Design, synthesis and biological evaluation of pyrazole-aromatic containing carboxamides as potent SDH inhibitors

Article abstract of DOI:10.1016/j.ejmech.2021.113230

To continue our ongoing studies on discovery of new potent antifungal leads, 43 novel pyrazole-aromatic containing carboxamides were rationally designed and synthesized. Bioassays indicated that most target compounds displayed good in vitro antifungal activities against Botrytis cinerea, Rhizoctonia cerealis and Sclerotinia sclerotiorum and in vivo antifungal activity against R. solani. Compound 11ea exhibited the most significant in vitro activity against R. cerealis (EC₅₀ = 0.93 μg/mL) with about 2-fold more potent than a previously reported lead compound A1 (EC₅₀ = 2.01 μg/mL), and about 11-fold more potent than the positive control/commercial succinate dehydrogenase inhibitor thifluzamide (EC₅₀ = 23.09 μg/mL). Structure-activity relationship analysis and molecular docking simulations indicated that the presence of difluoromethyl pyrazole-(m-benzene) carboxamide scaffold obviously increased the antifungal activity. The further enzymatic bioassay showed that both thifluzamide and compound 11ea displayed excellent SDH inhibitory effects, and fluorescence quenching analysis suggested that they may share the same target SDH.

Full text of DOI:10.1016/j.ejmech.2021.113230

European Journal of Medicinal Chemistry 214 (2021) 113230
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Design, synthesis and biological evaluation of pyrazole-aromatic
containing carboxamides as potent SDH inhibitors
,
*
Bin Yu a, Bin Zhao c, Zesheng Hao a, Lei Chen a, Lixin Cao a, Xiaofeng Guo b
,
,
, ***
Nailou Zhang a, Dongyan Yang a **, Liangfu Tang a, Zhijin Fan a
a State Key Laboratory of Elemento-Organic Chemistry, College of Chemistry, Nankai University, Tianjin, 300071, PR China
b College of Biology, Hunan University, Changsha, 410082, PR China
c College of Plant Protection, Hebei Agricultural University, Baoding, 071001, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
To continue our ongoing studies on discovery of new potent antifungal leads, 43 novel pyrazole-aromatic
containing carboxamides were rationally designed and synthesized. Bioassays indicated that most target
compounds displayed good in vitro antifungal activities against Botrytis cinerea, Rhizoctonia cerealis and
Sclerotinia sclerotiorum and in vivo antifungal activity against R. solani. Compound 11ea exhibited the
Received 28 March 2020
Received in revised form
4 January 2021
Accepted 23 January 2021
Available online 1 February 2021
most significant in vitro activity against R. cerealis (EC50 ¼ 0.93
than a previously reported lead compound A1 (EC50 ¼ 2.01 g/mL), and about 11-fold more potent than
g/mL).
mg/mL) with about 2-fold more potent
m
the positive control/commercial succinate dehydrogenase inhibitor thifluzamide (EC50 ¼ 23.09
m
Keywords:
Succinate dehydrogenase inhibitors
Antifungal activity
Structure-activity relationships
Molecular docking
Structure-activity relationship analysis and molecular docking simulations indicated that the presence of
difluoromethyl pyrazole-(m-benzene) carboxamide scaffold obviously increased the antifungal activity.
The further enzymatic bioassay showed that both thifluzamide and compound 11ea displayed excellent
SDH inhibitory effects, and fluorescence quenching analysis suggested that they may share the same
target SDH.
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
action and broad-spectrum antifungal activity, SDHIs have attrac-
ted a wide range of research in discovering novel antifungal
Plant diseases caused by phytopathogens pose a worldwide
threat to crop production and food security, fungicides played an
effective role in crop diseases control and plant protection [1].
Among the over 50 classes of fungicides categorized by the
Fungicide Resistance Action Committee (FRAC), succinate dehy-
drogenase inhibitor (SDHI) has been the fastest growing class of
fungicides, which have been discovered and launched into the
agrochemical market for the past decades [2,3]. The primary mode
of action of SDHIs is to block the tricarboxylic acid cycle (TCA cycle)
between succinate and fumarate thereby inhibiting the respiration
of fungi, and leading to fungi death [2,3]. Due to its unique mode of
products [4e9], and potent anticancer agents [10]. However, SDHIs
have been unrestrictedly used for plant protection since the first
commercial product carboxin launched in 1966 [11], directly
resulting in the development of fungi resistance [2,12,13]. There-
fore, it is always necessary for modern plant protection to develop
novel SDHI with different scaffolds to prevent cross-resistance.
Pyrazole derivatives, as an important class of nitrogen-
containing heterocyclic compounds, have exhibited a wide variety
of activities, including anti-inflammatory [14], anticancer [15],
antifungal activities [16], etc. [17,18]. The representative SDHI fun-
gicides including fluxapyroxad, benzovindiflupyr, bixafen, pen-
flufen, sedaxane, penthiopyrad and isopyrazam share a pyrazole
substructure (Fig. 1).
In our previous work, a series of novel pyrazole-thiazole
containing carboxamides were synthesized and discovered with
antifungal activity, especially against Rhizoctonia cerealis [19]. To
continue our ongoing work on the discovery of novel fungicide,
new pyrazole-thiazole carboxamides with various amine moi-
eties were widely investigated, and a previous representative
* Corresponding author. State Key Laboratory of Elemento-Organic Chemistry,
College of Chemistry, Nankai University, No. 94, Weijin Road, Nankai District,
Tianjin, 300071, PR China.
** Corresponding author.
*** Corresponding author.
E-mail addresses: guoxf@hnu.edu.cn (X. Guo), yangdy@nankai.edu.cn (D. Yang),
fanzj@nankai.edu.cn (Z. Fan).
https://doi.org/10.1016/j.ejmech.2021.113230
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

B. Yu, B. Zhao, Z. Hao et al.
European Journal of Medicinal Chemistry 214 (2021) 113230
Fig. 1. Structures of the representative pyrazole containing SDH inhibitors.
lead compound A1 was docked into the R. cerealis SDH active
pocket. It was found that difluoromethyl-pyrazole group played
an important role in the binding mode, because the eCHF2 group
formed an additional hydrogen bond with C-Arg81, and the
illustrated in Schemes 1-3. The commercially available compound 1
was used as a starting material to react with oxalyl chloride for
affording the corresponding acyl chloride. Amide 2 was obtained by
reaction of this acyl chloride with aqueous ammonia solution. The
oxygen atom of compound 2 was substituted by S atom via Law-
esson’s reagent to obtain the thioamide 3. Subsequently, the thio-
amide 3 and amide 2 were reacted with ethyl bromopyruvate by an
intermolecular cyclization to give intermediate thiazole 9a and
oxazole 9b, respectively (Scheme 1).
Carboxylic acid 1 was treated with diphenyl phosphorazidate
(DPPA) in the presence of t-BuOH by a Curtius rearrangement to
form the intermediate 4 [20] for further deprotection under an
acidic condition to yield the amine 5, followed by a Sandmeyer
reaction, the intermediate 6 was obtained. Afterwards, the com-
pound 7 was synthesized by reacting compound 6 with iso-
propoxyboronic acid pinacol ester in the presence of n-C4H9Li.
Boronic esters 7 was subjected to the Suzuki coupling in the pres-
ence of Pd(PPh3)4 to form the intermediate 9c (Scheme 1).
Commercially available compounds 8 and corresponding phe-
nylboronic acids were subjected to Suzuki coupling in the presence
of PdCl2(dppf) to form the intermediates 9d and 9e, respectively.
pyrazole-thiazole moiety formed
and B-Trp203, respectively (Fig. 2). On this basis, we supposed
that the interactions between the key residues and inhibitors
p-p interactions with B-His246
p-p
might be a main contribution to increase antifungal activity. To
prove our hypothesis, the pyrazole group was retained and the
thiazole ring conjugated with pyrazole was replaced to improve
the
p-p interactions with the key residues in the binding site,
thus, novel pyrazole-aromatic containing carboxamides (Fig. 3)
were designed and synthesized. Experimental results discovered
a new more potent compound 11ea with better antifungal ac-
tivity against R. cerealis than the positive control thifluzamide.
2. Results and discussion
2.1. Chemistry
The synthetic procedures of the key intermediates 10a-10e are
Fig. 2. Predicted binding mode of compound A1 (A and B) with R. cerealis SDH.
2

B. Yu, B. Zhao, Z. Hao et al.
European Journal of Medicinal Chemistry 214 (2021) 113230
Fig. 3. Design strategy of the target compounds.
Scheme 1. Synthesis of the intermediates 10a-10b. Reagents and conditions: a. (COCl)2/DMF/CH2Cl2, NH3$H2O/CH2Cl2; b. EtOH, reflux; c. NaOH, EtOH/H2O; d. Lawesson’s reagent,
THF, reflux, 1 h; e. DPPA, DIPEA, t-BuOH; f. TFA, CH2Cl2; g. 4 M HCl, NaNO2, KI; h. n-C4H9Li, THF, ꢀ78 ꢁC; i. 2 N Na2CO3, Pd(PPh3)4, reflux.
Finally, intermediates 9a-9e were saponified with a 2 mol/L NaOH
solution and subsequently acidified with a 2 mol/L HCl solution to
give the carboxylic acids 10a-10e (Scheme 2).
The crystal structure of the compound 11ea (CCDC: 1962907)
was identified by single crystal X-ray diffraction analysis as shown
in Fig. 4 and Table S1 for further structure validation.
The target compounds 11aa-11eh were synthesized according
to the reaction route described in Scheme 3. The key intermediates
10a-10e were combined with corresponding amine to yield the title
compounds 11aa-11eh. The structures of all of the target com-
pounds were characterized by proton nuclear magnetic resonance
(1H NMR), carbon-13 nuclear magnetic resonance (13C NMR)
spectroscopies and high-resolution mass spectrometry (HRMS).
2.2. Biological evaluation
2.2.1. In vitro antifungal activity
All target compounds were evaluated for their in vitro antifungal
activities against the nine representative plant fungi at a concen-
tration of 50 mg/mL according to our previously reported method
3

B. Yu, B. Zhao, Z. Hao et al.
European Journal of Medicinal Chemistry 214 (2021) 113230
Scheme 2. Synthesis of the intermediates 10d-10e. Reagents and conditions: c. NaOH, EtOH/H2O; j. PdCl2(dppf), Cs2CO3, reflux.
[21]. Commercial fungicides thifluzamide and fluxapyroxad were
selected as the positive controls. The preliminary screening results
are listed in Table 1, indicating that all compounds showed various
degrees of inhibitory activities against the tested fungi.
Extensive investigation on the effects of various substituted
anilines and amines on pyrazole-thiazole carboxamide scaffold
(11aa-11ay) revealed that most compounds displayed moderate to
good antifungal activities against Botrytis cinerea, Cercospora
arachidicola, Rhizoctonia cerealis, and Sclerotinia sclerotiorum. For
B. cinerea, compounds 11aa, 11aj, and 11aw exhibited good activity
with inhibitory effects more than 75%. Notably, the inhibition rate
of compound 11aw reached 83.8%, which was comparable to that of
the positive control fluxapyroxad (87.3%). For S. sclerotiorum,
Scheme 3. Synthesis of the target compounds 11aa-11eh. Reagents and conditions: k.
PyBOP, DIPEA, CH2Cl2, r.t, overnight.
Fig. 4. Crystal structure of compound 11ea (CCDC: 1962907).
4

B. Yu, B. Zhao, Z. Hao et al.
European Journal of Medicinal Chemistry 214 (2021) 113230
Table 1
Chemical structures and in vitro antifungal activity of the target compounds 11aa-11eh at 50 mg/mL.
Compd
R1
Ar
R2
Inhibition ratea (%)
A.sb
B. c
C. a
G. z
P. i
P. p
P. s
R. c
S. s
11aa
11 ab
11ac
11ad
11ae
11af
11 ag
11ah
11ai
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CF3
thiazole
thiazole
thiazole
thiazole
thiazole
thiazole
thiazole
thiazole
thiazole
thiazole
thiazole
thiazole
thiazole
thiazole
thiazole
thiazole
thiazole
thiazole
thiazole
thiazole
thiazole
thiazole
thiazole
thiazole
thiazole
oxazole
oxazole
oxazole
thiophene
thiophene
p-benzene
p-benzene
p-benzene
p-benzene
p-benzene
m-benzene
m-benzene
m-benzene
m-benzene
m-benzene
m-benzene
m-benzene
m-benzene
2-Cl-Ph
3-Cl-Ph
4-Cl-Ph
48.1
22.2
66.7
33.3
14.8
3.7
70.4
18.5
25.9
39.1
14.3
18.5
74.1
0
55.6
37.0
22.2
40.7
77.8
3.7
78.4
13.5
45.9
51.4
18.9
62.2
73.0
67.6
29.7
77.8
34.5
45.9
67.6
45.9
13.5
56.8
0
67.6
67.6
73.0
73.0
67.6
83.8
73.0
67.6
58.7
65.1
34.9
66.7
65.1
74.6
71.4
34.9
33.3
28.6
74.6
49.2
57.1
58.7
36.1
29.4
45.4
74.5
87.3
58.7
44.8
27.6
51.7
51.7
10.3
17.2
75.9
24.1
10.3
59.0
15.5
13.8
69.0
3.4
62.1
72.4
17.2
34.5
72.4
10.3
13.8
20.7
58.6
24.1
0
50.0
44.4
88.9
44.4
22.2
33.3
66.7
50.0
50.0
39.2
42.9
27.8
66.7
22.2
61.1
27.8
27.8
44.4
66.7
27.8
38.9
22.2
55.6
11.1
11.1
27.5
31.4
15.7
37.3
37.3
56.9
41.2
33.3
13.7
5.9
12.0
25.3
38.7
46.7
17.3
49.3
57.3
36.0
12.0
44.3
0
12.0
17.3
14.7
30.7
49.3
25.3
30.7
57.3
9.3
14.7
20.0
52.0
12.0
25.3
29.9
13.4
0
38.1
36.1
36.1
19.6
7.2
35.7
0
25.3
32.5
54.2
49.4
18.1
13.3
42.2
18.1
0
51.5
28.1
22.9
25.3
0
44.6
42.2
32.5
13.3
30.1
37.3
8.4
25.3
25.3
3.6
13.3
21.2
21.2
9.1
36.4
39.4
45.5
27.3
15.2
9.1
21.2
53.0
54.5
0
15.2
15.2
60.6
45.5
0
88.9
41.8
10.6
57.6
12.1
63.6
45.5
50.0
0
53.0
12.1
15.2
15.2
33.3
22.7
45.5
73.3
64.4
31.1
82.2
90.0
82.2
84.4
22.2
31.1
16.7
94.4
51.1
42.2
64.4
28.6
28.6
94.9
90.8
41.1
55.6
35.3
23.5
35.3
47.1
17.6
23.5
52.9
52.9
0
83.3
71.4
5.9
47.1
17.6
17.6
29.4
0
41.2
64.7
94.1
64.7
17.6
41.2
17.6
5.9
73.6
62.5
62.5
83.3
80.6
93.1
76.4
66.7
72.2
72.2
95.8
91.7
70.8
66.7
50.0
67.9
85.7
76.9
83.3
87.5
21.7
38.5
21.7
14.7
56.6
7.7
2-OMe-Ph
3-OMe-Ph
4-OMe-Ph
2-OCF3-Ph
3-OCF3-Ph
4-OCF3-Ph
2-iPr-Ph
3,5-Br2-Ph
3,4-Cl2-Ph
3,5-Cl2-Ph
cyclopropane
cyclohexane
1-naphthalen
1-(1,2,3,4-tetrahydronaphthalene)
2-(5,6,7,8-tetrahydronaphthalene)
2-pyridine
4-pyridine
3-Br-4-pyridine
4-pyrimidine
2-pyrazine
2-thiazole
4-Me-2-thiazole
3-Br-Ph
3-Me-Ph
2-CF3-Ph
3-Br-Ph
3-Me-Ph
3-Br-Ph
3-Me-Ph
2-CF3-Ph
PhCH2
3-CF3-Ph
3-Br-Ph
3-Me-Ph
2-CF3-Ph
PhCH2
3-Br-Ph
3-Me-Ph
2-iPr-Ph
7.7
11aj
53.6
13.4
13.3
63.6
18.9
46.9
0.7
21.7
13.3
60.8
18.9
59.4
28.7
27.3
13.3
7.7
38.1
29.8
32.1
28.6
45.2
40.5
16.7
2.4
2.4
7.1
27.4
14.3
14.3
9.5
4.9
9.8
23.2
67.9
47.6
39.3
11ak
11 al
11am
11an
11ao
11ap
11aq
11ar
11as
11 at
11au
11av
11aw
11ax
11.1
3.7
59.3
3.7
11ay
29.6
43.5
30.4
15.2
47.8
52.2
56.5
41.3
10.9
8.7
13.0
71.7
39.1
30.4
30.4
28.6
23.8
85.7
63.0
73.9
65.2
11ba
11bb
11bc
38.5
28.2
10.3
59.0
48.7
28.2
38.5
7.7
11ca
11 cb
11da
11 db
11dc
11dd
11de
11ea
11eb
11ec
11ed
11ee
11ef
11eg
11eh
Fluxapyroxad
Thifluzamide
5.1
1.0
1.0
46.4
9.3
9.3
19.6
0
0
39.3
40.4
13.4
13.4
17.9
66.7
33.3
28.2
28.2
15.5
15.5
83.1
65.7
100
82.1
15.2
42.4
3.0
56.9
35.3
45.1
17.6
17.9
25.0
0
56.8
41.2
35.3
15.2
39.4
15.6
21.9
53.1
32.7
15.2
15.2
CF3
CF3
CH3
3-Me-Ph
a
Values are the mean of three replicates.
b
Abbreviations: A. s: Alternaria solani; B. c: Botrytis cinerea; C. a: Cercospora arachidicola; G. z: Gibberella zeae; P. i: Phytophthora infestans (Mont) de Bary; P. p: Physalospora
piricola; P. s: Pellicularia sasakii; R. c: Rhizoctonia cerealis; and S. s: Sclerotinia sclerotiorum.
compounds 11aj, 11ak, and 11 at showed more than 70% inhibitory
activity. Particularly, the compound 11 at showed the highest in-
hibition effect (94.1%), which was superior to that of the positive
controls thifluzamide (87.5%) and fluxapyroxad (83.3%). Moreover,
compound 11aj displayed remarkably higher antifungal activity
(88.9%) than that of thifluzamide (55.6%) against R. cerealis. For
C. arachidicola, although compound 11 ag exhibited the highest
antifungal activity (75.9%), it was still lower than those of thi-
fluzamide (82.1%) and fluxapyroxad (100%).
Meanwhile, to further explore an optimal aromatic ring to
replace the middle thiazole ring, in vitro antifungal activities of the
synthesized compounds 11ba-11eh were also evaluated, and the
5

B. Yu, B. Zhao, Z. Hao et al.
European Journal of Medicinal Chemistry 214 (2021) 113230
results were illustrated in Table 1. It was worth noting that these
novel compounds showed good to excellent inhibitory effects
against B. cinerea, R. cerealis and S. sclerotiorum. Based on the results
of preliminary screening, all compounds 11ba-11eh and several
compounds from 11aa-11ay were chosen for their median effective
concentration (EC50) determination and comparison with the lead
compound A1. The results shown in Table 2 revealed that, these
compounds showed different inhibitory activities against the three
tested fungi. For R. cerealis, most compounds including 11aj, 11ba,
11ca, 11da, 11ea, 11eb, and 11eg showed significantly stronger
increased inhibitory activity against most of the tested fungi, but
reduced the inhibitory activity against S. sclerotiorum.
The in vitro antifungal activity of 11ba-11eh are listed in Tables 1
and 2, most of the compounds exhibited good to excellent inhibitory
activityagainstB. cinerea, R. cerealis andS. sclerotiorum. The results of
selected compounds in 11aa-11ay are listed in Table 2. 11ak
(R2 ¼ 3,5-Br2-Ph) showed a remarkably decrease in antifungal ac-
tivity against R. cerealis as compared with the compound A1, indi-
cating that the mono substituted group at ortho- or meta-position on
the phenyl ring might be more favorable for the inhibitory activity
than the disubstituted group. Notably,11aj exhibited similar activity
against R. cerealis as compared with A1, suggesting that introducing
a bulky hydrophobic group at ortho- or meta-position on the phenyl
ring may also afford potent inhibitors, its molecular mechanism
would be discussed in the following section. For 11ba-11eh, the EC50
values revealed that certain compounds such as 11 cb
(Ar ¼ thiophene), 11 db (Ar ¼ p-benzene) and 11ea, 11eb, 11ed
(Ar ¼ m-benzene) showed promising activity against R. cerealis as
comparedwith the corresponding pyrazole-thiazole compounds A1,
A2, A3, or A4. Among them, 11ea showed the best potency against
R. cerealis, which was superior to the positive control thifluzamide
and the lead compound A1, indicating that the benzene ring might
inhibitory activity with EC50 values ranging from 0.93 to 4.50
mL. They were more potent than thifluzamide with an EC50 value of
23.09 g/mL. Among them, 11ea was the most active one with the
lowest EC50 value of 0.93 g/mL. It was superior to thifluzamide and
about 2-fold more active than the lead compound A1 with an EC50
value of 2.01 g/mL. For B. cinerea, 11ba, 11ca, 11 cb, 11 db, and 11ea
exhibited stronger inhibitory activity with EC50 values ranging from
2.03 to 9.78 g/mL. They were more potent than thifluzamide with
an EC50 value of 11.65 g/mL. Among these, 11 cb showed the most
potent activity with an EC50 value of 2.03 g/mL. It was about 6-fold
mg/
m
m
m
m
m
m
more active than thifluzamide. For S. sclerotiorum, 11da, 11ea, 11ee,
and 11eg exhibited notably higher antifungal activity with EC50
values ranging from 0.51 to 1.64
than thifluzamide with an EC50 value of 4.88
11eg showed the strongest activity with the lowest EC50 value of
0.51 g/mL. It was about 9-fold more active than thifluzamide.
m
g/mL. They were more potent
increase the p-p interaction with key residues in RcSDH active site.
Surprisingly, 11ea also exhibited remarkably antifungal activities
against B. cinerea and S. sclerotiorum with EC50 values of 3.85 and
mg/mL. In particular,
m
0.85 mg/mL, respectively. These were superior to thifluzamide.
Moreover, 11ba (Ar ¼ oxazole) and 11da (Ar ¼ p-benzene) showed
stronger inhibitory activity against R. cerealis than thifluzamide. By
conclusion, SAR of 11ba-11eh against R. cerealis displayed a trend:
m-benzene > p-benzene > thiophene > oxazole, these results
2.2.2. SAR according to in vitro antifungal activity
Based on the results in Tables 1 and 2, SAR of novel derivatives
was analyzed. By comparing the inhibition rates of 11aa-11am in
Table 1, the antifungal activities were varied with regard to the
different groups and different substituents on the phenyl ring. The
compounds with a meta- or an ortho-substituent on the phenyl ring
exhibited better activity against most tested fungi than compounds
with a para-substituent. For example, trifluoromethoxy group in
11 ag and 11 ah at the ortho- or meta-position of the phenyl ring
were favorable for the inhibitory activity against all tested fungi.
Compound 11ad with a methoxy group at the ortho-position of the
phenyl group also showed better inhibitory activity against the
most tested fungi as compared with the compound 11af bearing
such a group on para-position. Moreover, an introduction of 3,5-Cl2
groups in 11am showed significant activity improvement against
almost all tested fungi as compared with the monosubstituted
compound 11 ab. In contrast, the presence of 3,4-Cl2 in 11 al
obviously decreased the inhibitory activity. These results indicated
that there may exist a steric hindrance between the para-substit-
uent of the benzene ring and SDH, which affected the bioactive
conformation of the inhibitor, thereby significantly reduced its
potency. Interestingly, an opposite trend was observed among
11aa-11ac. Compound 11ac containing para-substituted chlorine
displayed stronger inhibitory activity than 11aa and 11 ab against
almost all tested fungi. These results suggested that the steric effect
of chlorine appeared not to be the main factor affecting its anti-
fungal activity of monochlorinated compounds. In addition, the
structures of amines (11an-11ay) also played an important role on
the antifungal activity. It was concluded that compounds contain-
ing a six-membered aromatic ring generally exhibited higher ac-
tivity than compounds containing other rings such as an aliphatic
ring in 11an, a tetrahydronaphthalene ring in 11aq, 11ar, or a five-
membered aromatic ring in 11ax, 11ay. Among the six-membered
aromatic compounds, 11as (R2 ¼ 2-pyridine) showed a broad
spectrum of antifungal activity (exhibited >50% activity against
eight fungi). Compound 11 at (R2 ¼ 4-pyridine) exhibited excellent
activity against S. sclerotiorum (94.1%). In comparison with 11 at, an
introduction of 3-Br in 11au on the 4-pyridine group slightly
confirmed the importance of the
p-p interaction between the
pyrazole-aromatic moiety of the ligand and the key residues of SDH,
as hypothesized in our molecular design.
After in vitro antifungal activity confirmed that the scaffold of
pyrazole-(m-benzene) carboxamide could be selected as a lead
structure for further modification, we further modified the 3-
position of the pyrazole ring by replacing R1 ¼ eCHF2 with
R1 ¼ eCF3 or eCH3. Disappointedly, the inhibitory activity of 11ee-
11eh against R. cerealis significantly decreased, suggesting that the
difluoromethyl group at R1 would be more favorable for the anti-
fungal activity. Surprisingly, the inhibitory activity of 11eg against
S. sclerotiorum increased considerably.
To understand the structure-activity relationship of these novel
pyrazole-aromatic containing carboxamides at atomic level, the
positive control thifluzamide and selected compounds (11ea, 11ai,
11aj, and 11ak) were docked into the theoretical active site of
modeled RcSDH, respectively. As described in Fig. 5A, thifluzamide
was bound in the active pocket of RcSDH, the key residue B-Trp203
formed a hydrogen bond with the carbonyl oxygen atom, which
was crucial for the binding between the inhibitor and RcSDH. The
nitrogen atom of thiazole moiety formed an additional hydrogen
bond with C-Arg81. Meanwhile, the thiazole ring formed a weak
p-
p
stacking interaction with B-His246. The aniline moiety was
stretched into the hydrophobic pocket that consisted of the resi-
dues C-Phe65, C-Trp74, and C-Ile78. However, the trifluoromethoxy
group at the 4-position of the benzene ring existed steric repulsion
with the residues in the hydrophobic pocket. Moreover, the pres-
ence of a methyl substituent at the 2-position of the thiazole ring
led to unfavorable repulsion with D-Asp127, which may account for
its relative weaker antifungal potency.
The simulated binding mode of potent compound 11ea (Fig. 5B)
showed that the carbonyl oxygen atom also formed a hydrogen
bond with the key residue B-Trp203 and pyrazole ring formed a
stacking
p-p interaction with the residue B-His246, which were
similar to the thifluzamide. In addition, a fluorine atom of the
6

B. Yu, B. Zhao, Z. Hao et al.
European Journal of Medicinal Chemistry 214 (2021) 113230
Table 2
Chemical structures and antifungal potency of the target compounds 11aa-11eh against B. cinerea, R. cerealis and S. sclerotiorum.
R1
Ar
R2
EC50
(
m
g/mL)
a
Compd.
B. cinerea
R. cerealis
S. sclerotiorum
A1 [19]
CHF2
3-Br-Ph
-
2.01
-
A2 [19]
A3 [19]
A4 [19]
11aj
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
3-CH3-Ph
2- CF3-Ph
CH2Ph
-
6.23
10.04
20.07
2.77
30.99
-
-
-
-
-
-
2-iPr-Ph
3,5-Br2-Ph
4-pyridine
2-pyrazine
3-Br-Ph
24.94
>100
-
47.13
25.24
7.01
-
11ak
11 at
11aw
11ba
46.1
9.78
34.58
-
2.67
14.13
35.64
42.94
11bb
3-Me-Ph
(continued on next page)
7

B. Yu, B. Zhao, Z. Hao et al.
European Journal of Medicinal Chemistry 214 (2021) 113230
Table 2 (continued)
R1
Ar
R2
EC50 (mg/mL)
a
Compd.
B. cinerea
R. cerealis
S. sclerotiorum
11bc
CHF2
2-CF3-Ph
>100
>100
19.97
11ca
CHF2
CHF2
3-Br-Ph
4.18
2.03
4.50
6.41
16.67
17.39
11 cb
3-Me-Ph
11da
11 db
11dc
11dd
11de
11ea
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
3-Br-Ph
3-Me-Ph
2-CF3-Ph
PhCH2
12.73
4.86
2.19
7.09
>100
>100
8.60
0.93
1.32
5.19
65.38
12.83
5.16
0.85
>100
14.00
17.96
3.85
3-CF3-Ph
3-Br-Ph
11eb
11ec
11ed
11ee
11ef
11eg
11eh
CHF2
CHF2
CHF2
CF3
3-Me-Ph
2-CF3-Ph
PhCH2
>100
38.28
43.09
>100
>100
-
4.20
14.88
15.84
45.81
1.64
19.19
19.28
>100
>100
3.17
3-Br-Ph
3-Me-Ph
2-iPr-Ph
3-Me-Ph
CF3
12.62
0.51
CF3
CH3
33.17
11.65
9.94
16.15
4.88
Thifluzamide
23.09
a
Values are the mean of three replicates. “-” ¼ No test.
difluoromethyl group formed another hydrogen bond with C-Arg81
and pyrazole ring formed a T-shaped interaction with B-
Altogether, the aforementioned interactions may contribute to the
stabilization of 11ea and analogs in RcSDH active site.
p-p
Trp203. The 3-Br substituted aniline moiety was stretched through
the hydrophobic pocket that consists of C-Pro70 and C-Ile78, and its
The presence of trifluoromethoxy group on the benzene ring
(11ai) resulted in poor activity due to steric hindrance with C-
Tyr68, C-Gln69 and C-Pro70 (Fig. 5D). Besides, 11ai failed to form
the crucial hydrogen bond with B-Trp203. Notably, compared with
11ea, the high potency of compound 11aj (Fig. 5E) was attributed to
an additional hydrogen bond (C-F…H interaction) with C-Arg81,
although the hydrogen bond (C=O…H interaction) of its carbonyl
bromine atom formed a cation-
p interaction with C-Phe65. The
superimposition of 11ea and A1 (Fig. 5C) showed a similar pattern
in the SDH active site. The middle benzene ring of 11ea adjusted its
binding mode, which in favor of the
p-p interactions with both B-
His246 and B-Trp203 and the hydrogen bond with B-Trp203.
8

B. Yu, B. Zhao, Z. Hao et al.
European Journal of Medicinal Chemistry 214 (2021) 113230
Fig. 5. Simulated binding mode of (A) Thifluzamide (lime green sticks) with RcSDH, (B) compound 11ea (magenta sticks) with RcSDH; (C) the binding mode overlay of compound
11ea and A1 bonding with RcSDH (represented by magenta and cyan sticks); the binding mode of (D) compound 11ai (white sticks) with RcSDH, (E) compound 11aj (slate sticks)
with RcSDH; simulated binding mode overlay of compound 11aj and 11ak with RcSDH (represented by slate and yellow sticks).
oxygen atom was weaker than that of 11ea. Furthermore, the
presence of isopropyl group on the benzene ring of 11aj could
improve the hydrophobic interaction. As shown by superimposi-
tion of 11aj and 11ak (Fig. 5F), the decreased activity of 11ak was
due to the steric repulsion with D-D127.
Table 4 showed that most selected compounds showed a decrease
(at 10 g/mL) in enzyme activity as compared with the lead com-
m
pound A1 (14.13 U/mg). Among them, 11aj (10.96 U/mg) exhibited
better enzyme activity than 11ak (2.05 U/mg), 11eb (R1 ¼ eCHF2)
displayed higher enzyme activity than 11eh (R1 ¼ eCH3), the
enzyme activity showed the trends of 11da (Ar
¼
p-
benzene) > 11ba (Ar ¼ oxazole) z 11ca (Ar ¼ thiophene), these
trends were very similar to the results of their in vitro antifungal
activity against R. cerealis. However, 11ea (6.10 U/mg) did not show
the desired potency, the lower concentration determination for IC50
calculation shown in Table 5 indicated that 11ea exhibited SDH
2.2.3. In vivo antifungal activity
The in vivo protective activities of 11aa-11eh against E. graminis,
P. sorghi Schw, P. oryzae and R. solani were further evaluated. Thi-
fluzamide was chosen as a positive control. The results are listed in
Table 3. Although most compounds showed lower antifungal ac-
enzymatic inhibition activity with an IC50 value of 1.49
mg/mL, even
tivities (at 200
mg/mL) against E. graminis, P. sorghi Schw and
though it was less active than the positive control thifluzamide
P. oryzae than thifluzamide, some compounds still displayed good
inhibitions against P. sorghi Schw (11ba, 75%; 11bb, 98%; 11 db, 75%)
and P. oryzae (11af, 70%; 11ax, 70%; 11ay, 70%). Moreover, most
compounds also displayed good to excellent activities against
with its IC50 value of 0.49
at the same target SDH.
mg/mL, it was obvious that they might act
R. solani (11aa-11 ay at 200
those, 11ca, 11 cb, 11 db, 11eb, and 11eg exhibited over 80% inhib-
mg/mL,11ba-11eh at 100 mg/mL). Among
2.3. Fluorescence quenching of SDH
itory activity at 100
(70% inhibition at 10
10 mg/mL). Unfortunately, the potent compound 11ea with good
in vitro antifungal activity did not show the desired in vivo potency.
UV irradiation and/or metabolism may be impacting factors for
in vivo activity, which was worth for further study. These results
indicated that these compounds exhibited promising activities
against R. solani in vivo, and could be regarded as the new structural
scaffolds for further optimization.
m
m
g/mL, 11ca exhibited slightly lower activity
g/mL) than thifluzamide (80% inhibition at
To find the target of active compound with unknown mode of
action, gene identification combined with molecular biology by
bioinformatics and comprehensive omics is a good method [22,23].
Fluorescence quenching experiments can reveal the interactions
between a protein and a ligand and identify the target of the active
small molecule. Fluorescence quenching analysis of R. cerealis SDH
was conducted with the compound 11ea, thifluzamide and tricy-
clazole, which were used to validate interactions between protein
and compounds. As shown in Fig. 6A, the fluorescence intensity of
R. cerealis SDH was the strongest at 0
mg/mL (a, black curve) of 11ea,
2.2.4. SDH enzyme activity as influenced by target compounds
R. cerealis SDH activity as influenced by compound 11ea and
several representative compounds were tested. The results listed in
and the weakest at 5 g/mL (e, red curve) of 11ea, that was, the
fluorescence intensity of RcSDH was gradually quenched as the
concentration of compound 11ea increased, which was similar to
m
9

B. Yu, B. Zhao, Z. Hao et al.
European Journal of Medicinal Chemistry 214 (2021) 113230
Table 3
The in vivo antifungal activity of the target compounds 11aa-11eh.
Compd
Inhibition ratea (%)
b
b
b
b E. graminis
P. sorghi Schw
P. oryzae
R. solani
c R. solani
d R. solani
11aa
11 ab
11ac
11ad
11ae
11af
11 ag
11ah
11ai
0
0
70
0
0
0
0
0
20
0
0
0
0
70
90
10
30
40
-
70
40
-
-
-
0
50
-
-
-
-
-
-
0
10
10
0
0
0
0
0
50
30
0
100
80
70
100
50
60
30
10
100
50
10
40
10
80
70
90
0
20
30
-
0
30
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
20
50
0
0
0
50
0
0
70
0
0
0
0
0
0
0
0
0
0
0
0
50
50
50
0
0
70
70
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
100
40
100
100
90
70
70
100
0
0
20
20
50
20
0
0
0
0
0
0
0
0
0
50
0
0
11aj
11ak
11 al
11am
11an
11ao
11ap
11aq
11ar
0
0
0
0
0
0
0
0
0
20
0
0
0
0
-
-
-
-
-
-
-
-
-
100
70
100
50
60
60
80
100
100
100
100
60
100
100
-
-
-
-
-
-
-
-
-
11as
11 at
11au
11av
11aw
11ax
11ay
11ba
11bb
11bc
11ca
11 cb
11da
11 db
11dc
11dd
11de
11ea
11eb
11ec
11ed
11ee
11ef
0
75
98
20
40
20
45
75
0
-
70
60
0
60
-
-
-
-
0
-
-
-
-
70
50
80
0
-
-
-
-
-
-
-
-
20
10
0
40
50
10
0
10
40
100
-
-
-
-
-
-
-
-
11eg
11eh
Thifluzamide
-
98
-
50
-
100
a
Values are the mean of three replicates
b,c,d Test concentration was 200, 100 and 10
mg/mL, respectively; Abbreviations: E. graminis: wheat white powder; P. sorghi: corn rust; P. oryzae: rice blast; R. solani: rice sheath
blight.
“-” ¼ No test.
Table 5
Table 4
Inhibitory effect of 11ea against R. cerealis SDH.
R. cerealis SDH enzyme activity at 10 mg/mL.
Compd
IC50
(m
g/mL)
Regression equation
R2
Compd.
SDH activity (U/mg)
11ea
Thifluzamide
1.49
0.49
y ¼ 4.6768x þ 1.8735
y ¼ 5.4044x þ 1.3047
0.9949
0.9809
A1
11aj
14.13
10.96
2.05
6.89
6.52
12.56
6.10
10.63
5.68
8.46
5.68
2.48
11ak
11ba
11ca
11da
11ea
11eb
11ec
11ed
11eh
Thifluzamide
[24,25] (Fig. 6C). Compound 11ea showed a similar pattern of
fluorescence quenching as compared with thifluzamide, suggesting
that they have the same mode of action.
3. Conclusion
In order to further investigate the impact of various amine
moieties on pyrazole-thiazole carboxamide scaffold and different
pyrazole-aromatic scaffolds on antifungal activity, forty-three novel
pyrazole-aromatic containing carboxamides were designed, syn-
thesized and evaluated against different fungi. The results indicated
the pattern of the positive control thifluzamide (Fig. 6B). In another
aspect, there was no significant fluorescence change when treated
by a negative control tricyclazole, a melanin synthesis inhibitor
10

B. Yu, B. Zhao, Z. Hao et al.
European Journal of Medicinal Chemistry 214 (2021) 113230
Fig. 6. Fluorescence spectra of SDH with 11ea, thifluzamide or tricyclazole titration. (A) Fluorescence quenching analysis of compound 11ea with SDH. (B) Fluorescence quenching
analysis of thifluzamide with SDH. (C) Fluorescence quenching analysis of tricyclazole with SDH. The concentration of SDHI from “a to e” stands for “0e5 g/mL.
m
that most of the target compounds displayed satisfactory in vitro
antifungal activities, especially against B. cinerea, R. cerealis,
S. sclerotiorum and in vivo antifungal activity against R. solani. The
compounds with pyrazole-(m-benzene) carboxamide scaffold were
more suited for improving activity against R. cerealis. And SAR
analysis indicated that the promising antifungal potency of target
compounds could be affected by structural changes, including an
alteration of the amine moiety, replacement of the thiazole ring,
and the modification of difluoromethyl substitution on the pyrazole
ring. Among these compounds, compound 11ea exhibited the most
potent activity against R. cerealis, which was obviously better than
both thifluzamide and the previous lead structure A1. The further
enzymatic inhibitory activity and fluorescence quenching analysis
of 11ea suggested that SDH was the same target for both novel
active compounds and thifluzamide. In addition, molecular docking
simulations revealed that 11ea binds with the active site of RcSDH,
formed a hydrogen bond with B-Trp203. Meanwhile, its difluor-
omethyl group formed an additional hydrogen bond with C-Arg81
double of triplet (dt). High resolution mass spectra (HRMS) were
obtained from a Varion 7.0 T FTICR-MS instrument (Ion-Spec, USA).
Melting points (m.p.) were determined on an X-4 digital display
micro melting point apparatus (Henan Gongyi Yingyi Yuhua In-
strument Co., Ltd.) and were uncorrected. Single-crystal X-ray
diffraction analysis was performed on a Rigaku 007 Saturn 70 dif-
fractionmeter (Rigaku, Tokyo, Japan). Fluorescence quenching
analysis was conducted by a F-4500 fluorescence spectrophotom-
eter (Hitachi, Tokyo, Japan).
4.1.1. Synthetic procedure for compound 2
The synthesis procedure was adjusted according to the previ-
ously reported literature [19]. Oxalyl chloride (4.9 mL, 56.78 mmol)
and 0.15 mL of N, N-dimethylformamide (DMF) were added to a
suspension of compound 1 (5.0 g, 28.39 mmol) in dichloromethane
(50 mL). The reaction mixture was stirred for 30 min at room
temperature. After confirmation of the completion of the reaction,
the solvent was removed, the crude residue was diluted with dry
dichloromethane. Then the diluted solution was added dropwise to
a 25% aqueous ammonia solution (7.72 g, 113.56 mmol) using a
constant-pressure dropping funnel at 0 ꢁC and the reaction sus-
pension was stirred overnight at room temperature. After
completion of the reaction, the suspension was filtration and
evaporation to dryness. The crude product 2 (white solid, 4.6 g,
93%) was used directly in the next step without further purification.
and the pyrazole ring formed p-p interactions with both B-Trp203
and B-His246, thereby improving the potency of 11ea against
RcSDH. According to the above analysis, we concluded that the
difluoromethyl group at R1 position would increase the binding
affinity and the middle benzene ring could enhance the
p-p
interaction. Unfortunately, although there was no obvious phyto-
toxicity was observed, 11ea showed lower in vivo activity against
R. solani than thifluzamide, which may due to biological factors
(e.g., plant metabolism, systemic) or/and environmental factors
such as UV photolysis. Therefore, how to improve the in vivo anti-
fungal activity and retain the active scaffold would be the primary
concern in our future work. Overall, pyrazole-aromatic containing
carboxamides were worthy for further structural optimization as
potent SDHI fungicide leads.
4.1.2. Synthetic procedure for compound 3
The synthesis procedure was adjusted according to the previ-
ously reported literature [19]. A suspension of intermediate 2
(9.00 g, 51.39 mmol) and Lawesson’s reagent (12.73 g, 31.47 mmol)
in 50 mL of tetrahydrofuran (THF) was heated to reflux at 65 ꢁC for
30 min. After confirmation of the completion of the reaction by TLC,
the solvent was removed under reduced pressure. The residue was
diluted with ethyl acetate (30 mL) and sequentially washed with
saturated K2CO3 (30 mL ꢂ 2) and brine (15 mL), and dried over
anhydrous Na2SO4, then filtered and concentrated under reduced
pressure. The crude product was purified by silica gel flash column
chromatography with a solvent mixture of ethyl acetate and hexane
(2:1, v/v) to afford thioamide 3 [19].
4. Experimental section
4.1. Chemistry
All chemical reagents and solvents were commercially available
and were used without further purification. Thin-layer chroma-
tography (TLC) analysis was performed on silica gel F254 plates.
Flash column chromatography was carried out on silica gel
(200e300 mesh). 1H NMR (400 MHz) and 13C NMR (101 MHz)
spectra were recorded on a Bruker Avance-400MHz spectrometer
(Bruker, Switzerland) with TMS as an internal standard, deuterated
chloroform (CDCl3) or dimethyl sulfoxide (DMSO‑d6) as a solvent.
The chemical shifts for the NMR spectra were reported in parts per
million (ppm), the coupling constants (J) were described in hertz
(Hz). Peak multiplicities were expressed as singlet (s), doublet (d),
triplet (t), quartet (q), multiplet (m), double of doublet (dd) and
3-(Difluoromethyl)-1-methyl-1H-pyrazole-4-carbothioamide (3).
Yellow solid; yield, 88%; m.p.: 135e137 ꢁC.1H NMR (400 MHz,
CDCl3)
d
8.23 (s,1H), 7.76 (s,1H), 7.58 (s,1H), 6.79 (t, J ¼ 54.1 Hz,1H),
3.91 (s, 3H). 13C NMR (101 MHz, CDCl3)
d
191.4, 139.6 (t, J ¼ 29.9 Hz),
139.4, 121.7, 112.1 (t, J ¼ 232.7 Hz), 39.5. HRMS (ESI) m/z calcd for
C6H8F2N3S [M þ H]þ: 192.0402; found: 192.0404.
4.1.3. Synthetic procedure for compound 9a
The synthesis procedure was adjusted according to the previously
reported literature [19]. The ethyl bromopyruvate (4.9 mL,
11

B. Yu, B. Zhao, Z. Hao et al.
European Journal of Medicinal Chemistry 214 (2021) 113230
28.8mmol) wasadded dropwise to a solution of intermediate3 (5.0 g,
26.2mmol)inethanolatroomtemperature.Thereactionmixturewas
heatedtorefluxat80ꢁCfor2h.Afterconfirmingthecompletionofthe
reaction by TLC, the solutionwas cooled to ambient temperature. The
solvent was removed under reduced pressure, and then the residue
was diluted with ethyl acetate (30 mL) and sequentially washed with
saturated NaHCO3 (20 mL ꢂ 2) and brine (15 mL), and dried over
anhydrous Na2SO4. After the solution was filtered, the solvent was
removed under reduced pressure, and the crude product was purified
using silica gel flash column chromatography with a solvent mixture
of ethyl acetate and hexane (2:1, v/v) for elution. The solvent of the
corresponding fractions was evaporated under reduced pressure to
give compound 9a [19].
4.1.6. Synthetic procedure for compound 5
Compound 4 (6.31 g, 25.52 mmol) was dissolved in dichloro-
methane (50 mL). Trifluoroacetic acid (25 mL) was added dropwise
and the mixture was allowed to stir at room temperature for 2.5 h.
After confirmation of the completion of the reaction by TLC, the
solvent was removed under reduced pressure. The residue was
diluted with water (20 mL). The pH value of the solution was
adjusted to 8 with saturated a NaHCO3 solution. The solution was
extracted with ethyl acetate, and then the combined organic layers
were dried over anhydrous Na2SO4, filtered and concentrated un-
der the reduced pressure. The residue was purified using silica gel
flash column chromatography with a solvent mixture of ethyl ac-
etate and hexane (2:1, v/v) for elution to afford the compound 5.
3-(Difluoromethyl)-1-methyl-1H-pyrazol-4-amine (5). Brown
Ethyl 2-(3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)thiazole-
4-carboxylate (9a). Light yellow solid; yield, 83%; m.p.: 101e103 ꢁC.
liquid; yield, 96%. 1H NMR (400 MHz, CDCl3)
J ¼ 54.7 Hz, 1H), 3.77 (s, 3H), 3.30 (s, 2H).
d 6.97 (s, 1H), 6.67 (t,
1H NMR (400 MHz, CDCl3)
d
8.04 (s, 1H), 7.97 (s, 1H), 6.96 (t,
J ¼ 53.9 Hz, 1H), 4.35 (q, J ¼ 6.9 Hz, 2H), 3.89 (s, 3H), 1.34 (t,
J ¼ 6.5 Hz, 3H).13C NMR (101 MHz, CDCl3)
d
160.3, 157.7, 146.3, 141.8
4.1.7. Synthetic procedure for compound 6
(t, J ¼ 27.5 Hz), 131.1, 125.9, 113.9, 109.8 (t, J ¼ 234.7 Hz), 60.4, 38.5,
13.3. HRMS (ESI) m/z calcd for C11H11F2N3NaO2S [M þ Na]þ:
310.0432; found: 310.0435.
Compound 6 was synthesized according to a previously re-
ported procedure [26]. Compound 5 (1.5 g, 10.20 mmol) was dis-
solved in a 4 mol/L HCl solution (76.5 mL) and cooled down to 0 ꢁC.
Afterwards sodium nitrite (0.91 g, 13.25 mmol) dissolved in water
was added and the mixture was stirred for 1 h at 0 ꢁC. Then po-
tassium iodide (5.08 g, 30.59 mmol) was added portion-wise with
vigorous stirring, and the mixture was warmed to 20 ꢁC within
30 min. After confirmation of the completion of the reaction by TLC,
the reaction mixture was neutralized with saturated a NaHCO3
solution, extracted with dichloromethane (30 mL). The organic
layer was sequentially washed with saturated Na2S2O3 (20 mL ꢂ 2)
and brine (10 mL), and extracted with dichloromethane
(10 mL ꢂ 2). The combined organic layers were dried over anhy-
drous Na2SO4, filtered and concentrated under reduced pressure.
The residue was purified by silica gel flash column chromatography
with a solvent mixture of ethyl acetate and hexane (16:1, v/v) for
elution to yield compound 6.
4.1.4. Synthetic procedure for compound 9b
The synthesis procedure was adjusted according to the pro-
cedure of 9a. The ethyl bromopyruvate (1.07 mL, 6.85 mmol) was
added dropwise to a solution of amide 2 (1.00 g, 5.71 mmol) in
ethanol (20 mL) at room temperature. The solution was refluxed at
80 ꢁC for 8 h. After confirming the completion of the reaction by
TLC, the solution was cooled to room temperature. The solvent was
removed, and the residue was diluted with ethyl acetate (20 mL)
and sequentially washed with saturated NaHCO3 (10 mL ꢂ 2) and
brine (10 mL), and dried over anhydrous Na2SO4. Then the solution
was filtered and concentrated under reduced pressure. The crude
product was purified using silica gel flash column chromatography
with a solvent mixture of ethyl acetate and hexane (2:1, v/v) for
elution. The solvent of the corresponding fractions was evaporated
under reduced pressure to yield compound 9b.
3-(Difluoromethyl)-4-iodo-1-methyl-1H-pyrazole (6). Yellow
liquid; yield, 58%. 1H NMR (400 MHz, CDCl3)
d 7.47 (s, 1H), 6.66 (t,
Ethyl 2-(3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)oxazole-4-
J ¼ 53.8 Hz, 1H), 3.92 (s, 3H). 13C NMR (101 MHz, CDCl3)
d146.4 (t,
carboxylate (9b). Yellow solid; yield, 35%; m.p.: 80e82 ꢁC. 1H NMR
J ¼ 26.8 Hz), 136.7, 111.1 (t, J ¼ 235.1 Hz), 54.0 (t, J ¼ 1.8 Hz), 39.7.
HRMS (ESI) m/z calcd for C5H6F2IN2 [M þ H]þ: 258.9538; found:
258.9540.
(400 MHz, CDCl3)
d
8.23 (s, 1H), 8.08 (s, 1H), 7.13 (t, J ¼ 53.7 Hz, 1H),
4.41 (q, J ¼ 7.1 Hz, 2H), 4.00 (s, 3H), 1.40 (t, J ¼ 7.1 Hz, 3H). 13C NMR
(101 MHz, CDCl3)
d
161.1, 156.3, 143.7 (t, J ¼ 26.3 Hz), 143.2, 134.3,
132.5, 109.8 (t, J ¼ 236.2 Hz), 108.6, 61.4, 39.8, 14.3. HRMS (ESI) m/z
4.1.8. Synthetic procedure for compound 7
calcd for C11H12F2N3O3 [M þ H]þ: 272.0841; found: 272.0841.
The n-butyllithium (2.5 mol/L solution in hexane, 4.6 mL,
11.63 mmol) was added dropwise to a suspension of intermediate 6
(1.0 g, 3.88 mmol) in dry tetrahydrofuran (10 mL) via syringe
at ꢀ78 ꢁC under nitrogen. The resulting solution was stirred for
1 h at ꢀ78 ꢁC. Then the isopropoxyboronic acid pinacol ester (2.16 g,
11.63 mmol) was added dropwise via syringe to the solution. The
resulting mixture was stirred for 2 h. The reaction mixture was
carefully quenched with water (10 mL) and extracted with ethyl
acetate (10 mL ꢂ 2). The combined organic layers were washed
with brine (10 mL) and extracted with ethyl acetate (5 mL ꢂ 2). The
combined organic layers were dried over anhydrous Na2SO4,
filtered and concentrated under reduced pressure. The residue was
purified by silica gel flash column chromatography with a solvent
mixture of ethyl acetate and hexane (8:1, v/v) for elution to afford
product 7.
4.1.5. Synthetic procedure for compound 4
Compound 4 was prepared according to a previously reported
method with modifications [26]. Briefly, under an atmosphere of
argon, compound 3 (5.00 g, 28.39 mmol) was suspended in 100 mL
of tert-butanol. N, N-diisopropylethylamine (DIPEA, 9.89 mL,
56.78 mmol) and diphenyl phosphorazidate (DPPA, 10.16 g,
36.91 mmol) were added to the mixture with syringes. The
resulting mixture was stirred for 10 min at 20 ꢁC. Afterwards the
mixture was stirred for 4 h at 80e90 ꢁC. After confirmation of the
completion of the reaction by TLC, the solution was cooled to room
temperature, and the solvent was removed under reduced pres-
sure. The residue was purified using silica gel flash column chro-
matography with a solvent mixture of ethyl acetate and hexane
(4:1, v/v) for elution. The solvent of the corresponding fractions was
evaporated under reduced pressure to afford compound 4.
Tert-butyl (3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)carba-
mate (4). White solid; yield, 94%; m.p.: 85e87 ꢁC. 1H NMR
3-(Difluoromethyl)-1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)-1H-pyrazole (7). White solid; yield, 68%; m.p.:
87e89 ꢁC. 1H NMR (400 MHz, CDCl3)
d 7.66 (s, 1H), 6.96 (t,
J ¼ 54.4 Hz,1H), 3.94 (s, 3H),1.31 (s,12H). 13C NMR (101 MHz, CDCl3)
(400 MHz, CDCl3)
3.84 (s, 3H), 1.50 (s, 9H). 13C NMR (101 MHz, CDCl3)
J ¼ 29.1 Hz), 122.5, 119.9, 112.6 (t, J ¼ 231.5 Hz), 80.9, 39.4, 28.2.
d
7.86 (s, 1H), 6.70 (t, J ¼ 54.6 Hz, 1H), 6.66 (s, 1H),
d
151.3 (t, J ¼ 23.8 Hz), 138.2, 110.5 (t, J ¼ 235.2 Hz), 83.7, 39.1, 24.8.
d
152.6, 133.6 (t,
HRMS (ESI) m/z calcd for C11H18BF2N2O2 [M þ H]þ: 259.1424;
found: 259.1436.
12

B. Yu, B. Zhao, Z. Hao et al.
European Journal of Medicinal Chemistry 214 (2021) 113230
4.1.9. Synthetic procedure for compound 9c
4.1.11. General procedure for compounds 10a-10e
Under a nitrogen atmosphere, compound 7 (0.5 g, 1.94 mmol),
ethyl 5-bromothiophene-2-carboxylate (0.55 g, 2.32 mmol) and
Pd(PPh3)4 (0.11 g, 0.05 mmol) were dissolved in THF (40 mL). 2 mol/
L Na2CO3 (1.94 mL, 3.87 mmol) was added and the mixture was
stirred overnight at 100 ꢁC. After confirmation of the completion of
the reaction by TLC, the solution was cooled to room temperature,
the precipitate was removed by filtering through a pad of celite and
washed with ethyl acetate. The filtrate was concentrated under
reduced pressure. The residue was purified by silica gel flash col-
umn chromatography with a solvent mixture of ethyl acetate and
hexane (8:1, v/v) for elution to afford the compound 9c.
According to the procedure previously reported [27], the in-
termediates 9a-9e (1 equiv.) were dissolved in a mixed solution of
2 mol/L NaOH/EtOH (4:1, v/v) and heated to reflux for 30 min at
80 ꢁC. After completion of the reaction, the solution was cooled to
room temperature, then acidified to pH ¼ 2 with a 2 mol/L HCl
solution. The precipitated solid was filtered and dried to give the
corresponding carboxylic acids 10a-10e (56e93%).
2-(3-(Difluoromethyl)-1-methyl-1H-pyrazol-4-yl)thiazole-4-
carboxylic acid (10a) [19]. Off-white solid; yield, 91%; m.p.:
213e215 ꢁC. 1H NMR (400 MHz, DMSO‑d6)
d 13.14 (s, 1H), 8.59 (s,
1H), 8.45 (s, 1H), 7.41 (t, J ¼ 53.6 Hz, 1H), 3.98 (s, 3H). 13C NMR
Ethyl 5-(3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)thiophene-
(101 MHz, DMSO‑d6)
d
162.3, 158.8, 147.9, 141.9 (t, J ¼ 25.4 Hz),
2-carboxylate (9c). Yellow solid; yield, 74%; m.p.: 56e58 ꢁC. 1H NMR
133.6, 128.5, 114.5, 111.0 (t, J ¼ 233.5 Hz), 39.8. HRMS (ESI) m/z calcd
for C9H7F2N3NaO2S [M þ Na]þ: 282.0119; found: 282.0122.
2-(3-(Difluoromethyl)-1-methyl-1H-pyrazol-4-yl)oxazole-4-
carboxylic acid (10b). Yellow solid; yield, 56%; m.p.: 224e226 ꢁC. 1H
(400 MHz, CDCl3)
d
7.72 (d, J ¼ 3.8 Hz, 1H), 7.61 (s, 1H), 7.20 (d,
J ¼ 3.8 Hz, 1H), 6.76 (t, J ¼ 53.9 Hz, 1H), 4.35 (q, J ¼ 7.1 Hz, 2H), 3.95
(s, 3H), 1.38 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d 162.2,
142.3 (t, J ¼ 28.3 Hz), 139.1, 134.1, 132.4, 130.7, 126.4, 114.5, 111.4 (t,
NMR (400 MHz, DMSO)
d
13.19 (s, 1H), 8.80 (d, J ¼ 2.7 Hz, 1H), 8.56
J
C
¼
234.2 Hz), 61.2, 39.4, 14.4. HRMS (ESI) m/z calcd for
(d, J ¼ 2.9 Hz, 1H), 7.30 (t, J ¼ 53.5 Hz, 1H), 3.97 (s, 3H). 13C NMR
12H13F2N2O2S [M þ H]þ: 287.0660; found: 287.0664.
(101 MHz, DMSO)
d
162.4, 156.0, 145.1, 142.7 (t, J ¼ 25.5 Hz), 134.5,
133.9, 110.4 (t, J ¼ 234.2 Hz), 108.0 (t, J ¼ 2.8 Hz), 39.8. HRMS (ESI)
m/z calcd for C9H8F2N3O3 [M þ H]þ: 244.0528; found: 244.0531.
5-(3-(Difluoromethyl)-1-methyl-1H-pyrazol-4-yl)thiophene-2-
carboxylic acid (10c). Yellow solid; yield, 79%; m.p.: 185e187 ꢁC. 1H
4.1.10. General procedure for compounds 9d-9e
Compound 8 (1 equiv.), corresponding boronic acid (1.2 equiv.),
Cs2CO3 (2 equiv.), PdCl2(dppf) (0.05 equiv.) were dissolved in a
mixed solution of dioxane/H2O (10:1, v/v) under a nitrogen atmo-
sphere. The reaction mixture was heated to reflux overnight at
110 ꢁC. After confirmation of the completion of the reaction by TLC,
the solution was cooled to room temperature. The mixture was
filtered through a pad of celite and the filtrate was concentrated
under reduced pressure. The residue was purified by silica gel flash
column chromatography with a solvent mixture of ethyl acetate
and hexane (8:1, v/v) for elution to afford compounds 9d-9e (63-
89%).
NMR (400 MHz, DMSO)
d 13.14 (s, 1H), 8.32 (s, 1H), 7.73e7.67 (m,
1H), 7.24e7.22 (m,1H), 7.12 (t, J ¼ 53.2 Hz,1H), 3.92 (s, 3H). 13C NMR
(101 MHz, DMSO)
d
163.2, 141.3 (t, J ¼ 28.0 Hz), 139.5, 134.4, 133.2,
132.9, 126.4, 113.4, 112.2 (t, J ¼ 231.9 Hz), 39.3. HRMS (ESI) m/z calcd
for C10H9F2N2O2S [M þ H]þ: 259.0347; found: 259.0352.
4-(3-(Difluoromethyl)-1-methyl-1H-pyrazol-4-yl)benzoic
acid
(10d). White solid; yield, 91%; m.p.: 194e196 ꢁC. 1H NMR (400 MHz,
DMSO)
d
12.96 (s, 1H), 8.26 (s, 1H), 7.97 (d, J ¼ 8.2 Hz, 2H), 7.62 (d,
J ¼ 8.3 Hz, 2H), 7.11 (t, J ¼ 53.6 Hz, 1H), 3.93 (s, 3H). 13C NMR
Ethyl 4-(3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)benzoate
(101 MHz, DMSO)
d
167.0, 141.2 (t, J ¼ 27.7 Hz), 135.6, 132.3, 129.7,
(9d). White solid; yield, 65%; m.p.: 139e141 ꢁC. 1H NMR (400 MHz,
129.0, 127.5, 119.6, 112.0 (t, J ¼ 232.1 Hz), 39.3. HRMS (ESI) m/z calcd
CDCl3)
d
7.97 (d, J ¼ 8.1 Hz, 2H), 7.48 (d, J ¼ 2.9 Hz, 2H), 7.45 (s, 1H),
for C12H11F2N2O2 [M þ H]þ: 253.0783; found: 253.0786.
6.72 (t, J ¼ 54.1 Hz, 1H), 4.29 (q, J ¼ 7.0 Hz, 2H), 3.81 (s, 3H), 1.31 (t,
3-(3-(Difluoromethyl)-1-methyl-1H-pyrazol-4-yl)benzoic
acid
J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d
166.2, 142.2 (t,
(10ea). White solid; yield, 87%; m.p.: 141e142 ꢁC. 1H NMR
(400 MHz, DMSO) 13.06 (s, 1H), 8.24 (s, 1H), 8.10 (s, 1H), 7.88 (d,
J ¼ 27.8 Hz), 135.7, 130.7, 129.8, 129.0, 127.8, 120.9, 111.9 (t,
d
J
C
¼
233.8 Hz), 60.9, 39.1, 14.2. HRMS (ESI) m/z calcd for
J ¼ 7.6 Hz, 1H), 7.73 (d, J ¼ 7.6 Hz, 1H), 7.55 (t, J ¼ 7.7 Hz, 1H), 7.07 (t,
14H15F2N2O2 [M þ H]þ: 281.1096; found: 281.1096.
J ¼ 53.7 Hz, 1H), 3.93 (s, 3H). 13C NMR (101 MHz, DMSO)
d 167.1,
Ethyl 3-(3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)benzoate
141.0 (t, J ¼ 27.6 Hz), 132.0, 131.8, 131.6, 131.3, 129.0, 128.4, 127.7,
119.7, 112.2 (t, J ¼ 231.6 Hz), 39.3. HRMS (ESI) m/z calcd for
(9ea). Yellow oil; yield, 63%. 1H NMR (400 MHz, CDCl3)
d 8.13 (s, 1H),
7.97 (d, J ¼ 7.8 Hz, 1H), 7.68 (d, J ¼ 7.7 Hz, 1H), 7.56 (s, 1H), 7.45 (t,
C
12H11F2N2O2 [M þ H]þ: 253.0783; found: 253.0787.
3-(1-Methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)benzoic
J ¼ 7.7 Hz, 1H), 6.76 (t, J ¼ 54.1 Hz, 1H), 4.37 (q, J ¼ 7.1 Hz, 2H), 3.93
acid
(s, 3H), 1.39 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d
166.4,
(10eb). White solid; yield, 93%; m.p.: 162e164 ꢁC. 1H NMR
142.3 (t, J ¼ 27.6 Hz), 132.6 (t, J ¼ 2.1 Hz), 131.5, 130.9, 130.6, 129.1,
128.7, 128.2, 121.2, 111.8 (t, J ¼ 233.9 Hz), 39.3, 14.3. HRMS (ESI) m/z
calcd for C14H15F2N2O2 [M þ H]þ: 281.1096; found: 281.1102.
Ethyl 3-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)benzoate
(400 MHz, DMSO)
J ¼ 7.6 Hz, 1H), 7.66 (d, J ¼ 7.6 Hz, 1H), 7.58 (t, J ¼ 7.5 Hz, 1H), 3.98 (s,
3H). 13C NMR (101 MHz, DMSO)
d 13.15 (s, 1H), 8.27 (s, 1H), 8.02 (s, 1H), 7.95 (d,
d
167.0, 136.2 (q, J ¼ 35.5 Hz), 132.9,
132.3, 131.2, 130.7, 129.0, 128.7, 128.3, 121.8 (q, J ¼ 268.9 Hz), 120.1,
39.3. HRMS (ESI) m/z calcd for C12H10F3N2O2 [M þ H]þ: 271.0689;
found: 271.0688.
(9eb). Yellow oil; yield, 89%. 1H NMR (400 MHz, CDCl3)
d 8.07 (s,
1H), 8.02 (d, J ¼ 7.7 Hz, 1H), 7.60 (d, J ¼ 7.6 Hz, 1H), 7.55 (s, 1H), 7.46
(t, J ¼ 7.7 Hz, 1H), 4.39 (q, J ¼ 7.1 Hz, 2H), 3.99 (s, 3H), 1.40 (t,
3-(1,3-Dimethyl-1H-pyrazol-4-yl)benzoic acid (10ec). White
J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d 166.3, 138.5 (q,
J ¼ 36.5 Hz), 132.9, 131.1, 130.9, 129.6, 128.7, 128.6, 121.5 (q,
J ¼ 269.2 Hz), 121.5, 61.1, 39.6, 14.3. HRMS (ESI) m/z calcd for
solid; yield: 92%; m.p.: 183e185 ꢁC; 1H NMR (400 MHz, DMSO)
d 13.03 (s, 1H), 8.01e7.98 (m, 2H), 7.83e7.79 (m, 1H), 7.67 (d,
J ¼ 7.7 Hz, 1H), 7.51 (t, J ¼ 7.7 Hz, 1H), 3.80 (s, 3H), 2.32 (s, 3H). 13
C
C
14H14F3N2O2 [M þ H]þ: 299.1002; found: 299.1006.
NMR (101 MHz, DMSO) d 167.8, 144.5, 134.4, 131.7, 131.3, 130.4,
Ethyl 3-(1,3-dimethyl-1H-pyrazol-4-yl)benzoate (9ec). Yellow oil;
129.4, 127.7, 127.0, 119.2, 38.7, 13.7. HRMS (ESI) m/z calcd for
yield, 86%. 1H NMR (400 MHz, CDCl3)
d
8.06 (s, 1H), 7.92 (d,
C
12H13N2O2 [M þ H]þ: 217.0972; found: 217.0975.
J ¼ 7.7 Hz, 1H), 7.57 (d, J ¼ 7.7 Hz, 1H), 7.49 (s, 1H), 7.44 (t, J ¼ 7.7 Hz,
1H), 4.39 (q, J ¼ 7.1 Hz, 2H), 3.89 (s, 3H), 2.41 (s, 3H), 1.41 (t,
4.1.12. General procedure for target compounds 11aa-11eh
The carboxylic acids 10a-10e (1 equiv.) were dissolved in
dichloromethane, N, N-diisopropylethylamine (DIPEA, 2 equiv.) and
J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d 166.6,145.7,133.9,131.6,
130.9, 129.1, 128.6, 128.4, 127.1, 120.2, 61.0, 38.7, 14.4, 13.2. HRMS
(ESI) m/z calcd for C14H17N2O2 [M þ H]þ: 245.1285; found:
245.1284.
Benzotriazol-1-yl-
oxytripyrrolidinophosphonium
hexa-
fluorophosphate (PyBOP, 1.1 equiv.) were added at 0 ꢁC. After
13

B. Yu, B. Zhao, Z. Hao et al.
European Journal of Medicinal Chemistry 214 (2021) 113230
15 min, the corresponding amine (1.2 equiv.) was added. And then,
the reaction mixture was stirred overnight at room temperature.
The completion of the reaction was monitored by TLC, the solution
was sequentially washed with water and brine, and extracted with
dichloromethane. The combined organic layers were dried over
anhydrous Na2SO4, then filtered and concentrated under reduced
pressure. The crude residues were purified by silica gel column
chromatography to afford title compounds 11aa-11eh (30e99%).
N-(2-chlorophenyl)-2-(3-(difluoromethyl)-1-methyl-1H-pyrazol-
4-yl)thiazole-4-carboxamide (11aa). White solid; yield: 50%; purity:
yield: 30%; purity: 100%; m.p.: 140e142 ꢁC; 1H NMR (400 MHz,
CDCl3)
d
9.71 (s, 1H), 8.65 (d, J ¼ 8.1 Hz, 1H), 8.16 (s, 1H), 7.92 (s, 1H),
7.34 (dd, J ¼ 14.3, 7.3 Hz, 2H), 7.15 (t, J ¼ 7.7 Hz, 1H), 7.10 (t,
J ¼ 53.9 Hz, 1H), 4.01 (s, 3H). 13C NMR (101 MHz, CDCl3)
d 158.6,
158.6, 150.0, 143.0 (t, J ¼ 26.8 Hz), 138.2, 131.6, 130.7, 127.7, 124.2,
123.5, 121.2, 120.7 (q, J ¼ 258.8 Hz), 120.6, 114.9, 110.4 (t,
J ¼ 235.7 Hz), 39.7. HRMS (ESI) m/z calcd for C16H12F5N4O2S [M þ
H]þ: 419.0596; found: 419.0596.
2-(3-(Difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-N-(3-(tri-
fluoromethoxy)phenyl) thiazole-4-carboxamide (11ah). White solid;
yield: 90%; purity: 100%; m.p.: 139e141 ꢁC; 1H NMR (400 MHz,
100%; m.p.: 177e178 ꢁC; 1H NMR (400 MHz, CDCl3)
d 9.93 (s, 1H),
8.61 (d, J ¼ 8.2 Hz, 1H), 8.15 (s, 1H), 7.93 (s, 1H), 7.43 (dd, J ¼ 8.0,
CDCl3) d 9.29 (s, 1H), 8.16 (s, 1H), 7.97 (s, 1H), 7.78 (s, 1H), 7.53 (dd,
0.8 Hz, 1H), 7.33 (t, J ¼ 7.8 Hz, 1H), 7.16 (t, J ¼ 53.9 Hz, 2H), 7.11e7.02
J ¼ 8.2, 1.2 Hz, 1H), 7.38 (t, J ¼ 8.2 Hz, 1H), 7.14e6.99 (m, 1H), 6.99 (t,
(m, 1H), 4.01 (s, 3H). 13C NMR (101 MHz, CDCl3)
d
158.6,158.5,150.2,
J ¼ 53.8 Hz, 1H), 4.02 (s, 3H). 13C NMR (101 MHz, CDCl3)
d 158.7,
143.1 (t, J ¼ 26.6 Hz), 134.7, 131.5, 129.2, 127.8, 124.7, 123.3, 123.0,
158.3, 149.8, 149.7, 143.0 (t, J ¼ 27.9 Hz), 139.1, 131.8, 130.1, 123.7,
121.8, 117.7, 116.5, 114.7, 112.5, 110.8 (t, J ¼ 235.3 Hz), 39.7. HRMS
(ESI) m/z calcd for C16H12F5N4O2S [M þ H]þ: 419.0596; found:
419.0595.
120.9, 114.9, 110.4 (t, J ¼ 235.9 Hz), 39.7. HRMS (ESI) m/z calcd for
C
15H12ClF2N4OS [M þ H]þ: 369.0383; found: 369.0383.
N-(3-chlorophenyl)-2-(3-(difluoromethyl)-1-methyl-1H-pyrazol-
4-yl)thiazole-4-carboxamide (11 ab). White solid; yield: 87%; purity:
2-(3-(Difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-N-(4-(tri-
fluoromethoxy)phenyl) thiazole-4-carboxamide (11ai). White solid;
yield: 93%; purity: 100%; m.p.: 151e153 ꢁC; 1H NMR (400 MHz,
100%; m.p.: 141e143 ꢁC; 1H NMR (400 MHz, CDCl3)
d
9.22 (s, 1H),
8.14 (s, 1H), 7.95 (s, 1H), 7.82 (t, J ¼ 1.9 Hz, 1H), 7.53 (dd, J ¼ 8.1,
1.2 Hz, 1H), 7.29 (t, J ¼ 8.1 Hz, 1H), 7.12 (d, J ¼ 8.1 Hz, 1H), 6.99 (t,
CDCl3)
d
9.27 (s, 1H), 8.15 (s, 1H), 7.95 (s, 1H), 7.74 (d, J ¼ 9.0 Hz, 2H),
J ¼ 49.2 Hz, 1H), 4.00 (s, 3H). 13C NMR (101 MHz, CDCl3)
d
158.6,
7.23 (d, J ¼ 8.6 Hz, 2H), 7.00 (t, J ¼ 53.8 Hz, 1H), 4.01 (s, 3H). 13C NMR
158.3, 149.9, 142.9 (t, J ¼ 27.7 Hz), 138.8, 134.8, 131.7, 130.1, 124.5,
123.6, 119.7, 117.7, 114.8, 110.8 (t, J ¼ 235.3 Hz), 39.7. HRMS (ESI) m/z
calcd for C15H11ClF2N4NaOS [M þ Na]þ: 391.0202; found: 391.0206.
N-(4-chlorophenyl)-2-(3-(difluoromethyl)-1-methyl-1H-pyrazol-
4-yl)thiazole-4-carboxamide (11ac). Yellow solid; yield: 73%; purity:
(101 MHz, CDCl3)
d
158.6, 158.3, 149.9, 145.4, 143.0 (t, J ¼ 27.7 Hz),
136.3, 131.7, 123.5, 121.9, 120.8, 119.2, 114.8, 110.8 (t, J ¼ 235.3 Hz),
39.7. HRMS (ESI) m/z calcd for C16H11F5N4NaO2S [M þ Na]þ:
441.0415; found: 441.0418.
2-(3-(Difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-N-(2-
isopropylphenyl)thiazole-4-carboxamide (11aj). Yellow solid; yield:
87%; purity: 100%; m.p.: 109e111 ꢁC; 1H NMR (400 MHz, CDCl3)
99%; m.p.: 179e180 ꢁC; 1H NMR (400 MHz, CDCl3)
d 9.20 (s, 1H),
8.11 (s, 1H), 7.93 (s, 1H), 7.64 (d, J ¼ 8.3 Hz, 2H), 7.32 (d, J ¼ 8.3 Hz,
2H), 7.00 (t, J ¼ 53.8 Hz, 1H), 3.99 (s, 3H). 13C NMR (101 MHz, CDCl3)
d
9.38 (s,1H), 8.20 (d, J ¼ 7.9 Hz,1H), 8.12 (s, 1H), 7.88 (s,1H), 7.33 (d,
d
158.6, 158.3, 149.9, 142.9 (t, J ¼ 27.5 Hz), 136.3, 131.7, 129.3, 129.1,
J ¼ 7.5 Hz, 1H), 7.25 (d, J ¼ 7.9 Hz, 1H), 7.18 (t, J ¼ 7.3 Hz, 1H), 7.11 (t,
J ¼ 53.9 Hz, 1H), 3.97 (s, 3H), 3.20 (dt, J ¼ 13.5, 6.7 Hz, 1H), 1.34 (d,
123.4, 120.9, 114.7, 110.8 (t, J ¼ 235.3 Hz), 39.6. HRMS (ESI) m/z calcd
for C15H11ClF2N4NaOS [M þ Na]þ: 391.0202; found: 391.0205.
2-(3-(Difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-N-(2-
J ¼ 6.8 Hz, 6H). 13C NMR (101 MHz, CDCl3)
d 158.7,158.4,150.8,142.8
(t, J ¼ 26.6 Hz), 138.6, 134.3, 131.6, 126.5, 125.6, 125.3, 122.8, 122.4,
115.0, 110.4 (t, J ¼ 235.8 Hz), 39.7, 28.0, 22.9. HRMS (ESI) m/z calcd
for C18H19F2N4OS [M þ H]þ: 377.1242; found: 377.1249.
methoxyphenyl)thiazole-4-carboxamide (11ad). White solid; yield:
84%; purity: 100%; m.p.: 194e196 ꢁC; 1H NMR (400 MHz, CDCl3)
d
9.97 (s, 1H), 8.53 (dd, J ¼ 7.9, 1.5 Hz, 1H), 8.06 (s, 1H), 7.85 (s, 1H),
N-(3,5-dibromophenyl)-2-(3-(difluoromethyl)-1-methyl-1H-pyr-
azol-4-yl)thiazole-4-carboxamide (11ak). Yellow solid; yield: 64%;
7.30 (t, J ¼ 53.9 Hz, 1H), 7.08 (td, J ¼ 7.8, 1.6 Hz, 1H), 7.00 (td, J ¼ 7.7,
1.1 Hz, 1H), 6.92 (dd, J ¼ 8.0, 1.0 Hz, 1H), 3.98 (s, 3H), 3.97 (s, 3H). 13
C
purity: 100%; m.p.: 154e156 ꢁC; 1H NMR (400 MHz, CDCl3)
d 9.21 (s,
NMR (101 MHz, CDCl3)
d
158.3, 158.2, 150.8, 148.3, 143.1 (t,
1H), 8.17 (s, 1H), 7.98 (s,1H), 7.87 (d, J ¼ 1.4 Hz, 2H), 7.44 (s, 1H), 6.98
J ¼ 25.3 Hz), 131.1, 127.6, 124.0, 122.2, 121.1, 119.3, 115.1, 110.2 (t,
(t, J ¼ 53.8 Hz, 1H), 4.02 (s, 3H). 13C NMR (101 MHz, CDCl3)
d 158.6,
J
C
¼
236.1 Hz), 110.0, 55.8, 39.7. HRMS (ESI) m/z calcd for
158.5, 149.4, 143.0 (t, J ¼ 26.1 Hz), 139.8, 131.8, 129.8, 124.0, 123.1,
16H14F2N4NaO2S [M þ Na]þ: 387.0698; found: 387.0702.
121.1, 114.6, 110.9 (t, J ¼ 235.3 Hz), 39.7. HRMS (ESI) m/z calcd for
2-(3-(Difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-N-(3-
C
15H11Br2F2N4OS [M þ H]þ: 490.8983; found: 490.8973.
methoxyphenyl)thiazole-4-carboxamide (11ae). Light yellow solid;
yield: 98%; purity: 100%; m.p.: 165e167 ꢁC; 1H NMR (400 MHz,
N-(3,4-dichlorophenyl)-2-(3-(difluoromethyl)-1-methyl-1H-pyr-
azol-4-yl)thiazole-4-carboxamide (11 al). White solid; yield: 88%;
CDCl3)
d
9.20 (s, 1H), 8.12 (s, 1H), 7.94 (s, 1H), 7.50 (t, J ¼ 2.1 Hz, 1H),
purity: 99%; m.p.: 173e175 ꢁC; 1H NMR (400 MHz, CDCl3)
d
9.23 (s,
7.26 (t, J ¼ 8.1 Hz, 1H), 7.14 (dd, J ¼ 7.6, 1.4 Hz, 1H), 7.01 (t,
1H), 8.15 (s, 1H), 7.96 (s, 1H), 7.95 (d, J ¼ 2.5 Hz, 1H), 7.52 (dd, J ¼ 8.7,
J ¼ 53.9 Hz, 1H), 6.71 (dd, J ¼ 8.2, 1.9 Hz, 1H), 3.99 (s, 3H), 3.84 (s,
2.5 Hz, 1H), 7.42 (d, J ¼ 8.7 Hz, 1H), 6.99 (t, J ¼ 53.8 Hz, 1H), 4.02 (s,
3H). 13C NMR (101 MHz, CDCl3)
d
160.3, 158.6, 158.2, 150.3, 142.9 (t,
3H). 13C NMR (101 MHz, CDCl3)
d 157.6, 157.4, 148.5, 141.9 (t,
J ¼ 27.6 Hz), 138.9, 131.7, 129.8, 123.2, 114.8, 111.9, 110.8 (t,
J ¼ 27.9 Hz), 136.1, 131.9, 130.7, 129.6, 126.5, 122.8, 120.2, 117.9, 113.6,
J ¼ 235.4 Hz), 110.4, 105.3, 55.4, 39.6. HRMS (ESI) m/z calcd for
109.8 (t,
J
¼
235.2 Hz), 38.7. HRMS (ESI) m/z calcd for
C
16H14F2N4NaO2S [M þ Na]þ: 387.0703; found: 387.0702.
2-(3-(Difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-N-(4-
C
15H11Cl2F2N4OS [M þ H]þ: 402.9993; found: 402.9993.
N-(3,5-dichlorophenyl)-2-(3-(difluoromethyl)-1-methyl-1H-pyr-
methoxyphenyl)thiazole-4-carboxamide (11af). Yellow solid; yield:
azol-4-yl)thiazole-4-carboxamide (11am). Light yellow solid; yield:
99%; purity: 100%; m.p.: 170e172 ꢁC; 1H NMR (400 MHz, CDCl3)
58%; purity: 100%; m.p.: 181e183 ꢁC; 1H NMR (400 MHz, CDCl3)
d
9.11 (s, 1H), 8.12 (s, 1H), 7.95 (s, 1H), 7.62 (d, J ¼ 8.9 Hz, 2H), 7.03 (t,
d
9.22 (s, 1H), 8.14 (s, 1H), 7.95 (s, 1H), 7.66 (d, J ¼ 1.6 Hz, 2H), 7.12 (t,
J ¼ 53.8 Hz, 1H), 6.92 (d, J ¼ 8.9 Hz, 2H), 4.01 (s, 3H), 3.82 (s, 3H). 13
C
J ¼ 1.6 Hz, 1H), 6.97 (t, J ¼ 53.8 Hz, 1H), 4.00 (s, 3H). 13C NMR
NMR (101 MHz, CDCl3)
d
158.4, 158.1, 156.5, 150.4, 142.9 (t,
(101 MHz, CDCl3)
d
158.6, 158.5, 149.4, 143.0 (t, J ¼ 27.9 Hz), 139.5,
J ¼ 27.4 Hz), 131.7, 130.8, 122.9, 121.4, 114.9, 114.3, 110.7 (t,
J ¼ 235.3 Hz), 55.5, 39.7. HRMS (ESI) m/z calcd for C16H14F2N4NaO2S
[M þ Na]þ: 387.0698; found: 387.0702.
135.4, 131.8, 124.3, 124.0, 117.9, 114.6, 110.9 (t, J ¼ 235.2 Hz), 39.7.
HRMS (ESI) m/z calcd for C15H11Cl2F2N4OS [M þ H]þ: 402.9993;
found: 402.9996.
N-cyclopropyl-2-(3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)
thiazole-4-carboxamide (11an). White solid; yield: 96%; purity:
2-(3-(Difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-N-(2-(tri-
fluoromethoxy)phenyl) thiazole-4-carboxamide (11 ag). Yellow solid;
14

B. Yu, B. Zhao, Z. Hao et al.
European Journal of Medicinal Chemistry 214 (2021) 113230
100%; m.p.: 116e118 ꢁC; 1H NMR (400 MHz, CDCl3)
d
8.06 (s, 1H),
69%; purity: 99%; m.p.: 205e207 ꢁC; 1H NMR (400 MHz, CDCl3)
7.91 (s, 1H), 7.34 (s, 1H), 7.00 (t, J ¼ 53.9 Hz, 1H), 4.00 (s, 3H), 2.92
d
10.05 (s,1H), 8.71 (s,1H), 8.57 (d, J ¼ 5.5 Hz,1H), 8.49 (d, J ¼ 5.5 Hz,
(tq, J ¼ 7.3, 3.7 Hz, 1H), 0.94e0.86 (m, 2H), 0.70e0.63 (m, 2H). 13
C
1H), 8.21 (s, 1H), 7.95 (s, 1H), 7.18 (t, J ¼ 53.8 Hz, 1H), 4.03 (s, 3H). 13
C
NMR (101 MHz, CDCl3)
d
162.2, 158.1, 150.2, 142.9 (t, J ¼ 27.0 Hz),
NMR (101 MHz, CDCl3) d 159.0, 158.9, 151.9, 149.8, 149.3, 143.0 (t,
131.5, 122.5, 115.0, 110.6 (t, J ¼ 235.3 Hz), 39.6, 22.5, 6.8. HRMS (ESI)
m/z calcd for C12H13F2N4OS [M þ H]þ: 299.0773; found: 299.0774.
N-cyclohexyl-2-(3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)
thiazole-4-carboxamide (11ao). Light yellow solid; yield: 99%; pu-
J ¼ 26.7 Hz), 142.5, 131.6, 124.5, 114.7, 114.5, 110.8, 110.4 (t,
J ¼ 235.8 Hz), 39.8. HRMS (ESI) m/z calcd for C14H11BrF2N5OS [M þ
H]þ: 413.9830; found: 413.9830.
2-(3-(Difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-N-(pyrimidin-
4-yl)thiazole-4-carboxamide (11av). Light yellow solid; yield: 75%;
rity: 100%; m.p.: 115e117 ꢁC; 1H NMR (400 MHz, CDCl3)
d 8.03 (s,
1H), 7.93 (s, 1H), 7.24 (d, J ¼ 8.1 Hz, 1H), 7.02 (t, J ¼ 53.9 Hz, 1H), 3.99
(s, 3H), 2.05e1.97 (m, 2H), 1.80e1.71 (m, 2H), 1.68e1.60 (m, 1H),
1.50e1.38 (m, 2H), 1.37e1.19 (m, 4H). 13C NMR (101 MHz, CDCl3)
purity: 100%; m.p.: 208e210 ꢁC; 1H NMR (400 MHz, CDCl3)
d 9.78
(s, 1H), 8.95 (s, 1H), 8.70 (d, J ¼ 5.7 Hz, 1H), 8.34 (d, J ¼ 5.6 Hz, 1H),
8.25 (s, 1H), 8.07 (s, 1H), 6.98 (t, J ¼ 53.8 Hz, 1H), 4.02 (s, 3H). 13
C
d
159.9, 157.9, 150.7, 142.8 (t, J ¼ 27.0 Hz), 131.6, 122.3, 115.1, 110.6 (t,
NMR (101 MHz, CDCl3) d 159.5, 158.9, 158.6, 158.5, 156.8, 148.7,
J ¼ 235.3 Hz), 48.0, 39.6, 33.0, 25.6, 24.7. HRMS (ESI) m/z calcd for
142.9 (t, J ¼ 28.2 Hz), 132.2, 125.3, 114.5, 111.0 (t, J ¼ 234.9 Hz), 110.3,
39.7. HRMS (ESI) m/z calcd for C13H11F2N6OS [M þ H]þ: 337.0678;
found: 337.0681.
C
15H18F2N4NaOS [M þ Na]þ: 363.1062; found: 363.1062.
2-(3-(Difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-N-(naph-
thalen-1-yl)thiazole-4-carboxamide (11ap). White solid; yield: 72%;
2-(3-(Difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-N-(pyrazin-2-
yl)thiazole-4-carboxamide (11aw). Yellow solid; yield: 32%; purity:
purity: 99%; m.p.: 172e174 ꢁC; 1H NMR (400 MHz, CDCl3)
d
9.37 (s,
1H), 8.41 (s, 1H), 8.16 (s, 1H), 7.93 (s, 1H), 7.88e7.77 (m, 3H), 7.63 (d,
J ¼ 7.9 Hz, 1H), 7.45 (dd, J ¼ 18.3, 7.1 Hz, 2H), 7.05 (t, J ¼ 54.0 Hz, 1H),
99%; m.p.: 241e243 ꢁC; 1H NMR (400 MHz, CDCl3)
d 9.73 (d,
J ¼ 1.2 Hz, 1H), 9.70 (s, 1H), 8.40 (d, J ¼ 2.5 Hz, 1H), 8.37e8.33 (m,
3.98 (s, 3H). 13C NMR (101 MHz, CDCl3)
d 158.8,158.2,150.3,142.9 (t,
1H), 8.25 (s, 1H), 8.04 (s, 1H), 7.00 (t, J ¼ 53.8 Hz, 1H), 4.02 (s, 3H).
J ¼ 27.4 Hz), 135.1, 133.9, 131.7, 130.7, 128.9, 127.8, 127.6, 126.6, 125.1,
123.3, 119.7, 116.4, 114.9, 110.8 (t, J ¼ 235.3 Hz), 39.6. HRMS (ESI) m/z
calcd for C19H15F2N4OS [M þ H]þ: 385.0929; found: 385.0931.
2-(3-(Difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-N-(1,2,3,4-
tetrahydronaphthalen-1-yl)thiazole-4-carboxamide (11aq). Green
solid; yield: 80%; purity: 100%; m.p.: 136e138 ꢁC; 1H NMR
13C NMR (101 MHz, CDCl3)
d 158.7, 148.9, 148.0, 142.9 (d,
J ¼ 27.9 Hz), 142.4, 140.3, 136.9, 132.0, 124.9, 114.6, 110.9 (t,
J ¼ 235.0 Hz), 100.0, 39.7. HRMS (ESI) m/z calcd for C13H11F2N6OS
[M þ H]þ: 337.0678; found: 337.0681.
2-(3-(Difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-N-(thiazol-2-
yl)thiazole-4-carboxamide (11ax). Yellow solid; yield: 78%; purity:
(400 MHz, CDCl3)
d
8.11 (s, 1H), 7.90 (s, 1H), 7.56 (d, J ¼ 8.9 Hz, 1H),
99%; m.p.: 214e216 ꢁC; 1H NMR (400 MHz, CDCl3)
d 10.50 (s, 1H),
7.39e7.35 (m, 1H), 7.23e7.12 (m, 3H), 6.92 (t, J ¼ 53.9 Hz, 1H), 5.39
8.26 (s, 1H), 8.03 (s, 1H), 7.53 (d, J ¼ 3.5 Hz, 1H), 7.06 (d, J ¼ 3.5 Hz,
(dd, J ¼ 14.6, 5.8 Hz, 1H), 3.96 (s, 3H), 2.94e2.77 (m, 2H), 2.23e2.13
1H), 6.97 (t, J ¼ 53.8 Hz, 1H), 4.02 (s, 3H). 13C NMR (101 MHz, CDCl3)
(m, 1H), 1.97e1.87 (m, 3H). 13C NMR (101 MHz, CDCl3)
d
160.3,158.1,
d
159.0, 158.0, 157.5, 147.80, 142.9 (t, J ¼ 28.3 Hz), 138.0, 132.2, 125.1,
150.3, 142.8 (t, J ¼ 27.5 Hz), 137.6, 136.7, 131.7, 129.2, 128.7, 127.3,
126.3, 122.9, 115.0, 110.7 (t, J ¼ 235.2 Hz), 47.5, 39.6, 30.4, 29.3, 20.3.
HRMS (ESI) m/z calcd for C19H19F2N4OS [M þ H]þ: 389.1242; found:
389.1242.
2-(3-(Difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-N-(5,6,7,8-
tetrahydronaphthalen-2-yl)thiazole-4-carboxamide (11ar). White
solid; yield: 95%; purity: 99%; m.p.: 188e190 ꢁC; 1H NMR (400 MHz,
114.6, 114.1, 110.9 (t, J ¼ 234.8 Hz), 39.7. HRMS (ESI) m/z calcd for
C
12H10F2N5OS2 [M þ H]þ: 342.0289; found: 342.0290.
2-(3-(Difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-N-(4-
methylthiazol-2-yl) thiazole-4-carboxamide (11ay). Light yellow
solid; yield: 84%; purity: 98%; m.p.: 128e130 ꢁC; 1H NMR
(400 MHz, CDCl3)
d 10.43 (s, 1H), 8.24 (s, 1H), 8.00 (s, 1H), 6.96 (t,
J ¼ 53.8 Hz, 1H), 6.60 (d, J ¼ 1.0 Hz, 1H), 4.01 (s, 3H), 2.39 (d,
CDCl3)
d
9.27 (s, 1H), 8.13 (s, 1H), 8.10 (d, J ¼ 8.0 Hz, 1H), 7.90 (s, 1H),
J ¼ 0.9 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d 157.9, 156.9, 155.7,
146.9, 146.7, 141.8 (t, J ¼ 28.4 Hz), 131.1, 124.0, 113.6, 109.9 (t,
7.18 (t, J ¼ 7.8 Hz,1H), 7.08 (t, J ¼ 53.9 Hz,1H), 6.93 (d, J ¼ 7.6 Hz,1H),
4.02 (s, 3H), 2.78 (dt, J ¼ 20.7, 6.3 Hz, 4H), 1.97e1.87 (m, 2H),
J
¼
234.8 Hz), 107.6, 38.6, 16.1. HRMS (ESI) m/z calcd for
1.85e1.76 (m, 2H). 13C NMR (101 MHz, CDCl3)
d
158.4, 158.1, 150.9,
C
13H12F2N5OS2 [M þ H]þ: 356.0446; found: 356.0448.
142.9 (d, J ¼ 26.9 Hz), 138.1, 135.4, 131.5, 126.8, 125.9, 125.7, 122.7,
118.5, 115.0, 110.5 (t, J ¼ 235.7 Hz), 39.7, 29.9, 24.1, 22.9, 22.5. HRMS
(ESI) m/z calcd for C19H19F2N4OS [M þ H]þ: 389.1242; found:
389.1244.
N-(3-bromophenyl)-2-(3-(difluoromethyl)-1-methyl-1H-pyrazol-
4-yl)oxazole-4-carboxamide (11ba). White solid; yield: 71%; purity:
100%; m.p.: 151e153 ꢁC, 1H NMR (400 MHz, CDCl3)
d
8.65 (s, 1H),
8.25 (s, 1H), 7.99 (s, 1H), 7.93 (s, 1H), 7.59 (d, J ¼ 7.8 Hz, 1H), 7.28 (d,
2-(3-(Difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-N-(pyridin-2-
yl)thiazole-4-carboxamide (11as). Yellow solid; yield: 99%; purity:
J ¼ 1.4 Hz,1H), 7.22 (t, J ¼ 7.9 Hz,1H), 7.13 (t, J ¼ 55.3 Hz,1H), 4.01 (s,
3H). 13C NMR (101 MHz, CDCl3)
d 158.2, 155.6, 155.3, 143.7 (t,
100%; m.p.: 115e117 ꢁC; 1H NMR (400 MHz, CDCl3)
d
9.76 (s, 1H),
J ¼ 26.4 Hz), 141.0, 138.6, 136.8,132.2, 130.4, 127.6, 122.7, 118.3, 109.7
8.38 (d, J ¼ 4.7 Hz, 1H), 8.31 (d, J ¼ 8.3 Hz, 1H), 8.19 (s, 1H), 8.09 (s,
(t, J ¼ 236.8 Hz), 108.5 (t, J ¼ 2.5 Hz), 39.8. HRMS (ESI) m/z calcd for
1H), 7.80e7.73 (m, 1H), 7.09 (dd, J ¼ 7.2, 5.1 Hz, 1H), 6.99 (t,
C
15H12BrF2N4O2 [M þ H]þ: 397.0106; found: 397.0113.
J ¼ 53.8 Hz, 1H), 4.01 (s, 3H). 13C NMR (101 MHz, CDCl3)
d
159.1,
2-(3-(Difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-N-(m-tolyl)
158.6, 151.0, 149.5, 148.1, 142.8 (t, J ¼ 28.1 Hz), 138.6, 132.3, 124.3,
120.1, 114.7, 114.3, 111.0 (t, J ¼ 234.9 Hz), 39.6. HRMS (ESI) m/z calcd
for C14H12F2N5OS [M þ H]þ: 336.0725; found: 336.0729.
oxazole-4-carboxamide (11bb). Yellow solid; yield: 93%; purity:
99%; m.p.: 146e148 ꢁC; 1H NMR (400 MHz, CDCl3)
d
8.64 (s, 1H),
8.23 (s, 1H), 7.97 (s, 1H), 7.48 (d, J ¼ 6.6 Hz, 2H), 7.24 (t, J ¼ 8.2 Hz,
2-(3-(Difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-N-(pyridin-4-
yl)thiazole-4-carboxamide (11 at). White solid; yield: 68%; purity:
1H), 7.15 (t, J ¼ 53.7 Hz, 1H), 6.96 (d, J ¼ 7.4 Hz,1H), 3.97 (s, 3H), 2.36
(s, 3H). 13C NMR (101 MHz, CDCl3)
d 158.1, 155.2, 143.6 (t,
100%; m.p.: 183e185 ꢁC; 1H NMR (400 MHz, CDCl3)
d
9.36 (s, 1H),
J ¼ 26.1 Hz), 140.8, 139.0, 137.2, 132.1, 128.9, 125.5, 120.5, 117.0, 109.7
(t, J ¼ 236.3 Hz),108.6 (t, J ¼ 2.4 Hz), 39.7, 21.5. HRMS (ESI) m/z calcd
for C16H15F2N4O2 [M þ H]þ: 333.1158; found: 333.1157.
8.56 (dd, J ¼ 4.9,1.4 Hz, 2H), 8.18 (s,1H), 7.97 (s,1H), 7.64 (dd, J ¼ 4.8,
1.5 Hz, 2H), 6.99 (t, J ¼ 53.8 Hz, 1H), 4.01 (s, 3H). 13C NMR (101 MHz,
CDCl3)
d
159.1, 158.5, 150.8, 149.3, 144.6, 142.9 (t, J ¼ 27.9 Hz), 131.8,
2-(3-(Difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-N-(2-(tri-
fluoromethyl)phenyl) oxazole-4-carboxamide (11bc). White solid;
yield: 58%; purity: 100%; m.p.: 157e159 ꢁC; 1H NMR (400 MHz,
124.3, 114.6, 113.6, 110.9 (t, J ¼ 235.2 Hz), 39.7. HRMS (ESI) m/z calcd
for C14H12F2N5OS [M þ H]þ: 336.0725; found: 336.0728.
N-(3-bromopyridin-4-yl)-2-(3-(difluoromethyl)-1-methyl-1H-
pyrazol-4-yl) thiazole-4-carboxamide (11au). White solid; yield:
CDCl3)
d
9.38 (s, 1H), 8.52 (d, J ¼ 8.3 Hz, 1H), 8.27 (s, 1H), 7.99 (s, 1H),
7.66 (d, J ¼ 7.9 Hz, 1H), 7.60 (t, J ¼ 7.8 Hz,1H), 7.28e7.22 (m, 1H), 7.17
15

B. Yu, B. Zhao, Z. Hao et al.
European Journal of Medicinal Chemistry 214 (2021) 113230
(t, J ¼ 53.8 Hz, 1H), 4.02 (s, 3H). 13C NMR (101 MHz, CDCl3)
d
158.3,
1H), 7.97 (s, 1H), 7.85 (d, J ¼ 8.0 Hz, 3H), 7.55 (d, J ¼ 8.3 Hz, 3H), 7.45
(t, J ¼ 7.8 Hz,1H), 7.39 (d, J ¼ 7.5 Hz,1H), 6.74 (t, J ¼ 54.1 Hz,1H), 3.95
155.3, 143.9 (t, J ¼ 25.9 Hz), 140.8, 136.8, 135.0, 133.0, 132.0, 126.2 (q,
J ¼ 5.3 Hz), 124.1 (q, J ¼ 273.1 Hz), 124.3, 122.8, 119.5 (q, J ¼ 29.9 Hz),
109.6 (t, J ¼ 236.7 Hz), 108.5 (t, J ¼ 2.7 Hz), 39.8. HRMS (ESI) m/z
calcd for C16H12F5N4O2 [M þ H]þ: 387.0875; found: 387.0879.
N-(3-bromophenyl)-5-(3-(difluoromethyl)-1-methyl-1H-pyrazol-
4-yl)thiophene-2-carboxamide (11ca). Yellow solid; yield: 98%; pu-
(s, 3H). 13C NMR (101 MHz, CDCl3)
d
165.4, 142.6 (t, J ¼ 28.2 Hz),
138.5, 135.3, 132.9, 131.6 (q, J ¼ 32.4 Hz), 130.6, 129.7, 128.6, 127.5,
123.9 (q, J ¼ 272.4 Hz), 123.2, 121.1 (q, J ¼ 3.7 Hz), 121.0, 116.9 (q,
J ¼ 4.0 Hz), 111.9 (t, J ¼ 234.0 Hz), 39.4. HRMS (ESI) m/z calcd for
C
19H15F5N3O [M þ H]þ: 396.1130; found: 396.1144.
rity: 99%; m.p.: 103e105 ꢁC; 1H NMR (400 MHz, CDCl3)
d
8.26 (s,
N-(3-bromophenyl)-3-(3-(difluoromethyl)-1-methyl-1H-pyrazol-
1H), 7.86 (s, 1H), 7.59e7.54 (m, 2H), 7.50 (d, J ¼ 8.1 Hz, 1H), 7.22 (d,
4-yl)benzamide (11ea). Yellow solid; yield: 56%; purity: 99%; m.p.:
J ¼ 7.9 Hz, 1H), 7.15 (dd, J ¼ 9.5, 6.5 Hz, 2H), 6.74 (t, J ¼ 53.9 Hz, 1H),
150e152 ꢁC; 1H NMR (400 MHz, CDCl3)
d
7.98 (s, 1H), 7.93 (s, 2H),
3.91 (s, 3H). 13C NMR (101 MHz, CDCl3)
d
160.2,142.1 (t, J ¼ 28.5 Hz),
7.75 (d, J ¼ 7.8 Hz,1H), 7.69 (d, J ¼ 7.8 Hz,1H), 7.55 (d, J ¼ 7.5 Hz, 2H),
7.49 (t, J ¼ 7.7 Hz, 1H), 7.28 (d, J ¼ 8.1 Hz, 1H), 7.22 (t, J ¼ 8.0 Hz, 1H),
6.76 (t, J ¼ 54.1 Hz, 1H), 3.95 (s, 3H). 13C NMR (101 MHz, CDCl3)
138.9, 138.5, 137.1, 130.9, 130.3, 129.5, 127.6, 126.4 (t, J ¼ 2.8 Hz),
123.4,122.6,119.0, 114.2, 111.6 (t, J ¼ 234.1 Hz), 39.4. HRMS (ESI) m/z
calcd for C16H13BrF2N3OS [M þ H]þ: 411.9925; found: 411.9924.
5-(3-(Difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-N-(m-tolyl)
thiophene-2-carboxamide (11 cb). Yellow solid; yield: 96%; purity:
d
165.6, 142.4 (d, J ¼ 28.0 Hz), 139.2, 134.9, 132.0, 131.8, 130.6, 130.4,
129.2, 127.6, 126.8, 125.7, 123.1, 122.7, 121.0, 118.6, 112.0 (t,
J ¼ 233.7 Hz), 39.4. HRMS (ESI) m/z calcd for C18H15BrF2N3O [M þ
H]þ: 406.0361; found: 406.0361.
99%; m.p.: 115e117 ꢁC. 1H NMR (400 MHz, CDCl3)
d 8.08 (s, 1H), 7.55
(d, J ¼ 2.2 Hz, 2H), 7.45 (s, 1H), 7.40 (d, J ¼ 8.1 Hz, 1H), 7.19 (t,
J ¼ 7.8 Hz, 1H), 7.14 (d, J ¼ 3.8 Hz, 1H), 6.93 (d, J ¼ 7.5 Hz, 1H), 6.74 (t,
J ¼ 53.9 Hz, 1H), 3.89 (s, 3H), 2.29 (s, 3H). 13C NMR (101 MHz, CDCl3)
3-(3-(Difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-N-(m-tolyl)
benzamide, white solid (11eb). White solid; yield: 50%; purity: 100%;
m.p.: 161e163 ꢁC; 1H NMR (400 MHz, CDCl3)
d 7.94 (s, 2H), 7.76 (d,
d
160.1, 142.1 (t, J ¼ 28.3 Hz), 139.0, 137.8, 137.8, 137.6, 130.8, 129.1,
J ¼ 7.8 Hz,1H), 7.67 (d, J ¼ 7.8 Hz,1H), 7.55 (s,1H), 7.53e7.41 (m, 3H),
128.9, 126.3 (t, J ¼ 2.7 Hz), 125.4, 121.1, 117.6, 114.3, 111.5 (t,
J ¼ 234.0 Hz), 39.4, 21.4. HRMS (ESI) m/z calcd for C17H16F2N3OS
[M þ H]þ: 348.0977; found: 348.0978.
7.25 (t, J ¼ 7.8 Hz,1H), 6.97 (d, J ¼ 7.5 Hz,1H), 6.76 (t, J ¼ 54.1 Hz,1H),
3.94 (s, 3H), 2.36 (s, 3H). 13C NMR (101 MHz, CDCl3)
d 165.5, 142.4 (t,
J ¼ 28.1 Hz), 139.1, 137.8, 135.5, 131.9, 131.5, 130.6, 129.1, 128.9, 126.8,
125.7, 125.4, 121.2, 120.8, 117.3, 111.9 (t, J ¼ 233.8 Hz), 39.3, 21.5.
HRMS (ESI) m/z calcd for C19H18F2N3O [M þ H]þ: 342.1412; found:
342.1417.
N-(3-bromophenyl)-4-(3-(difluoromethyl)-1-methyl-1H-pyrazol-
4-yl)benzamide (11da). Yellow solid; yield: 91%; purity: 100%; m.p.:
110e112 ꢁC. 1H NMR (400 MHz, CDCl3)
d 8.35 (s, 1H), 7.91 (s, 1H),
7.79 (d, J ¼ 7.1 Hz, 2H), 7.58e7.44 (m, 4H), 7.24 (d, J ¼ 7.6 Hz, 1H),
3-(3-(Difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-N-(2-(tri-
fluoromethyl)phenyl) benzamide (11ec). White solid; yield: 32%;
7.16 (t, J ¼ 7.3 Hz, 1H), 6.73 (t, J ¼ 54.1 Hz, 1H), 3.92 (s, 3H). 13C NMR
(101 MHz, CDCl3)
d
165.8, 142.4 (t, J ¼ 28.0 Hz), 139.3, 135.0, 132.9,
purity: 100%; m.p.: 126e128 ꢁC; 1H NMR (400 MHz, CDCl3)
d 8.43
130.7, 130.3, 128.3, 127.6, 127.5, 123.3, 122.6, 120.9, 118.9, 111.9 (t,
J ¼ 233.9 Hz), 39.4. HRMS (ESI) m/z calcd for C18H15BrF2N3O [M þ
H]þ: 406.0361; found: 406.0351.
(d, J ¼ 8.2 Hz, 1H), 8.28 (s, 1H), 8.00 (s, 1H), 7.81 (d, J ¼ 7.7 Hz, 1H),
7.72 (d, J ¼ 7.7 Hz, 1H), 7.68e7.63 (m, 1H), 7.61 (d, J ¼ 7.4 Hz, 2H),
7.55 (t, J ¼ 7.8 Hz,1H), 7.29 (d, J ¼ 7.6 Hz,1H), 6.77 (t, J ¼ 54.1 Hz,1H),
4-(3-(Difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-N-(m-tolyl)
benzamide (11 db). White solid; yield: 95%; purity: 100%; m.p.:
3.98 (s, 3H). 13C NMR (101 MHz, CDCl3)
d
165.3,142.5 (t, J ¼ 28.2 Hz),
135.4, 134.7, 133.0, 132.2, 132.0, 130.6, 129.4, 126.8, 126.2 (q,
J ¼ 5.3 Hz), 125.7, 124.6,124.3, 124.2 (q, J ¼ 272.9 Hz), 121.0, 120.2 (q,
J ¼ 29.6 Hz), 111.9 (t, J ¼ 233.8 Hz), 39.4. HRMS (ESI) m/z calcd for
133e135 ꢁC, 1H NMR (400 MHz, CDCl3)
d 8.25 (s, 1H), 7.80 (d,
J ¼ 8.2 Hz, 2H), 7.49 (s, 2H), 7.47 (d, J ¼ 8.3 Hz, 2H), 7.43 (d, J ¼ 8.1 Hz,
1H), 7.19 (t, J ¼ 7.8 Hz,1H), 6.93 (d, J ¼ 7.4 Hz,1H), 6.72 (t, J ¼ 54.1 Hz,
C
19H15F5N3O [M þ H]þ: 396.1130; found: 396.1134.
1H), 3.89 (s, 3H), 2.29 (s, 3H). 13C NMR (101 MHz, CDCl3)
d
165.6,
N-benzyl-3-(3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)ben-
142.4 (t, J ¼ 27.9 Hz), 139.0, 138.0, 134.6, 133.6, 130.7, 128.8, 128.2,
127.5, 125.4, 121.1, 121.1, 117.5, 111.8 (t, J ¼ 233.9 Hz), 39.3, 21.5.
HRMS (ESI) m/z calcd for C19H18F2N3O [M þ H]þ: 342.1412; found:
342.1418.
4-(3-(Difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-N-(2-(tri-
fluoromethyl)phenyl)benzamide (11dc). Light yellow solid; yield:
49%; purity: 100%; m.p.: 153e155 ꢁC; 1H NMR (400 MHz, CDCl3)
zamide (11ed). Yellow solid; yield: 90%; purity: 100%; m.p.:
100e102 ꢁC; 1H NMR (400 MHz, CDCl3)
d
7.88 (s, 1H), 7.69 (d,
J ¼ 7.8 Hz, 1H), 7.62 (d, J ¼ 7.7 Hz, 1H), 7.47 (s, 1H), 7.39 (t, J ¼ 7.7 Hz,
1H), 7.31 (d, J ¼ 4.3 Hz, 4H), 7.26 (dt, J ¼ 6.1, 3.8 Hz, 1H), 6.90 (t,
J ¼ 4.9 Hz, 1H), 6.70 (t, J ¼ 54.1 Hz, 1H), 4.58 (d, J ¼ 5.7 Hz, 2H), 3.86
(s, 3H). 13C NMR (101 MHz, CDCl3)
138.2,134.8,131.7,131.3,130.6,128.9,128.7,127.8,127.5,126.9,125.7,
d
167.3, 142.3 (t, J ¼ 27.8 Hz),
d
8.41 (d, J ¼ 8.2 Hz, 1H), 8.27 (s, 1H), 7.90 (dd, J ¼ 8.4, 2.3 Hz, 2H),
7.68e7.59 (m, 5H), 7.27 (t, J ¼ 7.4 Hz, 1H), 6.77 (t, J ¼ 54.1 Hz, 1H),
3.97 (s, 3H). 13C NMR (101 MHz, CDCl3)
121.2, 111.8 (t, J ¼ 233.9 Hz), 44.0, 39.2. HRMS (ESI) m/z calcd for
C
19H18F2N3O [M þ H]þ: 342.1412; found: 342.1409.
d
165.1, 142.6 (t, J ¼ 28.1 Hz),
N-(3-bromophenyl)-3-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-
135.5, 135.4, 133.0, 132.8, 130.7, 128.6, 127.5, 126.2 (q, J ¼ 5.3 Hz),
124.6, 124.3, 124.3 (q, J ¼ 273.0 Hz), 120.9, 120.2 (q, J ¼ 29.6 Hz),
111.8 (t, J ¼ 234.0 Hz), 39.4. HRMS (ESI) m/z calcd for C19H15F5N3O
[M þ H]þ: 396.1130; found: 396.1136.
4-yl)benzamide (11ee). White solid; yield: 58%; purity: 100%; m.p.:
177e179 ꢁC; 1H NMR (400 MHz, CDCl3)
d
7.97 (s, 1H), 7.93 (t,
J ¼ 1.9 Hz, 1H), 7.85 (t, J ¼ 1.6 Hz, 1H), 7.80e7.76 (m, 1H), 7.60 (d,
J ¼ 7.8 Hz, 1H), 7.57e7.53 (m, 2H), 7.51 (t, J ¼ 7.7 Hz, 1H), 7.31e7.27
(m, 1H), 7.23 (t, J ¼ 8.0 Hz, 1H), 3.98 (s, 3H). 13C NMR (101 MHz,
N-benzyl-4-(3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)ben-
zamide (11dd). White solid; yield: 94%; purity: 100%; m.p.:
CDCl3)
d
165.4, 139.1, 138.6 (q, J ¼ 36.6 Hz), 134.9, 132.2, 132.2, 131.4,
142e144 ꢁC; 1H NMR (400 MHz, CDCl3)
d
7.80 (d, J ¼ 8.1 Hz, 2H),
131.1, 130.4, 129.2, 127.7, 127.3, 126.0, 123.1, 122.8, 121.5 (q,
7.53 (d, J ¼ 7.2 Hz, 2H), 7.50 (s, 1H), 7.34 (d, J ¼ 4.2 Hz, 4H), 7.31e7.25
(m, 1H), 6.72 (t, J ¼ 54.1 Hz, 1H), 6.64 (s, 1H), 4.63 (d, J ¼ 5.6 Hz, 2H),
J ¼ 269.4 Hz), 121.3, 118.6, 39.7. HRMS (ESI) m/z calcd for
C
18H14BrF3N3O [M þ H]þ: 424.0267; found: 424.0264.
3.94 (s, 3H). 13C NMR (101 MHz, CDCl3)
d
167.0, 142.5 (t, J ¼ 27.8 Hz),
3-(1-Methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)-N-(m-tolyl)
138.2, 134.5, 133.0, 130.6, 128.8, 128.3, 127.9, 127.6, 127.4, 121.2, 111.8
(t, J ¼ 234.0 Hz), 44.1, 39.4. HRMS (ESI) m/z calcd for C19H18F2N3O
[M þ H]þ: 342.1412; found: 342.1415.
benzamide (11ef). Light yellow solid; yield: 83%; purity: 100%; m.p.:
171e173 ꢁC; 1H NMR (400 MHz, CDCl3)
d
8.01 (s, 1H), 7.86 (s, 1H),
7.78 (d, J ¼ 7.3 Hz, 1H), 7.57 (d, J ¼ 7.3 Hz, 1H), 7.53e7.41 (m, 4H),
4-(3-(Difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-N-(3-(tri-
fluoromethyl)phenyl) benzamide (11de). White solid; yield: 23%;
7.23 (d, J ¼ 7.5 Hz, 1H), 6.97 (d, J ¼ 7.3 Hz, 1H), 3.95 (s, 3H), 2.35 (s,
3H). 13C NMR (101 MHz, CDCl3)
137.8, 135.5, 131.8, 131.3, 131.2, 129.0, 128.9, 127.3, 126.0, 125.5, 121.5
d
165.5, 139.1, 138.5 (q, J ¼ 37.1 Hz),
purity: 98%; m.p.: 136e138 ꢁC; 1H NMR (400 MHz, CDCl3)
d
8.27 (s,
16

B. Yu, B. Zhao, Z. Hao et al.
European Journal of Medicinal Chemistry 214 (2021) 113230
(q, J ¼ 269.5 Hz), 121.4, 120.9, 117.3, 39.6, 21.5. HRMS (ESI) m/z calcd
for C19H17F3N3O [M þ H]þ: 360.1318; found: 360.1321.
extraction buffer (5 mL of reagent 1 and 50 mL of reagent 2 (BC0955,
solarbio, Beijing)). After shaking and mixing at 4 ꢁC for 2 min, the
supernatant was collected for protein interaction analysis by
centrifugation (11000g, 4 ꢁC for 10 min, 2 times). The mitochondrial
suspensions were diluted in Phosphate Buffered Saline (PBS) buffer.
And the tested compounds concentrations ranged between 0.05
and 20 mg/L (six concentrations þ DMSO control). The SDH enzy-
matic activity was measured according to our previously reported
method [5]. The IC50 values of representative compounds were
calculated by GraphPad Prim version 6.02 program.
N-(2-isopropylphenyl)-3-(1-methyl-3-(trifluoromethyl)-1H-pyr-
azol-4-yl) benzamide (11eg). White solid; yield: 79%; purity: 100%;
m.p.: 142e144 ꢁC; 1H NMR (400 MHz, CDCl3)
d 7.90 (s, 2H), 7.83 (d,
J ¼ 7.5 Hz, 1H), 7.78 (s, 1H), 7.61e7.54 (m, 2H), 7.50 (t, J ¼ 7.7 Hz, 1H),
7.35e7.30 (m, 1H), 7.24e7.18 (m, 2H), 3.97 (s, 3H), 3.11 (dt, J ¼ 13.6,
6.8 Hz, 1H), 1.28 (s, 3H), 1.26 (s, 3H). 13C NMR (101 MHz, CDCl3)
d
165.8, 140.8, 138.5 (q, J ¼ 36.6 Hz), 135.4, 134.1, 131.8, 131.3, 131.2,
129.2, 127.2, 126.5, 126.3, 126.2, 125.8, 124.8, 121.6 (q, J ¼ 269.4 Hz),
121.4, 39.7, 28.2, 23.0. HRMS (ESI) m/z calcd for C21H21F3N3O [M þ
H]þ: 388.1631; found: 388.1632.
4.4. Fluorescence quenching analysis of SDH
3-(1,3-Dimethyl-1H-pyrazol-4-yl)-N-(m-tolyl)benzamide (11eh).
White solid; yield: 92%; purity: 100%; m.p.: 107e109 ꢁC; 1H NMR
The discovery and identification of new targets is an effective
basis for the development of novel pesticides [29]. Target validation
is also an important mean to expand the diversity of lead com-
pounds [30]. The mitochondria of R. cerealis were extracted ac-
cording to the operation instruction (BC0955, solarbio, Beijing). The
mitochondrial suspensions were diluted in PBS buffer (pH ¼ 7.4,
136.89 m mol/L NaCl, 2.67 m mol/L KCl, 8.1 m mol/L Na2HPO4, and
1.76 m mol/L KH2PO4), and then add 1 mL of the diluted mito-
chondrial suspension to the quartz cuvette. Fluorescence emission
spectra were recorded at a wavelength of 300e420 nm with an
excitation wavelength of 280 nm at 4 ꢁC, and the emission band
width was set at 10 nm with medium sensitivity. The solution of
compound 11ea and controls were added into the suspension for
(400 MHz, CDCl3)
d
8.46 (s, 1H), 7.83 (s, 1H), 7.67 (d, J ¼ 7.5 Hz, 1H),
7.54 (s, 1H), 7.48 (d, J ¼ 14.2 Hz, 1H), 7.46 (d, J ¼ 14.6 Hz, 1H), 7.39 (t,
J ¼ 7.7 Hz, 1H), 7.36 (s, 1H), 7.21 (t, J ¼ 7.8 Hz, 1H), 6.95 (d, J ¼ 7.4 Hz,
1H), 3.75 (s, 3H), 2.36 (s, 3H), 2.32 (s, 3H). 13C NMR (101 MHz,
CDCl3)
d 166.1, 145.6, 139.0, 138.1, 135.5, 134.2, 130.3, 129.2, 128.9,
126.3, 125.3, 124.4, 121.0, 120.1, 117.5, 38.6, 21.5, 13.2. HRMS (ESI) m/
z calcd for C19H20N3O [M þ H]þ: 306.1601; found: 306.1601.
4.2. Crystal structure determination
Single crystal X-ray diffraction data of compound 11ea was
collected on a Rigaku 007 Saturn 70 diffractionmeter using Mo K
a
radiation (
l
¼ 0.71073 Å). Atotalof10241 reflectionswerecollectedin
fluorescence determination respectively, 1
each intermission for 1 min. The concentrations of 11ea and con-
trols were within a range of 0e5
g/mL, with uniform 2 ꢂ dilution
factor steps (0
mL each time for 5 times,
the range of 1.920ꢁ
ꢃ
q
ꢃ 27.874ꢁ (ꢀ10 ꢃ h ꢃ 10, ꢀ12 ꢃ k
ꢃ 12, ꢀ15 ꢃ l ꢃ 15) with 3976 unique reflections (Rint ¼ 0.0674), of
m
which 1694 had I > 2s(I) for refinements. The structure was directly
m
g/mL þ four concentrations). Thifluzamide and
solved by Olex2 1.2 program.
tricyclazole were used as a positive and a negative control,
respectively.
4.3. Evaluation of biological activity
4.3.1. In vitro antifungal assay
4.5. Homology modeling and molecular docking
The antifungal activity of the title compounds 11aa-11eh against
Alternaria solani, Botrytis cinerea, Cercospora arachidicola, Gibberella
zeae, Phytophthora infestans (Mont.) de Bary, Physalospora piricola,
Pellicularia sasakii, Rhizoctonia cerealis, Sclerotinia sclerotiorum were
The crystal structure of R. cerealis SDH has not been reported
yet; therefore, a SDH structure of R. cerealis was constructed by
homology modeling. The target protein sequences of SDHB, SDHC
and SDHD (entry code: A0A173DSZ5, A0A173DSZ3 and
A0A173DT05, respectively) were obtained from uniprot website
(https://www.uniprot.org/). Then, the template proteins (entry
code: 4YXD and 1YQ3) were selected from the PDB (Protein Data
Bank) through BLASTp. Homology modeling was performed on
Modeller 9.18 [31,32]. Since the binding site of SDH was just con-
structed by SDHB, SDHC and SDHD, SDHA of R. cerealis SDH was not
constructed in this model. The individual sub-models (SDHB, SDHC
and SDH) were then assembled by superimposing on the template
(4YXD) using Chimera 1.13.1 [33].
The docking binding center of predicted RcSDH was defined by
Autodock Tools-1.5.6 according to the co-crystallized ligand fluto-
lanil (4YXD) [34,35]. The docking pocket was centered on the
ligand, which grid size was set 20 ꢂ 20 ꢂ 20 and the grid space was
0.375 Å. The crystal structure of 11ea was applied in this experi-
ment. Thifluzamide and other selected compounds were optimized
according to the ligand minimization protocol. During the process,
the ligands were allowed to be flexible while the receptor was held
rigid [36]. The molecular docking analysis was operated on PyMOL.
evaluated in vitro at 50 mg/mL according to our reported method
[21]. The commercial SDHI fungicide thifluzamide was used as a
positive control. Compounds with good to excellent inhibitory ac-
tivities were further assessed for their median effective concen-
tration (EC50) values according to our published procedures [21].
Three replicates were set up for each treatment.
4.3.2. In vivo antifungal assay
The in vivo antifungal activities of the target compounds against
Erysiphe graminis, Puccinia sorghi Schw., Pyricularia oryzae Cav. and
Rhizoctonia solani were assessed according to a previously reported
method [28]. Thifluzamide was selected as a positive control.
4.3.3. SDH enzyme activity analysis
SDH catalyzed the dehydrogenation of succinic acid to fumaric
acid. The removed hydrogen was transferred and reduced by
phenazine dimethyl sulfuric acid (PMS) to reduce 2,6-
dichlorophenol indophenol (DCPIP). The reduction rate of 2,6-
DCPIP was determined by the change of absorbance at 600 nm,
which represented the enzyme activity of SDH. The enzyme activity
was calculated as per nmol 2,6-DCPIP consumed/min/mg protein as
one unit (U/mg prot).
Declaration of competing interest
R. cerealis cultures were grown in Potato Dextrose Broth (PDB)
medium on a reciprocal shaker (25 ꢁC) for 5 days. One gram of the
mycelium was obtained from PDB medium and ground in liquid
nitrogen. The resultant powder was resuspended in mitochondrial
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
17

B. Yu, B. Zhao, Z. Hao et al.
European Journal of Medicinal Chemistry 214 (2021) 113230
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This work was supported in part by the National Natural Science
Foundation of China (No. 31872007), the Fundamental Research
Funds for the Central Universities, Nankai University (No.
63191743), the Tianjin Natural Science Foundation (No.
18JCZDJC33500), the International Science & Technology Coopera-
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Dedicated to the 100th Anniversary of Chemistry at Nankai
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