Design, synthesis and fungicidal activity evaluation of novel pyrimidinamine derivatives containing phenyl-thiazole/oxazole moiety

Article abstract of DOI:10.1016/j.bmc.2019.05.029

Diflumetorim is a member of pyrimidinamine fungicides that possess excellent antifungal activities. Nevertheless, as reported that the activity of diflumetorim to corn rust (Puccinia sorghi) was not ideal (EC₅₀ = 53.26 mg/L). Herein, a series of novel pyrimidinamine derivatives containing phenyl-thiazole/oxazole moiety were designed based on our previous study and the structural characteristics of diflumetorim, synthesized and bioassayed to discover novel fungicides with excellent antifungal activities. Among these compounds, T18 gave the optimal fungicidal activity, which respectively offers control effects with EC₅₀ values of 0.93 mg/L against P. sorghi and 1.24 mg/L against E. graminis, significantly superior to commercial fungicides diflumetorim, tebuconazole, and flusilazole. Cell cytotoxicity results suggested that compound T18 has lower toxicities than diflumetorim. Furthermore, DFT calculation indicated that the phenyl-thiazole/oxazole moiety plays an unarguable role in the improvement of activity, which will contribute to designing and developing more potent compounds in the future.

Full text of DOI:10.1016/j.bmc.2019.05.029

Accepted Manuscript
Design, synthesis and fungicidal activity evaluation of novel pyrimidinamine
derivatives containing phenyl-thiazole/oxazole moiety
Zhongzhong Yan, Aiping Liu, Yingcan Ou, Jianming Li, Haibo Yi, Ning Zhang,
Minhua Liu, Lu Huang, Jianwei Ren, Weidong Liu, Aixi Hu
PII:
S0968-0896(19)30182-8
https://doi.org/10.1016/j.bmc.2019.05.029
BMC 14916
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 February 2019
2 May 2019
18 May 2019
Please cite this article as: Yan, Z., Liu, A., Ou, Y., Li, J., Yi, H., Zhang, N., Liu, M., Huang, L., Ren, J., Liu, W.,
Hu, A., Design, synthesis and fungicidal activity evaluation of novel pyrimidinamine derivatives containing phenyl-
thiazole/oxazole moiety, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.
2019.05.029
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Design, synthesis and fungicidal activity evaluation of novel
pyrimidinamine derivatives containing phenyl-thiazole/oxazole
moiety
Zhongzhong Yan,a, b, 1 Aiping Liu,b, c, 1 Yingcan Ou,d Jianming Li,b, c Haibo Yi,a Ning
Zhang, b Minhua Liu,b, c Lu Huang,b, c Jianwei Ren,b, c Weidong Liu,b, c, *Aixi Hu a, **
a College of Chemistry and Chemical Engineering, Hunan University, No.1 Lushan
Road(s),
Changsha, 410082, Hunan Province, People’s Republic of China
bNational Engineering Research Center for Agrochemicals, Hunan Research Institute
of Chemical Industry, Changsha 410007, Hunan Province, People’s Republic of
China
cHunan Province Key Laboratory for Agrochemicals, Changsha 410014, People’s
Republic of China
dSchooll of Medicine, Hunan University of Chinese Medicine, Changsha 410208,
People’s Republic of China
*Corresponding author:
E-mail address: weidliu@yahoo.com (Weidong Liu); axhu@hnu.edu.cn (Aixi Hu)
1
Note: All the authors declare no competing financial interest. Zhongzhong Yan and
Aiping Liu contributed equally.
1

ABSTRACT
Diflumetorim is a member of pyrimidinamine fungicides that possess excellent
antifungal activities. Nevertheless, as reported that the activity of diflumetorim to
corn rust (Puccinia sorghi) was not ideal (EC50 = 53.26 mg/L). Herein, a series of
novel pyrimidinamine derivatives containing phenyl-thiazole/oxazole moiety were
designed based on our previous study and the structural characteristics of
diflumetorim, synthesized and bioassayed to discover novel fungicides with excellent
antifungal activities. Among these compounds, T18 gave the optimal fungicidal
activity, which respectively offers control effects with EC50 values of 0.93 mg/L
against P. sorghi and 1.24 mg/L against E. graminis, significantly superior to
commercial fungicides diflumetorim, tebuconazole, and flusilazole. Cell cytotoxicity
results suggested that compound T18 has lower toxicities than diflumetorim.
Furthermore, DFT calculation indicated that the phenyl-thiazole/oxazole moiety plays
an unarguable role in the improvement of activity, which will contribute to designing
and developing more potent compounds in the future.
Keywords: Diflumetorim; Pyrimidinamine; Antifungal activities; Puccinia sorghi;
DFT calculation
2

1. Introduction
Plant diseases, mostly caused by fungal and bacterial pathogens, lead to severe losses
for agriculture and horticulture crop production worldwide and constitute a great
threat to global food security.1,2 Fungicides are of value for global food security,
attributed to its essential role in controlling the growth and reproduction of plant
pathogens. However, due to the urgent need for currently available fungicides, they
are continuously supplied or even used without adequate restriction, making these
gradually display several drawbacks, such as pesticide related toxicity, severe
pesticide resistance, and serious pesticide interactions;3,4 thus, there is an urgent need
to discover new antifungal agents with high activity, low resistance, and less toxicity.
Because of their outstanding performance in biological activity, pyrimidine
derivatives have been an attractive topic for many years in the fields of medicinal and
agricultural
chemistry,
e.g.
anti-cancer,5,6 anti-HIV,7,8
anti-influenza,9,10
anti-microbial,11,12
insecticides,13,14
herbicides,15,16 fungicides,17,18
etc.
Pyrimidinamines, the main member of pyrimidine derivatives, act as mitochondrial
complex I electron transport inhibitors (MET I). On the basis of the report from the
Fungicide Resistance Action Committee (FRAC) (www.frac.info), at least two-thirds
of the pyrimidine fungicides belong to pyrimidinamine derivatives. Among these,
diflumetorim is the typical pyrimidinamine fungicide targeting MET I, but its
structure is obviously different from commercially available fungicides.19 More
importantly, it has no cross-resistance with the existing conventional fungicides and is
safe to nontarget organisms.20 These properties indicate that diflumetorim may be a
3

promising template for the discovery of new fungicide candidates.
Furthermore, as reported by Guan et al.21,22 that the commercial pyrimidine
fungicide diflumetorim is not ideal for corn rust (Puccinia sorghi) (EC50 = 53.26
mg/L), which leaves us plenty of room to improve its activity. On the other hand, in
our previous work,23 we have designed and synthesized a series of novel
pyrazolecarboxamide derivatives containing thiazole or oxazole ring with a variety of
substitutes at the pyrazole ring and benzene ring; according to our data of fungicidal
activity and structure-activity relationship (SAR), the phenyl-thiazole/oxazole group
turned to be the considerable moiety for improving biological activity. Hence, in this
ongoing study, we performed thorough modification on diflumetorim by bioisosteric
replacement and synthesized a series of novel pyrimidinamine derivatives containing
phenyl-thiazole/oxazole moiety (Fig. 1). The detailed synthesis, bioassay results, and
structure-activity relationships (SAR) of these compounds are discussed below.
Furthermore, density functional theory (DFT) calculation has been performed to
determine the quantum chemical features that influence the fungicidal activity.
Fig 1. Design strategy of the target compounds.
4

2. Results and discussion
2.1. Synthesis
According to the synthetic routes shown in Schemes 1−3, thirty-six pyrimidinamine
derivatives containing phenyl-thiazole/oxazole moieties were synthesized and the
chemical structures of compounds O and T were shown in Table 1. The synthesized
1
compounds were characterized by H NMR, GC/HPLC-MS, and elemental analyses.
All spectral and analytical data were consistent with the assigned structures.
Scheme 1. General synthesis procedure for compound 4.
Scheme 2. General synthesis procedure for target compounds containing phenyloxazoles.
5

Scheme 3. General synthesis procedure for target compounds containing phenylthiazoles.
2.2 Fungicidal activities
The in vitro fungicidal activities of the target compounds were assessed at a
concentration of 25 mg/L, and the results of both compounds O and T series against
Gibberella zeae, Phytophythora capsici, Alternaria alternata, Borrytis cinerea, and
Sclerotonia sclerotiorum are listed in Table 1. The results of preliminary bioassays
indicated that these synthetic compounds had weak to moderate inhibitory ratios
against the test fungi. The results are disappointing since most compounds hold
inhibition rates of less than 50%.
Table 1. Chemical structures and initial fungicidal activities of the target compounds.α
NO.
Average inhibitory ratios / effects (%) β
R1
H
Z
O
S
R2
H
R3
H
R (R4 = H)
CH2CH3
CH2CH3
CH2CH3
R (R4 = CH3)
CH3
Pv
70+
70+
70-
Bc
Pi
70-
70+
70-
Ss
Pr
70-
70+
70+
Ps
Eg
70+
70+
70+
Eg
L1γ
L2γ
L3γ
H
H
H
4-Cl
R1
H
S
H
H
Z
O
O
R2
R3
Gz
Pc
Aa
H
H
41.0 30.6 40.8
0
16.7
95
100
50
O1
O2
H
H
H
CHClCH3
43.6 24.5 20.8 58.6 10.7
85
6

2-CH3
3-CH3
O
O
O
O
O
O
O
O
O
O
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
52.3 24.1 12.5
47.7 13.0 12.5
6.8
0
7.1
4.8
85
90
90
85
75
10
80
85
75
90
90
85
65
95
70
95
95
95
50
90
O3
O4
3-CH3
CH2CH3
CH3
56.8 29.0 34.5 17.4 15.0
O5
4-CH3
40.9
9.3
12.5
0
19.0
15.0
50
O6
4-CH3
CH2CH3
CH3
52.3 14.5 24.1
4.3
O7
4-CH2CH3
2,4-di CH3
2,4-di CH3
2,6-di F
2,6-di F
45.5 24.1 33.3 22.7
47.7 9.3 20.8 9.1
O8
CH3
2.4
O9
CH2CH3
CH3
45.5 27.8 28.8 13.6 31.0
O10
O11
O12
30.6 22.7 25.6
46.3 38.3 12.8
8.5
0
46.2
23.7
CH2CH3
2,6-di F
O
H
H
OCH(CH3)2
40.8 22.7 41.0 33.9 30.8
65
40
O13
2,4-di Cl
2,4-di Cl
H
O
O
O
O
S
S
S
S
S
S
S
S
S
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
CH2CH3
CH3
27.3
7.2
0
0
2.5
33.3
17.5
2.5
20
75
95
95
95
100
75
95
95
50
70
90
90
65
95
85
40
90
75
90
95
90
90
95
95
100
O14
O15
O16
O17
T1
45.5 13.0 12.5
14.5 17.2 51.4
6.8
4.5
CH3
CH3
H
H
CH2CH3
CH2CH3
CHClCH3
CH2CH3
CH3
7.2
17.2 29.7 11.4
H
52.3 56.8 38.6 38.6 12.8
30.8 26.5 6.9 42.9
H
H
0
T2
2-CH3
2-OCH3
2-OCH3
3-CH3
3-CH3
4-CH3
4-CH3
H
63.6 36.2 69.0 47.8 25.0
43.6 26.5 29.2 17.2 42.9
T3
H
T4
H
CH2CH3
CH3
38.5
34.1
0
0
0
0
0
0
9.1
22.7
0
14.3
19.0
19
T5
H
T6
H
CH2CH3
CH3
52.3 27.8
T7
H
58.5 27.7 20.5
41.5 27.7 20.5
5.3
T8
H
CH2CH3
0
5.3
T9
4-CH3
S
H
H
c-Pr
41.5 48.9 48.7
0
7.9
50
90
T10
2,4-di CH3
S
S
S
S
S
S
S
S
S
H
H
H
H
CH3
CH2CH3
CH3
29.4
0
0
0
11.4 15.4
43.6
90
90
75
90
T11
T12
T13
T14
T15
T16
T17
T18
T19
2,4-di CH3
38.2 18.5
0
H
H
H
CH3
CH3
H
51.3 20.4 37.5 41.4 35.5
51.3 20.2 20.8 51.7 46.4
51.5 20.4 33.3 62.1 32.1
80
90
H
CH2CH3
CH3
85
90
H
CH3
CH3
CH3
CH3
CH3
95
85
H
H
CH2CH3
CH2CH3
CH3
51.3
45.5 14.5 34.5
23.5 37.0 10.2 22.7 25.6
7.2 17.2 48.6 11.4 27.5
44.4 31.0 55.0
51
37.5 41.4 46.4
95
80
2-Cl
4-CH3
4-CH3
H
4.3 10.0
30
50
H
90
95
H
CH2CH3
95
40
diflumetorim
tebuconazole
flusilazole
5.7
6.6
75
90
100
100
100
100
95.2
100
100
100
100
82.2 87.9 87.3
100
100
α In vitro (25 mg/L): Gz, Gibberella zeae; Pc, Phytophythora capsici; Aa, Alternaria alternata; Bc,
Borrytis cinerea ; Ss, Sclerotonia sclerotiorum. In vivo (500 mg/L): Ps, Puccinia sorghi; Eg,
β
Erysiphe graminis. In vitro data are given as the mean of triplicate experiments; in vivo date are
given as the mean of twice experiments at this initial screening. Pr, Puccinia recondita; Pi,
Phytophthora infestans; Pv, Plasmopara viticola. γ Chemical structures and fungicidal activities of
compounds L1, L2, L3 are from the patent,24 and “70+” means 70% disease control or higher
while “70-” means disease control less than 70%.
7

The in vivo protective activities of all target compounds against P. sorghi and E.
graminis at a concentration of 500 mg/L were evaluated (Table 1). Most of the
compounds showed desired fungicidal activities though only a few compounds, such
as O10, O15, T17, etc., displayed unsatisfactory activities against P. sorghi or E.
graminis. Compounds T2 exhibited 100% inhibitory effect against P. sorghi,
meanwhile compounds O1and T9 all exhibited 100% control effects against E.
graminis. This indicated that the introduction of phenyl-thiazole/oxazole moiety could
remarkably increase in vivo activities against P. sorghi and E. graminis.
Subsequently, these compounds with good inhibitory effects were chosen for
further experiments in the greenhouse at the concentrations ranging 0.625-200 mg/L
(Table 2). We further assessed the in vivo activities against P. sorghi and E. graminis
for these compounds, and the results indicated most the target compounds exhibited
good or excellent fungicidal activities, especially against P. sorghi. Among these,
compounds O10, O17, T5, T15, T16, and T8 displayed more potent fungicidal
activities against P. sorghi compared with the commercial fungicide diflumetorim and
flusilazole. Compounds O4 and T18 were outstanding inhibitory effects for E.
graminis, which exhibited higher in vivo fungicidal activities than fungicide
flusilazole. It is noteworthy that compounds T18 demonstrated the most potent
bioactivity against P. sorghi and E. graminis than others that included above
commercial fungicides. Thus, T18 could be a promising candidate and warranted
further development.
Table 2. EC50 values of the compounds O and T series
EC50 (mg /L) α
Property
Compound
P. sorghi
95% d β
E. graminis
95% d
ClogP
TPSA
8

5.90
7.97
12.24
3.67
2.07
10.21
nd δ
nd
4.27−8.17
5.61−11.31
8.33−17.98
2.70−4.97
1.57−2.74
7.32−14.24
14.21
nd
9.52−21.22
2.97
3.93
3.37
3.40
3.97
3.42
3.99
3.89
3.80
4.37
3.20
3.78
4.26
4.23
4.80
3.53
4.10
4.19
4.57
4.59
3.62
4.20
4.04
4.61
4.06
4.63
4.43
4.44
5.01
3.86
4.43
4.17
4.75
5.38
4.62
5.19
4.11
3.59
3.96
63.84
63.84
63.84
63.84
63.84
63.84
63.84
63.84
63.84
63.84
63.84
63.84
63.84
63.84
63.84
63.84
63.84
50.70
50.70
50.70
59.94
59.94
50.70
50.70
50.70
50.70
50.70
50.70
50.70
41.91
41.91
50.70
50.70
50.70
50.70
50.70
47.05
50.95
30.72
O1
O2
−
>100
1.29 γ
nd
−
O3
0.97−1.71
O4
−
O5
>20
nd
−
O6
ε
−
−
O7
−
>20
>20
nd
−
O8
>20
1.77
>50
3.83
nd
−
1.35−2.31
−
−
O9
−
O10
O11
O12
O13
O14
O15
O16
O17
T1
>50
>20
nd
−
2.82−5.19
−
−
−
nd
−
nd
−
>20
3.75
1.33
9.27
4.80
nd
−
>20
>100
nd
−
2.75−5.13
1.01−1.76
2.40−4.51
3.38−6.80
−
−
−
>50
nd
−
−
T2
>50
nd
−
T3
5.64
1.84
>20
nd
3.93−8.09
1.40−2.42
−
−
T4
nd
−
T5
>20
nd
−
T6
−
−
T7
4.09
3.95
nd
2.97−5.64
2.88−5.43
−
>20
>20
>20
nd
−
T8
−
T9
−
T10
T11
2.27
3.70
>20
9.52
1.49
1.73
nd
1.71−3.02
2.74−5.04
−
−
3.87
nd
2.92−5.13
T12
T13
T14
T15
T16
T17
T18
T19
diflumetorim
tebuconazole
flusilazole
−
6.55−13.83
1.14−1.94
1.31−2.28
−
nd
−
12.4
nd
8.26−18.62
−
nd
−
0.71−1.21
1.68−3.05
−
0.96−1.61
0.93
2.26
53.26 ζ
2.03
nd
1.24
nd
−
nd
−
−
1.55−2.66
nd
−
1.62
1.23−2.13
β
α Average of three replicates. Confidence limit. γ Promising candidates with EC50 values lower
δ
than the corresponding positive controls are shown in bold red. Not determined. ε No relevant
data. ζ Value comes from references.21, 22
9

2.3 Structure−activity relationship
First, in order to verify the feasibility of our design concept, we simulated the
skeleton of diflumetorim and employed our formerly synthesized compound 7 and the
newly synthesized compound 4 to obtain the target compounds O1-15 (Scheme 1).
Bioactivity feedbacks showed that these compounds have good in vivo activities
against P. sorghi and E. graminis. Unexpectedly, the activity of compound O10
against P. sorghi was better than that of the positive controls diflumetorim and
tebuconazole, meanwhile, the activity against E. graminis of compound O4 was
superior to corresponding positive control flusilazole. With the encouragement of the
results, further structural optimization was in full swing. Next, inspired by the chiral
carbon atom of fungicide diflumetorim, we decided to introduce a chiral center into
the target compound as well. Subsequently, compounds O16 and O17 were
synthesized and tested for their fungicidal activity. Although compound O16 was not
significantly active against both fungi, and compound O17 was also less effective
against E. graminis (inhibitory effect 40%), compound O17 (EC50 = 1.33 mg/L) was
better than O10 (EC50 = 1.77 mg/L) in activity against P. sorghi.
Furthermore, after reviewing our previous work,23 we speculated that the
phenylthiazole moiety might play a better role than phenyloxazole moiety in
improving fungicidal activity. Hence, we synthesized twelve target compounds
(T1-12) containing a phenylthiazole moiety (Scheme 2). The results showed that these
compounds had also high efficacy against the two species of fungi at a concentration
of 500 mg/L. Further activity tests showed that their activity against P. sorghi was
significantly more prominent. However, the most active structures among the twelve
compounds (T5), had no breakthrough in activity. After that, we tried to introduce a
methyl group on the nitrogen atom attached to the pyrimidine ring in the target
10

compounds. Unfortunately, the results were not as expected since the synthesized
compounds (T13, T14) showed a pronounced decline in activity after losing a
hydrogen atom on the nitrogen. At this point, considering the structural features of the
currently optimal one (O17), we have to resume thinking about the chiral center.
Keeping Z = S, R2 = CH3 and R3 = H as constant, compounds T15-19 were
synthesized and screened (Table 2). Bioactivity results showed that most of the
compounds (except T17) gave excellent fungicidal activity, especially against P.
sorghi, in which the compound T18 achieved a major breakthrough in the activity
against two strains. The SAR indicated the small-volume groups at R position on the
pyrimidine ring are more beneficial for the fungicidal bioactivity (CH3 > CH2CH3;
T15 versus T16, T18 versus T19), while the electron-withdrawing substituent at the
ortho-position on the phenyl ring causes a serious decrease in biological activity ( H >
Cl; T16 versus T17).
In general, the comparisons of the EC50 values in Table 2 and the initial in vivo
activities in Table 1 showed that changes in the type and location of substituents had a
decisive influence on biological activity. The comprehensive comparison of structures
and activities of all synthesized compounds led to the following general trends: (1).
The presence of alkyl groups had a great effect on improving the activity against both
of the two fungi, in which some compounds with excellent activity, including the
optimal compounds T18 (R, R1, R2 = CH3), were obtained by optimization with an
alkyl group. (2). The substitution of a single group on the phenyl ring has an
inapparent effect on the activity, but when substituted by two identical substituents on
the phenyl ring, all substituents except the 2,4-(CH3)2 substituent are detrimental to
activity. (3). The introduction of bulky groups (c-Pr, i-Pr, etc.) at R position will lead
to a sharp decrease in bioactivity, regardless of the substituent of the benzene ring. (4).
11

The introduction of chiral center enhanced the activity against P. sorghi (Table 2).
Once the methyl group was introduced at the R2 position, the chiral center was
immediately produced, and the corresponding activity was increased as well. Perhaps
one of the enantiomers contributes more to the activity. (5). The hydrogen on the
secondary amine in these compounds is essential for the activity. When R3 = H was
replaced by R3 = CH3 (T13, T14), their fungicidal activities remarkably impaired. (6).
By supplementing the fungicidal activity of compounds from similar structures in the
patent24 in Table 1, we found that when R4 = CH3, the target compounds have better
effects against E. graminis than R4 = H. Therefore, maintaining R4 = CH3 may play an
important role in improving the activity of target compounds against E. graminis etc.
In addition, since inherently physiochemical and structural properties (i.e.,
permeability and size) play vital roles in pesticide likeness,25 the calculated
octanol-water partition coefficients (ClogP) and topological polar surface areas
(TPSA)
were
extracted
from
online
server
(http://www.molinspiration.com/cgi-bin/properties). As Li et al.26 reported that 90%
of the ClogP values of fungicides range from 1.0 to 6.0 and the mean value of ClogP
for commercial fungicides is 3.3, while the ClogP values of our positive controls
diflumetorim，tebuconazole，and flusilazole ranged from 3.59 to 4.11, which are
higher than the average value of ClogP for commercial fungicides. The optimized
candidates (O4, O10, O17, T5, T15, T16, and T18) with ClogP values ranged from
3.40 to 4.62 are similar to those of positive controls. Besides, the TPSA values of
these compounds containing phenylthiazole moiety were generally lower than that of
the compounds containing phenyloxazole moiety. It was apparent from the data in
Table 2 that the most suitable TPSA value may be 50.70, which was very close to that
of tebuconazole (50.95). Therefore, keeping the ClogP values of the designed
12

compounds ranging from 3.40 to 4.62 and maintaining TPSA values near 50.7 may
help further optimize the structure for compounds with high fungicidal activity.
2.4 Cell cytotoxicity
The effects of the synthesized compounds on cell viability were evaluated in a panel
of A549 (human alveolar adenocarcinoma cell line), together with a healthy
noncancerous 16HBE14o- (human bronchial epithelial cell). Cell viability was
assessed using Cell Counting Kit 8 (CCK 8) as described and diflumetorim as positive
control. The results were summarized in Table 3. As observed from Table 3, for both
cells, the cell viability of phenyloxazoles O4 and O17 at 10 mg/L was significantly
worse than that of phenylthiazoles T15 and T18. Among them, the cell viability of
T18 on both cell lines exceeded 90%, which was higher than the positive control
diflumetorim. When the results were expressed in terms of half-maximal inhibitory
concentration (IC50) that represents the drug concentration required to inhibit cell
growth by 50%, the results still indicated that compound T18 has lower cell
cytotoxicity than others (including diflumetorim) to these two different types of cells.
Thus, compound T18 has an advantage of further development as a candidate
fungicide from the viewpoint of cytotoxicity.
Table 3. In vitro cytotoxicity activity of some potential compounds.
A549
Cell viability (%) α
16HBE14o-
Compounds
IC50
(μg/mL)
10.88
12.58
23.47
32.82
28.91
Cell viability (%) α
IC50 (μg/mL)
45.4
59.3
78.7
91.6
78.8
9.14
14.36
23.35
32.78
24.11
52.0
56.9
79.3
90.4
84.9
O4
O17
T15
T18
diflumetorim
α Cell viability is the average of three replicates at 10 μg/mL.
2.4 DFT calculation
13

Molecular total energy and frontier orbital energy levels of compound T18 are listed
in Table 4. Since T18 contained chiral centers, its R and S configurations were
optimized simultaneously. The results of geometric optimization and frequency
calculation shows that the R-configuration of T18 possesses lower total molecular
energy and a larger energy gap of frontier molecular orbital than S-configuration,
indicating that (R)-T18 may be more stable than (S)-T18.
Table 4. Total energy and frontier orbital energy of T18.
DFT α
(R)-T18
(S)-T18
−1775.079873
Etotal / Eh
−1775.08115
EHOMO / Eh
ELUMO / Eh
ΔE / Eh
− 0.226659
− 0.063580
0.163079
−0.228933
−0.067768
0.161165
α Eh : Hartree = 4.35974417×10-18 J =27.2113845 eV; ΔE = ELUMO − EHOMO
.
According to the frontier molecular orbital theory, the characteristics of the HOMO
and LUMO are two of the most important factors that affect bioactivity.27−29 The
HOMO will donate electrons, whereas the LUMO can accept electrons first, hence
studying the frontier molecular orbit (FMO) in this way can provide useful
information about the biological mechanism of a compound. From Fig. 2, it is clear
that the HOMO and LUMO of (R)-T18 are both mainly located on the phenylthiazole
moiety, and the LUMO of (S)-T18 is located on the same structural fragment as well;
whereas the HOMO of (S)-T18 is located across the whole molecule. According to
the distribution patterns of FMO, we note that there may exist a correlation with the
HOMO and LUMO shifting toward phenyl-thiazole/oxazole moieties, away from
pyrimidine ring, and the fungicidal activity. These results indicate that the
phenylthiazole moiety is critical to the biological activity.
14

Fig. 2 The HOMO and LUMO distribution patterns of the compound T18.
Electrostatic potential (ESP) analysis on a molecular van der Waals (vdW) surface
is critical for studying and predicting intermolecular interaction.30,31 Besides, by
determining the ESP surface, we can ascertain the spatial regions in the molecular
structure at which the molecular electrostatic potential is negative or positive, which
helps visualize charged regions of a molecule and is qualitatively useful in the
analysis of electrostatic interactions between the receptor and the title compounds.32
Therefore, lead compound diflumetorim, representative compound O1, and (R)-T18
were selected to conduct ESP analysis. As shown in Fig. 3, the calculated ESP
surfaces depict the size, configuration and charge density of (S)-diflumetorim (Fig.
3A), (R)-diflumetorim (Fig. 3B), O1 (Fig. 3C), and (R)-T18 (Fig. 3D), respectively.
Obviously, the variation of compound geometry may be an important factor leading to
the difference of fungicidal activity. Furthermore, the negative potentials around the
nitrogen atoms on the pyrimidine ring may play a key role in the interaction of the
15

compounds with the receptor. Meanwhile, the positive potentials around the hydrogen
on the secondary amine nitrogen atom of these compounds also have a non-negligible
contribution in the interaction with negative potentials of the acceptor. Additionally,
as observed in Fig. 3C and Fig. 3D, the thiazole moiety can increase the negative
potential of the molecule, meanwhile, the benzene ring moiety helps to enlarge the
positive potential area of the molecule in the region, which benefits interaction with
the potential receptors and improves activity.
Fig. 3 (A). ESP-mapped molecular vdW surface of (S)-diflumetorim; (B). ESP-mapped molecular
vdW surface of (R)-diflumetorim; (C). ESP-mapped molecular vdW surface of compound O1; (D).
ESP-mapped molecular vdW surface of compound (R)-T18. The local maxima and minima of
some vital electrostatic potentials were marked with calculated data.
2.5. Conclusion
In summary, we could conclude that T18, as a potent fungicide candidate, was the
optimal structure with desired fungicidal activity against P. sorghi and E. graminis. It
respectively offered control with EC50 values of 0.93 mg/L against P. sorghi and 1.24
mg/L against E. graminis, significantly superior to commercial fungicides
diflumetorim, tebuconazole, and flusilazole. DFT calculation indicates that the
16

phenyl-thiazole/oxazole moiety played a critical role in the improvement of these
activities, which provided meaningful clues as to the structural features of this new
family of fungicides. Further synthesis of analogs, structure optimization studies,
environmental toxicity tests, and even resolution of enantiomers of compound T18 or
synthesis of (R)-T18 from raw materials in the R configuration are in progress.
3. Expreiment section
3.1 Materials and instrumentation
All of the starting materials and reagents were commercially available and used without
further purification. Melting points were determined on a WPS-1B melting point
apparatus. 1H NMR spectra were recorded in CDCl3 with a Varian INOVA-300, using
TMS as the internal standard. Mass spectras were acquired using an Agilent 5973-6890
gas chromatography-mass spectrometer (GC-MS) and an Agilent 1100 series Liquid
Chromatography-Mass Spectrometer (LC-MS). Fixed mass percentages of elements in
all compounds were performed on a CHN-O-Rapid instrument. Column
chromatography was performed using 200-300 mesh silica gel. Anhydrous sodium
sulfate was used to dry all organic extracts.
3.2 Chemical synthesis
3.2.1 General procedure for the synthesis of compound 2.
Compound 1 (100 mmol) was placed in a round bottom flask and dissolved in 100 mL
dichloromethane, before a mixture of sulfonyl chloride (120 mmol, 16.2 g) and 50 mL
dichloromethane was slowly added by dropping funnel with stirring at −5 to 0 ℃.
Subsequently, the mixture was stirred for 10 h at the same temperature until the
starting material was almost consumed. The reacting was treated with water and then
extracted with dichloromethane twice. The organic layers were combined, and
17

concentrated under reduced pressure to obtain compound 2 in a 89−95% yield.
3.2.2 General procedure for the synthesis of compound 3.
The acetamidine hydrochloride (85 mmol) was dissolved with methanol, and then a
solution of sodium methoxide (80 mmol, 15.3 g, 30 wt.%) was added by dropping
funnel. After that, the mixture was stirred at room temperature for 30 minutes.
Compound 2 (85 mmol) was added by dropping funnel and the reaction mixture was
stirred at −5 to 0 ℃ for 30 minutes. The mixture was heated to reflux for 3 h before it
was cooled to room temperature. The reaction mixture was filtered and the filtrate was
concentrated under reduced pressure. Recrystallization with ethyl alcohol yielded a
purified solid in a 53−60% yield.
3.2.3 General procedure for the synthesis of compound 4.
Compound 3 (45 mmol) was dissolved in a small amount of toluene in 250 mL round
bottom flask, phosphorus oxychloride (135 mmol, 20.7 g) was added to the reaction
mixture by a dropping funnel, maintained at −5 to 0 ℃, and then triethylamine (90
mmol, 9.1 g) was added slowly. The mixture was stirred for 12 h at room temperature
before it was added to crushed ice dropwise under stirring. Potassium carbonate was
added to pH 9-10, and the reaction mixture was extracted with ethyl acetate three
times (3×60 mL). The combined organic layers were concentrated under reduced
pressure to to obtain compound 4 in a 42−55% yield (Scheme 1).
3.2.4 General synthesis procedure for target compounds O1-17.
Compound 5 was synthesized as described.33 Compounds 6 and 7 were synthesized in
our previous work.34 Compounds 4 (5 mmol), 7 (5 mmol) and anhydrous potassium
carbonate (5 mmol, 0.69 g) were added to a mixed solvent of ethyl alcohol (10 mL)
18

and water (10 mL), and then refluxed for 2−4 h. The progress was monitored by TLC.
After the reaction was complete, the reaction solution was poured into saturated saline
and extracted with ethyl acetate (3×80 mL). The extract was dried, filtered, and the
solvent was removed under reduced pressure to give a crude product.
Recrystallization from the mixed solvent of petroleum ether and ethyl acetate gave
1
pure target compounds O1-17 (Scheme 2). The yields, physical properties, and H
NMR, GC/HPLC-MS, elemental analyses of the target compounds are as follows:
1
Data for O1: yield, 75%; purity, 95.5%; yellow solid; mp, 101.9−103.9°C. H NMR
(CDCl3) δ 2.450 (s, 3H, CH3), 2.531 (s, 3H, CH3), 4.663 (q, J = 0.6 Hz, 2H, CH2),
5.899 (s, 1H, NH), 7.443−7.491 (m, 3H, Ph H), 7.664 (t, J = 0.9 Hz, 1H, oxzole H),
8.003−8.049 (m, 2H, Ph H); GC-MS M+ = 314, base peak 182. Anal. Calcd(%) for
C16H15ClN4O: C, 61.05; H, 4.80; N, 17.80. Found: C, 61.07; H, 4.79; N, 17.83.
1
Data for O2: yield, 78%; purity, 93.2%; white solid; mp, 140.0−141.1 °C. H NMR
(CDCl3) δ 1.810 (d, J=6.6 Hz, 3H, CH3), 2.564 (s, 3H, CH3), 4.668 (dd, J = 0.6, 5.4
Hz, 2H, CH2), 5.356 (q, J = 6.6 Hz, 1H, CH), 5.972 (br, 1H, NH), 7.442−7.495 (m,
3H, Ph H), 7.671 (t, J = 0.9 Hz, 1H, oxazole H), 8.006−8.047 (m, 2H, Ph H); GC-MS
M+ = 362, base peak 326. Anal. Calcd(%) for C17H16Cl2N4O: C, 56.21; H, 4.44; N,
15.42. Found: C, 56.20; H, 4.43; N, 15.39.
1
Data for O3: yield, 81%; purity, 99.5%; white solid; mp, 120.2−121.3 °C. H NMR
(CDCl3) δ 2.443 (s, 3H, CH3), 2.525 (s, 3H, CH3), 2.681 (s, 3H, CH3), 4.669 (dd, J =
1.2, 5.1 Hz, 2H, CH2), 5.912 (bs, 1H, NH), 7.262 (s, 1H, oxazole H), 7.277−7.978 (m,
4H, Ph H); HPLC-MS Pos[M+1]+ = 329. Anal. Calcd(%) for C17H17ClN4O: C, 62.10;
H, 5.21; N, 17.04. Found: C, 62.05; H, 5.24; N, 17.04.
19

1
Data for O4: yield, 82%; purity, 98.5%; white solid; mp, 103.1−103.9 °C. H NMR
(CDCl3) δ 2.414 (s, 3H, CH3), 2.425 (s, 3H, CH3), 2.508 (s, 3H, CH3), 4.659 (dd, J =
5.7, 3.9 Hz, 2H, CH2), 5.829 (br, 1H, NH), 7.251−7.370 (m, 2H, Ph H), 7.265 (s, 1H,
oxazole H), 7.643 (dd, J = 8.4, 0.9 Hz, 1H, Ph H), 7.804 (m, J = 7.5 Hz, 1H, Ph H);
GC-MS M+ = 328, base peak 328. Anal. Calcd(%) for C17H17ClN4O: C, 62.10; H, 5.21;
N, 17.04. Found: C, 62.12; H, 5.23; N, 17.01.
1
Data for O5: yield, 79%; purity, 99.1%; faint yellow solid; mp, 63.8−66.0 °C. H
NMR (CDCl3) δ 1.253 (t, J = 7.5 Hz, 3H, CH3), 2.417 (s, 3H, CH3), 2.526 (s, 3H,
CH3), 2.750 (q, J = 7.5 Hz, 2H, CH2), 4.656 (dd, J = 0.6, 5.4 Hz, 2H, CH2), 5.858 (bs,
1H, NH), 7.802 (s, 1H, oxazole H), 7.278−7.873 (m, 5H, Ph H); GC-MS M+ = 342.
base peak 342. Anal. Calcd(%) for C18H19ClN4O: C, 63.06; H, 5.59; N, 16.34. Found:
C, 63.06; H, 5.59; N, 16.34.
1
Data for O6: yield, 82%; purity, 98.3%; white solid; mp, 145.2−146.6 °C. H NMR
(CDCl3) δ 2.405 (s, 3H, CH3), 2.440 (s, 3H, CH3), 2.520 (s, 3H, CH3), 4.647 (dd, J =
1.2, 5.7 Hz, 2H, CH2), 5.861 (bs, 1H, NH), 7.262 (s, 1H, oxazole H), 7.625−7.928 (m,
4H, Ph H); HPLC-MS Pos [M+1]+ = 329. Anal. Calcd(%) for C17H17ClN4O: C, 62.10;
H, 5.21; N, 17.04. Found: C, 62.20; H, 5.19; N, 17.09.
1
Data for O7: yield, 80%; purity, 99.2%; white solid; mp, 82.3−83.1 °C. H NMR
(CDCl3) δ 1.210 (t, J = 7.5 Hz, 3H, CH3), 2.404 (s, 3H, CH3), 2.524 (s, 3H, CH3),
2.773 (q, J = 7.5 Hz, 2H, CH2), 4.646 (dd, J = 5.4, 0.9 Hz, 2H, CH2), 5.855 (bs, 1H,
NH), 7.637 (s, 1H, oxazole H), 7.250−7.928 (m, 4H, Ph H); HPLC-MS Pos [M+1]+ =
20

343. Anal. Calcd(%) for C18H19ClN4O: C, 63.06; H, 5.59; N, 16.34. Found: C, 63.01;
H, 5.61; N, 16.32.
1
Data for O8: yield, 72%; purity, 99.4%; yellow solid; mp, 101.4−103.9 °C. H NMR
(CDCl3) δ 1.237−1.336 (m, 5H, CH2CH3), 2.426 (s, 3H, CH3), 2.509 (s, 3H, CH3),
2.660 (d, J = 7.5 Hz, 2H, CH2), 2.660 (dd, J = 5.7, 0.6 Hz, 2H, CH2), 5.854 (br, 1H,
NH),7.266 (d, J = 8.1 Hz, 2H, Ph H), 7.917 (dd, J = 6.6, 1.6 Hz, 2H, Ph H); GC-MS
M+ = 342, base peak 342. Anal. Calcd(%) for C18H19ClN4O: C, 63.06; H, 5.59; N,
16.34. Found: C, 63.12; H, 5.57; N, 16.31.
1
Data for O9: yield, 75%; purity, 96.3%; white solid; mp, 113.7−116.4 °C. H NMR
(CDCl3) δ 2.362 (s, 3H, CH3), 2.480 (s, 3H, CH3), 2.518 (s, 3H, CH3), 2.642 (s, 3H,
CH3), 4.666 (dd, J = 5.2, 1.6 Hz, 2H, CH2), 5.889 (br, 1H, NH), 7.078 (s, 1H, oxazole
H), 7.102 (s, 1H, Ph H), 7.646 (s, 1H, Ph H), 7.856 (s, 1H, Ph H); GC-MS M+ = 342,
base peak 342. Anal. Calcd(%) for C18H19ClN4O: C, 63.06; H, 5.59; N, 16.34. Found:
C, 63.10; H, 5.56; N, 16.31.
1
Data for O10: yield, 78%; purity, 95.8%; faint yellow solid; mp, 80.8−83.8 °C. H
NMR (DMSO-d6) δ 1.245 (t, J = 7.5 Hz, 3H, CH3), 2.313 (s, 3H, CH3), 2.357 (s, 3H,
CH3), 2.566 (s, 3H, CH3), 2.651(q, J = 7.5 Hz, 2H, CH2), 4.532 (dd, J = 5.1, 0.9 Hz,
2H, CH2), 6.028 (br, 1H, NH), 7.165 (s, 1H, oxazole H), 7.572 (t, J = 5.7 Hz, 1H, Ph
H), 7.670 (d, J = 7.8 Hz, 1H, Ph H), 7.941(s, 1H, Ph H); GC-MS M+ = 356, base peak
356. Anal. Calcd(%) for C19H21ClN4O: C, 63.95; H, 5.93; N, 15.70. Found: C, 63.92;
H, 5.90; N, 15.81.
21

1
Data for O11: yield, 71%; purity, 94.1%; yellow solid; mp, 176.8−178.1°C. H NMR
(CDCl3) δ 2.445 (s, 3H, CH3), 2.531 (s, 3H, CH3), 4.717 (q, J = 0.9 Hz, 2H, CH2),
5.941(bs, 1H, NH), 6.995−7.092 (m, 2H, Ph H), 7.393−7.489 (m, 1H, Ph H) , 7.799 (s,
1H, oxzole H); GC-MS M+ = 350, base peak 209. Anal. Calcd(%) for C16H13ClF2N4O:
C, 54.79; H, 3.74; N, 15.97. Found: C, 54.82; H, 3.72; N, 15.93.
1
Data for O12: yield, 69%; purity, 95.7%; faint yellow solid; mp, 104.2−105.1 °C. H
NMR (CDCl3) δ 1.213 (t, J = 7.8 Hz, 3H, CH3), 2.535 (s, 3H, CH3), 2.730 (q, J = 7.8
Hz, 2H, CH2), 4.715 (dd, J = 5.7, 0.9 Hz, 2H, CH2), 5.910 (br, 1H, NH), 7.008−7.087
(m, 2H, Ph H), 7.386−7.483 (m, 1H, Ph H),7.796 (t, J = 0.9 Hz, 1H, oxazole H);
GC-MS M+ = 364, base peak 364. Anal. Calcd(%) for C17H15ClF2N4O: C, 55.98; H,
4.14; N, 15.36. Found: C, 55.96; H, 4.11; N, 15.39.
1
Data for O13: yield, 72%; purity, 97.9%; yellow solid; mp, 73.6−75.1 °C. H NMR
(CDCl3) δ 1.335 (t, J = 3.0 Hz, 6H, 2CH3), 2.386 (s, 3H, CH3), 4.702 (q, J = 0.6Hz,
2H, CH2), 5.163 (m, 1H, CH), 5.902 (s, 1H, NH), 7.010−7.090 (m, 2H, Ph H),
7.392−7.489 (m, 1H, Ph H), 7.757 (s, 1H, oxzole H); GC-MS M+ = 394, base peak
166. Anal. Calcd(%) for C18H17ClF2N4O2: C, 57.07; H, 4.52; N, 14.79. Found: C,
57.15; H, 4.49; N, 14.83.
1
Data for O14: yield, 70%; purity, 98.0%; off-white solid; mp, 145.2−146.9 °C. H
NMR (CDCl3) δ 2.459 (s, 3H, CH3), 2.537 (s, 3H, CH3), 4.691 (dd, J = 0.9, 5.7 Hz,
2H, CH2), 5.919 (bs, 1H, NH), 7.529 (s, 1H, oxazole H), 7.332−7.949 (m, 3H, Ph H);
HPLC-MS Pos [M+1]+ = 384. Anal. Calcd(%) for C16H13Cl3N4O: C, 50.09; H, 3.42;
N, 14.60. Found: C, 50.13; H, 3.46; N, 14.57.
22

1
Data for O15: yield, 79%; purity, 98.8%; white solid; mp, 86.8−88.9 °C. H NMR
(CDCl3) δ 1.211 (t, J = 7.5 Hz, 3H, CH3), 2.523 (s, 3H, CH3), 2.772 (q, J = 7.5 Hz, 2H,
CH2), 4.682 (dd, J = 0.9, 5.7 Hz, 2H, CH2), 5.866 (bs, 1H, NH), 7.267 (s, 1H, oxazole
H), 7.327−7.950 (m, 3H, Ph H); HPLC-MS Pos[M+1]+ = 397. Anal. Calcd(%) for
C17H15Cl3N4O: C, 51.34; H, 3.80; N, 14.09. Found: C, 51.54; H, 3.92; N, 14.11.
3.2.5 General procedure for the synthesis of compound 8.
Compound 5 (50 mmol) and 1-bromobutane-2,3-dione (8.25 g, 50 mmol) were
dissolved in a mixture of 100 mL tetrahydrofuran and 100 mL ethanol and refluxed
for 5 h with stirring. The reaction mixture was concentrated and extracted with ethyl
acetate (3×100 mL). The combined organic layers were washed with water (100 mL)
and saturated brine (100 mL), filtered and concentrated under reduced pressure. The
crude product was recrystallized with petroleum ether and ethyl acetate yielding
compound 8 in a 72−85% yield.
3.2.6 General procedure for the synthesis of compound 9.
A mixture of compound 8 (30 mmol), hydroxylamine hydrochloride (45 mmol, 3.12
g), sodium acetate (75 mmol, 6.15 g), 40 mL ethyl alcohol and 60 mL water was
placed in 250 mL round-bottomed flask and heated to reflux. The progress was
monitored by TLC. After the reaction was complete, the reaction mixture was
concentrated and extracted with ethyl acetate (3×80 mL). The combined organic
layers were washed with water (100 mL) and saturated brine (100 mL), filtered and
concentrated under reduced pressure. Recrystallization with ethyl alcohol yielded
purified solid in a 88−95% yield.
23

3.2.7 General procedure for the synthesis of compound 10.
Zinc dust (4.29 g, 66 mmol) was added to a solution of compound 9 (22 mmol) in 50
mL formic acid (50 mL), and then the reaction mixture was stirred at room
temperature overnight. Subsequently, the reaction mixture was diluted with methanol
and filtered. The filtrate was concentrated in vacuo, and the resulting residue was
dissolved with 100 mL ethyl acetate. The residual acid was neutralized with saturated
a sodium bicarbonate solution. The organic layer was washed with water (100 mL)
and saturated brine (100 mL), dried, filtered, and concentrated under reduced pressure.
The crude product was recrystallized with petroleum ether and ethyl acetate to obtain
compound 10 in a 75−86% yield.
3.2.8 General synthesis procedure for target compounds O16 and O17.
Compounds O16-17 were obtained in the same manner as described for the above
1
O1-15. The yields, physical properties, and H NMR, HPLC-MS, elemental analyses
of them are as follows:
1
Data for O16: yield, 78%; purity, 92.2%; faint yellow solid; mp, 94.9−97.1 °C. H
NMR (CDCl3) δ 1.627 (d, J = 6.9 Hz, 3H, CH3), 2.433 (s, 3H, CH3), 2.659 (s, 3H,
CH3), 5.455 (q, J = 6.6 Hz, 1H, CH), 5.967 (bs, 1H, NH), 7.446−7.472 (m, 3H, Ph H),
7.606 (s, 1H, oxazole H ), 8.021−8.053 (m, 2H, Ph H); HPLC-MS Pos [M+1]+ = 328.
Anal. Calcd(%) for C17H17ClN4O: C, 62.10; H, 5.21; N, 17.04. Found: C, 62.15; H,
5.18; N, 17.11.
1
Data for O17: yield, 82%; purity, 99.1%; white solid; mp, 101.1−103.7°C. H NMR
(CDCl3) δ 1.222 (t, J = 4.5 Hz, 3H, CH3), 1.621 (d, J = 6.6 Hz, 3H, CH3), 2.506 (s, 3H,
CH3), 2.718 (q, J = 7.2 Hz, 2H, CH2), 5.440 (q, J = 7.2 Hz, 1H, CH), 5.782 (d, J = 7.5
Hz, 1H, NH), 7.446−7.478 (m, 3H, Ph H), 7.598 (s, 1H, oxazole H ), 8.025−8.061 (m,
2H, Ph H); HPLC-MS Pos[M+1]+ = 342. Anal. Calcd(%) for C18H19ClN4O: C, 63.06;
H, 5.59; N, 16.34. Found: C, 63.11; H, 5.63; N, 16.24.
24

3.2.9 General synthesis procedure for target compounds T1-12.
Compounds 11, 12, and 13 were prepared according to the method of our privious
report.23 Compounds T1-12 were synthesized in the same method as described for the
above O1-19 (Scheme 3). The yields, physical properties, and 1H NMR,
GC/HPLC-MS, elemental analyses of the target compounds are as follows:
Data for T1: yield, 81%; purity, 97.0%; yellow solid; 71.4−73.1 °C 1H NMR (CDCl3)
δ 1.224 (t, J = 7.5 Hz, 3H, CH3), 2.533 (s, 3H, CH3), 2.744 (q, J = 7.5 Hz, 2H, CH2),
4.841 (dd, J = 0.6, 5.7 Hz, 2H, CH2), 6.095 (bs, 1H, NH), 7.183 (s, 1H, thiazole H),
7.408−7.490 (m, 3H, Ph H), 7.924−7.968 (m, 2H, Ph H); GS-MS M+ = 344, base peak
309. Anal. Calcd(%) for C17H17ClN4S: C, 59.21; H, 4.97; N, 16.25. Found: C, 59.31;
H, 4.88; N, 16.35.
1
Data for T2: yield, 79%; purity, 98.8%; yellow solid; mp, 124.5−125.5 °C. H NMR
(CDCl3) δ 1.817 (d, J = 6.9 Hz, 3H, CH3), 2.561 (s, 3H, CH3), 4.843 (dd, J = 5.4, 0.3
Hz, 2H, CH2), 5.368 (q, J = 6.6 Hz, 1H, CH), 6.161 (bs, 1H, NH), 7.189 (s,1H,
thiazole H), 7.424−7.493 (m, 3H, Ph H), 7.922−7.966 (m, 2H, Ph H); GC-MS M+ =
378, base peak 342. Anal. Calcd(%) for C17H16Cl2N4S: C, 53.83; H, 4.25; N, 14.77.
Found: C, 53.86; H, 4.23; N, 14.79.
1
Data for T3: yield, 76%; purity, 99.1%; yellow solid; mp, 71.0−73.1 °C. H NMR
(CDCl3) δ 1.229 (t, J = 7.5 Hz, 3H, CH3), 2.409 (s, 3H, CH3), 2.556 (s, 3H, CH3),
2.602 (s, 3H, CH3), 2.812 (q, J = 7.8 Hz, 2H, CH2), 4.856 (dd, J = 1.2, 5.4 Hz, 2H,
CH2), 6.212 (bs, 1H, NH), 7.539 (s, 1H, thiazole H), 7.301−8.008 (m, 4H, Ph H);
GC-MS M+ = 358, base peak 323. Anal. Calcd(%) for C18H19ClN4S: C, 60.24; H, 5.34;
N, 15.61. Found: C, 60.31; H, 5.24; N, 15.58.
1
Data for T4: yield, 80%; purity, 98.2%; yellow solid; mp, 146.1−146.6 °C. H NMR
(CDCl3) δ 2.452 (s, 3H, CH3), 2.524 (s, 3H, CH3), 4.031 (s, 3H, CH3), 4.847 (dd, J =
5.4, 0.6 Hz, 2H, CH2), 6.151 (bs, 1H, NH), 7.028−7.130 (m, 2H, Ph H), 7.219 (s, 1H,
thiazole H), 7.379−7.436 (m, 1H, Ph H), 8.380−8.412 (m, 1H, Ph H); GC-MS M+ =
360, base peak 325. Anal. Calcd(%) for C17H17ClN4OS: C, 56.58; H, 4.75; N, 15.53.
25

Found: C, 56.63; H, 4.78; N, 15.54.
1
Data for T5: yield, 83%; purity, 96.2%; white solid; mp, 123.7−124.3 °C. H NMR
(CDCl3) δ 1.231 (t, J = 7.5 Hz, 3H, CH3), 2.544 (s, 3H, CH3), 2.781 (d, J = 7.5, 2H,
CH2), 4.033 (s, 3H, CH3), 4.852 (d, J = 5.4 Hz, 2H, CH2), 6.171 (bs, 1H, NH),
7.032−7.130 (m, 2H, Ph H), 7.229 (s, 1H, thiazole H), 7.380−7.438 (m, 1H, Ph H),
8.381−8.413 (m, 1H, Ph H); GC-MS M+ = 376, base peak 339. Anal. Calcd(%) for
C18H19ClN4OS: C, 57.67; H, 5.11; N, 14.95. Found: C, 57.55; H, 5.21; N, 14.96.
1
Data for T6: yield, 76%; purity, 99.6%; faint yellow solid; mp, 86.8−87.3 °C. H
NMR (CDCl3) δ 2.425 (s, 3H, CH3), 2.437 (s, 3H, CH3), 2.507 (s, 3H, CH3), 4.845
(dd, J=5.4, 0.9 Hz, 2H, CH2), 6.044 (br, 1H, NH), 7.165 (s, 1H, thiazole H),
7.233−7.358 (m, 2H, Ph H), 7.717−7.772 (m, 2H, Ph H); GC-MS M+ = 344, base
peak 344. Anal. Calcd(%) for C17H17ClN4S: C, 59.21; H, 4.97; N, 16.25. Found: C,
59.22; H, 4.98; N, 16.34.
1
Data for T7: yield, 73%; purity, 99.2%; white solid; mp, 61.4−63.6 °C. H NMR
(CDCl3) δ 1.242 (t, J = 7.5 Hz, 3H, CH3), 2.425 (s, 3H, CH3), 2.517 (s, 3H, CH3),
2.765 (q, J = 7.5 Hz , 2H, CH2), 4.846 (dd, J = 5.4, 0.6 Hz, 2H, CH2), 6.028 (br, 1H,
NH), 7.168 (s, 1H, thiazole H), 7.232−7.358 (m, 2H, Ph H), 7.720−7.777 (m, 2H, Ph
H); GC-MS M+ = 358, base peak 323. Anal. Calcd(%) for C18H19ClN4S: C, 60.24; H,
5.34; N, 15.61. Found: C, 60.31; H, 5.28; N, 15.66.
1
Data for T8: yield, 77%; purity, 98.8%; yellow solid; mp, 116.9−119.0 °C. H NMR
(CDCl3) δ 2.400 (s, 3H, CH3), 2.443 (s, 3H, CH3), 2.514 (s, 3H, CH3), 4.822 (dd, J =
5.4, 0.9 Hz, 2H, CH2), 6.058 (br, 1H, NH), 7.129 (t, J = 0.9 Hz, 1H, thiazole H), 7.227
(d, J = 8.4 Hz, 2H, Ph H), 7.814 (d, J = 8.1 Hz, 2H, Ph H); GS-MS M+ = 344, base
peak 309. Anal. Calcd(%) for C17H17ClN4S: C, 59.21; H, 4.97; N, 16.25. Found: C,
59.14; H, 4.98; N, 16.32.
1
Data for T9: yield, 80%; purity, 99.8%; yellow solid; mp, 80.9−81.8 °C. H NMR
(CDCl3) δ 1.228 (t, J = 7.5 Hz, 3H, CH3), 2.403 (s, 3H, CH3), 2.542 (s, 3H, CH3),
2.755 (q, J = 7.5 Hz, 2H, CH2), 4.830 (dd, J = 5.4, 0.6 Hz, 2H, CH2), 6.120 (br, 1H,
NH), 7.143 (t, J = 0.9 Hz, 1H, thiazole H), 7.237 (d, J = 8.1 Hz, 2H, Ph H), 7.817 (d,
26

J = 8.4 Hz, 2H, Ph H); GC-MS M+ = 358, base peak 323. Anal. Calcd(%) for
C18H19ClN4S: C, 60.24; H, 5.34; N, 15.61. Found: C, 60.31; H, 5.34; N, 15.62.
1
Data for T10: yield, 76%; purity, 98.8%; yellow solid; mp, 89.9−91.2 °C. H NMR
(CDCl3) δ 0.074−0.999 (m, 2H, CH2), 1.104−1.152 (m, 2H, CH2), 2.265−2.365 (m,
1H, CH), 2.398 (s, 3H, CH3), 2.427 (s, 3H, CH3), 4.817 (dd, J = 5.4, 0.6 Hz, 2H, CH2),
5.958 (br, 1H, NH), 7.119 (s, 1H, thiazole H), 7.231 (d, J=8.4 Hz, 2H, Ph H),
7.815−7.856 (m, 2H, Ph H); HPLC-MS Pos[M+1]+ = 371. Anal. Calcd(%) for
C19H19ClN4S: C, 61.53; H, 5.16; N, 15.11. Found: C, 61.44; H, 5.19; N, 15.21.
1
Data for T11: yield, 81%; purity, 98.7%; white solid; mp, 124.0−124.4 °C. H NMR
(CDCl3) δ 2.367 (s, 3H, CH3), 2.455 (s, 3H, CH3), 2.530 (s, 3H, CH3), 2.574 (s, 3H,
CH3), 4.845 (dd, J = 5.4 Hz , J = 0.9 Hz, 2H, CH2), 6.146 (s, 1H, NH), 7.074 (d, J =
7.8 Hz, 1H, Ph H), 7.125 (s, 1H, thiazole H), 7.190 (s, 1H, Ph H), 7.617 (d, J = 8.1 Hz,
1H, Ph H); GS-MS M+ = 358, base peak 323. Anal. Calcd(%) for C18H19ClN4S: C,
60.24; H, 5.34; N, 15.61. Found: C, 60.22; H, 5.35; N, 15.66.
1
Data for T12: yield, 82%; purity, 92.0%; brown solid; mp, 55.8−58.2 °C. H
NMR(CDCl3) δ 1.220 (t, J = 7.5 Hz, 3H, CH3), 2.395 (s, 3H, CH3), 2.478 (s, 3H,
CH3), 2.574 (s, 3H, CH3), 2.729 (q, J = 7.5 Hz, 2H, CH2), 4.650 (d, J = 5.4 Hz, 2H,
CH2), 6.151 (br, 1H, NH), 7.259 (s, 1H, thiazole H), 7.072–7.143 (m, 1H, Ph H),
7.586–7.630 (m, 2H, Ph H); GS-MS M+ = 372, base peak 337. Anal. Calcd(%) for
C19H21ClN4S: C, 61.20; H, 5.68; N, 15.02. Found: C, 61.25; H, 5.66; N, 15.07.
3.2.10 General procedure for the synthesis of compound 14.
Compound 12 (20 mmol) was dissolved in 30 mL of methylamine (40 wt.% methanol
solution) and refluxed for 1 h. The reaction solution was concentrated and extracted
with ethyl acetate (3×50 mL). The combined organic layers were washed with water
(50 mL) and then saturated brine (50 mL). After filtration of anhydrous sodium sulfate,
the solvent was evaporated and the residue was directly used for the next reaction
without further purification.
27

3.2.11 General synthesis procedure for target compounds T13 and T14.
Target compounds T13 and T14 were also synthesized in the same method as the
1
above description for analogues. The yields, physical properties, and H NMR,
GC-MS, elemental analyses of them are as follows:
1
Data for T13: yield, 84%; purity, 98.7%; yellow solid; mp, 68.4−69.9 °C. H NMR
(CDCl3) δ 2.486 (s, 3H, CH3), 2.503 (s, 3H, CH3), 3.265 (s, 3H, CH3), 4.962 (s, 2H,
CH2), 7.166 (d, J = 0.6 Hz, 1H, thiazole H), 7.415−7.457 (m, 3H, Ph H), 7.918−7.950
(m, 2H, Ph H); GC-MS M+ = 344, base peak 175. Anal. Calcd(%) for C17H17ClN4S: C,
59.21; H, 4.97; N, 16.25. Found: C, 59.52; H, 4.94; N, 16.27.
1
Data for T14: yield, 81%; purity, 99.7%; yellow solid, 75.3~77.5 °C. H NMR
(CDCl3) δ 1.231 (t, J = 7.5 Hz, 3H, CH3), 2.515 (s, 3H, CH3), 2.792 (q, J = 7.5 Hz, 2H,
CH2), 3.254 (s, 3H, CH3), 4.948 (s, 2H, CH2), 7.183 (s, 1H, thiazole H), 7.410−7.439
(m, 3H, Ph H), 7.917−7.949 (m, 2H, Ph H); GC-MS M+ = 358, base peak 175. Anal.
Calcd(%) for C18H19ClN4S: C, 60.24; H, 5.34; N, 15.61. Found: C, 60.21; H, 5.64; N,
15.56.
3.2.12 General synthesis procedure for target compounds T15-19.
Compounds 15, 16 and 17 were obtained by the same synthesis method as compounds
8, 9 and 10, respectively. Unless otherwise mentioned, the target compounds were all
synthesized according to the same procedure described above. The yields, physical
properties, and 1H NMR, GC/HPLC-MS, elemental analyses of them are as follows:
1
Data for T15: yield, 77%; purity, 91.3%; white solid; mp, 53.2−56.1°C. H NMR
(CDCl3) δ 1.660 (d, J = 6.6 Hz, 3H, CH3), 2.478 (s, 3H, CH3), 2.539 (s, 3H, CH3),
5.523−5.617 (m, 1H, CH), 6.239 (br, 1H, NH), 7.124 (d, J = 0.6 Hz, 1H, thiazole H),
7.427−7.473 (m, 3H, Ph H), 7.932−7.986 (m, 2H, Ph H); GC-MS M+ = 344, base
peak 309. Anal. Calcd(%) for C17H17ClN4S: C, 59.21; H, 4.97; N, 16.25. Found: C,
59.32; H, 4.88; N, 16.28.
28

1
Data for T16: yield, 74%; purity, 98.5%; white solid; mp, 70.4−72.1 °C. H NMR
(CDCl3) δ 1.217 (t, J = 7.5 Hz, 3H, CH3), 1.656 (d, J = 6.9 Hz, 3H, CH3), 2.508 (s, 3H,
CH3), 2.730 (q, J = 7.5 Hz, 2H, CH2), 5.543−5.590 (m, 1H, CH), 6.106 (d, J = 7.8 Hz,
1H, NH), 7.111 (d, J = 0.6 Hz, 1H, thiazole H), 7.416−7.486 (m, 3H, Ph H),
7.948−7.988 (m, 2H, Ph H); GC-MS M+ = 358, base peak 323. Anal. Calcd(%) for
C18H19ClN4S: C, 60.24; H, 5.34; N, 15.61. Found: C, 60.33; H, 5.44; N, 15.68.
1
Data for T17: yield, 71%; purity, 99.3%; faint yellow solid; mp, 98.5−100.8°C. H
NMR (CDCl3) δ 1.227 (t, J = 7.5 Hz, 3H, CH3), 2.539 (s, 3H, CH3), 2.747 (q, J = 7.5
Hz, 2H, CH2), 4.878 (d, J = 4.8 Hz, 2H, CH2), 6.097 (bs, 1H, NH), 7.501(s, 1H,
thiazole H), 7.263−8.236 (m, 4H, Ph H); HPLC-MS Pos[M+1]+ = 394. Anal. Calcd(%)
for C18H18Cl2N4S: C, 54.97; H, 4.61; N, 14.24. Found: C, 54.87; H, 4.68; N, 14.34.
1
Data for T18: yield, 73%; purity, 96.1%; yellow solid; mp, 79.0−82.7 °C. H NMR
(CDCl3) δ 1.284 (d, 3H, CH3), 1.649 (d, J = 6.9 Hz, 3H, CH3), 2.444 (s, 3H, CH3),
2.506 (s, 3H, CH3), 5.457 (m, 1H, CH), 6.188 (br, 1H, NH), 7.062 (s, 1H, thiazole H),
7.232 (dd, J = 6.3, 1.6 Hz, 2H, Ph H), 7.856 (dd, J = 6.3, 1.6 Hz, 2H, Ph H); GC-MS
M+ = 358, base peak 323. Anal. Calcd(%) for C18H19ClN4S: C, 60.24; H, 5.34; N,
15.61. Found: C, 60.33; H, 5.24; N, 15.78.
1
Data for T19: yield, 78%; purity, 97.2%; yellow solid; mp, 93.8−95.7 °C. H NMR
(CDCl3) δ 1.252 (t, J = 7.2 Hz, 3H, CH3), 1.647 (d, J = 6.9 Hz, 3H, CH3), 2.401 (s, 3H,
CH3), 2.504 (s, 3H, CH3), 2.773 (q, J = 7.5 Hz, 2H, CH2), 5.550 (q, J = 6.9 Hz, 1H,
CH), 6.094 (d, J = 7.8 Hz, 1H, thiazole H), 7.069 (s, 1H, NH), 7.070−7.233 (m, 2H,
Ph H), 7.830−7.871 (m, 2H, Ph H); GC-MS M+ = 373, base peak 338. Anal. Calcd(%)
for C19H21ClN4S: C, 61.20; H, 5.68; N, 15.02. Found: C, 61.26; H, 5.74; N, 15.13.
3.3 Fungicidal assay
The fungi were provided by the National Engineering Research Center for
Agrochemicals, Hunan Research Institute of Chemical Industry (Changsha, China).
The fungicidal activities of the target compounds were tested against the seven plant
pathogenic fungi as the methods reported in our previous studies.23,35 The compounds
29