Synthesis and antifungal activity of novel sulfoxide derivatives containing trimethoxyphenyl substituted 1,3,4-thiadiazole and 1,3,4-oxadiazole moiety

Article abstract of DOI:10.1016/j.bmc.2008.02.006

Selective oxidation of sulfides 7 or 8 to sulfoxides 9 or 10 is achieved by mCPBA. The structures of the compounds 9 or 10 are confirmed by elemental analysis, IR, and ¹H NMR. The bioassay results showed that title compound 10a possess high ant

Full text of DOI:10.1016/j.bmc.2008.02.006

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3632–3640
Synthesis and antifungal activity of novel sulfoxide
derivatives containing trimethoxyphenyl substituted
1,3,4-thiadiazole and 1,3,4-oxadiazole moiety
Fang Liu, Xiao-Qiong Luo, Bao-An Song,* Pinaki S. Bhadury, Song Yang, Lin-Hong Jin,
Wei Xue and De-Yu Hu
Center for Research and Development of Fine Chemicals, Key Laboratory of Green Pesticide and Bioengineering,
Ministry of Education, Guizhou University, Guiyang 550025, PR China
Received 5 December 2007; revised 1 February 2008; accepted 5 February 2008
Available online 8 February 2008
Abstract—Selective oxidation of sulfides 7 or 8 to sulfoxides 9 or 10 is achieved by mCPBA. The structures of the compounds 9 or 10
are confirmed by elemental analysis, IR, and 1H NMR. The bioassay results showed that title compound 10a possess high antifungal
activities with EC50 values ranging from 19.91 to 63.97 lg/mL. The mechanism of action of 10a against Sclerotinia sclerotiorum was
studied. After treating with compound 10a at 100 lg/mL for 12 h, the mycelial reducing sugar, D-GlcNAc, soluble protein and pyru-
vate content, chitinase activity showed declining tendency.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
ically active heterocyclic compounds. Recently, we
reported the fungicidal activity of novel sulfone deriva-
tives, some of which were found to possess good fungi-
cidal bioactivity.11
Sulfoxide derivatives represent one of the most active
class of compounds possessing a wide spectrum of bio-
logical activity. They are widely used in pharmaceuticals
and agrochemicals,1–4,10 as fungicide,5,6 herbicide,7 and
antitumor agent.8,9 Some compounds with broad spec-
trum of bioactivity have been commercialized, for exam-
ple, Fipronil which is highly effective against Myzus
persicae, Empoasca, larvae of Lepidoptera, Musca
domestica, and Hymenoptera pests.10 Thus, their synthe-
sis has been of great interest in the elaboration of biolog-
Fascinated by these findings and in an attempt to eval-
uate the modification of the fungicidal profile induced
by the change of the substituents at the alkyl group,
we designed and synthesized a series of sulfoxide deriv-
atives with various substituents and measured their fun-
gicidal activities. The synthetic route is shown in Scheme 1.
Starting from the key intermediate 5-(3,4,5-trimethoxy-
phenyl)-1,3,4-thiadiazole-2-thiol (4) or the oxadiazole
analogue (5), the title compounds 9 and 10 were synthe-
sized in two steps. Thioetherification reaction of 4 or 5
with organohalide catalyzed by indium or indium tribro-
mide first affords appropriate sulfides 7 or 8, which were
then converted into title compounds 9 or 10 by mCPBA
oxidation in dichloromethane. The structures of 9 and
Abbreviations: DCM, dichloromethane; DCE, 1,2-dichloroethane;
[bmim]PF6, 1-butyl-3-methylimidazolium hexafluorophosphate; 13C
NMR, 13C nuclear magnetic resonance; mCPBA, meta-chloroperoxy-
benzoic acid; IR, infra-red; 1H NMR, 1H nuclear magnetic resonance;
PDA, potato dextrose agar culture; DNS, 3,5-dinitrosalicylic acid;
DMAB, 3,20-dimethyl-4-aminobiphenyl; EC50, 50% effective concen-
tration; LDH, lactate dehydrogenase; G. zeae, Gibberella zeae; C. man-
dshurica, Cytospora mandshurica; F. oxysporium, Fusarium oxysporium;
P. infestans, Phytophthora infestans; R. solani, Rhizoctonia solani;
T. cucumeris, Thanatephorus cucumeris; C. gloeosporioides, Colletotri-
chum gloeosporioides; B. cinerea, Botrytis cinerea; S. sclerotiorum, Scle-
rotinia sclerotiorum.
1
10 were firmly established by well-defined IR, H NMR,
13C NMR, and elemental analysis. Preliminary bioassay
tests showed that some compounds possess certain
degree of antifungal activity against three phytopatho-
genic fungi at 50 mg/L in vitro as shown in Table 5,
however, with a degree of variation. The bioassay results
showed that title compound 10a possessed high
antifungal activities against nine kinds of fungi and the
EC50 values ranging from 19.91 to 63.97 lg/mL. After
Keywords: Sulfoxides; Thioether; Antifungal bioactivity; Mechanism
of action.
*
Corresponding author. Tel.: +86 851 362 0521; fax: +86 851 362
2211; e-mail: songbaoan22@yahoo.com
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.006

F. Liu et al. / Bioorg. Med. Chem. 16 (2008) 3632–3640
3633
H3CO
H3CO
H3CO
HO
HO
a
b
c
(CH ) SO
H3CO
H3CO
COOH
COOCH3
+
COOH
H3CO
3 2
4
H3CO
HO
1
2
H3CO
H3CO
N N
X
for 7: i
for 4:(1)e;(2) f
for 5:(1)g;(2) h
d
SH
H3CO
H3CO
CONHNH2
for 8: j
H3CO
4
: X=S
5: X=O
3
H3CO
H3CO
H3CO
H3CO
H3CO
N
O
S R
N
N
N
k
S R
X
X
H3CO
9a-9g: X=S
10a-10h: X=O
7a-7g: X=S
8a-8h: X=O
Scheme 1. Synthetic route to title compounds 9 and 10. Reagents and conditions: (a) (CH3)2SO4, 10% NaOH; (b) 35% HCl; (c) CH3OH, 98% H2SO4,
reflux; (d) NH2NH2ÆH2O, CH3OH, reflux for 5 h; (e) KOH, CS2, C2H5OH, rt; (f) 98% H2SO4, 0–5 ꢁC; (g) KOH, CS2, C2H5OH, reflux for 6 h; (h) 5%
HCl, ice-bath; (i) In, 3% NaOH, H2O, RX (6), rt; (j) InBr3, 3% NaOH, H2O, RX (6), rt; (k) mCPBA, dichloromethane, 4 ꢁC, 2 h and then rt, 5 h.
treating Sclerotinia sclerotiorum with compound 10a at
100 lg/mL, only 5.4% of its spore bourgeoned, the perme-
ability of the cell membrane increased and most of its hy-
phal became distorted, malformed, and got entangled.
atom-efficient and waste-free methods for sulfoxide syn-
thesis are known and numerous methods for sulfide oxi-
dation by H2O2 in the presence of transition metals as
catalysts have been developed. In order to oxidize sulfide
to its sulfoxide selectively, we need to select suitable cat-
alyst to prevent sulfoxide from overoxidizing to sulfone.
The synthesis of 10a was carried out under several differ-
ent synthesis methods, and the results are summarized in
Table 1.
2. Results and discussion
2.1. Chemistry
The synthetic route designed for the sulfoxide analogues 9
and 10 is summarized in Scheme 1. Following the reported
method,12 5-(3,4,5-trimethoxyphenyl)-1,3,4-thiadiazole-
2-thiol 4 was synthesized from gallic acid in five steps
including etherification, esterification, hydrazidation, salt
formation, and cyclization. 5-(3,4,5-Trimethoxyphenyl)-
1,3,4-oxadiazole-2-thiol 5 was easily prepared by the reac-
tion of 5-trimethoxyphenylhydride 3, potassium hydrox-
ide, and carbon disulfide in water-free ethanol under
reflux condition. Then, 5-(3,4,5-trimethoxyphenyl)-
1,3,4-thiadiazole-2-thiol 4 and the oxadiazole analogue
5 were converted to thioether derivatives containing thia-
diazole 7 or oxadiazole moiety 8 by a thioetherification
reaction with halide (RX) catalyzed by indium or indium
tribromide. Treatment of sulfides 7 and 8 with mCPBA in
dichloromethane afforded the heterocyclic sulfoxides 9
and 10 with moderate yields.13–15
As could be observed from Table 1 that the oxidation of
sulfides to sulfoxides by hydrogen peroxide or potassium
periodate has proved to be unsuccessful methods. The
oxidation of sulfides to sulfoxides by mCPBA as oxidant
and dichloromethane as solvents has proved to be the
best oxidation method. So we tested this method.
In order to optimize the reaction conditions for the
preparation of 9 and 10, the synthesis of 9a was carried
out under several different conditions. We took mCPBA
as the oxidant and varied the reaction conditions. The
effects of reaction time, reaction temperature, and oxi-
dant amount on the reaction were investigated, and
the results are summarized in Table 2.
As can be seen from Table 2, treatment of 7a with
1.1 equiv of mCPBA (with respect to the substrate) at
4 ꢁC for 2 h followed by warming to room temperature
and stirring for another 5 h gave the highest yield
(43.7%). Further investigation indicated that the addi-
tion of mCPBA in excess to 1.1 equiv resulted in the for-
mation of sulfone. On the other hand, addition of less
than 1.1 equiv of mCPBA led to poor conversion of
the substrate into product 9a. Addition of different cat-
alysts in the reaction mixture had little or negligible ef-
fect in terms of improving the product yield. Under
the optimized conditions as described above, different
sulfides were converted by mCPBA oxidation into their
corresponding sulfoxides and the results are depicted in
Tables 3 and 4.
Recently, the oxidation of sulfides to sulfoxides by
hydrogen peroxide or mCPBA has proved to be tradi-
tional methods. Over the past few years, the importance
of hydrogen peroxide and its derivatives as oxidizing
agents has become an emerging part recently. In con-
trast to other commonly employed oxidizing agents,
for example, hypochlorite, hypobromite, potassium per-
manganate, and potassium chromate, hydrogen perox-
ide is easy to handle, store, transport, and is relatively
cheap. Also, Hydrogen peroxide is a kind of pro-nuclear
oxidant and the oxidation of sulfides to sulfoxides with-
out catalysts is generally slow. So a number of new,

3634
F. Liu et al. / Bioorg. Med. Chem. 16 (2008) 3632–3640
Table 1. The effect of yield in different methods
Entry
Oxidant (mmol)
Catalyst (mmol)
Temperature (ꢁC)
Reaction time (h)
Solvents
Yield (%)
1
2
3
4
5
6
KIO4 (1)
H2O2 (5)
—
Acetic acid (5)
InBr3 (10%)
0
7
Methanol
DCE
DCM
0
0
12
30
2
0
H2O2 (10)
H2O2 (1.5)
H2O2 (2)
19
10
20
4
0
(NH4)6Mo7O24 (2%) and [bmim]PF6 (20)
Lactic acid (20%) and [bmim]PF6 (20)
—
Ethanol
DCM
DCM
0
16
2
0
17.4
mCPBA (1.1)
Table 2. The yield of 9a under different reaction conditions
Table 4. Oxidation of oxadiazole sulfides to the corresponding
products
Entry
mCPBA
(mmol)
Reaction
temperatures (ꢁC)
Reaction
times (h)
Yield
(%)
R
Product
Yield (%) (mp ꢁC)
1
2
1.0
1.0
1.0
1.0
1.1
1.1
1.1
1.1
1.1
1.2
1.5
ꢀ10
0
0.5
0.5
0.5
0.5
0.5
1
0
8.7
10a
42.0 (126–128)
3
4
12.4
10.9
24.6
31.5
38.4
38.0
43.7
36.6
27.0
4
20
4
OCH3
F
5
10b
10c
47.30 (103–105)
51.0 (121–123)
6
4
7
4
2
8
4
3
9a
10a
11a
4
2 + 5
2 + 5
2 + 5
4
4
a Reaction was conducted at 4 ꢁC for 2 h, and then at room tempera-
ture for 5 h.
10d
46.0 (128–130)
Cl
10e
9f
48.5 (147–149)
45.3 (99–101)
42.4 (154–156)
Table 3. Oxidation of thiadiazole sulfides to the corresponding
product
N
Cl
O
R
Product
Yield (%) (mp ꢁC)
CH2COCH2CH3
9a
43.7 (106–108)
10g
F
O
9b
44.9 (124–126)
CH2COCH2CH3
10h
39.8 (134–136)
NO2
OCH3
F
H3CO
9c
9d
9e
42.1 (168–170)
50.4 (123–125)
50.4 (116–118)
heterocyclic sulfides with 1,3,4-oxadiazole or 1,3,4-thia-
diazole moiety were converted by this procedure into
their corresponding sulfoxides in moderate yields.
2.2. Antifungal activity and structure–activity relationship
Three fungi, for example, Gibberella zeae, Fusarium oxys-
porium, and Cytospora mandshurica were employed in the
fungicidal bioassay using mycelial growth rate method.16
The results of preliminary bioassays were compared with
that of a commercial agricultural fungicide, Hymexazol.
As indicated in Table 5, most of the synthesized com-
pounds showed certain antifungal activities against the
tested fungi. The data provided in Table 5 indicate that
introduction of 1,3,4-oxadiazole in sulfoxide might im-
prove their antifungal activities. The interesting thing is
the conversion of the 1,3,4-oxadiazole in sulfoxides 10a–
10h into 1,3,4-thiadiazole in sulfoxides 9a–9g weakened
their antifungal activities. Among these compounds con-
taining 1,3,4-oxadiazole moiety, 10a (R = benzyl) is much
more active against G. zeae, F. oxysporum, and C. mand-
shurica than the other ones 10b–10h (R = 3-methoxyben-
9f
50.4 (133–134)
51.5 (77–79)
F
9g
CH2CH2OCH2CH3
As could be observed from Tables 3 and 4 that the alkyl
sulfide is, however, more easily oxidized to the corre-
sponding sulfoxide as compared to the aralkyl sulfides.
The yield was low with p-fluorobenzyl sulfide due to
easy over oxidation into sulfone. In order to avoid side
product and over oxidation of the sulfide to sulfone, we
preferred to settle for a relatively lower yield of the
product. It should be noted, however, that due to their
differences in polarity, it is easy to remove sulfone from
sulfoxide. Thus, a wide range of aromatic, aliphatic, and

F. Liu et al. / Bioorg. Med. Chem. 16 (2008) 3632–3640
3635
Table 5. Inhibitiona effect of sulfoxides derivatives at 50 lg/mL against
phytopathogenic fungi
F. oxysporum, C. mandshurica, Rhizoctonia solani, Tha-
natephorus cucumeris, Phytophthora infestans, S. sclero-
tiorum, Botrytis cinerea, Colletotrichum gloeosporioides,
were 28.84 lg/mL, 58.48 lg/mL, 63.97 lg/mL, 44.67 lg/
mL, 23.44 lg/mL, 33.11 lg/mL, 21.63 lg/mL, 19.91 lg/
mL, 39.45 lg/mL, respectively (Table 6). Compound
10a had more potent antifungal activities against most
of the tested fungi, and showed a broad-spectrum
bioactivity.
Compound
Inhibition ratea (%)
G. zeae
F. oxysporum
C. mandshurica
Hymexazolb
9a
53.68 1.03
26.88 1.33
34.83 0.89
5.45 1.09
8.29 0.67
14.93 0.76
6.40 0.72
16.82 1.17
53.26 2.21
37.69 1.46
7.29 1.17
7.79 1.21
31.91 1.44
18.09 1.37
11.31 1.24
29.90 1.50
52.11 0.97
15.54 0.97
4.58 1.17c
3.31 0.85
8.65 1.02
11.70 0.87
2.54 0.77
8.14 0.87
52.28 1.46
30.50 1.94
ꢀ0.88 1.27
7.04 0.86
14.95 1.04
1.47 1.08
4.40 0.63
32.26 1.07
52.02 1.12
15.99 1.00
8.42 0.85
6.63 1.14
10.97 0.87
11.99 1.13
3.83 0.99
16.07 0.75
56.09 1.43
25.71 0.94
ꢀ0.31 0.79
ꢀ0.33 0.88
11.91 1.26
6.90 1.16
2.82 1.03
18.18 1.02
9b
9c
9d
9e
2.2.1. The morphology changes of hyphal. As shown in
Figure 1, we can find that the hyphal of the control
was slippy, vimineous, and branched normally. The
endosome of the cell distributed evenly. But after treat-
ment of S. sclerotiorum with compound 10a at the con-
centration of 100 lg/mL for 24 h, most of its hyphal
became distorted, malformed, and got entangled. In addi-
tion, the cell of the hypha swelled and some of its endo-
some condensed, leaked out, and formed blanks (Fig. 1).
9f
9g
10a
10b
10c
10d
10e
10f
10g
10h
a Average of three replicates.
2.2.2. Effect of compound 10a on sporule germination of
S. sclerotiorum. From Figure 2 we can observe that
when S. sclerotiorum was treated with 10a and a com-
mercial agricultural fungicide—Hymexazol, the sporule
germination ratio (%) showed declining tendency with
the increase of the concentration. When the concentra-
tion was lower than 40 lg/mL, the sporule germination
ratio with 10a treatment was found to be higher than
that treated by Hymexazol. On the other hand, when
the concentration was higher than 40 lg/mL, the sporule
germination ratio with 10a treatment was lower than
that of the Hymexazol. At 80 lg/mL concentration,
the ratio was 12.6% and showed a more rapid decreasing
tendency as compared to that observed with Hymexazol
treatment. When the concentration reached 100 lg/mL,
the sporule germination ratio of the treated was 5.4%,
39.3% lower than that obtained with Hymexazol treat-
ment (Fig. 2).
b The commercial agricultural fungicide, Hymexazol was used for the
comparison of activity.
c The values were estimated statistically by SPSS 11.5 software using a
personal computer.
zyl, 3-fluorobenzyl, 2-chlorobenzyl, (2-chloropyridine-5-
yl)methyl, 2-ethoxycarbonylmethyl, 2-fluoro-benzyl, 2-
methoxybenzyl). Compound 10a at 50 lg/mL inhibited
the growth of G. zeae, F. oxysporum, and C. mandshurica
at 53.26%, 52.28%, and 56.09%, respectively, which is
close to that of Hymexozole (53.68% against G. zeae,
52.11% against F. oxysporum, 59.02 % against C. mand-
shuric at 50 lg/mL).
Further bioassays disclosed that compound 10a showed
remarkable inhibitory effect on nine kinds of plant patho-
genic fungi. The EC50 of compound 10a on G. zeae,
Table 6. Toxicity of 10a on nine kinds of fungi
Compound
Fungi
Toxic regression equationa
EC50 (lg/mL)a
r
10a
G. zeae
Y = 1.81x + 2.36
Y = 1.59x + 2.19
Y = 0.98x + 3.23
Y = 1.65x + 2.28
Y = 2.43x + 1.67
Y = 1.43x + 2.82
Y = 2.75x + 1.33
Y = 1.67x + 2.83
Y = 1.61x + 2.43
28.84 5.6
58.48 7.1
63.97 4.9
44.67 5.9
23.44 7.7
33.11 3.5
21.63 4.3
19.91 3.3
39.45 5.1
0.991
0.989
0.996
0.966
0.983
0.965
0.978
0.991
0.987
F. oxysporum
C. mandshurica
R. solani
T. cucumeris
P. infestans
B. cinerea
S. sclerotiorum
C. gloeosporioides
Hymexazolb
G. zeae
Y = 1.92x + 2.27
Y = 1.05x + 3.46
Y = 0.87x + 3.84
Y = 2.73x + 1.33
Y = 0.95x + 3.48
Y = 1.60x + 2.76
Y = 3.48x + 2.57
Y = 3.55x + 2.63
Y = 0.91x + 3.91
26.4 7.1
29.1 7.6
21.4 9.3
52.1 6.1
40.4 9.7
25.1 9.3
5.12 4.5
4.63 3.9
15.8 8.2c
0.969
0.994
0.961
0.995
0.965
0.941
0.931
0.926
0.983
F. oxysporum
C. mandshurica
R. solani
T. cucumeris
P. infestans
S. sclerotiorum
B. cinerea
C. gloeosporioides
a Average of three replicates.
b The standard compound was used for the comparison of activity.
c The values were estimated statistically by SPSS 11.5 software using a personal computer.

3636
F. Liu et al. / Bioorg. Med. Chem. 16 (2008) 3632–3640
Figure 1. Microphotograph of the hyphal morphology of S. sclerotiorum treated with compound 10a (800·).
Figure 2. The effect of compound 10a on sporule germination of
S. sclerotiorum (solid lines) is compared with Hymexazol (dashed lines).
Figure 3. The effect of compound 10a on membrane permeability of
S. sclerotiorum is compared with the Control (dashed blue lines) and
Hymexazol (dashed red lines).
2.2.3. Membrane permeability of S. sclerotiorum. After
S. sclerotiorum was treated with compound 10a
(100 lg/mL), the relative permeability rate of the cell
membrane was always higher than that of the control
and the Hymexazol treatment. After being treated with
10a for 240 min, the relative permeability rate was found
to be 26.7% while the rate under identical conditions
with control and Hymexazol treated were 21.9% and
24.6%, respectively. With longer treatment time, the rel-
ative permeability of the control rose gradually, but the
one treated with compound 10a and Hymexazol did not
rise at the same rate. The results showed that when S.
sclerotiorum was incubated with compound 10a, the cell
membrane was destroyed quickly and the relative per-
meability of S. sclerotiorum treated with Hymexazol
was a little higher than that of the control. The results
may be attributed to the fact that compound 10a attacks
the cell membrane of S. sclerotiorum (Fig. 3).
Figure 4. Changes of mycelial reducing sugar content, 10a (solid lines),
Control (dashed blue lines), and Hymexazol (dashed red lines).
lower than that of the control and 8.8% higher than that
with the Hymexazol treated one (Fig. 4).
2.2.4. Changes of mycelial reducing sugar content. In the
beginning up to 3 h, mycelial reducing sugar content of
S. sclerotiorum for the treated one was higher than the
control, being 0.30 mg/mL for the control against
0.34 mg/mL for the treated which was 13.3% higher than
that of the control. After 3 h, although the content of
control continued to rise, but the one treated with 10a
reached its peak value after 12 h and then started to fall.
At the end of 24 h, the mycelial reducing sugar content
of the 10a treated was 0.37 mg/mL, which was 11.9%
2.2.5. Changes of mycelial chitosan content. From Figure
5, we can find that when S. sclerotiorum was treated with
compound 10a, the mycelial chitosan content, namely
the content of D-GlcNAc in the mycelial cell, started
to fall at the beginning before showing a rising tendency
that continued till 12 h, and then began to decline again.
On the other hand, the one treated with control showed
an increasing tendency throughout and always main-
tained a lead compared to the one treated with 10a ex-

F. Liu et al. / Bioorg. Med. Chem. 16 (2008) 3632–3640
3637
Figure 5. Changes of mycelial D-GlcNAc content, 10a (solid lines),
Control (dashed blue lines), and Hymexazol (dashed red lines).
Figure 8. Changes of mycelial pyruvate content, 10a (solid lines),
Control (dashed blue lines), and Hymexazol (dashed red lines).
cept at the end of 1 h. The mycelial chitosan content was
0.12 mg/mL after being cultivated with compound 10a
at the end of 6 h, which was 7.7% lower than that of
the control (the control was 0.13 mg/mL). After treat-
ment with compound 10a after 24 h, the D-GlcNAc con-
tent was 0.12 mg/mL, which was 14.3% and 7.7% lower
than that of the control and the Hymexazol, respectively
(the control was 0.14 mg/mL, the Hymexazol was
0.13 mg/mL) (Fig. 5).
2.2.7. Changes of mycelial soluble protein content. As we
can see from Figure 7, when S. sclerotiorum was treated
with compound 10a for 0.5 h, the mycelial soluble pro-
tein content was 0.72 mg/mL, 43% higher than that of
the control (0.50 mg/mL). After 6 h of inoculation, the
mycelial soluble protein content for the control was al-
ways higher but similar as compared to the one treated
with Hymexazol. Although the compound treated one
showed an irregular pattern up to 12 h, the mycelial sol-
uble protein content revealed a gradual but uniform
decreasing tendency thereafter. At the end of 24 h, the
mycelial soluble protein content of the treated one was
0.69 mg/mL, for the control the value was 0.83 mg/
mL, and the one with commercial fungicide Hymexazol
it was 0.91 mg/mL. So the results seem to indicate that
compound 10a can inhibit the synthesis of protein while
the acting site of Hymexazol may not be necessarily the
soluble protein (Fig. 7).
2.2.6. Changes of mycelial chitinase activity. When trea-
ted with compound 10a, the mycelial chitinase activity
of S. sclerotiorum showed a fluctuating tendency. After
being cultured for 12 h, the mycelial chitinase activity
of S. sclerotiorum began to fall. Both control and the
treated one showed similar behavioral pattern up to
6 h and reached their respective lowest values at the
end of this period, the control began to rise constantly
thereafter. After being inoculated for 24 h, the D-Glc-
NAc content that was catalyzed by chitinase without
treatment was found to be 0.129 mg/mL. The corre-
sponding values for the one treated with compound
10a and Hymexazol were 0.125 mg/mL and 0.124 mg/
mL, respectively (Fig. 6).
2.2.8. Changes of mycelial pyruvate content. As has been
shown in Figure 8, the mycelial pyruvate content of
S. sclerotiorum treated with compound 10a was lower
than that of the control throughout the experiment.
With Hymexazol treated one, the changes were irregular
up to 12 h before showing a uniform decreasing pattern.
After being cultivated for 24 h, the mycelial pyruvate
content of S. sclerotiorum treated with 10a was found
to be 0.19 mg/mL, which was 13.6% lower than the con-
trol and 9.52% lower than with the Hymexazol treat-
ment, respectively. These data indicate that compound
10a can inhibit the activity of lactate dehydrogenase
(LDH) thereby inducing the decrease of the pyruvate
content (Fig. 8).
Figure 6. Changes of mycelial chitinase activity, 10a (solid lines),
Control (dashed blue lines), and Hymexazol (dashed red lines).
3. Conclusion
In summary, the present method of the formation of sulf-
oxide derivatives by mCPBA oxidation in dichlorometh-
ane offers several advantages such as fast reaction rate
and moderate yield. It was also found that title compound
10a displayed good antifungal activity and had a wide
spectrum of bioactivity. Using the mycelial growth rate
method in the laboratory, the mechanisms of action of
10a against S. sclerotiorum in vitro were studied. The re-
sults showed that 10a had high inhibitory effect on the
growth of most of the fungi with the EC50 values ranging
from 13.9 to 63.9 lg/mL. After S. sclerotiorum was trea-
ted with compound 10a at 100 lg/mL, the permeability
Figure 7. Changes of mycelial soluble protein content, 10a (solid lines),
Control (dashed blue lines), and Hymexazol (dashed red lines).

3638
F. Liu et al. / Bioorg. Med. Chem. 16 (2008) 3632–3640
of the cell membrane rose and most of its hyphal became
distorted, malformed, and got entangled. The cell of the
hypha swelled and its endosome condensed, malformed,
leaked out, and formed blanks. From these results, it
can be concluded that the cell membrane and the mor-
phology of S. sclerotiorum may be destroyed and changed
by compound 10a. After treatment with compound 10a at
100 lg/mL for 12 h, the soluble protein content began to
fall and perhaps the activity of proteolytic enzymes
started to decrease. But these were not obvious when S.
sclerotiorum was treated with Hymexazol, perhaps due
to the fact that the main site of attack by Hymexazol
was not the same. In addition, the mycelial reducing sugar
and D-GlcNAc content also showed decreasing trends
indicating that the production of energy probably could
be inhibited by compound 10a. At the same time, the
mycelial pyruvate content and chitinase activity showed
decreasing tendency that may be attributed to suppres-
sion of lactate dehydrogenase and chitinase activities by
compound 10a. The fact that only 5.4% of its spore bour-
geoned, could possibly explain the results of high inhibi-
tion rate (%) while using compound 10a against nine
kinds of fungi (Table 6). Although, some preliminary
studies were conducted, the precise way in which com-
pound 10a efficiently inhibits the growth of the hypha still
remains to be ascertained. So, further investigation re-
quires to be done in the future research.
compound 5. The structure was confirmed by 1H
NMR, 13C NMR, IR, and elemental analysis (see Sup-
porting Information).
4.1.2. Preparation of 2-substituted methylthio-5-(3,4,5-
trimethoxyphenyl)-1,3,4-thiadiazole (7a–7g). To a 50 mL,
three-necked, round-bottomed flask equipped with a
magnetic stirrer were added 1.5 mmol of 4, 20 mL of dis-
tilled water, and 2 mL (3%, w/w) of NaOH solution. The
mixture was stirred at room temperature for 10 min.
Then 1.5 mmol of halide 6 and 17.2 mg (0.15 mmol) of
indium were added. The resulting mixture was stirred
at room temperature for 4 h, and then filtered. The white
solid resulted was washed with 5% Na2CO3 solution and
distilled water, dried under vacuum, and recrystallized
from ethanol to give compound 7 (for 7g, after the com-
pletion of the reaction, extracted with CH2Cl2 and dried
over MgSO4. The solvent was removed and the crude
product was purified by chromatography (petroleum/
ether, 2/1)) (see Supporting Information).
4.1.3. Preparation of 2-substituted methylthio-5-(3,4,5-
trimethoxyphenyl)-1,3,4-oxadiazole (8a–8h). A 50 mL
round-bottomed flask equipped with a magnetic stirrer
was charged with 5 (1.5 mmol) and 2 mL (3%, w/w) of
sodium hydroxide solution. The mixture was dissolved
in 20 mL of distilled water. The flask was stirred at room
temperature for 10 min, and then halide 6 (1.5 mmol)
and indium tribromide (0.15 mmol) were added to the
reaction mixture and stirred at room temperature for
4 h. The mixture was filtered and the white solid ob-
tained was washed with 5% Na2CO3 solution and dis-
tilled water, dried under vacuum, and recrystallized
from ethanol to give compound 8 (see Supporting
Information).
4. Experimental
4.1. Analysis and instruments
The melting points of the products were determined on a
XT-4 binocular microscope (Beijing Tech Instrument
Co., China) and were not corrected. The IR spectra were
recorded on a Bruker VECTOR22 spectrometer in KBr
4.1.4. Preparation of 2-substituted sulfinyl-5-(3,4,5-trime-
thoxyphenyl)-1,3,4-thiadiazole (oxadiazole) (9a–9g, 10a–
10h). To a three-necked round-bottomed flask equipped
1
disks. H and 13C NMR (solvent CDCl3) spectra were
recorded on a JEOL-ECX 500 NMR spectrometer at
room temperature using TMS as an internal standard.
Elemental analysis was performed on an Elementar Var-
io-III CHN analyzer. The reagents were all of analytical
grade or chemically pure. Analytical TLC was per-
formed on silica gel GF254. Column chromatographic
purification was carried out using silica gel. 3,4,5-Tri-
with
a
magnetic stirrer and containing Sulfide
(1.0 mmol) and dichloromethane (10 mL) was added
dropwise 1.6 mmol mCPBA dissolved in 10 mL dichlo-
romethane over a period of 15 min. The reaction mix-
ture was first stirred at 0 ꢁC for 2 h, and then stirring
was continued for another 5 h at room temperature.
The mixture was washed with 0.25 mol/L Na2HPO4
solution (3· 20 mL), dried over MgSO4. The solvent
was removed and the crude product was purified by col-
umn chromatography (petroleum/ether, 1:1) (see Sup-
porting Information).
methoxyphenylhydrides
phenyl)-1,3,4-thiadiazole-2-thiol
3
and 5-(3,4,5-trimethoxy-
were prepared
4
according to the literature method as described.12 The
optical density (OD) was determined on WFZUV-2100
UV–Vis Spectrophotometer. The conductivity was done
by DDS-11A Conductivity Meter. The microphoto-
graph of the hyphal morphology was taken from a
OLYMOPUS Microscope.
4.2. Antifungal assay
The antifungal activity of all synthesized compounds were
tested against three pathogenic fungi, namely G. zeae,
F. oxysporum, C. mandshurica by the poison plate
technique.16
4.1.1. Preparation of 5-(3,4,5-trimethoxyphenyl)-1,3,4-
oxadiazole-2-thiol (5). A mixture of 0.56 g (10 mmol) of
potassium hydroxide, 2.26 g (10 mmol) of compound 3,
and 1.14 g (15 mmol) of carbon disulfide in 50 mL of
absolute ethanol was refluxed for 8 h. After the solvent
was evaporated in vacuum, the residue was dissolved
in ice-cold water and acidified with dilute hydrochloric
acid. The precipitate was filtered off, washed with water,
dried, and recrystallized from absolute ethanol to give
Compounds were dissolved in 1 mL acetone before mix-
ing with 90 mL potato dextrose agar (PDA). The final
concentration of the compounds in the medium was
tested at 50 lg/mL. All kinds of fungi were incubated
in PDA at 27 1 ꢁC for 4 days to get new mycelium

F. Liu et al. / Bioorg. Med. Chem. 16 (2008) 3632–3640
3639
for antifungal assay. Then mycelia dishes of approxi-
mately 4 mm diameter were cut from culture medium
and one of them was picked up with a sterilized inocula-
tion needle and inoculated in the center of PDA plate
aseptically. The inoculated plates were incubated at
27 1 ꢁC for 5 days. Acetone in sterile distilled water
served as control, while Hymexazole severed as positive
control. For each treatment, three replicates were con-
ducted. The radial growth of the fungal colonies was mea-
sured and the data were statistically analyzed. The
inhibiting effects of the test compounds in vitro on these
fungi were calculated by the formula: I(%) = [(C ꢀ T)/
(C ꢀ 0.4)] · 100, where C represents the diameter of fungi
growth on untreated PDA, and T represents the diameter
of fungi on treated PDA while I means inhibition rate.
centration was 100 lg/mL. Then mycelium was filtered,
collected, and washed orderly at 0.5, 1, 3, 6, 12, and
24 h. After soaking the water with filter paper, the dried
mycelium was weighed and then preserved at ꢀ20 ꢁC.
The dry mycelium (1.0 g) in cold mortar was mixed with
Tris–HCl buffer (2.5 mL, 0.05 mol/L, pH 7.5), triturated
into paste quickly, and then centrifuged at 4 ꢁC, 15,000g
for 30 min. The clean upper layer was preserved at
ꢀ20 ꢁC. Every treatment had three repetitions.
4.2.4. Effect of 10a on the relative permeability rate of cell
membrane.20 Collected the mycelial of S. sclerotiorum
which incubated in a few days and washed with double
distilled water. 1 g of the mycelial was placed in 15 mL
centrifuge tubes containing compound 10a, at 100 lg/
mL concentration. Then measured the conductivity at
0 (J0), 5, 10, 30, 60, 180, 360 min (J1), gradually. After
boiling which was followed by cooling, the conductivity
Compound 10a was tested against nine pathogenic fungi
namely G. zeae, F. oxysporum, C. mandshurica, P. infe-
stans, R. solani, T. cucumeris, C. gloeosporioides, B. cine-
rea, S. sclerotiorum at different concentrations of 100,
50, 25, 12.5, and 6.25 lg/mL.The EC50 values were esti-
mated statistically by Probit analysis with the help of
Probit package of SPSS 11.5 software using a personal
computer. The average EC50 (lg/mL) was taken (effec-
tive dose for 50% inhibition lg mLꢀ1) from at least three
separate analyses for inhibition of growth using the Ba-
sic LD50 program version 1.1.16
(J2) was measured. In the end, calculated the permeabil-
J1ꢀJ0
ity by the formula: P% ¼
ꢁ 100%.
J2ꢀJ0
4.2.5. Detection of mycelial reducing sugar content.21 The
200 lL of upper clear layer of mycelial extract was
mixed with 3,5-dinitrosalicylic acid (DNS, 400 lL),
and then the mixture was boiled for 5 min. It was then
quickly cooled to room temperature and diluted to a
volume of 2.5 mL using distilled water. Absorbance va-
lue of the mixture measured at 540 nm was converted
into the value of glucose content (mg) by the standard
curve of glucose. Tris–HCl buffer served as the control.
4.2.1. The hyphal morphology observation of S. sclerotio-
rum. Compound 10a was added in sterilized Czapek med-
ia (0.2% NaNO3, 0.131% K2HPO4Æ3H2O, 0.05% KCl,
0.05% MgSO4Æ7H2O, 0.00183% FeSO4Æ7H2O, 3% su-
crose, pH 6.8)17 which had incubated S. sclerotiorum for
a few days, and the final concentration was 100 lg/mL.
Then incubated together at 27 ꢁC. After 24 h, observed
under microscope. Acetone (1.0 mL) served as the
control.
4.2.6. Detection of mycelial chitosan content.22 The upper
clear layer (200 lL) of mycelial extract was mixed with
K2B4O7 solution (100 lL), and then the mixture was
boiled for 3 min. After quickly cooling to room temper-
ature, the mixture was added into 3 mL of 1% 3,20-di-
methyl-4-aminobiphenyl (DMAB). Subsequently, it
was incubated for 20 min at 36 ꢁC, and then cooled
down to room temperature again. The absorbance value
of the mixture was measured immediately at 544 nm.
Tris–HCl buffer served as the control.
4.2.2. Effect of compound 10a on sporule germination of
S. sclerotiorum.18 Compound 10a was mixed with PDA
medium at the concentrations of 0 (Control), 20, 40, 60,
80, and 100 lg/mL, for each treatment, three replicates
were conducted. Poison media plates were prepared using
9 cm Petri dishes, 0.5 mL sporule in suspension was
spread at poison media plates. This was followed by incu-
bation at 27 ꢁC and then after 24 h, observed at every 2 h.
When the germination of the control bourgeoned over
90%, investigated the germination of every concentration,
and observed three eyeshots per Petri dish. The germina-
tion rate was calculated using the following formula:
4.2.7. Detection of mycelial chitinase activity.23,24 The
mixture of the upper clear layer (400 lL) with chitin col-
loid (200 lL) was dripped into clear centrifugal tube.
The tube was incubated for 1 h (37 ꢁC), and then boiled
for 5 min. After being centrifuged at 5000 rpm for
10 min, the mixture (400 lL) mixed with K2B4O7 solu-
tion (200 lL, 0.8 M) was boiled, and then cooled down
quickly. This was followed by the addition of 3 mL of
1% DMAB. The mixture was kept at 36 ꢁC for 20 min,
and then cooled down to room temperature. The absor-
bance value of the mixture was measured immediately at
544 nm. The control was boiled before incubating.
Germination rate ð%GRÞ ¼ 100 ꢁ G=S
where GR represents the germination rate, G represents
the number of germinated sporule and S represents the
number of all observed sporule.
4.2.3. Preparation of the crude extract of mycelium
(S. sclerotiorum).19 The six mycelial discs (4 mm diame-
ter) taken from a starting colony growing on PDA were
placed in an Erlenmeyer flask containing 90 mL of ster-
ilized Czapek. Then these were incubated in a whirly
shaker (140 rpm, 27 ꢁC). After 15 days, compound 10a
was dripped into the culture media and the tested con-
4.2.8. Detection of mycelial soluble protein content.21 The
Coomassie brilliant blue G-250 dye-binding technique
was adopted to observe the changes of protein content.
The upper clear layer of mycelial extract (100 lL) was
mixed with coomassie brilliant blue G-250 solution
(3 mL), and then the mixture was kept still for 5 min.

3640
F. Liu et al. / Bioorg. Med. Chem. 16 (2008) 3632–3640
3. Gadepalli, R. S. V. S.; Rimoldi, J. M.; Fronczek, F. R.;
Nillos, M.; Gan, J.; Deng, X.; Rodriguezfuentes, G.;
Schlenk, D. Chem. Res. Toxicol. 2007, 20, 257.
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The absorbance value of the mixture was measured at
595 nm. Tris–HCl buffer served as the control.
4.2.9. Detection of mycelial pyruvate content.25 The upper
clear layer (100 lL) of mycelial extract was mixed with
2,4-dinitrophenylhydrazine (500 lL, 2.5 mM), and then
the mixture was swayed evenly and incubated for
10 min (37 ꢁC), this was followed by the addition of
NaOH (5000 lL, 0.4 M), then swayed uniformly. The
absorbance value of the mixture measured at 520 nm
was converted into the value of pyruvate content (mg)
by the standard curve of pyruvate. Tris–HCl buffer served
as the control.
10. Caboni, P.; Sammelson, E.; Casida, J. E. J. Agric. Food
Chem. 2003, 51, 7055.
4.3. Statistical analysis
11. Chen, C.-J.; Song, B.-A.; Yang, S.; Xu, G.-F.; Bhadury, P.
S.; Jin, L.-H.; Hu, D.-Y.; Li, Q.-Z.; Liu, F.; Xue, W.; Lu,
P.; Chen, Z. Bioorg. Med. Chem. 2007, 15, 3981.
12. Song, B.-A.; Chen, C.-J.; Yang, S.; Jin, L.-H.; Xue, W.;
Zhang, S.-M.; Zou, Z.-H.; Hu, D.-Y. Acta Chim. Sinica
2005, 63, 1720.
All the statistical analyses were done under SPSS 11.5
using personal computer. Each experiment had three
replicates and all experiments were run three times with
similar results. Measurements from all the replicates
were combined and treatment effects analyzed.
13. Chich, D.; Neelamkavil, S. Tetrahedron 2002, 58, 3865.
14. Liu, Z.-M.; Yang, G.-F.; Qing, X.-H. J. Chem. Technol.
Biotechnol. 2001, 76, 1154.
Acknowledgments
15. Yang, G.-F.; Liu, Z.-M.; Qing, X.-H. Chin. Chem. Lett.
2001, 12, 877.
16. Tarun, K. C.; Prem, D. J. Agric. Food Chem. 2006, 54,
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18. Overy, D. P.; Blunt, J. W. J. Nat. Prod. 2004, 67,
1850.
We are grateful for the financial support from the Na-
tional Key Project for Basic Research (2003CB114404),
Key Technologies R&D Program (2006BAE01A01-15)
and the National Natural Science Foundation of China
(20562003).
19. Tao, W.; Svetlana, Z.; Ann, F. D.; William, S. C.; Carl, E.
S. J. Agric. Food Chem. 2005, 53, 3888.
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Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.
2008.02.006.
22. Shi, Z.-Q.; Shen, S.-G.; Xu, L.-L.; Fan, Y.-J. Chin. J. Pest.
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