Synthesis and antimicrobical evaluation of a novel class of 1,3,4-thiadiazole: Derivatives bearing 1,2,4-triazolo[1,5-a]pyrimidine moiety

Article abstract of DOI:10.1016/j.ejmech.2013.04.014

A series of novel 1,3,4-thiadiazole derivatives bearing 1,2,4-triazolo[1,5-a]pyrimidine moiety were synthesized by the method of splicing active substructures. Among these derivatives, compounds 12, 13, 15-22 and 24-31 were firstly reported. All the compounds were assayed for antimicrobial activities against five fungi strains and four bacteria strains. The preliminary results indicated that compounds 25 and 28-31 showed good antifungal activities against Physaclospora piricola and Rhizoctonia solani. Compound 26 exhibited good antifungal activities against Cercospora beticola and R. solani. Most of the compounds showed better antibacterial activities against Gram-negative bacteria strains than Gram-positive bacteria strains. Compounds 25 and 28 showed the best activities against Pseudomonas fluorescence while compounds 30-31 showed good activities against Escherichia coli.

Full text of DOI:10.1016/j.ejmech.2013.04.014

European Journal of Medicinal Chemistry 64 (2013) 54e61
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antimicrobical evaluation of a novel class
of 1,3,4-thiadiazole: Derivatives bearing 1,2,4-triazolo[1,5-a]
pyrimidine moiety
,
,
,
,
c
,
b
Yin Luo a 1, Shuai Zhang a 1, Zhi-Jun Liu a, Wu Chen a b, Jie Fu a , Qing-Fu Zeng ,
*
Hai-Liang Zhu a
,
*
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of China
b Engineering Research Center for Clean Production of Textile Dyeing and Printing, Ministry of Education, Wuhan 430073, People’s Republic of China
c State Key Laboratory of Pollution Control & Resource Reuse, Nanjing University, Nanjing 210046, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 1,3,4-thiadiazole derivatives bearing 1,2,4-triazolo[1,5-a]pyrimidine moiety were syn-
thesized by the method of splicing active substructures. Among these derivatives, compounds 12,13,15e22
and 24e31 were firstly reported. All the compounds were assayed for antimicrobial activities against five
fungi strains and four bacteria strains. The preliminary results indicated that compounds 25 and 28e31
showed good antifungal activities against Physaclospora piricola and Rhizoctonia solani. Compound 26
exhibited good antifungal activities against Cercospora beticola and R. solani. Most of the compounds
showed better antibacterial activities against Gram-negative bacteria strains than Gram-positive bacteria
strains. Compounds 25 and 28 showed the best activities against Pseudomonas fluorescence while
compounds 30e31 showed good activities against Escherichia coli.
Received 28 December 2012
Received in revised form
3 April 2013
Accepted 3 April 2013
Available online 15 April 2013
Keywords:
1,3,4-Thiadiazole
Antifungal
Ó 2013 Elsevier Masson SAS. All rights reserved.
Antibacterial
1. Introduction
medicines because of their strong biological activities [12e14]. Some
derivatives were synthesized as potential anti-infective, anti-inflam-
Recently, some publications reported the multidrug-resistant
(MDR) bacteria and fungi drastically impaired the efficacy of anti-
microbial agents and limited their clinical uses [1e4]. The emergence
of Gram-negative Enterobacteriaceae with resistance to carbapenem
matory and antiproliferative agents [12,13]. Many derivatives also had
antifungal activity [14]. Besides, some derivatives combining both
1,3,4-thiadiazole and 1,2,4-triazole moieties had been reported to
have various biological activities such as anticancer, antitubercular,
anti-inflammatory, analgesic or anticonvulsant activities [15e20],
especially antimicrobial activity [21e23].
conferred by New Delhi metallo-b-lactamase 1 (NDM-1) had drawn
global attention [5]. Due to the reason, the exploration of novel
antibacterial agents or the improvement of previous reported drugs
is crucial to overcome MDR infections [6].
In our previous study, we had introduced 1,2,4-triazole
moiety into 1,2,4-triazolo[1,5-a]pyrimidine moiety and got novel
lead compounds with fairly good antifungal activity [24]. In this
paper, in order to study the biological activities of diheterocyclic
compounds containing 1,2,4-triazolo[1,5-a]pyrimidine and 1,3,4-
thiadiazole moieties and to find potential antimicrobial activities,
we have connected these two moieties using thioether bond by the
method of splicing active substructures. Thus, a series of novel 1,3,4-
thiadiazole derivatives bearing 1,2,4-triazolo[1,5-a]pyrimidine moi-
ety were synthesized. Their antibacterial activities against Escher-
ichia coli, Pseudomonas fluorescence, Bacillus subtilis and Staphy
lococcus aureus, and antifungal activities against Fusarium oxysporum
f.sp. vasinfectum, Gibberella sanbinetti, Cercospora beticola Sacc,
Physaclospora piricola and Rhizoctonia solani were evaluated. The
results of this study might be useful to researchers attempting to find
new potential antimicrobials.
Synthesis and biological activities of novel heterocyclic com-
pounds bearing triazolopyrimidine moiety have been studied widely
for many years [7e11]. Some were demonstrated as A2a and A3 re-
ceptor ligands [7,8]. Some were potent and selective dihydroorotate
dehydrogenase inhibitors with antimalarial activity [9]. Many aceto-
lactate synthase-inhibiting herbicides, such as florasulam, were tri-
azolopyrimidine-2-sulfonamide derivatives while some derivatives
exhibited antifungal activity [10,11]. On the other hand, thiadiazole
derivatives were also widely applied in the fields of pesticides and
* Corresponding authors. State Key Laboratory of Pharmaceutical Biotechnology,
Nanjing University, Nanjing 210093, People’s Republic of China. Tel./fax: þ86 25
83592572.
E-mail addresses: jiefu@nju.edu.cn (J. Fu), zhuhl@nju.edu.cn (H.-L. Zhu).
These authors contributed equally to the paper.
1
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.04.014

Y. Luo et al. / European Journal of Medicinal Chemistry 64 (2013) 54e61
55
2. Results and discussion
fungicides triadimefon, validamycin and carbendazim were
selected as positive controls. The results were summarized in
Table 2.
2.1. Chemistry
The results showed that most of the synthesized compounds
exhibited good antifungal activities except 15 and 23, however, all
the compounds showed different activities against different fungi
strains. For example, most compounds showed very good inhibi-
tory activities against P. piricola and activities of 11e14, 16, 18e21,
24, 25 and 28e31 could be comparable with Grade B (inhibition
rate ¼ 70%e89%). Many compounds showed good antifungal ac-
tivities against R. solani, such as 25e31, however, only several
compounds (17, 22 and 26) showed good inhibitory activities
against C. beticola. The inhibitory activities of these compounds
against F. oxysporum and G. sanbinetti were rather poor and only
the activity of 24 against G. sanbinetti was comparable with Grade
B. On the other hand, many compounds, whose activities were
comparable with Grade B, showed good inhibitory activities on
two fungi strains. 25 and 28e31 showed good activities against
P. piricola and R. solani while 26 showed good activities against
C. beticola and R. solani. The compounds, whose activities could be
comparable with Grade B, were more potent than that of positive
controls.
As previously reported, 1,2,4-triazolo[1,5-a]pyrimidine de-
rivatives exhibited many biological activities [7e11]. The tri-
azolopyrimidine structure including the S atom, which is a large
delocalized electron-rich system, might play an important role in
the various activities. The biological activities were possibly related
to the spatial orientation and electron donating capacity of the
triazolopyrimidine [25]. On the other hand, thiadiazole derivatives
had strong antifungal activity. The antifungal effect was mainly
caused by an activation of phospholipases in the mitochondrial
This series of novel 1,3,4-thiadiazole derivatives bearing 1,2,4-
triazolo[1,5-a]pyrimidine moiety were synthesized according to
the synthetic route outlined in Scheme 1.
The precursor, 2-(5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-
2-ylthio)acetohydrazide, compound 6, was prepared via five steps
which began with hydrazine hydrate, according to the reported
method [24,25]. The synthesized 6 was then dissolved in ethanol
and reacted with carbon disulfide (CS2) at the presence of potas-
sium hydroxide (KOH) to afford compound 7. After that compound
10, was obtained from 7 via three steps as white powder.
Target compounds 11e31, 1,3,4-thiadiazole derivatives bearing
1,2,4-triazolo[1,5-a]pyrimidine moiety, were synthesized by nucleo
philic substitution reaction of compound 10 and halogen com-
pounds as shown in Scheme 1. Among these compounds,12,13,15e
22 and 24e31 were firstly reported.
The structure elucidations of the newly synthesized compounds
were carried out by different spectroscopic techniques like 1H NMR,
13C NMR and EI-MS. Further confirmations of the compounds were
carried out by elemental analysis (ꢀ0.4%). The elemental analysis
data and some physical properties of these compounds were re-
ported in Table 1.
2.2. Antifungal activity
The antifungal activities of compounds 11e31 against Fusarium
vasinfectum, G. sanbinetti, C. beticola, P. piricola and R. solani were
evaluated by our previous method [24]. Three commercial
H
N
SH
S
S
H2N
NH2NH2 . H2O+H2SO4
refluxed, 100-103oC, 3h
CH3
NaOH, refluxed, 3h
NH4SCN
1
NH2CNHNHCNH2
2 (69.4%)
N
HCl, pH=1
N
3(83.6%)
CH3
CH3COCH2COCH3
piperidine, acetic acid
ethyl bromoacetate
methanol
N
N
N
N
SH
N
refluxed, 20h
stirred, r.t., 2h
SCH2COOC2H5
H3C
N
N
H3C
N
4 (87.6%)
5(83.0%)
CH3
CH3
NH2NH2 . H2O
refluxed, 6h
N
O
S
KOH/ethanol+CS2
stirred, r.t., >24h
H3C
N
N
N
N
SCH2CONHNH2
SCH2CNHNHCS- K+
N
H3C
N
N
6(74.0%)
7(94.3%)
CH3
SH
S- Na+
S
CH3
S
H2SO4
N
NaOH
H2SO4
N
N
N
N
stirred, 0 oC, 24h
N
S
N
N
S
N
H3C
N
N
H3C
N
8
9
CH3
SH
CH3
S
S
S
R
NaOH, H2O
N
HCl
N
N
N
N
N
N
N
S
R-X, methanol/water (DMF/water)
stirred, r.t., >18h
N
S
N
H3C
N
H3C
N
10 (47.0%)
11-31
Scheme 1. Synthesis of target compounds 11e31. If ReX was liquid under room temperature, methanol/water was used as mixed solvent; if ReX was solid, DMF/water was used.

56
Y. Luo et al. / European Journal of Medicinal Chemistry 64 (2013) 54e61
Table 1
Characterization data of compounds 11e31.
Compd
R
Molecular formula
Molecular mass
M.p. (ꢁC)
Yield (%)
Elemental analysis found (calc.)
C
H
N
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
2-CH3eC6H4CH2e
3-CH3eC6H4CH2e
4-CH3eC6H4CH2e
2-CleC6H4CH2e
3-CleC6H4CH2e
2-BreC6H4CH2e
3-BreC6H4CH2e
4-BreC6H4CH2e
2-FeC6H4CH2e
3-FeC6H4CH2e
C18H18N6S3
C18H18N6S3
C18H18N6S3
414.57
414.57
414.57
434.99
434.99
479.44
479.44
479.44
418.53
418.53
418.53
445.54
430.57
426.58
459.57
459.57
436.52
436.52
436.52
436.52
497.43
93e96
67.6
67.3
60.1
77.3
80.1
74.6
88.1
84.7
82.5
88.3
81.3
92.2
66.5
71.9
66.9
45
52.12(52.15)
52.13(52.15)
52.19(52.15)
46.95(46.94)
46.95(46.94)
42.55(42.59)
42.57(42.59)
42.58(42.61)
48.73(48.78)
48.76(48.78)
48.80(48.78)
45.81(45.83)
50.22(50.21)
53.55(53.50)
47.00(47.04)
47.06(47.04)
46.73(46.77)
46.71(46.77)
46.73(46.77)
46.80(46.77)
41.07(41.05)
4.36(4.38)
4.35(4.38)
4.37(4.38)
3.49(3.48)
3.46(3.48)
3.12(3.15)
3.18(3.15)
3.15(3.17)
3.57(3.61)
3.63(3.61)
3.60(3.61)
3.34(3.39)
4.19(4.21)
4.29(4.25)
3.70(3.73)
3.71(3.73)
3.27(3.23)
3.27(3.23)
3.22(3.23)
3.27(3.23)
2.79(2.84)
20.23(20.27)
20.25(20.27)
20.28(20.27)
19.36(19.32)
19.36(19.32)
17.50(17.53)
17.56(17.53)
17.50(17.55)
20.12(20.08)
20.04(20.08)
20.13(20.08)
22.07(22.01)
19.61(19.52)
19.73(19.70)
21.36(21.33)
21.37(21.33)
19.23(19.25)
19.26(19.25)
19.30(19.25)
19.29(19.25)
16.85(16.89)
138e140
131e133
165e166
139e140
171e173
152e154
160e162
133e135
164e166
140e142
156e158
103e106
112e113
153e154
152e155
196e198
184e187
131e133
103e104
104e106
C17H15ClN6S3
C17H15ClN6S3
C17H15BrN6S3
C17H15BrN6S3
C17H15BrN6S3
C17H15FN6S3
C17H15FN6S3
C17H15FN6S3
C17H15N7O2S3
C18H18N6OS3
C19H18N6S3
C18H17N7O2S3
C18H17N7O2S3
C17H14F2N6S3
C17H14F2N6S3
C17H14F2N6S3
C17H14F2N6S3
C17H14BrFN6S3
4-FeC6H4CH2e
3-NO2eC6H4CH2e
3-OCH3-C6H4CH2e
4-CH2]CHeC6H4CH2e
2-NO2e4-CH3eC6H3CH2e
3-NO2e4-CH3eC6H3CH2e
3,5-2FeC6H3CH2e
2,6-2FeC6H3CH2e
3,4-2FeC6H3CH2e
2,4-2FeC6H3CH2e
2-Fe4-BreC6H3CH2e
88.6
80.8
82.6
76.7
65.1
membranes. The formation of lipid peroxide of membranes could
be the primary toxic effect [26].
steric hindrance might play an important role. However, the com-
pounds with substituents at position 3 of benzene ring, such as 12,
15, 17, 20, 22 and 23, showed worse activity than others, indicating
that steric hindrance at position 3 could decrease the activity. The
substituents at position 5 and 6 were also disfavored. For example,
27 and 29 displayed worse activity than 28 and 30 respectively, so
the F atom at position 5 and 6 might decrease the activity.
From Table 2, we had also found that many compounds
exhibited good inhibitory activities against R. solani and benzene
ring with two substituents increased the antifungal activity. For
example, 25e31 with two substituents on benzene ring exhibited
better inhibitory activities than other compounds with only one
substituent. It indicated that the steric effect might play an
important role.
Table 2 showed that some compounds (17, 22 and 26) displayed
better inhibitory activities against C. beticola than positive controls.
It might be related to the Br atom or nitro (eNO2) group at position
3 of benzene ring. Most compounds displayed low inhibitory ac-
tivities against G. sanbinetti and F. oxysporum, however, 24 showed
good activity against G. sanbinetti (78%) comparable with carben-
dazim (75%) and triadimefon (81%). The substituents of benzyl
might play an important role.
The antifungal activities of all the compounds were quite
different, however, their structures were similar; thus the activities
were related to the influence of different substituents on the ben-
zene ring with different electrostatic and steric properties. For the
inhibitory activities against P. piricola, the target compounds
bearing electron-withdrawing groups at position 2 and 4 of the
benzene ring exhibited better activities. 14, 16, 18, 19, 21, 25, 30 and
31 showed better activity than the others. 30 showed the best ac-
tivity (86%) which was also much better than carbendazim (74%) so
the two electron-withdrawing F atoms at position 2 and 4 with low
Table 2
Antifungal activity of compounds 11e31.
Compd
Inhibition rate (%) (concentration: 50 ppm)
F. oxysporum G. sanbinetti C. beticola P. piricola R. solani
11
12
13
14
15
16
17
18
19
20
21
32
23
24
25
26
50 ꢀ 3a
55 ꢀ 2
65 ꢀ 3
40 ꢀ 1
57 ꢀ 2
40 ꢀ 1
56 ꢀ 5
62 ꢀ 3
55 ꢀ 2
45 ꢀ 5
42 ꢀ 2
28 ꢀ 1
44 ꢀ 1
42 ꢀ 1
54 ꢀ 5
40 ꢀ 5
38 ꢀ 4
36 ꢀ 2
66 ꢀ 3
56 ꢀ 1
38 ꢀ 3
37 ꢀ 2
35 ꢀ 2
48 ꢀ 5
45 ꢀ 5
32 ꢀ 1
45 ꢀ 5
15 ꢀ 2
44 ꢀ 1
47 ꢀ 1
25 ꢀ 1
45 ꢀ 4
17 ꢀ 4
18 ꢀ 3
64 ꢀ 3
67 ꢀ 4
78 ꢀ 3
66 ꢀ 3
41 ꢀ 2
41 ꢀ 2
33 ꢀ 4
62 ꢀ 4
64 ꢀ 3
35 ꢀ 2
81 ꢀ 2
37 ꢀ 3
75 ꢀ 1
36 ꢀ 3
43 ꢀ 5
38 ꢀ 5
43 ꢀ 5
57 ꢀ 2
45 ꢀ 5
77 ꢀ 5
43 ꢀ 5
39 ꢀ 5
41 ꢀ 3
60 ꢀ 5
70 ꢀ 1
62 ꢀ 1
54 ꢀ 3
64 ꢀ 3
78 ꢀ 4
61 ꢀ 4
60 ꢀ 1
64 ꢀ 2
64 ꢀ 2
62 ꢀ 5
35 ꢀ 3
42 ꢀ 3
66 ꢀ 1
73 ꢀ 2b
72 ꢀ 2
74 ꢀ 2
77 ꢀ 5
66 ꢀ 2
75 ꢀ 4
58 ꢀ 2
76 ꢀ 1
78 ꢀ 2
71 ꢀ 2
79 ꢀ 2
63 ꢀ 5
69 ꢀ 3
72 ꢀ 1
78 ꢀ 2
68 ꢀ 2
68 ꢀ 2
73 ꢀ 3
75 ꢀ 2
86 ꢀ 2
82 ꢀ 2
55 ꢀ 1
47 ꢀ 4
74 ꢀ 1
55 ꢀ 5
54 ꢀ 2
57 ꢀ 5
61 ꢀ 2
53 ꢀ 5
63 ꢀ 5
67 ꢀ 5
51 ꢀ 5
68 ꢀ 1
60 ꢀ 5
55 ꢀ 2
64 ꢀ 5
49 ꢀ 3
50 ꢀ 1
80 ꢀ 3
89 ꢀ 2
75 ꢀ 2
83 ꢀ 4
78 ꢀ 2
79 ꢀ 5
75 ꢀ 1
40 ꢀ 4
75 ꢀ 5
62 ꢀ 3
Moreover, it was worthy of notice that some compounds dis-
played multiple good antifungal activities. 25 and 28e31 showed
good antifungal activities against P. piricola and R. solani while 26
exhibited good antifungal activities against C. beticola and R. solani.
2.3. Antibacterial activity
In this study, attention was also focused on the evaluation of the
antibacterial activities. Compounds 11e31 were evaluated for their
antibacterial activities against two Gram-positive bacteria strains
(B. subtilis ATCC 6633 and S. aureus ATCC 6538) and two Gram-
negative bacteria strains (E. coli ATCC 35218 and P. fluorescence
ATCC 13525) by the serial dilution method recommended by
Clinical Laboratory Standards Institute (CLSI) [27], taking kana-
mycin as positive control.
27
28
29
30
31
Triadimefon
Validamycin
Carbendazim 48 ꢀ 4
The results were shown in Table 3 and the minimum inhibitory
concentrations (MICs) of the compounds differed greatly, ranging
a
Each value is the mean ꢀ S.D. (n ¼ 3).
b
from 1.56 to over 100
mg/mL. Overall, most of the compounds
Bold numbers represent the efficacies reach the scope of Grade B (inhibition
rate ¼ 70%e89%).
exhibited better inhibitory activities against Gram-negative

Y. Luo et al. / European Journal of Medicinal Chemistry 64 (2013) 54e61
57
Table 3
Overall, in most cases when the substituent number on the
phenyl group was two and the substituent was F atom, the com-
pound would show better antibacterial activity.
Antibacterial activity of compounds 11e31.
Compd
Minimum inhibitory concentrations (mg/mL)
Gram-negative
Gram-positive
3. Conclusions
E. coli ATCC
P. fluorescence
B. subtilis
S. aureus
35218
ATCC 13525
ATCC 6633
ATCC 6538
In this study we reported the synthesis, characterization and
antimicrobial activities evaluation of new 1,3,4-thiadiazole de-
rivatives bearing 1,2,4-triazolo[1,5-a]pyrimidine moiety. The target
compounds from 1,2,4-triazole and 1,3,4-thiadiazole class were
obtained from thiourea’s cyclization. To study the potential anti-
microbial activities of the synthesized compounds, screening ex-
periments were performed for four bacteria and five fungi strains.
The best antibacterial activity was presented by 25 against
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
12.5
12.5
12.5
12.5
25
25
50
50
100
100
25
50
100
25
50
50
>100
>100
>100
>100
50
>100
12.5a
>100
>100
>100
50
25
50
50
25
12.5
50
50
25
25
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
25
12.5
12.5
25
>100
>100
>100
50
25
>100
3.13
12.5
50
12.5
12.5
12.5
12.5
25
P. fluorescence with MIC of 1.56 mg/mL. 25 and 28e31 showed good
antifungal activities against P. piricola and R. solani and 26 exhibited
good antifungal activities against C. beticola and R. solani which
were comparable with the controls. The most antifungal activity
was presented by the compound 26 against R. solani (inhibition
rate ¼ 89 ꢀ 2%).
25
25
>100
100
1.56
12.5
12.5
6.25
12.5
12.5
12.5
3.13
12.5
12.5
25
27
28
29
30
25
Structureeactivity relationship (SAR) study indicated a general
appreciation of the relationship between the molecular structures
and biological activities of the synthesized compounds, it might be
concluded that in most cases:
12.5
12.5
3.13
6.25
1.56
31
50
0.78
Kanamycin
a
- the presence of F substituent in molecule increases the anti-
microbial efficiency of corresponding compounds;
- the number of substituents can influence the efficiency of the
studied compounds;
Bold numbers represent the best efficacies of compounds against each bacterial
strain.
bacteria strains than Gram-positive bacteria strains. These com-
pounds had not significant inhibitory activities against S. aureus
- to some degree, substituents on ortho- and para-position can
improve the antimicrobial activity of the tested compounds;
- most of the compounds exhibited better inhibitory activity
against Gram-negative bacteria than Gram-positive bacteria.
Most of the compounds exhibited relatively better activity
against P. piricola and R. solani.
supported by the fact that most of their MICs were over 100
25 (MIC ¼ 1.56 g/mL) and 28 (MIC ¼ 6.25 g/mL) exhibited potent
antibacterial activities against P. fluorescence, with MICs compa-
g/
mg/mL.
m
m
rable to kanamycin. 30 (MIC ¼ 3.13
m
g/mL) and 31 (MIC ¼ 6.25
m
mL) exhibited potent activities against E. coli comparable to
kanamycin.
4. Experimental section
As shown in Table 3, 23 (MIC
¼
12.5 mg/mL) and 24
(MIC ¼ 12.5
mg/mL) exhibited the best activities against S. aureus.
4.1. Chemistry
This indicated that OCH3 and C]C groups could improve the
electron donating ability which might do some good to S. aureus
All the reagents were analytical reagent or chemical pure. 1H
NMR and 13C NMR spectra were recorded at 300 MHz on 1H-Varian-
Mercury-300 spectrometers in CDCl3, using TMS as internal stan-
dard. MS spectra were recorded with a Finnigan Trace MS spec-
trometer. Elementary analyses were performed on a Vario EL III
elementary analysis instrument. Melting points were obtained by
using an Electrothermal digital melting point apparatus. TLC was
run on the silica gel coated aluminum sheets (silica gel 60 GF254, E.
Merck, Germany) and visualized in UV light (254 nm).
inhibitory activity. For B. subtilis, compounds 17 (MIC ¼ 12.5
mg/mL)
and 26 (MIC ¼ 12.5 g/mL) with 3-Br and 3-NO2e4-CH3 groups
m
respectively exhibited better activities. This result indicated that
the position of substituent might influence the activity. The pres-
ence of Br and NO2 substituents in the meta-positions on the phenyl
group, would be beneficial for the activity against B. subtilis
comparing to the situation when the substituent was linked in the
ortho- and para-positions of the phenyl group.
For P. fluorescence, 25 (MIC ¼ 1.56
mg/mL) exhibited the best
inhibitory activity. Compounds owning substituents in the ortho-
position showed better antibacterial activities than those com-
pounds with substituents in meta- and para-positions, for example,
4.1.1. General procedure for synthesis of 2-(5,7-dimethyl-[1,2,4]
triazolo[1,5-a]pyrimidin-2-ylthio)acetohydrazide (6)
(1) 1 (500 g, 6.58 mol) was dissolved in water (960 mL).
NH2NH2$H2O (161.2 g, 4.28 mol, 85%) and H2SO4 (179.3 mL, 98%)
was then added. The solution was heated to 100e103 ꢁC and
refluxed for 3 h. After cooling, solid was precipitated and recrys-
tallization from methanol gave 2 as colorless crystals. (2) NaOH
(225 g, 5.62 mol) was dissolved in water (960 mL). 2 was then
added and the solution was refluxed for 3 h. After cooling, the pH
value of the solution was adjusted to 1 using hydrochloric acid.
After that the solid was precipitated and recrystallization from
methanol gave 3 as colorless crystals. (3) 3 (60.6 g, 0.52 mol),
CH3COCH2COCH3 (0.52 mol), piperidine (4.8 mL) and acetic acid
(1500 mL) were mixed and refluxed for 20 h. After cooling, crystals
were precipitated and filtrated by the pump, affording 4 as colorless
11 (MIC ¼ 25
mL) and 13 (MIC ¼ 50
activity than 17 (MIC ¼ 50
(MIC ¼ 25
and 21(MIC ¼ 50
m
g/mL) showed better activity than 12 (MIC ¼ 50
mg/
m
g/mL); 16 (MIC ¼ 25
m
g/mL) showed better
mg/mL) and 18 (MIC ¼ 100
mg/mL); 19
m
g/mL) showed better activity than 20 (MIC ¼ 50
m
m
g/mL)
g/mL)
mg/mL). Overall, 25e31 (MIC ¼ 1.56e12.5
showed relatively better inhibitory activities against P. fluorescence,
so it could say that owning two substituents on benzene ring and
introducing F or NO2 groups are helpful for compounds to improve
antibacterial activity.
For E. coli, all the compounds exhibited good inhibitory activity.
30 and 31 with two substituents in ortho- and para-positions
showed the best inhibitory activity against E. coli.

58
Y. Luo et al. / European Journal of Medicinal Chemistry 64 (2013) 54e61
crystals. (4) 4 (72 g, 0.40 mol) and NaOH (16 g, 0.40 mol) were
dissolved in H2O (1000 mL) and the solution was stirred to clarified
solution. Ethyl bromoacetate (44.5 mL, 0.40 mol) þ methanol
(100 mL) was then added slowly. The solution was stirred at room
temperature for 2 h. Then solid was precipitated and filtrated by
pump, affording 5. (5) 5 (107 g, 0.40 mol) was dissolved in ethanol
(1300 mL) and NH2NH2$H2O (320 mL) was added. The solution was
refluxed for 6 h and cooled. Crystals were then precipitated and
filtrated by pump, affording 6 as colorless crystals. Yield: 74.0%;
(C18H18N6S3): C 52.15%, H 4.38%, N 20.27%; found: C 52.12%, H 4.36%,
N 20.23%.
4.1.4.2. 2-((5,7-Dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylthio)
methyl)-5-(3-methylbenzylthio)-1,3,4-thiadiazole (12). Pale yellow
powder, m.p. 138e140 ꢁC, yield 67.3%. 1H NMR (300 MHz, CDCl3):
d
¼ 2.33 (s, 3H, 3-CH3Ph), 2.68 (s, 3H, 5-CH3), 2.75 (s, 3H, 7-CH3),
4.49 (s, 2H, eCH2Ph), 4.88 (s, 2H, eSCH2e), 6.81 (s, 1H, 6-H), 7.08e
7.28 (m, 4H, AreH ꢂ4) ppm; 13C NMR (300 MHz, CDCl3):
¼ 17.92,
d
m.p.: 208e209 ꢁC; 1H NMR (300 MHz, CDCl3):
d
¼ 2.33 (s, 3H, 5-
21.32, 25.87, 34.92, 39.31, 109.60, 115.43, 127.69, 128.58, 137.87,
140.22, 156.72, 164.46, 166.02, 169.18 ppm; EI-MS: m/z (%) ¼ 416
[M þ 2]þ (1.0), 415 [M þ 1]þ (2.9), 237 (5.2), 221 (0.8), 220 (6.9), 193
(1.8), 181 (20.7),180 (13.8), 108 (8.4), 107 (15.7), 105 (100), 77 (18.4);
Anal. (C18H18N6S3): C 52.15%, H 4.38%, N 20.27%; found: C 52.13%, H
4.35%, N 20.25%.
CH3), 2.52 (s, 3H, 7-CH3), 4.05 (s, 2H, eCH2e), 4.23 (s, 2H, eNH2e
), 7.08 (s, 1H, 6-H), 9.08 (s, 1H, eNHe) ppm; Anal. (C9H12N6OS): C
42.85%, H 4.79%, N 33.31%; found: C 42.63%, H 4.95%, N 33.24%.
4.1.2. General procedure for the synthesis of potassium 2-(2-(5,7-
dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylthio)acetyl)hydrazine
carbodithioate (7)
6 (2.02 g, 8 mmol), KOH (0.8 g) and ethanol (60 mL) were
placed in a three necked bottle (100 mL) and the solution was
stirred to dissolve the solid. CS2 (5.8 g, dissolved in 10 mL ethanol)
were then added in drops and the solution was stirred under room
temperature for more than 24 h. After that solid was precipitated
and filtrated. Recrystallization from DMF/H2O gave compound 7
as white powder. Yield: 94.3%; m.p.: 117e119 ꢁC. 1H NMR
4.1.4.3. 2-((5,7-Dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylthio)
methyl)-5-(4-methylbenzylthio)-1,3,4-thiadiazole (13). Pale red crys
tal, m.p. 131e133 ꢁC, yield 60.1%. 1H NMR (300 MHz, CDCl3):
d
¼ 2.32 (s, 3H, 4-CH3Ph), 2.67 (s, 3H, 5-CH3), 2.74 (s, 3H, 7-CH3),
4.59 (s, 2H, eCH2Ph), 4.86 (s, 2H, eSCH2e), 6.79 (s, 1H, 6-H), 7.09e
7.29 (m, 4H, AreH ꢂ4) ppm; 13C NMR (300 MHz, CDCl3):
d
¼ 17.07,
21.44, 24.91, 34.27, 38.14, 108.62, 119.41, 128.32, 129.51, 135.03,
136.45, 146.32, 157.27, 165.14, 166.79, 168.34 ppm; EI-MS: m/z
(%) ¼ 416 [M þ 2]þ (0.5), 415 [M þ 1]þ (1.64), 237 (2.3), 221 (1.2),
220 (6.0), 193 (0.8), 181 (14.3), 180 (8.6), 108 (4.9), 107 (11.2), 105
(100), 77 (13.4); Anal. (C18H18N6S3): C 52.15%, H 4.38%, N 20.27%;
found: C 52.19%, H 4.37%, N 20.28%.
(300 MHz, CDCl3):
(s, 2H, eCH2e), 4.56 (s, 1H, eNHe), 7.06 (s, 1H, 6-H), 9.34 (s, 1H, e
NHe) ppm.
d
¼ 2.33 (s, 3H, 5-CH3), 2.50 (s, 3H, 7-CH3), 4.01
4.1.3. General procedure for the synthesis of 5-((5,7-dimethyl-[1,2,4]
triazolo[1,5-a]pyrimidin-2-ylthio)methyl)-1,3,4-thiadiazole-2-thiol
(10)
Concentrated sulfuric acid (67 mL) was added in an Erlenmeyer
flask (100 mL) placed in ice bath. 7 (16.2 g) was added in batch and
the solution was stirred for 24 h. After pouring the solution into ice
water, solids were precipitated and filtrated. The solids were dis-
solved in NaOH solution and the residues were removed. After an
acidification of the filtrate by hydrochloride, crystals were precip-
itated and filtrated, affording to 10 as orange yellow crystals. Yield:
4.1.4.4. 2-(2-Chlorobenzylthio)-5-((5,7-dimethyl-[1,2,4]triazolo[1,5-
a]pyrimidin-2-ylthio)methyl)-1,3,4-thiadiazole (14). Pale yellow crys
tal, m.p. 165e166 ꢁC, yield 77.3%. 1H NMR (300 MHz, CDCl3):
d
¼ 2.66 (s, 3H, 5-CH3), 2.74 (s, 3H, 7-CH3), 4.64 (s, 2H, eCH2Ph),
4.86 (s, 2H, eSCH2e), 6.80 (s, 1H, 6-H), 7.18e7.56 (m, 4H, AreH ꢂ4)
ppm; 13C NMR (300 MHz, CDCl3):
d
¼ 18.13, 24.96, 34.35, 38.28,
108.84, 119.18, 127.44, 128.86, 129.33, 132.09, 136.93, 146.17, 157.12,
161.24, 165.45, 166.82, 168.72 ppm; EI-MS: m/z (%) ¼ 438 [M þ 4]þ
(0.6), 437 [M þ 3]þ (1.4), 251 (5.9), 221 (3.9), 220 (4.3), 193 (3.2), 181
(10.9),180 (13.7),149 (2.0),125 (100),108 (21.0),107 (38.2), 77 (3.9);
Anal. (C17H15ClN6S3): C 46.94%, H 3.48%, N 19.32%; found: C 46.95%,
H 3.49%, N 19.36%.
47.0%; m.p.: 217e220 ꢁC; 1H NMR (300 MHz, CDCl3):
d
¼ 2.53 (s, 3H,
5-CH3), 2.58 (s, 3H, 7-CH3), 4.69 (s, 2H, eCH2e), 7.16 (s, 1H, 6-H),
14.20 (s, 1H, eSH) ppm; EI-MS: m/z (%) ¼ 310.0 [M]þ (97.5), 193.1
(32.1), 181.1 (14.1), 180.0 (100.0), 149.1 (98.8), 108.1 (23.0), 107.0
(28.2), 67.0 (44.6); Anal. (C10H10N6S3): C 38.69%, H 3.25%, N 27.07%;
found: C 38.87%, H 3.09%, N 27.27%.
4.1.4.5. 2-(3-Chlorobenzylthio)-5-((5,7-dimethyl-[1,2,4]triazolo[1,5-
a]pyrimidin-2-ylthio)methyl)-1,3,4-thiadiazole (15). Pale yellow crys
tal, m.p. 139e140 ꢁC, yield 80.1%. 1H NMR (300 MHz, CDCl3):
4.1.4. General experimental procedure for the synthesis of compounds
11e31
d
¼ 2.67 (s, 3H, 5-CH3), 2.74 (s, 3H, 7-CH3), 4.48 (s, 2H, eCH2Ph),
4.86 (s, 2H, eSCH2e), 6.80 (s, 1H, 6-H), 7.21e7.39 (m, 4H, AreH ꢂ4)
ppm; 13C NMR (300 MHz, CDCl3):
10 (1.55 g, 5 mmol) and NaOH (0.2 g, 5 mmol) were dissolved in
water (15 mL) and the solution was stirred to clarify solution. Then
methanol or DMF (3 mL) with different halohydrocarbon (6 mmol)
was added carefully and the solution was stirred at room temper-
ature for more than 18 h. Solid was precipitated and filtrated.
Recrystallization from different organic solvents gave target com-
pounds as different physical states.
d
¼ 18.45, 25.33, 34.92, 38.74,
107.34, 119.97, 128.32, 129.17, 129.56, 132.24, 135.24, 147.23, 158.67,
162.15, 166.24, 166.73, 168.83 ppm; EI-MS: m/z (%) ¼ 438 [M þ 4]þ
(0.5), 437 [M þ 3]þ (2.4), 251 (3.1), 221 (0.9), 220 (6.5), 193 (3.6), 181
(17.9), 180 (20.4), 149 (23.0), 125 (100), 108 (21.0), 107 (38.2), 77
(3.9); Anal. (C17H15ClN6S3): C 46.94%, H 3.48%, N 19.32%; found: C
46.95%, H 3.46%, N 19.36%.
4.1.4.1. 2-((5,7-Dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylthio)
4.1.4.6. 2-(2-Bromobenzylthio)-5-((5,7-dimethyl-[1,2,4]triazolo[1,5-
methyl)-5-(2-methylbenzylthio)-1,3,4-thiadiazole (11). Gray crystal,
a]pyrimidin-2-ylthio)methyl)-1,3,4-thiadiazole (16). Yellow crystal,
m.p. 93e96 ꢁC, yield 67.6%. 1H NMR (300 MHz, CDCl3):
d
¼ 2.39 (s,
m.p. 171e173 ꢁC, yield 74.6%. 1H NMR (300 MHz, CDCl3):
d
¼ 2.68 (s,
3H, 2-CH3Ph), 2.66 (s, 3H, 5-CH3), 2.74 (s, 3H, 7-CH3), 4.55 (s, 2H, e
CH2Ph), 4.87 (s, 2H, eSCH2e), 6.81 (s, 1H, 6-H), 7.12e7.34 (m, 4H,
3H, 5-CH3), 2.75 (s, 3H, 7-CH3), 4.67 (s, 2H, eCH2Ph), 4.87 (s, 2H, e
SCH2e), 6.81 (s, 1H, 6-H), 7.14e7.59 (m, 4H, AreH ꢂ4) ppm; 13C
AreH ꢂ4) ppm; 13C NMR (300 MHz, CDCl3):
d
¼ 18.09, 19.43, 25.14,
NMR (300 MHz, CDCl3):
d
¼ 18.54, 24.24, 34.76, 38.14, 107.93,
34.57, 36.72, 107.44, 106.31, 127.32, 128.93, 137.40, 140.35, 154.93,
160.77,166.38,169.25 ppm; EI-MS: m/z (%) ¼ 416 [M þ 2]þ (0.8), 415
[M þ 1]þ (3.2), 237 (6.6), 221 (2.2), 220 (11.0), 193 (1.6), 181 (31.7),
180 (14.3), 108 (7.1), 107 (17.0), 105 (100), 77 (23.7); Anal.
119.22, 127.32, 128.98, 129.23, 132.25, 136.18, 146.92, 157.69, 162.35,
166.19, 166.74, 168.23 ppm; EI-MS: m/z (%) ¼ 481 [M þ 4]þ (0.8),
479 [M þ 1]þ (1.0), 251 (11.4), 237 (20.6), 220 (5.5), 193 (5.7), 181
(22.2), 180 (26.7), 169 (66.7), 149 (31.7), 108 (21.7), 107 (57.6), 89

Y. Luo et al. / European Journal of Medicinal Chemistry 64 (2013) 54e61
59
(100), 77 (3.9); Anal. (C17H15BrN6S3): C 42.59%, H 3.15%, N 17.53%;
found: C 42.55%, H 3.12%, N 17.50%.
(14.8); Anal. (C17H15FN6S3): C 48.78%, H 3.61%, N 20.08%; found: C
48.80%, H 3.60%, N 20.13%.
4.1.4.7. 2-(3-Bromobenzylthio)-5-((5,7-dimethyl-[1,2,4]triazolo[1,5-
a]pyrimidin-2-ylthio)methyl)-1,3,4-thiadiazole (17). Pale yellow
crystal, m.p. 152e154 ꢁC, yield 88.1%. 1H NMR (300 MHz, CDCl3):
4.1.4.12. 2-((5,7-Dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylthio)
methyl)-5-(3-nitrobenzylthio)-1,3,4-thiadiazole (22). Yellow crystal,
m.p.156e158 ꢁC, yield 92.2%. 1H NMR (300 MHz, CDCl3):
d
¼ 2.69 (s,
d
¼ 2.66 (s, 3H, 5-CH3), 2.73 (s, 3H, 7-CH3), 4.46 (s, 2H, eCH2Ph),
4.85 (s, 2H, eSCH2e), 6.79 (s, 1H, 6-H), 7.15e7.53 (m, 4H, AreH ꢂ4)
ppm; 13C NMR (300 MHz, CDCl3):
3H, 5-CH3), 2.75 (s, 3H, 7-CH3), 4.60 (s, 2H, eCH2Ph), 4.85 (s, 2H, e
SCH2e), 6.81 (s, 1H, 6-H), 7.28e7.80 (m, 4H, AreH ꢂ4) ppm; 13C
d
¼ 17.36, 25.13, 34.89, 38.13,
NMR (300 MHz, CDCl3):
d
¼ 18.26, 24.03, 34.25, 38.77, 108.32,
107.24, 120.34, 127.68, 128.13, 129.56, 132.07, 135.87, 146.37, 158.22,
161.48, 165.41, 166.80, 169.03 ppm; EI-MS: m/z (%) ¼ 481 [M þ 4]þ
(4.1), 479 [M þ 1]þ (4.0), 251 (5.1), 237 (34.0), 220 (7.4), 193 (6.6),
181 (28.8), 180 (33.2), 169 (55.7), 149 (36.3), 108 (32.3), 107 (62.5),
89 (100), 77 (6.4); Anal. (C17H15BrN6S3): C 42.59%, H 3.15%, N
17.53%; found: C 42.57%, H 3.18%, N 17.56%
119.68, 127.04, 128.79, 129.65, 132.99, 137.58, 146.53, 158.22, 160.09,
165.93, 166.91, 168.82 ppm; EI-MS: m/z (%) ¼ 278 (15.2), 206 (48.6),
149 (55.0), 109 (25.3), 108 (26.9), 107 (58.7), 89 (22.9), 77 (15.3), 67
(68.1), 44 (100.0); Anal. (C17H15N7O2S3): C 45.83%, H 3.39%, N
22.01%; found: C 45.81%, H 3.34%, N 22.07%.
4.1.4.13. 2-((5,7-Dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylthio)
methyl)-5-(3-methoxybenzylthio)-1,3,4-thiadiazole (23). Pale yellow
crystal, m.p. 103e106 ꢁC, yield 66.5%. 1H NMR (300 MHz, CDCl3):
4.1.4.8. 2-(4-Bromobenzylthio)-5-((5,7-dimethyl-[1,2,4]triazolo[1,5-
a]pyrimidin-2-ylthio)methyl)-1,3,4-thiadiazole (18). Gray crystal,
m.p.160e162 ꢁC, yield 84.7%. 1H NMR (300 MHz, CDCl3):
d
¼ 2.65 (s,
d
¼ 2.66 (s, 3H, 5-CH3), 2.74 (s, 3H, 7-CH3), 3.78 (s, 3H, 4-OCH3), 4.49
3H, 5-CH3), 2.72 (s, 3H, 7-CH3), 4.44 (s, 2H, eCH2Ph), 4.84 (s, 2H, e
(s, 2H, eCH2Ph), 4.86 (s, 2H, eSCH2e), 6.77 (s, 1H, 6-H), 6.80e7.29
SCH2e), 6.78 (s, 1H, 6-H), 7.25e7.41 (m, 4H, AreH ꢂ4) ppm; 13C
(m, 4H, AreH ꢂ4) ppm; 13C NMR (300 MHz, CDCl3):
¼ 17.54, 24.07,
d
NMR (300 MHz, CDCl3):
d
¼ 17.27, 24.02, 35.09, 38.27,108.01,119.44,
34.62, 39.20, 57.97, 108.97, 110.62, 128.75, 128.91, 129.04, 132.85,
137.26, 146.03, 158.17, 160.34, 165.93, 166.47, 168.08 ppm; EI-MS: m/
z (%) ¼ 432 [M þ 2]þ (1.4), 431 [M þ 1]þ (3.8), 237 (8.4), 220 (9.0),
193 (2.8), 181 (48.3), 180 (20.5), 149 (12.5), 121 (100), 108 (14.8), 107
(24.4), 89 (21.0), 77 (25.0), 67 (20.2); Anal. (C18H18N6OS3): C 50.21%,
H 4.21%, N 19.52%; found: C 50.22%, H 4.19%, N 19.61%.
127.78, 128.12, 129.64, 132.25, 135.08, 146.36, 157.09, 161.24, 163.83,
165.83, 166.09, 168.62 ppm; EI-MS: m/z (%) ¼ 481 [M þ 4]þ (3.6),
479 [M þ 1]þ (3.6), 251 (3.9), 237 (27.0), 220 (18.0), 193 (6.0), 181
(51.5), 180 (43.7), 169 (85.6), 149 (39.5), 108 (29.1), 107 (56.1), 89
(100), 77 (6.1); Anal. (C17H15BrN6S3): C 42.59%, H 3.15%, N 17.53%;
found: C 42.58%, H 3.15%, N 17.50%.
4.1.4.14. 2-((5,7-Dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylthio)
4.1.4.9. 2-((5,7-Dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylthio)
methyl)-5-(2-fluorobenzylthio)-1,3,4-thiadiazole (19). Pale yellow
crystal, m.p. 133e135 ꢁC, yield 82.5%. 1H NMR (300 MHz, CDCl3):
methyl)-5-(4-vinylbenzylthio)-1,3,4-thiadiazole
(24). Pale
gray
crystal, m.p. 112e113 ꢁC, yield 71.9%. 1H NMR (300 MHz, CDCl3):
d
¼ 2.67 (s, 3H, 5-CH3), 2.72 (s, 3H, 7-CH3), 4.52 (s, 2H, eCH2Ph),
d
¼ 2.66 (s, 3H, 5-CH3), 2.74 (s, 3H, 7-CH3), 4.56 (s, 2H, eCH2Ph),
4.86 (s, 2H, eSCH2e), 6.80 (s, 1H, 6-H), 7.00e7.49 (m, 4H, AreH ꢂ4)
ppm; 13C NMR (300 MHz, CDCl3):
4.87 (s, 2H, eSCH2e), 5.24 (q, J ¼ 7.5 Hz, 1H, eCH]), 5.77 (d,
J ¼ 7.5 Hz, 2H, eCH2]), 6.67 (s, 1H, 6-H), 7.28e7.35 (m, 4H, Are
d
¼ 17.43, 25.16, 34.94, 38.34,
H ꢂ4) ppm; 13C NMR (300 MHz, CDCl3):
d
¼ 17.27, 24.29, 34.33,
108.03, 120.34, 128.26, 129.65, 130.72, 132.69, 137.23, 145.29, 158.21,
162.04, 165.63, 166.84, 169.16 ppm; EI-MS: m/z (%) ¼ 420 [M þ 2]þ
(1.0), 419 [M þ 1]þ (4.7), 251 (1.6), 237 (13.4), 220 (3.2), 193 (2.4),
181 (8.9), 180 (7.8), 149 (12.0), 109 (100), 108 (12.2), 107 (22.9), 89
(16.7), 67 (15.0); Anal. (C17H15FN6S3): C 48.78%, H 3.61%, N 20.08%;
found: C 48.73%, H 3.57%, N 20.12%.
38.06, 108.18, 115.07, 119.36, 127.15, 128.47, 129.55, 132.03, 136.98,
137.53, 146.67, 158.05, 160.68, 165.83, 166.74, 168.02 ppm; EI-MS:
m/z (%) ¼ 427 [M þ 1]þ (1.8), 297 (3.8), 237 (3.1), 220 (7.4), 193
(6.4), 181 (26.1), 180 (13.8), 149 (9.6), 116 (100), 115 (31.2), 108 (6.3),
107 (13.6), 89 (22.1), 77 (2.3), 67 (15.0); Anal. (C19H18N6S3): C
53.50%, H 4.25%, N 19.70%; found: C 53.55%, H 4.29%, N 19.73%.
4.1.4.10. 2-((5,7-Dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylthio)
methyl)-5-(3-fluorobenzylthio)-1,3,4-thiadiazole (20). Yellow crys-
tal, m.p. 164e166 ꢁC, yield 88.3%. 1H NMR (300 MHz, CDCl3):
4.1.4.15. 2-((5,7-Dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylthio)
methyl)-5-(4-methyl-2-nitrobenzylthio)-1,3,4-thiadiazole
Pale gray crystal, m.p. 153e154 ꢁC, yield 66.9%. 1H NMR (300 MHz,
CDCl3):
(25).
d
¼ 2.67 (s, 3H, 5-CH3), 2.75 (s, 3H, 7-CH3), 4.51 (s, 2H, eCH2Ph),
4.87 (s, 2H, eSCH2e), 6.81 (s, 1H, 6-H), 7.00e7.34 (m, 4H, AreH ꢂ4)
ppm; 13C NMR (300 MHz, CDCl3):
d
¼ 2.57 (s, 3H, 4-CH3Ph), 2.67 (s, 3H, 5-CH3), 2.75 (s, 3H,
7-CH3), 4.54 (s, 2H, eCH2Ph), 4.85 (s, 2H, eSCH2e), 6.81 (s, 1H, 6-
d
¼ 18.09, 24.55, 35.32, 38.76,
H), 7.26e7.60 (m, 3H, AreH ꢂ3) ppm; 13C NMR (300 MHz, CDCl3):
108.03, 119.07, 127.17, 129.17, 130.25, 132.27, 137.04, 146.05, 157.24,
160.03, 166.74, 167.08, 168.93 ppm; EI-MS: m/z (%) ¼ 420 [M þ 2]þ
(1.0), 419 [M þ 1]þ (3.2), 251 (1.7), 237 (10.0), 220 (3.2), 193 (2.1),
181 (9.1), 180 (11.0), 149 (13.6), 109 (100), 108 (14.4), 107 (24.7), 89
(18.4), 67 (17.3); Anal. (C17H15FN6S3): C 48.78%, H 3.61%, N 20.08%;
found: C 48.76%, H 3.63%, N 20.04%.
d
¼ 17.02, 20.36, 24.03, 33.02, 36.17, 108.92, 119.45, 127.26, 128.07,
129.73, 132.05, 136.67, 146.23, 158.53, 161.58, 165.04, 166.76,
168.09 ppm; EI-MS: m/z (%) ¼ 460 [M þ 1]þ (10.1), 309 (16.3), 237
(75.7), 220 (22.1), 193 (15.4), 181 (5.6), 180 (11.6), 149 (86.8), 134
(71.0), 108 (62.5), 107 (100), 89 (57.8), 77 (72.3), 67 (25.0); Anal.
(C18H17N7O2S3): C 47.04%, H 3.73%, N 21.33%; found: C 47.00%, H
3.70%, N 21.36%.
4.1.4.11. 2-((5,7-Dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylthio)
methyl)-5-(4-fluorobenzylthio)-1,3,4-thiadiazole (21). Colorless nee-
dle, m.p. 140e142 ꢁC, yield 81.3%. 1H NMR (300 MHz, CDCl3):
4.1.4.16. 2-((5,7-Dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylthio)
methyl)-5-(4-methyl-3-nitrobenzylthio)-1,3,4-thiadiazole
Yellow crystal, m.p. 152e155 ꢁC, yield 45.0%. 1H NMR (300 MHz,
CDCl3):
(26).
d
¼ 2.66 (s, 3H, 5-CH3), 2.74 (s, 3H, 7-CH3), 4.47 (s, 2H, eCH2Ph), 4.85
(s, 2H, eSCH2e), 6.79 (s, 1H, 6-H), 6.93e7.36 (m, 4H, AreH ꢂ4) ppm;
13C NMR (300 MHz, CDCl3):
d
¼ 2.57 (s, 3H, 4-CH3Ph), 2.67 (s, 3H, 5-CH3), 2.75 (s, 3H,
d
¼ 18.02, 24.57, 34.02, 38.63, 108.06,
7-CH3), 4.54 (s, 2H, eCH2Ph), 4.86 (s, 2H, eSCH2e), 6.81 (s, 1H, 6-
H), 7.27e7.60 (m, 3H, AreH ꢂ3) ppm; 13C NMR (300 MHz, CDCl3):
119.34, 127.45, 128.42, 129.98, 132.27, 136.11, 146.05, 158.39, 161.02,
165.25, 166.02, 167.34 ppm; EI-MS: m/z (%) ¼ 420 [M þ 2]þ (1.1), 419
[M þ 1]þ (4.8), 251 (1.0), 237 (10.2), 220 (7.2), 193 (2.1), 181 (14.5),
180 (11.9), 149 (13.4), 109 (100), 108 (12.7), 107 (22.0), 89 (15.6), 67
d
¼ 17.53, 20.83, 24.09, 33.16, 36.24, 108.04, 119.42, 127.85, 128.55,
129.63, 132.01, 136.35, 146.86, 158.28, 161.72, 165.05, 166.79,
168.25 ppm; EI-MS: m/z (%) ¼ 461 [M þ 2]þ (2.1), 460 [M þ 1]þ

60
Y. Luo et al. / European Journal of Medicinal Chemistry 64 (2013) 54e61
(8.7), 309 (15.0), 237 (59.6), 220 (20.5), 193 (9.6), 181 (47.1), 180
(74.4), 149 (66.8), 134 (70.6), 108 (52.9), 107 (100), 89 (75.1), 77
(93.0); Anal. (C18H17N7O2S3): C 47.04%, H 3.73%, N 21.33%; found:
C 47.06%, H 3.71%, N 21.37%.
(%) ¼ 499 [M þ 3]þ (3.4), 497 [M þ 1]þ (2.5), 237 (36.9), 206
(24.4), 193 (6.0), 187 (72.3), 181 (25.2), 180 (23.5), 149 (32.7), 108
(91.2), 107 (100), 89 (40.6), 77 (1.7), 67 (43.0); Anal.
(C17H14BrFN6S3): C 41.05%, H 2.84%, N 16.89%; found: C 41.07%, H
2.79%, N 16.85%.
4.1.4.17. 2-(3,5-Difluorobenzylthio)-5-((5,7-dimethyl-[1,2,4]triazolo
[1,5-a]pyrimidin-2-ylthio)methyl)-1,3,4-thiadiazole (27). Pale yellow
powder, m.p. 196e198 ꢁC, yield 88.6%. 1H NMR (300 MHz, CDCl3):
4.2. Antifungal activity
F. oxysporum, G. sanbinetti, C. beticola, P. piricola and R. solani
were provided through the courtesy of the Center for Bioassay,
Central China Normal University. Three commercial fungicides,
triadimefon, validamycin and carbendazim, were selected as posi-
tive controls. The tested samples were dissolved in DMF (0.5 mL) at
a concentration of 500 mg/L. The solutions (1 mL) were mixed
rapidly with thawed potato glucose agar culture medium (9 mL) at
50 ꢁC. The mixtures were poured into Petri dishes. After the dishes
were cooled, the solidified plates were incubated with 4 mm
mycelium disk, inverted, and incubated at 28 ꢁC for 48 h. The mixed
medium without sample was used as the blank control. Three
replicates of each test were carried out. The mycelia elongation
radius (mm) of fungi settlements was measured after 48 h of cul-
ture. The growth inhibition rates were calculated with the
following equation:
d
¼ 2.70 (s, 3H, 5-CH3), 2.77 (s, 3H, 7-CH3), 4.50 (s, 2H, eCH2Ph),
4.88 (s, 2H, eSCH2e), 6.72 (s, 1H, 6-H), 7.00e7.30 (m, 3H, AreH ꢂ3)
ppm; 13C NMR (300 MHz, CDCl3):
d
¼ 18.24, 24.07, 34.46, 38.78,
108.36, 119.27, 128.33, 129.53, 132.28, 136.11, 146.92, 158.03, 161.43,
161.52, 165.56, 166.19, 167.72 ppm; EI-MS: m/z (%) ¼ 438 [M þ 2]þ
(2.0), 437 [M þ 1]þ (6.7), 237 (22.0), 206 (17.1), 193 (3.5), 181 (7.5),
180 (13.6),149 (20.6),127 (100.0),108 (16.1),107 (40.4), 89 (21.7), 77
(4.8), 67 (27.0); Anal. (C17H14F2N6S3): C 46.77%, H 3.23%, N 19.25%;
found: C 46.73%, H 3.27%, N 19.23%.
4.1.4.18. 2-(3,4-Difluorobenzylthio)-5-((5,7-dimethyl-[1,2,4]triazolo
[1,5-a]pyrimidin-2-ylthio)methyl)-1,3,4-thiadiazole (28). Pale yellow
crystal, m.p. 184e187 ꢁC, yield 80.8%. 1H NMR (300 MHz, CDCl3):
d
¼ 2.69 (s, 3H, 5-CH3), 2.76 (s, 3H, 7-CH3), 4.48 (s, 2H, eCH2Ph),
4.88 (s, 2H, eSCH2e), 6.82 (s, 1H, 6-H), 7.04e7.30 (m, 3H, AreH ꢂ3)
ppm; 13C NMR (300 MHz, CDCl3):
d
¼ 18.04, 24.25, 34.67, 38.29,
104.23, 119.02, 128.97, 129.09, 132.35, 136.09, 146.24, 158.51, 161.92,
162.38, 165.06, 166.43, 167.06 ppm; EI-MS: m/z (%) ¼ 438 [M þ 2]þ
(0.6), 437 [M þ 1]þ (4.4), 237 (14.8), 206 (11.7), 193 (2.3), 181 (13.5),
180 (13.7),149 (16.4),127 (100.0),108 (15.7),107 (35.6), 89 (17.6), 77
(7.8), 67 (20.7); Anal. (C17H14F2N6S3): C 46.77%, H 3.23%, N 19.25%;
found: C 46.71%, H 3.27%, N 19.26%.
I ¼ ½ðC ꢃ TÞ=Cꢄ ꢂ 100%
Here, I is the growth inhibition rate (%), C is the control settle-
ment radius (mm), and T is the treatment group fungi settlement
radius (mm).
The grades of efficacies were Grade A (inhibition rate ꢅ 90%),
Grade
B
(inhibition rate
¼
70%e89%), Grade C (inhibition
4.1.4.19. 2-(2,6-Difluorobenzylthio)-5-((5,7-dimethyl-[1,2,4]triazolo
rate ¼ 50%e69%) and Grade D (inhibition rate < 50%).
[1,5-a]pyrimidin-2-ylthio)methyl)-1,3,4-thiadiazole
crystal, m.p. 131e133 ꢁC, yield 82.6%. 1H NMR (300 MHz, CDCl3):
¼ 2.69 (s, 3H, 5-CH3), 2.75 (s, 3H, 7-CH3), 4.59 (s, 2H, eCH2Ph), 4.88
(s, 2H, eSCH2e), 6.88 (s, 1H, 6-H), 7.26e7.30 (m, 3H, AreH ꢂ3) ppm;
13C NMR (300 MHz, CDCl3):
(29). Colorless
4.3. Antibacterial activity
d
The antibacterial activities of the synthetic compounds were
tested against three Gram-negative bacterial strains: E. coli ATCC
35218 and P. fluorescens ATCC 13525, three Gram-positive bacterial
strains: B. subtilis ATCC 6633 and S. aureus ATCC 6538, using
method recommended by Clinical Laboratory Standards Institute
(CLSI) [27].
In vitro activities of the compounds were tested in Nutrient
broth (NB) for bacteria by the two-fold serial dilution method.
Seeded broth (broth containing microbial spores) was prepared in
NB from 24 h old bacterial cultures on nutrient agar (Hi-media) at
37 ꢀ 1 ꢁC. The bacterial suspension was adjusted with sterile saline
to a concentration of 1 ꢂ 104e105 CFU/mL. The tested compounds
and reference drugs were prepared by two-fold serial dilution to
d
¼ 18.78, 24.09, 34.37, 38.98, 104.35,
119.83, 128.64, 129.35, 132.44, 136.96, 146.27, 158.46, 161.08, 161.89,
165.37, 166.28, 167.88 ppm; EI-MS: m/z (%) ¼ 438 [M þ 2]þ (0.8), 437
[M þ 1]þ (3.5), 237 (14.7), 206 (11.4),193 (2.3),181 (4.8),180 (7.4),149
(13.7), 127 (100.0), 108 (11.4), 107 (29.2), 89 (16.1), 77 (5.6), 67 (19.2);
Anal. (C17H14F2N6S3): C 46.77%, H 3.23%, N 19.25%; found: C 46.73%, H
3.22%, N 19.30%.
4.1.4.20. 2-(2,4-Difluorobenzylthio)-5-((5,7-dimethyl-[1,2,4]triazolo
[1,5-a]pyrimidin-2-ylthio)methyl)-1,3,4-thiadiazole (30). Yellow green
crustal, m.p. 103e104 ꢁC, yield 76.7%. 1H NMR (300 MHz, CDCl3):
d
¼ 2.68 (s, 3H, 5-CH3), 2.76 (s, 3H, 7-CH3), 4.52 (s, 2H, eCH2Ph), 4.87
(s, 2H, eSCH2e), 6.77 (s, 1H, 6-H), 7.30e7.50 (m, 3H, AreH ꢂ3) ppm;
13C NMR (300 MHz, CDCl3):
obtain the required concentrations of 100, 50, 25,12.5, 6.25, 3.13 mg/
d
¼ 18.22, 24.16, 34.25, 38.07, 103.23,
mL and even lower concentration. The tubes were incubated in BOD
incubators at 37 ꢀ 1 ꢁC for bacteria. The MICs were recorded by
visual observations after 24 h (for bacteria) of incubation. Kana-
mycin was used as standards for bacterial.
112.68, 129.71, 132.75, 136.05, 146.26, 157.21, 160.34, 161.54, 162.44,
165.91, 166.43, 168.24 ppm; EI-MS: m/z (%) ¼ 438 [M þ 2]þ (0.8), 437
[M þ 1]þ (3.7), 237 (7.4), 206 (7.4), 193 (1.7), 181 (5.4), 180 (7.0), 149
(11.6), 127 (100.0), 108 (10.4), 107 (22.3), 89 (13.6), 77 (3.2), 67 (11.2);
Anal. (C17H14F2N6S3): C 46.77%, H 3.23%, N 19.25%; found: C 46.80%, H
3.27%, N 19.29%.
Conflict of interest
The material is original and has not been submitted for publi-
cation elsewhere. There is no conflict of interest in the manuscript.
4.1.4.21. 2-(4-Bromo-2-fluorobenzylthio)-5-((5,7-dimethyl-[1,2,4]
triazolo[1,5-a]pyrimidin-2-ylthio)methyl)-1,3,4-thiadiazole
Yellow crystal, m.p. 104e106 ꢁC, yield 65.1%. 1H NMR (300 MHz,
CDCl3):
¼ 2.66 (s, 3H, 5-CH3), 2.74 (s, 3H, 7-CH3), 4.50 (s, 2H, e
(31).
d
Acknowledgments
CH2Ph), 4.86 (s, 2H, eSCH2e), 6.81 (s, 1H, 6-H), 7.18e7.42 (m, 3H,
AreH ꢂ3) ppm; 13C NMR (300 MHz, CDCl3):
d
¼ 17.97, 24.23,
This work was supported by China Postdoctoral Science Foun-
dation (D) Funds (No. 2012M510131), and Jiangsu Planned Projects
for Postdoctoral Research Funds (No. 1102002C).
34.18, 38.75, 103.34, 112.73, 129.07, 132.63, 136.26, 146.44, 157.07,
160.09, 161.04, 162.23, 165.98, 166.25, 168.13 ppm; EI-MS: m/z

Y. Luo et al. / European Journal of Medicinal Chemistry 64 (2013) 54e61
61
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