Synthesis, antifungal activity and CoMFA analysis of novel 1,2,4-triazolo[1,5-a]pyrimidine derivatives

Article abstract of DOI:10.1016/j.ejmech.2007.04.021

In order to search novel agrochemicals with higher antifungal activity, a series of new 1,2,4-triazolo[1,5-a]pyrimidine derivatives bearing 1,3,4-oxadiazole moieties were designed and synthesized. Their antifungal activities against Rhizoctonia solani wer

Full text of DOI:10.1016/j.ejmech.2007.04.021

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 595e603
http://www.elsevier.com/locate/ejmech
Original article
Synthesis, antifungal activity and CoMFA analysis of novel
1,2,4-triazolo[1,5-a]pyrimidine derivatives
1
Qiong Chen , Xiao-Lei Zhu , Li-Li Jiang, Zu-Ming Liu, Guang-Fu Yang
1
*
Key Laboratory of Pesticide and Chemical Biology, Ministry of Education, College of Chemistry,
Central China Normal University, Wuhan 430079, PR China
Received 24 February 2007; received in revised form 26 April 2007; accepted 27 April 2007
Available online 27 May 2007
Abstract
In order to search novel agrochemicals with higher antifungal activity, a series of new 1,2,4-triazolo[1,5-a]pyrimidine derivatives bearing
1,3,4-oxadiazole moieties were designed and synthesized. Their antifungal activities against Rhizoctonia solani were evaluated in vitro. By de-
termining the EC50 values of all the newly synthesized compounds and 10 formerly synthesized compounds, compound 8r, 2-((5-(sec-butylthio)-
1,3,4-oxadiazol-2-yl)-methylthio)-5-dimethyl-1,2,4-triazolo-[1,5-a]pyrimidine, was found to display the highest antifungal activity
(EC50 ¼ 6.57 mg mLꢀ1). Based on the quantitative structureeactivity relationships analyses, 2-(1-(5-(sec-butylthio)-1,3,4-oxadiazol-2-yl)-
ethylthio)-5,7-dimethyl-1,2,4-triazolo[1,5-a]pyrimidine (9j) was designed and synthesized, which was found to display much higher activity
(EC50 ¼ 3.34 mg mLꢀ1) than compound 8r and the control. To further explore the comprehensive structureeactivity relationships, a 3D-
QSAR analysis using the method of comparative molecular field analysis (CoMFA) was performed and a statistically reliable model with
good predictive power (r2 ¼ 0.929, q2 ¼ 0.588) was achieved on the basis of the common substructure-based alignment. According to the
CoMFA model, the structureeantifungal activity relationship was explained reasonably.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: 1,2,4-Triazolo[1,5-a]pyrimidine; 1,3,4-Oxadiazole; Antifungal activity; CoMFA
1. Introduction
diheterocyclic compounds displayed good antifungal activity
against Rhizoctonia solani. For example, compound 1h
(R ¼ p-NO2C6H4) displayed better in vivo antifungal activity
against R. solani at the concentration of 200 mg mLꢀ1 than car-
bendazim and validamycin A, two commercial fungicides. As
a continuation of our ongoing project related to the discovery
and optimization of pesticide leads [11e15], we synthesized
a series of new 1,3,4-oxadiazole-containing 1,2,4-triazolo[1,
5-a]pyrimidines with the aim of finding potent antifungal activ-
ities. In addition, a comparative molecular field analysis
(CoMFA) was also performed to understand the quantitative
structureeactivity relationships of this family.
The synthesis and biological activities of 1,2,4-triazolo[1,5-
a]pyrimidine derivatives have been an interesting topic in the
fields of medicinal and agricultural chemistry for many years
[1e9]. Some triazolopyrimidine derivatives are known as car-
diovascular vasodilators [1], dual thrombin/factor Xa inhibitors
[2], and human adenosine A2a and A3 receptor ligands [3e5].
Additionally, many triazolopyrimidine-2-sulfonamide deriva-
tives, such as flumetsulam [6], metosulam [7] and florasulam
[8], are commercially available as acetolactate synthase-inhib-
iting herbicides [9]. Previously, we designed and synthesized
a series of diheterocyclic compounds as shown in Fig. 1 by in-
tegrating 1,3,4-oxadiazole moiety into the scaffold of 1,2,4-
triazolo[1,5-a]pyrimidine [10]. It was found that these
2. Results and discussion
2.1. Chemistry
* Corresponding author. Tel.: þ86 27 67867800; fax: þ86 27 67867141.
E-mail address: gfyang@mail.ccnu.edu.cn (G.-F. Yang).
Co-first authors due to their equal contribution.
The target compounds 8aes and 9aej were synthesized by
a multiple-step procedure as shown in Scheme 1. Firstly, the
1
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.04.021

596
Q. Chen et al. / European Journal of Medicinal Chemistry 43 (2008) 595e603
with various halides to afford the target compounds 8aes and
9aej in yields of 32e90%.
N
N
N
N
N
S
The reaction of 7 with halide is a typical nucleophilic sub-
stitution process. The experimental results indicated that the
reactivity of the halide determined the reaction time and the
yields of title compounds. For example, at room temperature,
intermediate 7a reacted with p-fluorobenzyl chloride under ba-
sic condition for 1 h to give the title compound 8j in a yield of
90%. But no product was observed when intermediate 7 re-
acted with 1-bromocyclohexane under the same condition.
The structures of all new compounds were characterized by
1H NMR, MS, and elemental analyses.
SR
O
N
1
R = alkyl, substituted benzyl, allyl, ester group, et al.
Fig. 1.
starting material 2 reacted with ethyl bromoacetate to afford in-
termediate 4, which cyclized with acetylacetaldehyde dimethyl
acetal to give ethyl 2-(5-methyl-1,2,4-triazolo-[1,5-a]pyrimi-
din-2-ylthio)acetate 5a in a yield of 80%. Ethyl 2-(5,7-
dimethyl-1,2,4-triazolo[1,5-a]-pyrimidin-2-ylthio)propanoate
5b was prepared in a high yield (96%) by the reaction of 2-
mercapto-5,7-dimethyl-1,2,4-triazolo[1,5-a]pyrimidine 3 with
ethyl 2-bromopropionate under basic condition. It should be
noted that the reaction of compound 5a with an excess of hydra-
zine hydrate did not give the desired product, but compound 5b
reacted smoothly with an excess of hydrazine hydrate to afford
compound 6b in a yield of 79%. Interestingly, the desired com-
pound 6a could be prepared in a yield of 68% by the reaction of
compound 5a with an equivalent of hydrazine hydrate. Then,
compounds 6 were refluxed with carbon sulfide in ethanol
in the presence of KOH to give 5-(1-(5-methyl-1,2,4-
triazolo[1,5-a]pyrimidin-2-ylthio)ethyl)-1,3,4-oxadiazole-2-thiol
7a in a yield of 96% and 5-(1-(5,7-dimethyl-1,2,4-triazolo[1,
5-a]pyrimidin-2-ylthio)ethyl)-1,3,4-oxadiazole-2-thiol 7b in
a yield of 81%, respectively. Compounds 7a and 7b reacted
2.2. Fungicidal activity and SAR
The EC50 values against R. solani of compounds 8aes and
9aej are listed in Table 1. Carbendazim, a commercial fungi-
cide, was used as a control. In order to discuss the structuree
activity relationships, we also determined the EC50 values
against R. solani of the previously synthesized compounds
1aej [10], which are also listed in Table 1. For the sake of
clarity, compounds 8aes, 1aej and 9aej are named as mono-
methyl, dimethyl and trimethyl derivatives, respectively,
throughout the text. Results showed (Table 1) that compound
8r [R1 ¼ CH3CH2(CH3)CH, EC50 ¼ 6.57 mg mLꢀ1] exhibited
the highest antifungal activity, which is higher than that of car-
bendazim (EC50 ¼ 7.62mg mLꢀ1). Except 9h and 8b, trimethyl
and dimethyl derivatives always displayed higher activities
N
N
SH
N
N
b
3
X
N
R
HN
N
HN
N
N
N
a
c
N
S
COOC2H5
H2N
H2N
SCH2COOC2H5
SH
N
N
2
4
5a (R = X = H)
5b (R = X = CH3)
d
X
N
X
N
X
R
R
R
N
N
N
N
N
N
N
f
N
N
e
N
N
N
S
S
S
CONHNH2
N
O
O
SR1
SH
N
8 (X = H, R = H)
9 (X = CH3, R = CH3)
7a (R = X = H)
7b (R = X = CH3)
6a (R = X = H)
6b (R = X = CH3)
a, BrCH2COOC2H5, NaOH, rt; b, BrCH(CH3)COOC2H5, NaOH, rt; c, CH3COCH2CH(OCH3)2, Na/C2H5OH, rt;
d, NH2NH2H2O, C2H5OH, reflux; e, CS2, KOH, C2H5OH; f, R1Y, CH3OH.
Scheme 1.

Q. Chen et al. / European Journal of Medicinal Chemistry 43 (2008) 595e603
597
Table 1
Determination of EC50 values against R. solani of compounds 8aes, 9aej and 1aej
N
N
N
N
N
N
N
N
N
N
N
N
N
N
S
N
S
S
O
SR1
N
O
SR1
N
O
SR1
N
1a–j
8a–s
9a–j
R1
y ¼ ax þ ba
rb
EC50 (mg mLꢀ1
)
pEC50
c
No
Exp.
Calcd.
8a
C6H5CH2
4-NO2C6H4CH2
3-NO2C6H4CH2
y ¼ 1.6594x þ 2.8463
y ¼ 2.0515x þ 2.5409
y ¼ 1.4101x þ 2.9754
y ¼ 2.2954x þ 1.8177
y ¼ 2.2591x þ 2.1229
y ¼ 1.4909x þ 2.3886
y ¼ 2.0494x þ 1.7855
y ¼ 1.4229x þ 3.1647
y ¼ 1.3072x þ 3.0296
y ¼ 1.5633x þ 3.0111
y ¼ 1.3627x þ 2.5929
y ¼ 1.5328x þ 3.1718
y ¼ 1.9301x þ 2.1145
y ¼ 1.6562x þ 2.5750
y ¼ 1.7878x þ 2.3871
y ¼ 1.8157x þ 2.5294
y ¼ 1.5043x þ 3.2459
y ¼ 1.6458x þ 3.6545
y ¼ 1.9045x þ 1.9933
y ¼ 1.2932x þ 3.4988
y ¼ 1.1875x þ 3.5896
y ¼ 0.8715x þ 4.1295
y ¼ 1.3647x þ 3.3614
y ¼ 1.8920x þ 2.9222
y ¼ 1.5223x þ 3.0315
y ¼ 1.4178x þ 3.2225
y ¼ 1.5668x þ 3.0735
y ¼ 1.4782x þ 3.2965
y ¼ 1.3117x þ 4.3136
y ¼ 1.9098x þ 2.7225
y ¼ 2.4355x þ 2.0425
y ¼ 2.2458x þ 2.0479
y ¼ 2.0106x þ 2.6763
y ¼ 1.5066x þ 3.4096
y ¼ 2.6486x þ 1.2071
y ¼ 2.8571x þ 1.3206
y ¼ 1.7035x þ 3.0604
y ¼ 1.8786x þ 2.8819
y ¼ 1.7533x þ 3.0182
y ¼ 3.8168x þ 1.6335
0.9763
0.9462
0.9480
0.9274
0.9985
0.9779
0.9982
0.9844
0.9270
0.8833
0.9439
0.9759
0.9796
0.9447
0.9589
0.9675
0.9473
0.9187
0.9586
0.9879
0.9410
0.8836
0.9724
0.9799
0.9559
0.9825
0.9384
0.9332
0.9963
0.9772
0.9722
0.9927
0.9657
0.9504
0.9803
0.9436
0.9509
0.9591
0.9581
0.9443
19.86
15.80
27.28
24.34
18.77
56.44
37.03
19.49
32.16
18.72
58.40
15.59
31.26
29.12
28.94
22.94
14.66
6.57
4.27
4.41
4.18
4.25
4.33
3.86
4.08
4.30
4.08
4.31
3.82
4.39
4.09
4.14
4.01
4.13
4.36
4.71
3.99
4.47
4.43
4.62
4.44
4.53
4.32
4.38
4.41
4.46
5.03
4.30
4.37
4.31
4.35
4.55
4.13
4.29
4.49
4.39
4.45
4.25
4.48
4.15
4.12
4.31
4.05
4.11
4.38
4.06
4.24
3.92
4.34
4.02
4.15
4.03
4.15
4.40
4.62
3.93
4.49
4.46
4.62
4.37
4.51
4.36
4.30
4.46
4.43
5.05
4.27
4.35
4.31
4.33
4.56
4.19
4.17
4.50
4.50
4.45
8b
8c
8d
8e
2-NO2-4-CH3C6H3CH2
2-ClC6H4CH2
3-ClC6H4CH2
4-BrC6H4CH2
2-FC6H4CH2
8f
8g
8h
8i
3-FC6H4CH2
4-FC6H4CH2
8j
8k
8l
4-CH3C6H4CH2
2-CH3C6H4CH2
3-CH3C6H4CH2
4-OCH3C6H5COCH2
CH3
8m
8n
8o
8p
8q
8r
CH3CH2
CH3CH2CH2CH2
CH3CH2(CH3)CH
CH3CH2OOCCH2
4-ClC6H4CH2
2-FC6H4CH2
8s
9a
37.91
14.48
15.41
9.97
9b
9c
4-FC6H4CH2
9d
9e
3-ClC6H4CH2
2-CH3C6H4CH2
3-CH3C6H4CH2
3-OCH3C6H4CH2
4-NO2C6H4CH2
4-CH3C6H4CH2
CH3CH2(CH3)CH
CH3
15.87
12.54
19.64
17.94
16.97
14.20
3.34
9f
9g
9h
9i
9j
1ad
1bd
1cd
1dd
1ed
1fd
1gd
1hd
1id
1jd
Carbendazim
a
15.58
16.38
20.63
14.31
11.37
27.04
19.40
13.76
13.41
13.50
7.62
C6H5CH2
4-ClC6H4CH2
CH3CH2
CH3(CH2)6CH2
CH3OOCCH2
CH3CH2OOCCH2
4-NO2C6H4CH2
CH3CH2CH2
CH3(CH2)4CH2
The correlation equations for the determination of EC50 values.
b
c
d
The correlation coefficients of the equations.
pEC50 ¼ log(MW/EC50), MW refers to molecular weight.
These compounds were synthesized previously [10].
than monomethyl derivatives bearing the same R1 group
(9b > 8h; 9c > 8j; 9d > 8f; 9e > 8l; 9f > 8m; 9i > 8k;
1a > 8o; 1b > 8a; 1d > 8p; 1g > 8s). Trimethyl derivatives
bearing p-chlorobenzyl group displayed higher activities
than the corresponding dimethyl derivatives (9a > 1c), how-
ever, trimethyl derivatives bearing p-nitrobenzyl group dis-
played lower activities than the corresponding dimethyl
derivatives (9h < 1h), which indicated that the effect of
a-methyl in the bridge on the antifungal activity is very com-
plex. In addition, among the monomethyl derivatives contain-
ing a substituted benzyl group, electron-withdrawing group on
4-position of the benzyl resulted in higher activity. For exam-
ple, compounds 8b (R1 ¼ 4-NO2C6H4CH2) and 8j (R1 ¼ 4-
FC6H4CH2) displayed higher activity than compounds 8g

598
Q. Chen et al. / European Journal of Medicinal Chemistry 43 (2008) 595e603
(R1 ¼ 4-BrC6H4CH2), 8k (R1 ¼ 4-CH3C6H4CH2), and 8n
(R1 ¼ 4-CH3OC6H4CH2). Compound 9c is the highest active
compound within the trimethyl derivatives, which indicated
that the electrostatic and steric properties have important ef-
fects on the antifungal activity.
Table 2
Summary of CoMFA analysis
q2cv r2n-cv
S
F
Comp. Contribution
Steric Electrostatic
6 53.2% 46.8%
CoMFA 0.588 0.929 0.066 69.864
According to the above SAR analysis, the best R1, R and X
groups should be sec-butyl, CH3 and CH3, respectively. Then,
compound 9j (R1 ¼ CH(CH3)CH2CH3, R ¼ X ¼ CH3) was
synthesized and tested its antifungal activity against R. solani.
The results indicated that the EC50 value of compound 9j is
3.34 mg mLꢀ1, which is much higher than that of compounds
8r and carbendazim.
branched methyl into this green region. The electrostatic con-
tour plot is shown in Fig. 3. The blue contour defines a region
where an increase in the positive charge will result in an in-
crease in the activity, whereas the red contour defines a region
of space where increasing electron density is favorable. As
shown in Fig. 3, the target compounds bearing an electron-do-
nating group at the ortho-position of the benzyl and an elec-
tron-withdrawing group at the other ortho-position displayed
higher activity. In addition, an electron-withdrawing group at
the other para-position will also improve the antifungal
activity.
2.3. CoMFA analysis
3D-QSAR methods [16e18], especially CoMFA [17], are
used widely in drug design, because they allow rapid genera-
tion of QSARs from which biological activity of newly de-
signed molecules can be predicted. The basic assumption in
CoMFA is that an appropriate sampling of the steric and elec-
trostatic fields around a set of aligned molecules might provide
all the information necessary for understanding their biologi-
cal activities. As listed in Table 2, a predictive CoMFA model
was established with the conventional correlation coefficient
r2 ¼ 0.929 and the cross-validated coefficient q2 ¼ 0.588, the
contribution of steric and electrostatic fields are 53.2% and
46.8%, respectively. In Figs. 2 and 3, the isocontour diagrams
of the steric and electrostatic field contributions (‘‘stdev*coeff’’)
obtained from the CoMFA analysis are illustrated together
with exemplary ligands. The observed and calculated activity
values for all the compounds are shown in Table 1, and the
plots of the predicted versus the actual activity values for all
the compounds are shown in Fig. 4.
The steric field contour map is plotted in Fig. 2. The green
displays2 positions where a bulky group would be favorable
for higher antifungal activity. In contrast, yellow indicates po-
sitions where a decrease in the bulk of the target molecules is
favored. As shown in Fig. 2, the CoMFA steric contour plots
obviously indicated that a yellow region is located around
the 3-position and 4-position of the benzyl group. This means
that the bulky substituents at 3-position and 4-position will de-
crease the antifungal activity. For example, some compounds
bearing substituents at 3-position of the benzyl group, such
as 8c, 8f, 8i, 8m, 9f, and 9g, displayed lower antifungal activ-
ity. Compound 9c displayed higher activity than compound
9h, which might due to the fact that the NO2 (Es ¼ ꢀ2.52)
group is more bulky than the F (Es ¼ ꢀ0.46) atom. In addition,
a green region near the 7-position of the 1,2,4-triazolo[1,5-
a]pyrimidine ring could be observed obviously, which
explained why trimethyl and dimethyl derivatives always
displayed higher activity than monomethyl derivatives. The
second green region was located near the 2-position of
the benzyl group, and the third green region was surrounded
the region above the benzyl plane. Compound 8r inserted its
3. Conclusion
In summary, by optimizing the substituents of the formerly
discovered lead compound and quantitative structureeactivity
relationship analysis, a new compound with high antifungal
activity (EC50 ¼ 3.34mg mLꢀ1) against R. solani, 2-(1-(5-
(sec-butylthio)-1,3,4-oxadiazol-2-yl)ethylthio)-5,7-dimethyl-1,2,
4-triazolo[1,5-a]pyrimidine (9j), was developed. Additionally,
a 3D-QSAR analysis using the method of CoMFAwas performed
to explore the comprehensive structureeactivity relationships and
a statistically reliable model with good predictive power
(r2 ¼ 0.929, q2 ¼ 0.588) was achieved on the basis of the com-
mon substructure-based alignment. According to the CoMFA
model, R1 group of the target compound should have an optimum
steric effect. When R1 are substituted benzyl groups, substituents
at 3-position of the benzyl are disfavored and substituents at 4-
position should be electron-withdrawing group having a small
steric effect. One of the ortho-substituents of the benzyl should
be an electron-withdrawing group; the other one should be an
electron-donating group.
Fig. 2. Steric map from the CoMFA model. Compounds 8r and 9c are shown
inside the field. Sterically favored areas (contribution level of 80%) are repre-
sented by green polyhedra. Sterically disfavored areas (contribution level of
20%) are represented by yellow polyhedra. (For interpretation of the refer-
ences to color in figure legends, the reader is referred to the web version of
this article.)
2
For interpretation of the references to color in the text, the reader is re-
ferred to the web version of this article.

Q. Chen et al. / European Journal of Medicinal Chemistry 43 (2008) 595e603
599
4.2.1. Ethyl 2-(5-amino-1H-1,2,4-triazol-3-ylthio)acetate 4
Sodium hydroxide 0.88 g (22 mmol) was dissolved in
50 mL of water and 2.32 g (20 mmol) of 5-amino-3-mer-
capto-1,2,4-triazole (2) was added. After stirring for 15 min,
a solution of 3.34 g (20 mmol) of ethyl bromoacetate in
5 mL of methanol was added dropwise over 20 min. After ad-
dition was completed, the solution was stirred at room temper-
ature for about 2 h. The solid was filtered and recrystallized
from ethanol to give pure products as a white crystal. Com-
Fig. 3. Electrostatic map from the CoMFA model. Compounds 8r and 9c are
shown inside the field. Blue contours (80% contribution) encompass regions
where an increase of positive charge will enhance affinity, whereas in red con-
toured areas (20% contribution) more negative charges are favorable for bind-
ing properties.
pound
4 e
yield: 86%; mp 125e126 ꢁC; 1H NMR
(400 MHz, DMSO-d6): d 11.87 (s, 1H), 6.04 (s, 2H), 4.05
(q, J ¼ 7.2 Hz, 2H), 3.82 (s, 2H), 1.15 (t, J ¼ 7.2 Hz, 3H).
4.2.2. Ethyl 2-(5-methyl-1,2,4-triazolo[1,5-a]pyrimidin-2-
ylthio)acetate 5a
4. Experimental section
A solution of sodium ethoxide in EtOH was prepared by
dissolving 0.55 g (24 mmol) of sodium metal in 120 mL of an-
hydrous EtOH, and 9.7 g (48 mmol) of 2-(5-amino-1H-1,2,4-
triazol-3-ylthio)acetate (4) was added. After stirring for
15 min at room temperature, 6.35 g (48.4 mmol) of acetylace-
taldehyde dimethyl acetal dissolved in 100 mL of absolute
EtOH was added dropwise. After the addition was complete,
the reaction mixture was stirred at room temperature for
24 h. The solid was filtered and recrystallized from ethanol
to give pure products as a white crystal. Compound 5a e
4.1. Materials
All chemical reagents were commercially available and
treated with standard methods before use. Solvents were dried
in a routine way and redistilled. 5-Amino-3-mercapto-1,2,
4-triazole 2 and 2-mercapto-5,7-dimethyl-1,2,4-triazolo[1,
5-a]pyrimidine 3 were prepared according to the previously
reported method [19,20]. R. solani was a gift from the courtesy
of the Center for Bioassay, The Environment and Plant Protec-
tion Institute of Chinese Academy of Tropical Agricultural
Sciences.
1
yield: 80%; mp 114e115 ꢁC; H NMR (400 MHz, CDCl3):
d 8.56 (dd, J ¼ 7.2 Hz, 1H), 6.91 (dd, J ¼ 7.2 Hz, 1H), 4.23
(q, J ¼ 7.2 Hz, 2H), 4.10 (s, 2H), 2.69 (s, 3H), 1.29 (t,
J ¼ 7.2 Hz, 3H).
4.2. Analysis and instruments
Melting points were uncorrected and determined with elec-
tro thermal digital melting point apparatus. MS spectra were
determined using a Finnigan Trace MS organic mass spec-
4.2.3. Ethyl 2-(5,7-dimethyl-1,2,4-triazolo[1,5-a]pyrimidin-
2-ylthio)propanoate 5b
1
Sodium hydroxide (22 mmol) dissolved in 50 mL of water
and 3.6 g (20 mmol) 5,7-dimethyl-2-mercapto-1,2,4-tria-
zolo[1,5-a]pyrimidine (3) was added. After stirring for
15 min, a solution of 3.62 g (20 mmol) of ethyl 2-bromopropi-
onate in 5 mL of ethanol was added dropwise over 20 min. Af-
ter addition was completed, the solution was stirred at room
temperature for about 2 h. The solid was filtered and recrystal-
lized from ethanol to give pure products as a white crystal.
Compound 5b e yield: 96%; mp 85e86 ꢁC; 1H NMR
(400 MHz, CDCl3): d 6.75 (s, 1H), 4.68 (q, J ¼ 7.2 Hz, 1H),
4.21 (q, J ¼ 7.2 Hz, 2H), 2.72 (s, 3H), 2.64 (s, 3H), 1.71 (d,
J ¼ 7.2 Hz, 3H), 1.27 (t, J ¼ 6.8 Hz, 3H).
trometry, and signals were given in m/z. H NMR was re-
corded in CDCl3 or DMSO on a Varian Mercury 400 MHz,
Varian Mercury 300 MHz spectrometer or Bruker AC-P
200 MHz spectrometer and resonance are given in parts per
million (d) relative to TMS. Elementary analyses (EA) were
performed on a Vario EL III elementary analysis instrument.
5.2
5.0
4.8
4.6
4.4
4.2
4.0
3.8
4.2.4. 2-(5-Methyl-1,2,4-triazolo[1,5-a]pyrimidin-2-ylthio)-
acetohydrazide 6a
A mixture of 12.6 g (0.05 mol) of 2-(5-methyl-1,2,4-tria-
zolo[1,5-a]pyrimidin-2-ylthio)acetate
(5a)
and
2.94 g
(0.05 mol) of 85% hydrazine monohydrate in 200 mL of
EtOH was heated at 60 ꢁC for 4 h. After the reaction mixture
was cooled to room temperature, the precipitate was filtered
and recrystallized from methanol to give pure product as
a white crystal. Compound 6a e yield: 68%; mp 170e
172 ꢁC; 1H NMR (400 MHz, DMSO-d6): d 9.36 (s, 1H),
9.12 (dd, J ¼ 6.8 Hz, 1H), 7.20 (dd, J ¼ 6.4 Hz, 1H), 4.33 (s,
2H), 3.95 (s, 2H), 2.60 (s, 3H).
3.8
4.3
4.8
5.3
Calculated (pEC50
)
Fig. 4. CoMFA predicted as experimental pEC50 values.

600
Q. Chen et al. / European Journal of Medicinal Chemistry 43 (2008) 595e603
4.2.5. 2-(5,7-Dimethyl-1,2,4-triazolo[1,5-a]pyrimidin-2-
ylthio)propanehydrazide 6b
Compound 8a e yield: 76%; yellowy solid, mp 120e
121 ꢁC; 1H NMR (300 MHz, CDCl3):
8.53 (dd,
d
A mixture of 14.0 g (0.05 mol) of 2-(5,7-dimethyl-1,2,4-tri-
azolo[1,5-a]pyrimidin-2-ylthio)propanoate (5b) and 20.6 g
(0.35 mol) of 85% hydrazine monohydrate in 200 mL of
EtOH was refluxed for 7 h. After the reaction mixture was
cooled to room temperature, the precipitate was filtered and re-
crystallized from methanol to give pure product as a white
J ¼ 6.9 Hz, 1H), 7.24e7.97 (m, 5H), 2.68 (s, 3H), 6.89 (dd,
J ¼ 6.9 Hz, 1H), 4.40 (s, 2H), 4.68 (s, 2H); EIMS (probe)
70 eV, m/z (rel. int.): 370 (Mþ, 3), 247 (91), 179 (41), 134
(20), 90 (100). Anal. Calcd for C16H14N6OS2: C, 51.87; H,
3.81; N, 22.69. Found: C, 51.61; H, 4.03; N, 22.93.
Compound 8b e yield: 46%; yellowy solid, mp 127e
1
1
crystal. Compound 6b e yield: 79%; mp 104e106 ꢁC; H
128 ꢁC; H NMR (300 MHz, CDCl3): d 8.57 (dd, J ¼ 6.6 Hz,
NMR (400 MHz, DMSO-d6): d 9.47 (s, 1H), 7.13 (s, 1H),
4.50 (q, J ¼ 6.8 Hz, 1H), 4.35 (s, 2H), 2.68 (s, 3H), 2.56 (s,
3H), 1.56 (t, J ¼ 7.2 Hz, 3H).
1H), 7.59e8.16 (m, 4H), 6.95 (dd, J ¼ 6.6 Hz, 1H), 4.69 (s,
2H), 4.47 (s, 2H), 2.71 (s, 3H); EIMS (probe) 70 eV, m/z (ret.
int.): 416 (M þ 1, 1), 247 (100), 179 (41), 135 (30), 94 (54).
Anal. Calcd for C16H13N7O3S2: C, 46.26; H, 3.15; N, 23.60.
Found: C, 46.01; H, 3.40; N, 23.33.
4.2.6. 5-(1-(5-Methyl-1,2,4-triazolo[1,5-a]pyrimidin-2-
ylthio)ethyl)-1,3,4-oxadiazole-2-thiol 7a
Compound 8c e yield: 79%; yellowy solid, mp 156e
158 ꢁC; 1H NMR (200 MHz, CDCl3):
KOH (1.35 g, 24 mmol) was dissolved in 160 mL of anhy-
drous ethanol and 20 mmol of 2-(5-methyl-1,2,4-triazolo[1,5-
a]pyrimidin-2-ylthio)acetohydrazide (6a) was added. To the
vigorously stirred solution, 2 g (0.026 mol) of methanedi-
thione dissolved in 20 mL of EtOH was added slowly at
room temperature. After addition was completed, the solution
was refluxed for 6 h. The solvent was removed under reduced
pressure, and residue was dissolved in 100 mL of water. Then
it was acidified to pH z 6 with glacial acetic acid and filtered
to give the crude product, which was recrystallized from meth-
anol to afford pure product as a yellowy crystal. Compound 7a
d
8.52 (dd,
J ¼ 6.0 Hz, 1H), 2.68 (s.3H), 7.41e8.23 (m, 4H), 6.89 (dd,
J ¼ 6.0 Hz, 1H), 4.66 (s, 2H), 4.46 (s, 2H); EIMS (probe)
70 eV, m/z (rel. int.): 415 (Mþ, 1), 247 (100), 179 (51), 94
(72). Anal. Calcd for C16H13N7O3S2: C, 46.26; H, 3.15; N,
23.60. Found: C, 46.42; H, 2.97; N, 23.84.
Compound 8d e yield: 90%; yellowy solid, mp 145e
146 ꢁC; 1H NMR (200 MHz, CDCl3): d 8.52 (dd, 1H,
J ¼ 6.0 Hz), 7.22e8.00 (m, 3H), 6.89 (dd, 1H, J ¼ 6.0 Hz),
4.69 (s, 2H), 4.66 (s, 2H), 2.67 (s, 3H), 2.39 (s, 3H); EIMS
(probe) 70 eV, m/z (rel. int.): 430 (7), 429 (Mþ, 1), 247
(100), 179 (67), 135 (46), 94(60). Anal. Calcd for
C17H15N7O3S2: C, 47.54; H, 3.52; N, 22.83. Found: C,
47.28; H, 3.80; N, 22.64.
1
e yield: 96%; mp 202e204 ꢁC; H NMR (400 MHz, DMSO-
d6): d 14.32 (s, 1H), 9.16 (dd, J ¼ 6.8 Hz, 1H), 7.23 (dd,
J ¼ 6.8 Hz, 1H), 4.64 (s, 2H), 2.61 (s, 3H).
Compound 8e e yield: 85%; yellowy solid, mp 124e
125 ꢁC; 1H NMR (200 MHz, CDCl3):
4.2.7. 5-(1-(5,7-Dimethyl-1,2,4-triazolo[1,5-a]pyrimidin-2-
ylthio)ethyl)-1,3,4-oxadiazole-2-thiol 7b
d
8.51 (dd,
J ¼ 6.0 Hz, 1H), 7.14e7.51 (m, 4H), 6.87 (dd, J ¼ 6.0 Hz,
1H), 4.66 (s, 2H), 4.49 (s, 2H), 2.67 (s, 3H); EIMS (probe)
70 eV, m/z (rel. int.): 405 (M þ 1, 9), 247 (100), 179 (72),
134 (25), 94 (69). Anal. Calcd for C16H13ClN6OS2: C,
47.46; H, 3.24; N, 20.76. Found: C, 47.35; H, 3.52; N, 21.02.
Compound 8f e yield: 84%; yellowy solid, mp 122e
This compound was synthesized in 81% yield from 2-(5,7-
dimethyl-1,2,4-triazolo[1,5-a]pyrimidin-2-ylthio)-propanehy-
drazide (6b) using the similar procedure to that described for
7a; mp 198e200 ꢁC; 1H NMR (400 MHz, DMSO-d6): d 14.38
(s, 1H), 7.16 (s, 1H), 5.12 (q, J ¼ 7.2 Hz, 2H), 2.67 (s, 3H),
2.57 (s, 3H), 1.77 (d, J ¼ 7.2 Hz, 3H).
123 ꢁC; 1H NMR (200 MHz, CDCl3):
d
8.51 (dd,
J ¼ 6.0 Hz, 1H), 7.17e7.34 (m, 4H), 6.87 (dd, J ¼ 6.0 Hz,
1H), 4.66 (s, 2H), 4.35 (s, 2H), 2.67 (s, 3H); EIMS (probe)
70 eV, m/z (rel. int.): 405 (M þ 1, 22), 279 (28), 247 (100),
134 (39), 94 (58). Anal. Calcd for C16H13ClN6OS2: C,
47.46; H, 3.24; N, 20.76. Found: C, 47.70; H, 3.04; N, 20.97.
Compound 8g e yield: 85%; yellowy solid, mp 120e
4.3. General procedure for the synthesis of the target
compounds 8aes
Sodium hydroxide (3.3 mmol) was dissolved in 15 mL of
water and 3 mmol of 7a was added. After stirring for
15 min, a solution of 3.3 mmol of substituted benzyl halide
or alkyl halide in 5 mL of ethanol was added dropwise. The
resulted mixture was stirred at room temperature for 2e30 h.
The precipitate was filtered off and recrystallized from petro-
leum ether/acetone to give the pure target compounds 8aes in
yields of 40e90%.
122 ꢁC; 1H NMR (200 MHz, CDCl3):
d
8.51 (dd,
J ¼ 6.0 Hz, 1H), 2.68 (s, 3H), 7.20e7.39 (m, 4H), 6.88 (dd,
1H, J ¼ 6.0 Hz), 4.66 (s, 2H), 4.32 (s, 2H); EIMS (probe)
70 eV, m/z (rel. int.): 449(Mþ, 1), 247 (21), 179 (26), 169
(100), 134 (7), 94 (29). Anal. Calcd for C16H13BrN6OS2: C,
42.77; H, 2.92; N, 18.70. Found: C, 42.96; H, 3.15; N, 18.52.
Compound 8h e yield: 88%; yellowy solid, mp 123e
4.4. General procedure for the synthesis of the target
compounds 9aej
124 ꢁC; 1H NMR (300 MHz, CDCl3):
d
8.57 (dd,
J ¼ 7.2 Hz, 1H), 7.02e7.49 (m, 4H), 6.93 (dd, J ¼ 7.2 Hz,
1H), 4.70 (s, 2H), 4.46 (s, 2H), 2.70 (s, 3H); EIMS (probe)
70 eV, m/z (rel. int.): 389 (27), 388 (Mþ, 8), 247 (90), 134
(26), 109 (100), 94 (68). Anal. Calcd for C16H13FN6OS2: C,
49.47; H, 3.37; N, 21.64. Found: C, 49.21; H, 3.50; N, 21.89.
Target compounds 9aej were synthesized in 59e82%
yield from 7b using the similar procedure to that described
for 8aes.

Q. Chen et al. / European Journal of Medicinal Chemistry 43 (2008) 595e603
601
Compound 8i e yield: 87%; yellowy solid, mp 121e
122 ꢁC; 1H NMR (200 MHz, CDCl3): d 8.52 (dd, 1H,
J ¼ 6.0 Hz), 6.95e7.24 (m, 4H), 6.88 (dd, J ¼ 6.0 Hz, 1H),
4.66 (s, 2H), 4.37 (s, 2H), 2.67 (s, 3H); EIMS (probe)
70 eV, m/z (rel. int.): 388 (Mþ, 1), 247 (48), 179 (25),
109 (100), 94 (28). Anal. Calcd for C16H13FN6OS2: C,
49.47; H, 3.37; N, 21.64. Found: C, 49.70; H, 3.12; N,
21.90.
C11H12N6OS2: C, 42.84; H, 3.92; N, 27.25. Found: C, 43.03;
H, 3.76; N, 27.07.
Compound 8q e yield: 34%; yellowy solid, mp 132e
134 ꢁC; 1H NMR (300 MHz, CDCl3):
d
8.58 (dd,
J ¼ 6.9 Hz, 1H), 6.93 (dd, 1H, J ¼ 6.9 Hz), 4.69 (s, 2H),
3.18 (t, J ¼ 7.2 Hz, 2H), 2.68 (s, 3H), 1.72 (m, J ¼ 7.5 Hz,
2H), 1.43 (m, J ¼ 7.5 Hz, 2H), 0.90 (t, J ¼ 7.2 Hz, 3H);
EIMS (probe) 70 eV, m/z (rel. int.): 336 (Mþ, 2), 247 (100),
179 (35), 135 (19), 94 (34). Anal. Calcd for C13H16N6OS2:
C, 46.41; H, 4.79; N, 24.98. Found: C, 46.54; H, 4.96; N,
25.10.
Compound 8j e yield: 90%; yellowy solid, mp 127e
129 ꢁC; 1H NMR (200 MHz, CDCl3):
d
8.51 (dd,
J ¼ 6.0 Hz, 1H), 6.94e7.35 (m, 4H), 6.87 (dd, J ¼ 6.0 Hz,
1H), 4.66 (s, 2H), 4.36 (s, 2H), 2.68 (s, 3H); EIMS (probe)
70 eV, m/z (rel. int.): 388 (Mþ, 1), 247 (92), 179 (25), 109
(100), 94 (14). Anal. Calcd for C16H13FN6OS2: C, 49.47; H,
3.37; N, 21.64. Found: C, 49.25; H, 3.50; N, 21.81.
Compound 8k e yield: 85%; yellowy solid, mp 94e95 ꢁC;
1H NMR (200 MHz, CDCl3): d 8.50 (dd, J ¼ 6.0 Hz, 1H),
7.03e7.23 (m, 4H), 6.87 (dd, J ¼ 6.0 Hz, 1H), 4.66 (s, 2H),
4.36 (s, 2H), 2.67 (s, 3H), 2.29 (s, 3H); EIMS (probe) 70 eV,
m/z (rel. int.): 384 (Mþ, 1), 280 (2), 247 (11), 179 (13), 138
(2), 134 (7), 125 (2), 120 (11), 105 (100), 94 (29), 77 (12).
Anal. Calcd for C17H16N6OS2: C, 53.11; H, 4.19; N, 21.86.
Found: C, 53.26; H, 4.00; N, 21.53.
Compound 8l e yield: 77%; yellowy solid, mp 89e90 ꢁC;
1H NMR (200 MHz, CDCl3): d 8.51 (dd, J ¼ 6.0 Hz, 1H),
7.12e7.30 (m, 4H), 6.87 (dd, J ¼ 6.0 Hz, 1H), 4.67 (s, 2H),
4.42 (s, 2H), 2.67 (s, 3H), 2.38 (s, 3H); EIMS (probe) 70 eV,
m/z (rel. int.): 384 (Mþ, 1), 247 (42), 105 (100), 94 (15).
Anal. Calcd for C17H16N6OS2: C, 53.11; H, 4.19; N, 21.86.
Found: C, 49.98; H, 4.32; N, 21.98.
Compound 8r e yield: 32%; yellowy solid, mp 149e
150 ꢁC; 1H NMR (300 MHz, CDCl3):
d
8.59 (dd,
J ¼ 6.6 Hz, 1H), 6.94 (dd, J ¼ 6.9 Hz, 1H), 4.72 (s, 2H),
3.70 (q, J ¼ 6.6 Hz, 2H), 2.71 (s, 3H), 1.72 (m, J ¼ 7.5 Hz,
2H), 1.4 (d, J ¼ 7.5 Hz, 2H), 1.02 (t, J ¼ 7.2 Hz, 3H); EIMS
(probe) 70 eV, m/z (rel. int.): 337 (56), 336 (Mþ, 45), 247
(100), 179 (66), 135 (57), 94 (55). Anal. Calcd for
C13H16N6OS2: C, 46.41; H, 4.79; N, 24.98. Found: C, 46.56;
H, 4.53; N, 24.61.
Compound 8s e yield: 73%; yellowy solid, mp 157e
158 ꢁC; 1H NMR (300 MHz, CDCl3):
d
8.59 (dd,
J ¼ 6.6 Hz, 1H), 6.94 (dd, J ¼ 6.9 Hz, 1H), 4.70 (s, 2H),
4.22 (q, J ¼ 7.2 Hz, 2H), 4.02 (s, 2H), 2.70 (s, 3H), 1.27
(t, J ¼ 7.2 Hz, 3H); EIMS (probe) 70 eV, m/z (rel. int.): 366
(Mþ, 24), 279 (34), 247 (100), 135 (29), 134 (46), 94 (42).
Anal. Calcd for C13H14N6O3S2: C, 42.61; H, 3.85; N, 22.94.
Found: C, 42.53; H, 4.02; N, 22.69.
Compound 9a e yield: 82%; white solid, mp 126e128 ꢁC;
1H NMR (400 MHz, CDCl3): d 7.25e7.35 (m, 4H), 6.76 (s,
1H), 5.39 (q, J ¼ 7.2 Hz, 1H), 4.37 (s, 2H), 2.70 (s, 3H),
2.67 (s, 3H), 1.91 (d, J ¼ 7.2 Hz, 3H); EIMS (probe) 70 eV,
m/z (rel. int.): 433 (Mþ, 8), 275 (76), 219 (55), 180 (73),
134 (73), 125 (100), 107 (65), 89 (62). Anal. Calcd for
C18H17ClN6OS2: C, 49.93; H, 3, 96; N, 19.41. Found: C,
49.78; H, 3.85; N, 19.72.
Compound 9b e yield: 80%; white solid, mp 130e132 ꢁC;
1H NMR (400 MHz, CDCl3): d 7.03e7.50 (m, 4H), 6.76 (s,
1H), 5.41 (q, J ¼ 7.2 Hz, 1H), 4.44 (s, 2H), 2.71 (s, 3H),
2.64 (s, 3H), 1.91 (d, J ¼ 7.2 Hz, 3H); EIMS (probe) 70 eV,
m/z (rel. int.): 417 (Mþ, 8), 275 (56), 219 (47), 180 (53),
134 (56), 109 (100), 89 (10). Anal. Calcd for C18H17FN6OS2:
C, 51.91; H, 4.11; N, 20.18. Found: C, 51.76; H, 4.35; N,
20.07.
Compound 9c e yield: 76%; white solid, mp 97e99 ꢁC;
1H NMR (400 MHz, CDCl3): d 6.96e7.40 (m, 4H), 6.77 (s,
1H), 4.39 (s, 2H), 5.40 (q, J ¼ 7.2 Hz, 1H), 2.71 (s, 3H),
2.65 (s, 3H), 1.91 (d, J ¼ 7.2 Hz, 3H); EIMS (probe)
70 eV, m/z (rel. int.): 417 (Mþ, 3), 275 (48), 219 (20),
180 (31), 109 (100), 89 (7). Anal. Calcd for C18H17FN6OS2:
C, 51.91; H, 4.11; N, 20.18. Found: C, 52.12; H, 4.25; N,
20.03.
Compound 8m e yield: 85%; yellowy solid, mp 92e93 ꢁC;
1H NMR (200 MHz, CDCl3): d 8.50 (dd, J ¼ 6.0 Hz, 1H),
7.04e7.22 (m, 4H), 6.87 (dd, J ¼ 6.0 Hz, 1H), 4.67 (s, 2H),
4.36 (s, 2H), 2.67 (s, 3H), 2.31 (s, 3H); EIMS (probe) 70 eV,
m/z (rel. int.): 384 (Mþ, 50), 279 (22), 247 (100), 179 (79),
135 (73), 94 (72). Anal. Calcd for C17H16N6OS2: C, 53.11;
H, 4.19; N, 21.86. Found: C, 53.39; H, 4.50; N, 21.67.
Compound 8n e yield: 58%; yellowy solid, mp 220e
222 ꢁC; 1H NMR (300 MHz, CDCl3):
d
8.58 (dd,
J ¼ 7.5 Hz, 1H), 6.94e8.00 (m, 4H), 6.93 (dd, J ¼ 7.5 Hz,
1H), 4.86 (s, 2H), 4.70 (s, 2H), 3.89 (s, 3H), 2.70 (s, 3H);
EIMS (probe) 70 eV, m/z (rel. int.): 428 (Mþ, 1), 247 (72),
135 (100), 94 (15). Anal. Calcd for C18H16N6O3S2: C,
50.54; H, 3.76; N, 19.61. Found: C, 50.09; H, 4.03; N, 19.36.
Compound 8o e yield: 57%; yellowy solid, mp 108e
109 ꢁC; 1H NMR (300 MHz, CDCl3):
d
8.59 (dd,
J ¼ 7.2 Hz, 1H), 6.93 (dd, J ¼ 7.2 Hz, 1H), 4.70 (s, 2H),
2.71 (s, 3H), 2.68 (s, 3H); EIMS (probe) 70 eV, m/z (rel.
int.): 294 (Mþ, 19), 247 (100), 179 (38), 135 (32), 94 (84).
Anal. Calcd for C10H10N6OS2: C, 40.80; H, 3.42; N, 28.55.
Found: C, 40.53; H, 3.58; N, 28.86.
Compound 8p e yield: 50%; yellowy solid, mp 103e
108 ꢁC; 1H NMR (300 MHz, CDCl3):
d
8.58 (dd,
Compound 9d e yield: 75%; white solid, mp 125e126 ꢁC;
1H NMR (400 MHz, CDCl3): d 7.23e7.40 (m, 4H), 6.76 (s,
1H), 5.40 (q, J ¼ 7.2 Hz, 1H), 4.38 (s, 2H), 2.71 (s, 3H),
2.64 (s, 3H), 1.91 (d, J ¼ 7.2 Hz, 3H); EIMS (probe) 70 eV,
m/z (rel. int.): 433 (Mþ, 1), 219 (3), 180 (6), 134 (5), 125
J ¼ 6.6 Hz, 1H), 6.93 (dd, J ¼ 6.6 Hz, 1H), 4.71 (s, 2H),
3.22 (q, 2H, J ¼ 7.2 Hz), 2.70 (s, 3H), 1.44 (t, 3H,
J ¼ 7.2 Hz); EIMS (probe) 70 eV, m/z (rel. int.):308 (Mþ, 1),
247 (70), 179 (26), 135 (17), 94(21). Anal. Calcd for

602
Q. Chen et al. / European Journal of Medicinal Chemistry 43 (2008) 595e603
(100), 89 (34). Anal. Calcd for C18H17ClN6OS2: C, 49.93; H,
3, 96; N, 19.41. Found: C, 49.69; H, 4.15; N, 19.25.
Compound 9e e yield: 78%; white solid, mp 126e128 ꢁC;
1H NMR (400 MHz, CDCl3): d 7.14e7.36 (m, 4H), 6.76 (s,
1H), 5.42 (q, J ¼ 7.2 Hz, 1H), 4.46 (s, 2H), 2.72 (s, 3H),
2.64 (s, 3H), 2.41 (s, 3H), 1.92 (d, J ¼ 7.2 Hz, 3H); EIMS
(probe) 70 eV, m/z (rel. Int.): 412 (Mþ, 1), 275 (20), 219 (8),
180 (11), 105 (100), 89 (3). Anal. Calcd for C19H20N6OS2:
C, 55.32; H, 4.89; N, 20.37. Found: C, 55.14; H, 4.78; N,
20.56.
Compound 9f e yield: 78%; white solid, mp 132e133 ꢁC;
1H NMR (400 MHz, CDCl3): d 7.09e7.27 (m, 4H), 6.76 (s,
1H), 5.41 (q, J ¼ 7.2 Hz, 1H), 4.39 (s, 2H2), 2.71 (s, 3H),
2.65 (s, 3H), 2.33 (s, 3H), 1.91 (d, J ¼ 7.2 Hz, 3H); EIMS
(probe) 70 eV, m/z (rel. int.): 412 (Mþ, 1), 219 (3), 180 (4),
134 (4), 105 (100), 89 (4); Anal. Calcd for C19H20N6OS2: C,
55.32; H, 4.89; N, 20.37; Found: C, 55.45; H, 4.98; N, 20.27.
Compound 9g e yield: 50%; white solid, mp 121e122 ꢁC;
1H NMR (400 MHz, CDCl3): d 6.94e7.27 (m, 4H), 6.76 (s,
1H), 5.41 (q, J ¼ 7.2 Hz, 1H), 4.40 (s, 2H), 3.80 (s, 3H),
2.71 (s, 3H), 2.64 (s, 3H), 1.91 (d, J ¼ 7.2 Hz, 3H); EIMS
(probe) 70 eV, m/z (rel. int.): 428 (Mþ, 3), 219 (5), 180 (14),
134 (15), 121 (100), 89 (9); Anal. Calcd for C19H20N6O2S2:
C, 53.25; H, 4.70; N, 19.61; Found: C, 53.56; H, 4.98; N,
19.38.
Compound 9h e yield: 80%; white solid, mp 128e129 ꢁC;
1H NMR (400 MHz, CDCl3): d 7.60e8.16 (m, 4H), 6.77 (s,
1H), 5.39 (q, J ¼ 7.2 Hz, 1H), 4.47 (s, 2H), 2.71 (s, 3H),
2.65 (s, 3H), 1.90 (d, J ¼ 7.2 Hz, 3H); EIMS (probe) 70 eV,
m/z (rel. int.):443 (Mþ, 1), 275 (15), 219 (18), 180 (37), 134
(37), 107 (37), 89 (59), 78 (100). Anal. Calcd for
C18H17N7O3S2: C, 48.75; H, 3.86; N, 22.11. Found: C,
48.98; H, 3.75; N, 22.23.
Compound 9i e yield: 59%; white solid, mp 97e99 ꢁC; 1H
NMR (400 MHz, CDCl3): d 7.10e7.29 (m, 4H), 6.76 (s, 1H),
5.40 (q, J ¼ 7.2 Hz, 1H), 4.39 (s, 2H), 2.71 (s, 3H), 2.64 (s,
3H), 2.32 (s, 3H), 1.91 (d, J ¼ 7.2 Hz, 3H); EIMS (probe)
70 eV, m/z (rel. int.): 412 (Mþ, 1), 275 (6), 219 (5), 180
(17), 134 (15), 105 (100), 89 (5). Anal. Calcd for
C19H20N6OS2: C, 55.32; H, 4.89; N, 20.37. Found: C, 55.56;
H, 5.08; N, 20.17.
agar culture medium (9 mL) under 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 me-
dium without sample was used as the blank control. Three rep-
licates of each test were carried out. The mycelial elongation
radius (mm) of fungi settlements was measured after 48 h of
culture. The growth inhibition rates were calculated with the
following equation: I ¼ [(C ꢀ T )/C] ꢂ 100%. Here, I is the
growth inhibition rate (%), C is the control settlement radius
(mm), and T is the treatment group fungi settlement radius
(mm). The molar concentration of each compound required
to inhibit the mycelial growth to half the length of the control
(EC50 value) was evaluated by the probit method [21]. The re-
sults are summarized in Table 2.
4.6. CoMFA analysis
The 3D structures of all compounds were built and mini-
mized by using SYBYL 7.0 on a Silicon Graphics Fuel
workstation. The geometries of all molecules involved in
this study were fully optimized by using the PM3 method.
The lowest-energy conformations were considered as the
bioactive conformations. A useful kind of net atomic charges
called electrostatic potential (ESP)-fitting charges were
derived from the PM3 calculated molecular electrostatic
potential distribution. All the nitrogen and oxygen atoms in
1,3,4-oxadiazole and 1,2,4-triazolo[1,5-a]-pyrimidine rings
were selected as the atoms to superimpose all the compounds
using an atom-by-atom least-squares fit implemented in the
‘‘SYBYL FIT’’ option in SYBYL. Compound 8r with the
best biological activity was selected as the reference molecule.
The CoMFA descriptors, steric and electrostatic field energies,
˚
were calculated using the SYBYL default parameters: 2 A grid
points spacing, an sp3 carbon probe atom with þ1 charge and
a minimum s (column filtering) of 2.0 kcal molꢀ1, and an
energy cutoff of 30.0 kcal molꢀ1
.
Acknowledgments
Compound 9j e yield: 52%; white solid, mp125e126 ꢁC;
1H NMR (400 MHz, CDCl3): d 6.67 (s, 1H), 5.41 (q,
J ¼ 7.2 Hz, 1H), 3.69 (q, J ¼ 6.8 Hz, 1H), 2.73 (s, 3H), 2.64
(s, 3H), 1.93 (d, J ¼ 7.2 Hz, 3H), 1.74 (m, 2H), 1.43 (d,
J ¼ 6.8 Hz, 3H), 1.00 (t, J ¼ 7.2 Hz, 3H); EIMS (probe)
70 eV, m/z (rel. int.): 365 ([M þ 1]þ, 3), 275 (42), 233 (6),
207 (19), 180 (41), 148 (38), 124 (5), 107 (84), 92 (17).
Anal. Calcd for C15H20N6OS2: C, 49.43; H, 5.53; N, 23.06.
Found: C, 49.56; H, 5.25; N, 23.29.
The present work was supported by National ‘‘973’’ Project
(2003CB114400), National NSFC (Nos. 20572030, 20528201,
and 20432010), Key project of Ministry of Education (Nos.
103116 and 104205), and Program for Excellent Research
Group of Hubei Province (No. 2004ABC002).
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