values against R. solanacearum,
g/mL displayed by Kocide 3000.
µ
µ
µ
µ
–
µ
Bismerthiazol (119.5 µg/mL). More over 5I-3 is the most active against X. axonopodis.
In particular, 5I-1 bearing 4-F substituted phenoxymethyl, acted best for combating X. oryzae
and R. solanacearum with EC50 values of 0.45 and 1.97
three commercial positive controls. Besides, the compounds 5I-2
0.72, 0.52 g/mL against X. oryzae and 13.42, 20.51, 7.75 g/mL against R. solanacearum, respectively,
appeared to be promising bactericides against plant bacterial diseases.
µg/mL respectively, which were far better than
,
5I-4 5I-7 with EC50 values of 1.44,
,
µ
µ

Molecules 2017, 22, 64
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Table 2. Antibacterial Activity of Compounds 5I-1–5I-7 against X. oryzae, R. solanacearu, and X. axonopodis a.
X. oryzae
R. solanacearum
X. axonopodis
Compd.
EC50 (µg/mL)
Regression Equation
r
EC50 (µg/mL)
Regression Equation
r
EC50 (µg/mL)
Regression Equation
r
5I-1
5I-2
5I-3
5I-4
5I-5
5I-6
5I-7
0.45 ± 0.06
1.44 ± 0.18
1.67 ± 0.22
0.72 ± 0.15
1.67 ± 0.16
1.86 ± 0.23
0.52 ± 0.16
92.61 ± 2.15
101.60 ± 5.12
121.82 ± 3.59
y = 0.90x + 5.32
y = 2.73x + 4.57
y = 2.96x + 4.40
y = 2.28x + 5.33
y = 2.78x + 4.38
y = 1.59x + 4.57
y = 0.99x + 5.28
y = 1.50x + 2.05
y = 160x + 1.80
y = 1.54x + 1.79
0.88
0.93
0.97
0.89
0.96
0.90
0.96
0.98
0.99
0.98
1.97 ± 0.23
13.42 ± 1.54
19.61 ± 0.98
20.51 ± 1.56
14.94 ± 1.27
13.55 ± 2.12
7.75 ± 1.01
59.69 ± 2.56
45.91 ± 6.6
>200
y = 2.25x + 4.34
y = 2.79x + 1.86
y = 1.44x + 3.14
y = 2.18x + 2.14
y = 3.43x + 0.97
y = 2.62x + 2.04
y = 1.04x + 4.08
y = 1.21x + 2.84
y = 4.87x − 3.10
/
0.98
0.92
0.93
0.98
0.95
0.91
0.99
0.98
0.98
/
80.46 ± 5.38
31.35 ± 3.56
24.89 ± 2.52
49.05 ± 2.34
96.83 ± 5.78
85.02 ± 4.32
52.23 ± 2.14
119.5 ± 5.1
>200
y = 0.99x + 2.77
y = 1.95x + 2.09
y = 1.52x + 2.87
y = 1.40x + 2.58
y = 1.36x + 2.29
y = 1.06x + 2.96
y = 1.67x + 2.14
y = 1.50x + 1.82
/
0.96
0.98
0.95
0.93
0.97
0.96
0.97
0.98
/
Bismerthiazol b
Kocide 3000 b
Thiodiazole-copper b
a
107.04 ± 1.96
y = 2.15x + 0.94
0.98
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); b The commercial
agricultural antibacterial agents Bismerthiazol, Kocide 3000, and Thiodiazole-copper were used as positive control.

Molecules 2017, 22, 64
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2.3. In Vivo Antibacterial Activity
Having established outstanding bactericidal activity of 5I-1
,
5I-2
,
5I-4 in vitro, we further explored
their antibacterial potency in vivo against rice bacterial leaf blight via the leaf-cutting method [22
,23]
under greenhouse condition at a concentration of 200 g/mL. Bismerthiazol and Thiodiazole-copper
µ
served as positive controls for this investigation. It was observed that all inoculated plants in 14 days
exhibited blight symptoms with 100% morbidity. Plants for blank control (no bacteria inoculations)
appeared absence of bacterial infection throughout the testing time. The plants for negative
control (inoculated but treated with no compounds) showed severe bacterial blight symptoms with
about 20.0 cm long and spreading blight lesions. While the plants treated with tested compounds were
relatively healthy and had obvious shorter lesions ranged from1.9 to 4.9 cm. 5I-1 afforded a maximum
of lesion reduction. It can be seen (Table 3) that, comparing to the untreated control, 5I-1, 5I-2 and 5I-4
reduced bacterial leaf blight incidence by 90.4%, 77.7% and 81.1% respectively, which were superior to
that of Bismerthiazol (25.6%) and Thiodiazole-copper (32.0%).
Table 3. In vivo Inhibitory Effect of Testing Compounds against X. oryzae at 200 µg/mL.
14 Days after Spraying
Compd.
Morbidity (%) c
Disease Index
Protective Efficiency (%) d
5I-1
5I-2
100.0
100.0
100.0
100.0
100.0
0.0
15.6
23.8
17.5
60.0
55.6
0.0
90.4 ± 2.8
77.7 ± 3.4
81.1 ± 1.9
25.6 ± 4.4
32.0 ± 3.0
100.0 ± 0.0
/
5I-4
Bismerthiazol
Thiodiazole-copper
CK1 a
CK2 b
100.0
91.1
a
b
c
CK1: blank control sample; CK2: negative control sample; Bacteria inoculation was successful and all
inoculated plants were infected; d 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).
2.4. Structure-Activity Relationship (SAR) Analyses
On the basis of the activity values shown in Tables 1 and 2, a preliminary conclusion of the
structure-activity relationship could be deduced. First, the antibacterial studies with both series
compounds against tested bacteria revealed that compounds 5I displayed significantly higher
antibacterial activity than the corresponding 1,3,4-thiadiazole 5II. Apparently the presence of
heterocyclic unit 1,3,4-oxadiazole was critical to achieve significant inhibitory effect. Thus, for example,
1,3,4-oxadiazole sulfones 5I-1–5I-7 may serve as more potent source of antibacterial compounds
compared to their corresponding 1,3,4-thiadiazole derivatives 5II. The difference may lie in that the
oxygen atom in oxadiazole is more electronegative but less bulky than sulfur atom in thiadiazole which
causing different interaction with the surrounding residues. However, the strategy of oxygen–sulfur
bioisosterism substitution may not necessarily lead to one way enhancement of structure bioactivity.
For example, a similar trend was documented that presence of 1,3,4-oxadiazole moiety is more potential
than 1,3,4-thiadiazole in imparting the antifungal activity [13]. While 1,3,4-thiadiazol-2(3H)-ones
displayed much higher inhibition activity than the corresponding 1,3,4-oxadiazol-2(3H)-ones as human
protoporphyrinogen oxidase inhibitors [24,25].
Further antibacterial tests on X. oryzae and R. solanacearum, have revealed that 4-substituted
mono halogenated phenoxymethyl derivatives (R1 = 4-Cl, 4-F, 4-Br) exhibited significant antibacterial
activity which appears to decrease with increase in the size of the halogen substituent. Thus the
order follows the trend 5I-1(F) > 5I-2(Cl) > 5I-3(Br) indicating that 4-F substituted derivative is most
favorable in imparting antibacterial activity. At this point, it is in line with the previous report [13,15]
that bulky groups are normally disfavored in similar structures. In addition, introduction of stronger
electron-withdrawing substituents (F > Cl > Br) in the para-position of the benzene ring might assist in
enhancing antibacterial activity. Specifically, when compound 5I-6(R1 = H) was concerned, it is not

Molecules 2017, 22, 64
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bound to be the most potent among the 4-halogenated compounds with taking account the above
two factors.
With regard to the nature of R2 substituent attached to the sulfonyl unit, similar trend in terms of
activity was noted with increase in size of the alkyl group:CH3 > CH2CH3 > CH2Ph, which means
that methyl-sulfonyl derivatives are most active. The ethyl-sulfonyl bearing oxadiazole showed
potent antibacterial activity, close to but slightly lower than the corresponding methylated one.
Whereas incorporation of relatively bulky benzyl group in the sulfonyl unit led to dramatic reduction
of antibacterial activity. This trend has been amply demonstrated in the observed decreasing order
of activity with two different series of halogen substituents e.g., 5I-1
> 5I-7 > 5I-9(R1 = F) and
5I-4 5I-8 5I-12(R1 = 2,4-di Cl). A similar decreasing behavior of activity with growing size
>
>
of alkyl group attached to the sulfonyl unit was also observed with 5II series of compounds wherein
oxadiazole moiety is replaced by thiadiazole skeleton.
Finally when the results are compared with those of previously reported sulfone analogs
(Table 4), where the positive controls were same and acted in identical level [
5I-1 provides an ideal example of being associated with exceptionally high activity against X.
oryzae (0.45 g/mL) and R. solanacearum (1.67 g/mL). In this regard, 2-(4-fluorophenoxymethyl)-
4,14–16], compound
µ
µ
5-(methylsulfonyl)-1,3,4-oxadiazole (5I-1) is even more potent than the previously reported fungicides
such as 2-(4-fluorophenzyl)-5-(methylsulfonyl)-1,3,4-oxadiazole [16] and 2-(4-fluorobenzyl)-5-
(methylsulfonyl)-1,3,4-oxadiazole [15]. It worth to note that when the molecular weights are
considered, the same mass concentration means lower molar concentration, for the present new
compounds possess the higher molecular weight with an -O-, or OCH2 group added. Similar
bioactive trend could be observed on 5I-2(4-Cl) or 5I-4(2,4-di Cl) with their corresponding analog
bearing 2-(4-chlorophenyl) or 2-(2,4-dichlorobenyl) substituent on heterocyclic side chain. It can be
further derived from earlier reported data and our present investigation (Table 4) that the nature of
2-substituent in the 1,3,4-oxadiazole ring also plays a significant role in the design of final desired
structure. The antibacterial activity with 2-substituents appears to follow the trend: phenyl < benzyl <
phenoxymethyl. The fact that the presence of substituted phenoxymethyl substituent at the 2-position
of 1,3,4-oxadiazole moiety helps to enhance biological activity may be attributed to the improved
structural flexibility offered by sp3-hybridized methylene (-CH2-), which helps to combine the receptor
protein of pathogenic bacteria [15]. Furthermore, in accordance with the prediction put forward by
CoMSIA 3D-QSAR, hydrogen-bond interaction offered by the oxygen atom of the alkyl aryl ether in
the designated region in between phenyl and heteroalkyl group could be responsible for the enhanced
bioactivity [14]. At the same time the electron withdrawing ability of the aroxymethyl group makes
the compound more potent by reducing electron density on the heterocyclic system and increasing
structure flexibility and stability.
Table 4. The Contribution of 2-Substituents in 1,3,4-Oxadiazole Ring towards Antibacterial Activity.
Plant Bacteria
EC50 (µg/mL) a
R
X = No Atom
X = CH2
X = OCH2
9.89 ± 1.52 16
23.21 ± 0.98 16
52.14 ± 1.05 16
1.07 ± 0.68 15
12.23 ± 1.45 15
1.96 ± 0.99 15
F
4-Cl
2,4-di Cl
0.45 ± 0.06 (5I-1)
1.44 ± 0.18 (5I-2)
0.72 ± 0.15 (5I-4)
1.97 ± 0.27 (5I-1)
13.42 ± 1.54 (5I-2)
20.51 ± 1.56 (5I-4)
X. oryzae
8.29 ± 0.56 14
120.90 ± 2.6 14
16.55 ± 1.12 14
/
b
F
4-Cl
2,4-di Cl
NA 4
R. solanacearum
59.9 26
a
Note: The EC50 values of the positive control for the reference compounds and present compounds were
determined under the same condition and are constantly in same level; “/”means no reported data.
b
NA, not active. Superscript [4,14–16,26] are the references cited.

Molecules 2017, 22, 64
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2.5. Phytotoxic Activity
We preliminarily assessed the possible toxicity of 5I-1
,
5I-2
,
5I-4 on rice germination and tobacco
leaf inoculation [27,28].
Initially, phytotoxic activity on tobacco leaves was assessed by infiltrating 50
µL of a 50, 100, 200
and 300 M solution of each compound into the mesophylls of the leaves. Bismerthiazol was also
µ
included for comparison purposes, and as a reference control. After 4-day of innoculation, a brown
necrotic area of around 0–0.3 cm diameter was observed (Figure 3). It appeared s no obvious difference
among the inoculation spots. 5I-1
effect on tobacco plant, with a necrosis between 0.2–0.3 cm at 300
,
5I-2
,
5I-4 and Bismerthiazol appeared no or negligible phytotoxic
M concentration. And there’s no
µ
dose-response effect with a clear phytotoxicity increase from 50 to 300 µM for all treatment.
Figure 3. Phytotoxicity assay on tobacco plant representative examples of tobacco leaves four days
after inoculation.
Further assays were carried out with rice germination test. Phytotoxic effects were quantified
through shoot lengths of seedlings grown in the presence of decreasing concentrations of compounds
(Table 5). Toxic effects were dose dependent. The high concentrations (300
severely affected seedling development (Figure 4). 5I-1 5I-2 and 5I-4 were divergent on the phenotypic
effects. Remarkably, 5I-4 severely inhibited both root growth and shoot development even at the
lowest concentration 1 M. 5I-2 predominantly inhibited root growth while allowing moderate
shoot development. While, 5I-1, barely effected root growth or inhibited shoot development at
concentration 1–100 M, similar to the appearance caused by Bismerthiazol. However, for the target
synthetic compounds, phytotoxicity was only observed at concentrations many times higher than
µM) of all chemicals
,
µ
µ
their bactericidal EC50 in terms of molar. While Bismerthiazol showed the phytotoxicity on 300 µM,
which is around same level as bactericidal EC50 119 µg/mL (about 333 µM).
Table 5. Phytotoxicity of target bactericidal compounds on rice germination a.
Inhibition on Rice Germination Shoot Length (cm)
Comp.
1 µM
10 µM
50 µM
100 µM
300 µM
5I-1
5I-2
5I-4
3.8 ± 0.5
3.7 ± 0.6
1.7 ± 0.3
4.2 ± 0.6
3.8 ± 0.5
3.2 ± 0.3
1.6 ± 0.2
3.5 ± 0.5
3.7 ± 0.6
3.2 ± 0.6
1.0 ± 0.1
3.7 ± 0.4
4.6 ± 0.2
2.8 ± 0.2
1.5 ± 0.0
1.1 ± 0.0
3.2 ± 0.3
0.1 ± 0.1
0.5 ± 0.1
0.5 ± 0.0
0.1 ± 0.1
Bismerthiazol b
H2O
a
Rice seeds germinated for seven days in H2O or increased concentrations of compounds. The size of rice
germination shoot length (cm) is considered as a measure of phytotoxicity. The numbers indicate confidence
b
interval of the mean. Antibacterial agents Bismerthiazol was used as comparative agent.

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Figure 4. Phytotoxicity assay on rice seeds germination.
3. Experimental
3.1. Chemicals and Instruments
All the reagents and solvents were purchased from Aladdin (Shanghai, China) and used without
further purification. The progress of the reactions and purity of compounds were assessed by thin-layer
chromatography (TLC) with GF254 silica-gel precoated sheets (Merck KGaA, Darmstadt, Germany)
using hexane/ethyl acetate as eluent; the melting points of the products were determined on an XT-4
1
binocular microscope (Beijing Tech Instrument Co., Beijing, China). H and 13C NMR (solvent CDCl3
or DMSO-d6) spectral studies were conducted on a JEOL-ECX 500 NMR spectrometer (JEOL, Tokyo,
Japan) using TMS as internal standard. Elemental analysis was performed on an Elemental Vario-III
CHN analyzer (Elementar Analysensysteme GmbH, Hanau, Germany). Mass spectral studies were
conducted on an Agilent LC/MSD Trap VL (Agilent, Santa Clara, CA, USA) mass spectrometer.
3.2. General Synthetic Procedure for the Target Compounds 5I and 5II
The synthetic route to target compounds is shown in Figure 2 which has been designed partly
based on previously reported methods [18–20].
3.2.1. Synthesis of Substituted Phenoxyacetohydrazide (2) as Intermediate
To an oven-dried round bottomed flask with a stirring bar was added a solution of substituted
phenol (0.01 mol, 1 eq.) and K2CO3 (1.2 eq.) in 50 mL of dry DMF. Excess ethyl chloroacetate
dissolved in 10 mL of DMF was then slowly added through a dropping funnel to the above solution.
The resulting mixture was stirred at 90 ◦C for 5–6 h. The reaction was quenched with saturated NaCl
solution, then extracted with dichloromethane(DCM), washed with brine, dried over MgSO4 and then
concentrated on a rotary evaporator affording methyl 2-phenoxyacetate
1 (80%–95% yield) which
was used for the next step without further purification [17].This product 1 (0.009 mol, 1 eq.) was
dissolved in 30 mL of ethanol and then placed in a round-bottomed flask equipped with a stirring bar;

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80% of hydrazine hydrate (0.018 mol, 2 eq.) was slowly added to this mixture at room temperature
through a dropping funnel. The resulting solution was heated up to reflux temperature and stirring
was continued under this condition for another 6 h. The reaction mixture was filtered through celite,
the filtrate was washed with brine, dried over MgSO4, and then concentrated in vacuo. The crude
phenoxyacetohydrazide 2 was used directly without further purification in the next step.
3.2.2. Synthesis of the Key Intermediate 2-Thiol-5-Substituted-1,3,4-Oxadiazole/Thiadiazole (3)
For X=O (3I), to a 100 mL three necked rounded-bottomed flask was added a solution of
intermediate 2 (0.009 mol, 1 eq.) and KOH (0.0108 mol, 1.2 eq.) in 50 mL absolute ethanol.
This mixture was first digested and then a diluted solution carbon disulfide (0.0108 mol, 1.2 eq) in 10 mL
of absolute ethanol was slowly added. The resulting mixture was stirred for 1–2 h and then refluxed
◦
at 80 C till a homogeneous solution was obtained [19]. The alcohol was removed under reduced
pressure on a rotary evaporator. The solution was washed with added brine followed by treatment
with dilute hydrochloric acid solution. The pH was adjusted to 5–6 when a precipitate appeared
which was subsequently filtered to afford a white solid 5-(phenoxymethyl)-1,3,4-oxadiazole-2-thiol (3I
)
(yield 60%–75%).
For X=S (3II), to a 100 mL three necked rounded-bottomed flask was added a solution of
intermediate 2 (0.009 mol, 1 eq.), and KOH (0.0108 mol, 1.2 eq.) in 50 mL of absolute ethanol.
This mixture was first digested and then a diluted solution carbon disulfide (0.0108 mol, 1.2 eq)
in 10 mL of absolute ethanol was slowly added. The resulting mixture was stirred for 5–6 h and then
filtered through celite. The filtrate was concentrated in vacuo. The crude potassium 2-(2-phenoxyacetyl)
hydrazinecarbodithioate (
2–
3) was washed with absolute ethanol alcohol and used directly in the next
step for the preparation of 3II. 2–3(0.01 mol) was crushed into small powder and then slowly added
to 50 mL H2SO4 (98%). An exothermic reaction ensued, the solution was stirred on an ice salt bath
for 2 h, and the temperature was kept temperature below 5 ◦C in order to control the violence of the
reaction. The progress of the reaction was monitored by TLC; the reaction was quenched by adding
crushed ice equivalent to 200 mL of water. A white precipitate appeared which was filtered and then
washed with water. The solid was subsequently dissolved in 10% NaOH solution and filtered again.
The filtrate was then acidified with hydrochloric acid (HCl) to afford a white solid which was filtered
to obtain the desired 5-(phenoxymethyl)-1,3,4-thiadiazole-2-thiol 3II.
3.2.3. Synthesis of the Intermediate 2-Thiol-5-Substituted-1,3,4-Oxadiazole/Thiadiazole (4)
To a solution of 2-thiol-5-substituted-1, 3, 4-oxadiazole/thiadiazole
3 (2 mmol) and sodium
hydroxide (2.4 mmol, 1.2 eq.) in water (10 mL), dimethyl sulfate/diethyl sulfate (2.4 mmol, 1.2 eq.)
was added drop wise with constant stirring at room temperature. The reaction was monitored
for its completion by thin layer chromatography (TLC), quenched with saturated NaCl solution,
then extracted with dichloromethane (3
Na2SO4 and then concentrated on a rotary evaporator affording a yellow oil, 2-thiol-5-substituted-1, 3,
×
20 mL). The solvent layer was washed with brine, dried over
4-oxadiazole/ thiadiazole (4) (70%–80% yield).
3.2.4. Synthetic Procedure for the Target Compounds 5
A 50 mL three-neck round-bottom flask equipped with a magnetic stirrer was charged with
intermediate
4 and glacial acetic acid (15 mL). The mixture was stirred for 10 min and then
7% potassium permanganate (KMnO4, 5 mmol) solution was slowly added with string at room
temperature [20]. After the completion of the reaction (assessed by TLC), 10% sodium bisulfite
(NaHSO3) solution was added to deoxidize the residual KMnO4. The reaction mixture were
subsequently filtered and the solid obtained was washed with distilled water, dried under vacuum,
and recrystallized from ethanol to give compounds 5.
2-(4-Fluorophenoxymethyl)-5-(methylsulfonyl)-1,3,4-oxadiazole (5I-1). White acicular crystal; m.p.:
◦
108–110 C; 1H-NMR (500 MHz, CDCl3) 3.50 (s, 3H), 5.32 (s, 2H), 7.12–6.89 (m, 4H); 13C-NMR (CDCl3,

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125 MHz) δ: 42.2, 61.6, 112.7 (d, J = 46.1 Hz), 116.2, 116.4 (d, J = 1.9 Hz), 116.5, 120.0 (d, J = 1.0 Hz),
155.5 (d, J = 468.7 Hz), 173.8, 178.9; Anal. calcd. for C10H9FN2O4S: C, 44.12; H, 3.33; N, 10.29; found:
C, 43.51; H, 3.53; N, 10.33. MS (ESI) (m/z): 273.2/[M + H]+.
◦
2-((4-Chlorophenoxy)methyl)-5-(methylsulfonyl)-1,3,4-oxadiazole (5I-2). White solid; m.p.: 113–114 C;
1H-NMR (500 MHz, CDCl3)
δ
7.50–7.40 (m, 2H, Ar-H), 6.96–6.87 (m, 2H, Ar-H), 5.33 (s, 2H, -CH2-),
3.50 (s, 3H, -CH3); 13C-NMR (125 MHz, CDCl3)
δ
164.3, 163.5, 156.3, 132.8, 116.7, 115.3, 59.9, 43.0;
Anal. calcd. for C10H9ClN2O4S: C, 36.23; H, 3.21; N, 8.42. Found: C, 36.05; H, 2.72; N, 8.41; MS (ESI)
(m/z): 357.2/[M + Na]+.
◦
2-((4-Bromophenoxy)methyl)-5-(methylsulfonyl)-1,3,4-oxadiazole (5I-3). White solid; m.p.: 97–99 C;
1H-NMR (500 MHz, CDCl3)
δ
7.30 (ddd, J = 8.8, 5.8, 2.9 Hz, 2H, Ar-H), 6.98–6.90 (m, 2H, Ar-H),
5.52 (s, 2H, -CH2-), 3.48 (s, 3H, -CH3); 13C-NMR (125 MHz, CDCl3)
δ
172.5, 170.1, 155.6, 129.95, 129.9,
127.9, 116.2, 116.2, 65.1, 43.1; Anal. calcd. for C10H9BrN2O4S: C, 41.50; H, 3.71; N, 9.71. Found: C, 41.60;
H, 3.14; N, 9.70; MS (ESI) (m/z): 311.2/[M + Na]+.
2-((2,4-Dichlorophenoxy)methyl)-5-(methylsulfonyl)-1,3,4-oxadiazole (5I-4). White solid; m.p.: 123–124 ◦C
1H-NMR (500 MHz, CDCl3)
7.42 (d, J = 2.5 Hz, 1H, Ar-H), 7.25–7.22 (m, 1H, Ar-H), 7.05
(d, J = 8.8 Hz, 1H, Ar-H), 5.39 (s, 2H, -CH2-), 3.50 (s, 3H, -CH3); 13C-NMR (125 MHz, CDCl3)
;
δ
δ
163.9, 163.6, 151.8, 130.8, 128.8, 128.1, 125.2, 116.4, 61.5, 43.0; Anal. calcd. for C10H8Cl2N2O4S: C, 37.54;
H, 2.96; N, 8.97. Found: C, 37.17; H, 2.50; N, 8.67; MS (ESI) (m/z): 323.3/[M + H]+.
2-((4-C◦hloro-2-methylphenoxy)methyl)-5-(methylsulfonyl)-1,3,4-oxadiazole (5I-5). White solid; m.p.:
1
96–98 C; H-NMR (500 MHz, CDCl3)
δ
7.17 (d, J = 2.5 Hz, 1H, Ar-H), 7.14 (dd, J = 8.6, 2.6 Hz,
1H, Ar-H), 6.86 (d, J = 8.6 Hz, 1H, Ar-H), 5.34 (s, 2H, -CH2-), 3.50 (s, 3H, -CH3), 2.23 (s, 3H, Ar-CH3);
13C-NMR (125 MHz, CDCl3)
δ
164.6, 163.4, 154.1, 131.2, 129.6, 127.5, 126.7, 112.9, 60.3, 43.0, 16.1; Anal.
calcd. for C11H11ClN2O4S: C, 43.91; H, 3.72; N, 9.35. Found: C, 43.64; H, 3.66; N, 9.25; MS (ESI) (m/z):
325.2/[M + Na]+.
◦
1
2-(Methylsulfonyl)-5-(phenoxymethyl)-1,3,4-oxadiazole (5I-6). White solid; m.p.: 66–67 C; H-NMR
(500 MHz, CDCl3) : 7.37–7.29 (m, 2H, Ar-H), 7.05 (t, J = 7.4 Hz, 1H, Ar-H), 7.00–6.97 (m, 2H, Ar-H),
4.94 (s, 2H, -CH2-), 3.46 (s, 3H, -CH3); 13C-NMR (125 MHz, CDCl3)
: 157.4, 154.8, 153.7, 129.8, 122.5,
δ
δ
115.0, 60.7, 49.0; Anal. calcd. for C10H10N2O4S: C, 47.51; H, 4.33; N, 11.33. Found: C, 47.24; H, 3.96; N,
11.02; MS (ESI) (m/z): 277.3/[M + Na]+.
◦
2-(Ethylsulfonyl)-5-((4-fluorophenoxy)methyl)-1,3,4-oxadiazole (5I-7). White solid; m.p.: 106–108 C;
1H-NMR (500 MHz, CDCl3)
δ
7.12–6.90 (m, 4H), 5.32 (s, 2H), 3.58 (q, J = 7.4 Hz, 2H), 1.51
(t, J = 7.4 Hz, 3H); 13C-NMR (125 MHz, CDCl3)
δ
164.6, 162.7, 153.4, 116.6, 116.5, 116.4, 116.4, 115.9, 60.6,
50.2, 6.9; Anal. calcd. for C11H11FN2O4S: C, 46.15; H, 3.87; N, 9.79. Found: C, 45.35; H, 3.84; N, 9.85;
MS (ESI) (m/z): 309.3/[M + Na]+.
◦
2-((2,4-Dichlorophenoxy)methyl)-5-(ethylsulfonyl)-1,3,4-oxadiazole (5I-8). White solid; m.p.: 88–89 C;
1H-NMR (500 MHz, DMSO-d6)
δ
7.62 (d, J = 2.5 Hz, 1H, Ar-H), 7.39 (dt, J = 21.6, 5.7 Hz, 2H, Ar-H),
5.66 (s, 2H, -CH2-), 3.74 (q, J = 7.3 Hz, 2H, -CH2CH3), 1.26 (t, J = 7.3 Hz, 3H, -CH3); 13C-NMR
(125 MHz, DMSO-d6) 164.6, 161.9, 151.7, 129.6, 128.2, 126.3, 122.9, 116.4, 61.1, 49.5, 6.6; Anal. calcd.
δ
for C11H10Cl2N2O4S: C, 39.49; H, 3.48; N, 8.65. Found: C, 39.18; H, 2.99; N, 8.31; MS (ESI) (m/z):
359.1/[M + Na]+.
2-(Benzylsulfonyl)-5-((4-fluorophenoxy)methyl)-1,3,4-oxadiazole (5I-9). White sold; m.p.: 94–96 ◦C;
1H-NMR (CDCl3, 500 MHz)
(CDCl3, 125 MHz)
δ
7.42–7.23 (m, 5H), 7.06–6.88 (m, 4H), 5.21 (s, 4H), 4.75 (s, 4H); 13C-NMR
164.7, 162.6, 158.4 (d, J = 240.9 Hz), 153.3, 131.2, 130.0, 129.3, 124.6, 116.5, 116.3
δ
(t, J = 4.1 Hz), 62.1, 60.4; Anal. calcd. for C16H13FN2O4S: C, 55.17; H, 3.76; N, 8.04. Found: C, 55.17;
H, 3.66; N, 8.29; MS (ESI) (m/z): 371.1/[M + Na]+.
2-(Benzylsulfonyl)-5-((4-chlorophenoxy)methyl)-1,3,4-oxadiazole (5I-10). White solid; m.p.: 126–128 ◦C;
1H-NMR (500 MHz, CDCl3)
δ 7.38–7.26 (m, 7H, Ar-H), 6.95–6.85 (m, 2H, Ar-H), 5.23 (s, 2H, -CH2-),

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4.75 (s, 2H, -CH2-); 13C-NMR (125 MHz, CDCl3)
δ
164.5, 162.6, 155.7, 131.2, 130.0, 129.9, 129.3, 127.8,
124.5, 116.2, 62.1, 59.8; Anal. calcd. for C16H13ClN2O4S: C, 52.27; H, 4.13; N, 7.61. Found: C, 52.68;
H, 3.59; N, 7.68; MS (ESI)(m/z): 387.2/[M + Na]+.
2-(Benzylsulfonyl)-5-((4-bromophenoxy)methyl)-1,3,4-oxadiazole (5I-11). White solid; m.p.: 142–143 ◦C;
1H-NMR (500 MHz, CDCl3)
δ
7.57–7.47 (m, 2H, Ar-H), 7.42–7.23 (m, 5H, Ar-H), 7.10–7.01 (m, 2H,
Ar-H), 5.55 (s, 2H, -CH2-), 5.20 (s, 2H, -CH2-); 13C-NMR (125 MHz, CDCl3)
δ
164.4, 162.7, 156.2, 132.8,
131.2, 130.0, 129.3, 124.5, 116.7, 115.2, 62.1, 59.7; Anal. calcd. for C16H13BrN2O4S: C, 46.83; H, 3.93;
N, 6.70. Found: C, 46.96; H, 3.20; N, 6.84; MS (ESI) (m/z): 433.1/[M + Na]+.
2-(Benzylsulfonyl)-5-((2,4-dichlorophenoxy)methyl)-1,3,4-oxadiazole (5I-12). White solid; m.p.: 121–122 ◦C;
1H-NMR (500 MHz, CDCl3)
δ
7.68 (d, J = 2.6 Hz, 1H, Ar-H), 7.46 (dd, J = 8.9, 2.6 Hz, 1H, Ar-H),
7.40–7.29 (m, 6H, Ar-H), 5.67 (s, 2H, -CH2-), 5.21 (s, 2H, -CH2-); 13C-NMR (125 MHz, CDCl3)
δ
164.6, 162.0, 151.6, 131.3, 129. 7, 129.3, 128.7, 128.2, 126.2, 125.7, 122.8, 116.0, 60.7, 60.5; Anal. calcd.
for C16H12Cl2N2O4S: C, 48.39; H, 3.43; N, 7.32. Found: C, 48.13; H, 3.03; N, 7.02; MS (ESI) (m/z):
399.4/[M + H]+.
2-(Benzylsulfonyl)-5-((4-chloro-2-methylphenoxy)methyl)-1,3,4-oxadiazole (5I-13). Yellow crystal; m.p.:
◦
1
144–145 C; H-NMR (500 MHz, CDCl3)
7.31–7.29 (m, 2H, Ar-H), 7.27 (s, 2H, Ar-H), 7.26–7.24 (m, 1H, Ar-H), 7.11 (d, J = 8.8 Hz, 1H, Ar-H),
5.55 (s, 2H, -CH2-), 5.21 (s, 2H, -CH2-), 2.17 (s, 3H, Ar-CH3); 13C-NMR (125 MHz, CDCl3)
174.6,
δ 7.39–7.35 (m, 1H, Ar-H), 7.32 (d, J = 1.4 Hz, 1H, Ar-H),
δ
169.8, 165.8, 162.4, 154.6, 131.8, 130.9, 129.8, 129.4, 129.2, 127.1, 126.3, 125.93, 114.4, 61.0, 60.6, 16.2; Anal.
calcd. for C17H15ClN2O4S: C, 53.31; H, 4.23; N, 7.26. Found: C, 53.90; H, 3.99; N, 7.39; MS (ESI) (m/z):
401.2/[M + Na]+.
◦
1
2-(Benzylsulfonyl)-5-(phenoxymethyl)-1,3,4-oxadiazole (5I-14). White solid; m.p.: 95–96 C; H-NMR
(500 MHz, CDCl3) 7.40–7.24 (m, 7H, Ar-H), 7.06 (ddd, J = 11.7, 8.3, 0.9 Hz, 3H, Ar-H), 5.53 (s, 2H,
-CH2-), 5.20 (s, 2H, -CH2-); 13C-NMR (125 MHz, CDCl3)
165.4, 161.9, 157.0, 131.3, 129.7, 129.3, 128.8,
δ
δ
125.8, 122.0, 114.8, 60.5, 59.5; Anal. calcd. for C16H14N2O4S: C, 58.52; H, 4.60; N, 8.66. Found: C, 58.17;
H, 4.27; N, 8.48; MS (ESI) (m/z): 353.2/[M + Na]+.
2-((4-Fluorophenoxy)methyl)-5-(methylsulfonyl)-1,3,4-thiadiazole (5II-1). White solid; m.p.: 107–109 ◦C;
1H-NMR (500 MHz, CDCl3)
δ
7.08–7.00 (m, 2H, Ar-H), 6.99–6.92 (m, 2H, Ar-H), 5.52 (s, 2H, -CH2-),
3.48 (s, 3H, -CH3); 13C-NMR (125 MHz, CDCl3)
δ
172.9, 170.0, 159.3, 157.4, 153.1, 116.6, 116.4, 116.2,
116.2, 65.5, 43.1; Anal. calcd. for C10H9FN2O3S2: C, 41.64; H, 3.49; N, 9.93. Found: C, 41.66; H, 3.15; N,
9.72; MS (ESI) (m/z): 311.2/[M + Na]+.
2-((4-Chlorophenoxy)methyl)-5-(methylsulfonyl)-1,3,4-thiadiazole (5II-2). White solid; m.p.: 119–120 ◦C;
1H-NMR (500 MHz, CDCl3)
δ
7.46–7.26 (m, 2H, Ar-H), 7.15 (dd, J = 5.8, 3.2 Hz, 2H, Ar-H), 5.73 (s, 2H,
-CH2-), 3.66 (s, 3H, -CH3); 13C-NMR (125 MHz, CDCl3)
δ
172. 7, 170.6, 156.5, 130.0, 126.3, 117.4, 64.9,
43.6; Anal. calcd. for C10H9ClN2O3S2: C, 39.53; H, 3.32; N, 9.20. Found: C, 39.41; H, 2.98; N, 9.19;
MS (ESI) (m/z): 327.1/[M + Na]+.
2-((4-Bromophenoxy)methyl)-5-(methylsulfonyl)-1,3,4-thiadiazole (5II-3). Yellow crystal; m.p.: 114–115 ◦C;
1H-NMR (500 MHz, CDCl3)
δ
7.49–7.40 (m, 2H, Ar-H), 6.94–6.85 (m, 2H, Ar-H), 5.52 (s, 2H, -CH2-),
3.47 (s, 3H, -CH3); 13C-NMR (125 MHz, CDCl3)
δ
172.5, 170.1, 156.1, 132.9, 116.7, 115.2, 65.0, 43.1;
Anal. calcd. for C10H9BrN2O3S2: C, 33.29; H, 2.99; N, 7.76. Found: C, 34.39; H, 2.60; N, 8.02; MS (ESI)
(m/z): 373.1/[M + Na]+.
2-((2,4-Dichlorophenoxy)methyl)-5-(methylsulfonyl)-1,3,4-thiadiazole (5II-4).
160–162 ◦C; 1H-NMR (500 MHz, CDCl3)
7.45 (d, J = 2.5 Hz, 1H, Ar-H), 7.25 (dd, J = 8.8, 2.5 Hz, 1H,
Ar-H), 6.98 (d, J = 8.8 Hz, 1H, Ar-H), 5.57 (s, 2H, -CH2-), 3.49 (s, 3H, -CH3); 13C-NMR (125 MHz, CDCl3)
Yellow crystal; m.p.:
δ
δ
172.0, 170.3, 151.6, 130.8, 128.1, 124.6, 120.0, 115.1, 66.2, 43.2; Anal. calcd. for C10H8Cl2N2O3S2: C,
35.37; H, 2.81; N, 8.26. Found: C, 35.41; H, 2.38; N, 8.26; MS (ESI) (m/z): 361.1/[M + Na]+.
2-((4-Chloro-2-methylphenoxy)methyl)-5-(methylsulfonyl)-1,3,4-thiadiazole (5II-5). White solid; m.p.:
◦
1
160–161 C; H-NMR (500 MHz, CDCl3)
δ 7.19 (d, J = 2.5 Hz, 1H, Ar-H), 7.16 (dd, J = 8.6, 2.6 Hz,

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1H, Ar-H), 6.81 (d, J = 8.6 Hz, 1H, Ar-H), 5.52 (s, 2H, -CH2-), 3.49 (s, 3H, -CH3), 2.27 (s, 3H, Ar-CH3);
13C-NMR (125 MHz, CDCl3)
172.9, 170.0, 153.8, 131.3, 129.0, 127.4, 126.8, 112.3, 65.2, 43.1, 16.3;
δ
Anal. calcd. for C11H11ClN2O3S2: C, 39.95; H, 3.95; N, 8.57. Found: C, 41.44; H, 3.48; N, 8.79; MS (ESI)
(m/z): 341.2/[M + Na]+.
◦
2-(Ethylsulfonyl)-5-((4-fluorophenoxy)methyl)-1,3,4-thiadiazole (5II-6). Yellow crystal; m.p.: 89–90 C;
1H-NMR (500 MHz, CDCl3)
δ
7.04 (dd, J = 9.1, 8.1 Hz, 2H, Ar-H), 6.95 (dd, J = 9.1, 4.2 Hz, 2H),
Ar-H, 5.51 (s, 2H, -CH2-), 3.60 (q, J = 7.4 Hz, 2H, -CH2CH3), 1.47 (t, J = 7.4 Hz, 3H, -CH3); 13C-NMR
(125 MHz, CDCl3) 172.9, 169.1, 159.3, 157.4, 153.2, 116.6, 116.4, 116.2, 116.2, 65.6, 50.3, 7.2; Anal. calcd.
δ
for C11H11FN2O3S2: C, 43.19; H, 4.09; N, 9.19. Found: C, 43.70; H, 3.67; N, 9.27; MS (ESI) (m/z):
325.2/[M + Na]+.
2-((2,4-Dichlorophenoxy)methyl)-5-(ethylsulfonyl)-1,3,4-thiadiazole (5II-7). White solid; m.p.: 127–128 ◦C;
1H-NMR (500 MHz, CDCl3) 7.45 (d, J = 2.5 Hz, 1H, Ar-H), 7.25 (dd, J = 8.8, 2.6 Hz, 1H, Ar-H), 6.97
δ
(d, J = 8.8 Hz, 1H, Ar-H), 5.57 (s, 2H, -CH2-), 3.61 (q, J = 7.4 Hz, 2H, -CH2CH3), 1.49 (t, J = 7.4 Hz, 3H,
-CH3); 13C-NMR (125 MHz, CDCl3)
δ
172.1, 169.3, 151.6, 130.7, 128.4, 128.1, 124.6, 115.1, 66.3, 50.2, 7.2;
Anal. calcd. for C11H10Cl2N2O3S2: C, 37.18; H, 3.37; N, 7.96. Found: C, 37.40; H, 2.85; N, 7.93; MS (ESI)
(m/z): 375.1/[M + Na]+.
◦
2-(Benzylsulfonyl)-5-((4-fluorophenoxy)methyl)-1,3,4-thiadiazole (5II-8). White solid; m.p.: 132–134 C;
1H-NMR (500 MHz, CDCl3)
δ
7.02 (dd, J = 9.0, 8.1 Hz, 2H, Ar-H), 6.89 (dd, J = 9.2, 4.2 Hz, 2H, Ar-H),
5.46 (s, 2H, -CH2-), 4.79 (s, 2H, -CH2-); 13C-NMR (125 MHz, CDCl3)
δ
173.4, 168.6, 159.3, 157.4, 153.1,
153.0, 131.3, 129.6, 129.2, 125.9, 116.6, 116.4, 116.2, 116.1, 65.5, 61.9; Anal. calcd. for C16H13FN2O3S2: C,
52.69; H, 3.89; N, 7.64. Found: C, 52.73; H, 3.60; N, 7.69; MS (ESI) (m/z): 387.2/[M + Na]+.
2-(Benzylsulfonyl)-5-((4-chlorophenoxy)methyl)-1,3,4-thiadiazole (5II-9). White solid; m.p.: 140–141 ◦C;
1H-NMR (500 MHz, CDCl3)
δ
7.33 (ddd, J = 5.6, 3.5, 1.6 Hz, 2H, Ar-H), 7.30–7.28 (m, 3H, Ar-H), 7.25
(d, J = 1.7 Hz, 2H, Ar-H), 6.87 (dd, J = 8.8, 1.8 Hz, 2H, Ar-H), 5.47 (s, 2H, -CH2-), 4.79 (s, 2H, -CH2-);
13C-NMR (125 MHz, CDCl3)
173.1, 168.7, 155.5, 131.3, 129.9, 129.6, 129.2, 127.8, 125.8, 116.2, 65.0, 61.9;
δ
Anal. calcd. for C16H13ClN2O3S2: C, 50.92; H, 3.93; N, 7.50. Found: C, 50.46; H, 3.44; N, 7.36; MS (ESI)
(m/z): 403.2/[M + Na]+.
2-(Benzylsulfonyl)-5-((4-bromophenoxy)methyl)-1,3,4-thiadiazole (5II-10). White solid; m.p.: 147–148 ◦C;
1H-NMR (500 MHz, CDCl3)
δ
7.48–7.39 (m, 2H, Ar-H), 7.39–7.22 (m, 5H, Ar-H), 6.88–6.78 (m, 2H,
Ar-H), 5.47 (s, 2H, -CH2-), 4.78 (s, 2H, -CH2-); 13C-NMR (125 MHz, CDCl3)
δ
173.0, 168.7, 156.0, 132.8,
131.3, 129.6, 129.2, 125.8, 116.7, 115.1, 64.9, 61.9; Anal. Calcd for C16H13BrN2O3S2: C, 45.37; H, 3.47; N,
6.62. Found: C, 45.18; H, 3.08; N, 6.59; MS (ESI) (m/z): 449.1/[M + Na]+.
◦
2-(Benzylsulfonyl)-5-((2,4-dichlorophenoxy)methyl)-1,3,4-thiadiazole (5II-11). White solid; m.p.: 124–125 C;
1H-NMR (500 MHz, CDCl3)
δ
7.43–7.41 (m, 1H, Ar-H), 7.37–7.27 (m, 5H, Ar-H), 7.23 (ddd, J = 8.8, 2.4,
1.7 Hz, 1H, Ar-H), 6.92 (dd, J = 8.8, 1.2 Hz, 1H, Ar-H), 5.52 (s, 2H, -CH2-), 4.79 (s, 2H, -CH2-); 13C-NMR
(125 MHz, CDCl3) 172.5, 168.9, 151.5, 131.3, 130.7, 130.0, 129.2, 128.3, 128.0, 125.8, 124.53, 115.1, 66.1,
δ
61.9; Anal. calcd. for C16H12Cl2N2O3S2: C, 46.74; H, 3.28; N, 6.80. Found: C, 46.27; H, 2.91; N, 6.75;
MS (ESI) (m/z): 437.1/[M + Na]+.
2-(Benzylsulfonyl)-5-((4-chloro-2-methylphenoxy)methyl)-1,3,4-thiadiazole (5II-12). White solid; m.p.:
◦
1
125–126 C; H-NMR (500 MHz, CDCl3)
(d, J = 8.6 Hz, 1H, Ar-H), 5.46 (s, 2H, -CH2-), 4.79 (s, 2H, -CH2-), 2.19 (s, 3H, Ar-CH3); 13C-NMR (125
MHz, CDCl3) 173.4, 168.5, 153.7, 131.3, 131.2, 129.6, 129.2, 129.0, 127.3, 126.8, 125.87, 112.3, 65.1, 61.9,
δ 7.39–7.26 (m, 5H, Ar-H), 7.18–7.10 (m, 2H, Ar-H), 6.75
δ
16.2; Anal. calcd. for C17H15ClN2O3S2: C, 51.93; H, 4.02; N, 7.17. Found: C, 51.71; H, 3.83; N, 7.09;
MS (ESI) (m/z): 417.2/[M + Na]+.

Molecules 2017, 22, 64
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3.3. X-ray Diffraction Analysis
Crystal structure of compound 5II-9 is shown in Figure 5. Colorless crystal of compound
5II-9 (0.12 mm 0.13 mm 0.13 mm) was mounted on a quartz fiber with protection oil.
Cell dimensions and intensities were measured at 293 K on an Xcalibur (Eos, Gemini) diffractometer
with graphite monochromated Cu K radiation ( = 1.54184 Å). A total of 23281 reflections were
measured, of which 3130 were unique (Rint = 0.0728) in the range of 4.66 < 13 to 13;
< 67.23◦ (h,
k, 10 to 5; l, 40 to 40), and 2142 observed reflections with I > 2 (I) were used in the refinement on
×
×
α
λ
θ
−
−
−
σ
F2. The structure was solved by direct method with the SHELXTL-97 program. All of the non-H atoms
were refined anisotropically by full-matrix least-squares to give the final R = 0.0459 and WR = 0.0459.
All hydrogen atoms were computed and refined using a riding model. The atomic coordinates for
5II-9 have been deposited at the Cambridge Crystallographic Data Centre. CCDC 1008894 contains
the supplementary crystallographic data for this paper.
Figure 5. Crystal structure of compound 5II-9.
3.4. Antibacterial Bioassay In Vitro
The antibacterial activity against X. oryzae, R. solanacearum, X. axonopodis in vitro were
evaluated according to the previously reported turbidmeter test [21]. The working solution
concentration was set at 200 and 100
µg/mL. Negative control(DMSO in sterile distilled water
containing 0.1% Tween 20) were set up identically to test for absence of compound; Bismerthiazol
(5,50-(methylenediimino)bis-1,3,4-Thiadiazole-2(3H)-thione, 20% WP, China Jiangxi Heyi Chem. Ind.
Co., Ltd., Pengze, China), Kocide 3000 (Copper hydroxide, 46.1%, DuPont, Wilmington, DE, USA)) and
Thiodiazole-copper (2-amino-5-mercapto-1,3,4-thiadiazole-copper, 20% SC, China Zhejiang Longwan
Chem. Ind. Co., Ltd., Wenzhou, China) served as positive controls. Approximately 1 mL of stock
solution was added to 4 mL nontoxic liquid medium nutrient broth (NB, 3 g of beef extract, 5 g of
peptone, 1 g of yeast powder, 10 g of glucose, and 1000 mL of distilled water, pH 7.0 to 7.2) in tubes.
Then, approximately 40
containing compounds, DMSO or Kocide 3000, Thiodiazole-copper. Then, the inoculated test tubes
were incubated at 30
1 ◦C with continuous shaking at 180 rpm for 24 h. The growth of bacterial
µL NB culture media containing bacteria was added to 5 mL of NB solution
±
culture was monitored with a spectrophotometer by measuring the optical density at 600 nm (OD600).
The inhibitory rate of bacterial culture growth was calculated using the following equation:
I (%) = (CK
−
T)/CK
×
100. Where “CK” implies the value of the corrected optical density of
bacterial growth on untreated NB (negative control), and “T” means the value of corrected optical
density of bacterial growth on treated NB; “I” denotes the inhibition rate.
The EC50 values of some well performed compounds were evaluated using the above-mentioned
method under 5 different concentrations (e.g., 50, 25, 12.5, 6.25, 3.12
R. solanacearum and 200, 100, 50, 25, 12.5 g/mL for X. axonopodis). The average EC50 was computed
from at least three separate analyses for growth inhibition with Log10 probit analysis of SPSS 17.0.
µg/mL for X. oryzae and
µ

Molecules 2017, 22, 64
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3.5. Antibacterial Activity Bioassay In Vivo
The in vivo preventive activities of compounds 5I-1
,
5I-2
,
5I-4 on lesion formation caused by
X. oryzae in rice plant were evaluated according to the following procedure under greenhouse
condition [22 23]. Rice seeds were soaked with water overnight and sown in small pots.
,
Then the rooted seedlings were transplanted when they were 20 days old in net-house. The rice
plants grown for 35–40 day bearing 3–4 leaves were ready to be treated. The tested compounds
were dissolved in 120
µL DMSO and diluted with 30 mL 0.1% Tween-20 to the final concentration
of 200 g/mL. For comparison, parallel test were performed on Bismerthiazol, Thiodiazole-copper
µ
(positive control), no-compound (negative control), distilled water (blank control, absence of bacterial
infection and compound treatment) with three replicates of each treatment. The seedlings on the 45th
day after sowing received foliar spray applications with 4 mL test compounds solution. One day later,
the rice plants were clip inoculated with a bacterial suspension of X. oryzae culture (108 CFU/mL).
Development of bacterial blight lesions was scored 14 days after the inoculation. The disease severity
including infection rate and disease index was determined in terms incidence of lesion length and
area. The percent disease suppression in the compounds treatment was calculated as compared to
disease index of the untreated control in three independent experiments. And the data were analyzed
for statistical significance using the least significant difference (LSD) test. Inhibitory effect afforded by
the chemical treatment from that of the untreated control was calculated with the following formula:
Disease suppression (%) = (C
leaves; T means disease index in compound treated rice leaves.
−
T)/C
×
100, where C implies disease index in untreated rice
3.6. Phytotoxic Bioassay
5I-1, 5I-2, 5I-4 were evaluated for their phytotoxicity on rice seed germination and tobacco plant
as reference described [27,28].
We initially assessed the possible toxicity on rice seed. For each treatment, a total of 4
surface-sterilized rice seeds (Fengyou Xiangzhan) were germinated in 200
solution at the appropriate concentration (0, 1, 10, 50,100, and 300 M) in each well of a 24-well
plate for seven days. And 200 L double-distilled water was implemented in the third day.
Five different concentrations of test compound, in triplicate, and reared in an incubator maintained
at 28
1 ◦C with a photoperiod of 14 h light/10 h dark. Bismerthiazol was set as comparative agent.
Seedling morphology was recorded and shoot length was measured. Phytotoxicity values in rice
µL water or compounds
µ
µ
±
germination assays are given as minimal compound concentrations (
of shoot length.
µM) causing significant alteration
Tobacco (Nicotiana benthamiana) plants were grown from seed in a heated glasshouse and used
between 20 and 30 days old. 50 L chemically solution at 50, 100, 200 or 300 M were infiltrated
µ
µ
into the mesophylls of fully expanded tobacco leaves (previously wounded with a needle) using
a micropipette. The plants were kept at standard glasshouse conditions for seven days. Up to six
independent inoculations were carried out in a single leaf, and at least three independent inoculations
were performed for each compound and concentration, randomly distributed in different leaves
and plants. Control infiltrations with water or comparative agent Bismerthiazol at the same molar
concentration were performed. The appearance of symptoms on the leaves was followed for four days
after infiltration. Toxicity was measured as the lesion diameter.
4. Conclusions
In summary, a series of novel aroxymethyl-1,3,4-oxadiazole/thiadiazole sulfone derivatives
were obtained via embedding the substituted phenoxymethyl group into scaffold of heterocyclic
substituted sulfone. The antibacterial activities were studied by the turbidimeter test against
pathogens X. oryzae, R. solanacearum, and X. axonopodis, respectively. 1,3,4-Oxadiazole sulfones 5I
are more potent as antibacterials compared to their corresponding 1,3,4-thiadiazole derivatives 5II
and positive controls. Interestingly, as compared to commercial bactericides or our previous reported
sulfone analogs (Table 4), compounds 5I-1–5I-7 possessed remarkably higher in vitro bactericidal

Molecules 2017, 22, 64
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activity against tested bacteria, which indicating that high-efficient target molecules were successfully
obtained as we proposed. In vivo tests also revealed that 5I-1 5I-2 and 5I-4 exhibited excellent
preventive effects against X. axonopodis with inhibitory effect of 90.4%, 77.7% and 81.1% respectively.
Compared to Bismerthiazol, 5I-1 5I-2 exhibited no apparently phytotoxicity in tobacco leaves and
,
,
rice seed germination with exception that 5I-4 highly suppressed both root and shoot development.
The structure-activity relationship (SAR) analyses have suggested that aroxymethyl is found extremely
useful to enhance the activity of the target structure and 1,3,4-oxadiazole sulfone derivatives bearing
suitably substituted phenoxymethyl group may lead to the development of potent bactericides for
crop protection.
Supplementary Materials: 1H and 13C NMR spectra of all the compounds are presented as Supporting
Information; crystallographic data of compound 5II-9 (CCDC 1008894) for this paper could be obtained free
of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk. The supplementary is
available online at www.mdpi.com/1420-3049/22/1/64/s1.
Acknowledgments: This work was supported by the National Natural Science Foundation of China (20962005)
and the Excellent Young Science and Technology Talent Cultivation Plan (201122).
Author Contributions: Jin conceived the project. Zhou & Su contributed equally to this work. Su performed
most of the experimental work while Zhou & Liao implemented the biological test protocols. Qi prepared and
participated in the phytotoxity assay. All authors analyzed the data and contributed to manuscript preparation.
Zhou wrote the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Not available.
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2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).