Design, synthesis, DFT study and antifungal activity of the derivatives of pyrazolecarboxamide containing thiazole or oxazole ring

Article abstract of DOI:10.1016/j.ejmech.2018.02.036

Pyrazolecarboxamide fungicides are one of the most important classes of agricultural fungicides, which belong to succinodehydrogenase inhibitors (SDHIS). To discover new pyrazolecarboxamide analogues with broad spectrum and high activity, a class of new compounds of pyrazole carboxamide derivatives containing thiazole or oxazole ring were designed by scaffold hopping and bioisosterism, and 36 pyrazole carboxamide derivatives with antifungal activity were synthesized. Those compounds were evaluated against five phytopathogenic fungi, Gibberella zeae, Phytophythora capsici, Sclerotonia sclerotiorum, Erysiphe graminis and Puccinia sorghi. The results indicated that most of the compounds displayed good fungicidal activities, especially against E. graminis. Theoretical calculations were carried out at the B3LYP/6-31G (d, p) level and the full geometry optimization was carried out using the 6-31G (d, p) basis set, and the frontier orbital energy, atomic net charges, molecular docking were discussed, and the structure-activity relationships were also studied.

Full text of DOI:10.1016/j.ejmech.2018.02.036

Accepted Manuscript
Design, synthesis, DFT study and antifungal activity of the derivatives of
pyrazolecarboxamide containing thiazole or oxazole ring
Zhongzhong Yan, Aiping Liu, Mingzhi Huang, Minhua Liu, Hui Pei, Lu Huang, Haibo
Yi, Weidong Liu, Aixi Hu
PII:
S0223-5234(18)30166-1
10.1016/j.ejmech.2018.02.036
EJMECH 10213
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 October 2017
Revised Date: 18 January 2018
Accepted Date: 10 February 2018
Please cite this article as: Z. Yan, A. Liu, M. Huang, M. Liu, H. Pei, L. Huang, H. Yi, W. Liu, A. Hu,
Design, synthesis, DFT study and antifungal activity of the derivatives of pyrazolecarboxamide
containing thiazole or oxazole ring, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.02.036.
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Design, Synthesis, DFT Study and Antifungal Activity of the Derivatives
of Pyrazolecarboxamide Containing Thiazole or Oxazole Ring
ACCEPTED MANUSCRIPT
Zhongzhong Yan a, b, Aiping Liu b, c, Mingzhi Huang b, c, Minhua Liu b, c, Hui Pei c, Lu Huang c,
Haibo Yi a, Weidong Liu b, c, *, Aixi Hu a, *
a College of Chemistry and Chemical Engineering, Hunan University, No.1 Lushan Road(s),
Changsha, 410082, China
b
National Engineering Research Center for Agrochemicals, Hunan Research Institute of
Chemical Industry, Changsha 410007, Hunan Province, People’s Republic of China
c Hunan Province Key Laboratory for Agrochemicals, Changsha 410014, People’s Republic of
China
Abstract
Pyrazolecarboxamide fungicides are one of the most important classes of agricultural
fungicides, which belong to succinodehydrogenase inhibitors (SDHIS). To discover
new pyrazolecarboxamide analogues with broad spectrum and high activity, a class of
new compounds of pyrazole carboxamide derivatives containing thiazole or oxazole
ring were designed by scaffold hopping and bioisosterism, and 36 pyrazole
carboxamide derivatives with antifungal activity were synthesized. Those compounds
were evaluated against five phytopathogenic fungi, Gibberella zeae, Phytophythora
capsici, Sclerotonia sclerotiorum, Erysiphe graminis and Puccinia sorghi. The results
indicated that most of the compounds displayed good fungicidal activities, especially
against E. graminis. Theoretical calculations were carried out at the B3LYP/6-31G (d, p)
level and the full geometry optimization was carried out using the 6-31G (d, p) basis
set, and the frontier orbital energy, atomic net charges, molecular docking were
discussed, and the structure-activity relationships were also studied.
Keywords
Pyrazole carboxamides; bioisosterism; SDHIS; antifungal activity; theoretical
calculations
*Corresponding author: E-mail: axhu@hnu.edu.cn (Aixi Hu); weidliu@yahoo.com (Weidong Liu).
Note: The authors declare no competing financial interest.

1. Introduction
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Plant diseases have been recognized as a worldwide threat to crop production and
the uses of fungicides were, are and will remain critical for the effective controls of
most plant diseases in agriculture [1]. Among more than 224 fungicides listed on
Fungicide Resistance Action Committee (FRAC) classification on mode of action 2017
(www.frac.info)，the sterol biosynthesis inhibitors and quinone-outside inhibitors (QoIs)
are two of the most relevant for disease control in commercial agriculture. Nonetheless,
the succinate dehydrogenase inhibitor (SDHI) class is the fastest growing in terms of
new compounds produced and launched into the market. As is well-known, many plant
pathogens have the propensity for developing resistance to fungicides, particularly to
those classes of compounds that act at a single defined target site [2-5]. In this light, the
SDHI fungicides are a fundamental cornerstone for resistance management but, in the
meantime, this class itself is also threatened by resistance [6]. Therefore, it is necessary
to use all available knowledge to protect their longevity by discovering new SDHI
fungicides to diminish the resistance of plant pathogenic fungi [7].
There is no doubt that heterocyclic structures are important feature in natural
products, synthetic medicines and pesticides for their high-efficiency, low toxicity and
diversity of possible substituents [8-11]. Pyrazole derivatives, as an important kind of
heterocyclic nitrogen compounds, have exhibited a wide range of biological activities
whether in the field of pesticides or medicine, such as anticancer [12],
anti-inflammatory [13], antifungal [14], insecticidal [15] et al. Structurally, it is not
difficult to find that the structure of pyrazole ring is the key part of antifungal activity:
bixafen, penflufen, sedaxane, fluxapyroxad, penthiopyrad, benzovindiflupyr, for
example. Owing to the importance of the pyrazole-4-carboxamide derivatives in the
aspect of antifungal activity and the application of bioisosterism in the design of
pyrazolecarboxamide fungicides (Figure 1), we tentatively designed a series of
pyrazole-5-carboxamide derivatives, looking forward to obtaining some compounds
with excellent fungicidal activities. Figure 1 revealed that bioisosterism can be
completely used in the design of pyrazole amide fungicides, which laid a theoretical
foundation for our design. Furthermore, considering the application of thiazole and
oxazole in commercial pesticides, such as thifluzamide, ethaboxam, thiabendazole,
fluesulfone, oxathiapiprolin, pyrisoxazole, isoxaflutole, etoxazole , metamifop et al.,
we introduced the thiazole ring structure or oxazole ring structures which are taken as
an active fragment being widely used in the field of pesticide and medicine as well
[16-19]. Moreover, benzothiazole and benzoxazole derivatives have been found many

applications in medicine and pesticides for their unique biological activity [20-25].
Expecting to get high antifungal activity compounds, we obtained bioisosteres by
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transforming benzothiazole and benzoxazole into phenyl thiazole and phenyl oxazole,
then we prepared a series of the target compounds by using the bioisosteres to replace
the tert-butylbenzene structure of tebufenpyrad (Figure 2).
2. Results and Discussion
2.1 Synthesis
The synthesis of the derivatives of pyrazole carboxamide containing thiazole or
oxazole ring is outline in Scheme 1. The target compounds of pyrazole carboxamide
1
derivatives were obtained by the reaction of a~j, and MS, H NMR and elemental
analysis date were applied to confirm the structures of the new synthesized compounds.
2.2 Antifungal activities
The antifungal activities were shown in table 1 and table 2. 36 compounds were
synthesized and tested the antifungal activities, thifluzamide and boscalid were selected
as the positive controls. Table 1 was the preliminary screening of antifungal activities
of the synthesized compounds and table 2 was the second screening of some
compounds with excellent activity against Erysiphe graminis. The in vitro fungicidal
activities of all synthesized compounds against Gibberella zeae, Phytophythora capsici
and Sclerotonia sclerotiorum were tested at the concentration of 25mg/L and the in
vivo fungicidal activities against E. graminis and Puccinia sorghi were tested at the
concentration of 500mg/L. According to the results of preliminary screening, most of
the synthesized compounds exhibited a certain degree of antifungal activities,
especially against E. graminis activity since one half of the compounds synthesized
exhibited their control effects were not lower than 90%. Among those compounds
containing thiazole, A2 and A4 presented 100% control effects against E. graminis at
500mg/L; B1 and B14 were containing oxazole ring, they showed the best antifungal
activities among those compounds containing oxazole ring. We preliminarily singled
out A1, A4, B1 and B14 to evaluate their EC50 values (Table 2).
The EC50 values expressed by molarity revealed that the antifungal activities of our
synthesized compounds were close to the positive control, thifluzamide (8.6 ꢀM),
while the EC50 values expressed by mass concentration of the A2, A4 and B1 were all
lower than thifluzamide (4.52 mg/L). Thereinto, compound A4 displayed the best
antifungal activity against E. graminis and the EC50(mg/L) of compound B14 was
equivalent to that of thifluzamide. Considering that a thifluzamide molecule contains

two bromine atoms and six fluorine atoms which lead to its large molecular weight
(MW=528), we believe that there are obvious problems of pesticide halogen residues.
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Therefore, the compounds we synthesized, especially compound A4, have obvious
advantages for their small molecular weight, low halogen residues and high antifungal
activities.
The results of the preliminary structure-activity relationship (SAR) analysis
suggested that the introduction of thiazole or oxazole ring, particularly thiazole ring,
was significantly strengthened antifungal activities.
2.3 DFT Calculation
Theoretical calculations carried out at the DFT-B3LYP/6-31G(d,p) [27,28] level and
the full geometry optimization carried out using the 6-31G(d,p) basis set were
implemented using the Gaussian 09 software [29].
To compare the changes in the three-dimensional structure, geometry optimizations
of lead compound (tebufenpyrad), target compound A4 and carboxin were carried out
(Figure 4). From the geometry optimization of tebufenpyrad and compound A4, we can
easily find out that the two planes of pyrazol ring and benzene ring in the lead
compound are almost perpendicular while the target compound A4, like fungicides
carboxin, is more like a plane structure. Besides, when we fix the amide bond it comes
to light that the pyrazol ring of the target compound A4 was 180°flip comparing to that
of the lead compound. Those configuration changes may be the key of the
transformation from insecticidal activity to antifungal activity.
Moreover, the total molecular energy and frontier orbital energy were calculated as
well. The total molecular energy and the energy of LUMO and HOMO of A4, lead
compound (tebufenpyrad), carboxin and thifluzamide are listed in Table 3. According
to the frontier molecular orbital theory, HOMO and LUMO are the most important
factors that affect the bioactivity [30,31]. HOMO has the priority to provide electrons,
while LUMO can accept electrons first. Thus, a study of the frontier orbital energy can
provide useful information about the biological mechanism of action [32] and the lower
value of the HOMO and LUMO energy gap can explains the eventual charge transfer
interaction taking place with the molecules [33]. We selected the most potent
compound A4 , lead compound (tebufenpyrad), carboxin, and thifluzamide, as models
to compare their total energy and frontier orbital energy (Table 3). From what showed
in the Table 3 and Figure 5, we can find that the total energy of above compounds had a
large gap, but their energy gap (ꢁE) were similar, especially our target compound A4
and the succinate dehydrogenase inhibitor carboxin, and their ꢁE value are 0.16572

and 0.17230, respectively. Taking the DFT results for example
(Figure 5), the LUMO
of lead compound is located on pyrazole ring and amide bond while the LUMO of
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target compound A4 is located on the fragment of phenylthiazole (bioisostere). The
HOMO of lead compound located not only on pyrazle ring, amide bond but also on
phenyl ring and tert-butyl while the HOMO of compound A4 is still mainly located on
the bioisostere and amide bond. This frontier orbital characters can also help to
understand the changes in the activity of the lead compounds. Figure 5 also exhibits
that the gap of ꢁE value between compound A4 and carboxin is extremely close to
which between thifluzamide and carboxin. The fact that the title compound has strong
affinity suggests the importance of the frontier molecular orbital in the π-π stacking or
hydrophobic interactions. This also implies that the introduction of phenyl ring and
thiazole ring had an important impact on the antifungal activity.
2.4 Mulliken Atomic Charges and Molecular Docking
Table 4 lists the calculated Mulliken atomic charges except for the H atoms. Taking
DFT for example again, five atoms 9C、11S、14C、15C and 18C are the most positively
charged ones, which can interact with negative charged part of the receptor easily.
While the negative charges are mainly located on atoms 8N、10C、13N、18C 、19N、
20N、21C and 22C, of which 8N、13N、16O、19N and 20N are the most negatively
charged ones, so they can interact easily with the positive part of the receptor.
Therefore, we supposed that this compound can combine the amino-acid residues on its
surface by the interaction of 16O-14C-13N, 8N-9C-10C and 19N-20V-15C.
In order to confirm our supposition, molecular docking of compound A4 and
carboxin to the binding site of SDH (PDB Code: 2FBW [34,35]) pdb was performed
by Ledock (http://www.lephar.com) and the results were analyzed and visualized
using PyMOL (http://www.pymol.org/pymol) (Figure 5). The three-dimensional
schematic diagrams clearly explained the possible optimal combination between the
ligands and receptor protein. Carboxin and compound A4 adopted similar
conformations and locations in the active site. Their docking total scores were -5.33
kcal /mol and -7.33 kcal /mol, respectively. Carboxin was well bound to the receptor
protein by forming three hydrogen bonds with TRP-173, TYR-58 and SER-39 (Figure
5b). While compound A4 can form four hydrogen bonds with TRP-173, TYR-58 and
SER-39, and therein amido linkage can simultaneously form two hydrogen bonds with
SER-39; at the same time, the benzene ring of compound A4 can form π-π stacking
interaction with HIS-216, and ARG-43 participated in the formation of polar
interaction or van der Waals interaction with the thiazole ring (Figure 5d). Therefore,

the interactions of hydrogen bonds, π-π stacking and van der Waals’ force played an
important role to form a stable complex between compound A4 and the SDH, which
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proved that the SDH enzyme is one of the significant action targets of our title
compounds and our previous supposition had been confirmed.
2.5 Conclusions
In conclusion, more than 36 pyrazole amide derivatives containing thiazole or
oxazole ring were synthesized and evaluated in this study. The results of bioassays
indicate that most of the target compounds exhibited satisfactory antifungal activities,
especially against E. graminis. Among these 36 compounds, compound A4 showed
the most potent activity against E. graminis, which is superior to the commercial
thifluzamide in terms of mass concentration. In addition, theoretical calculations and
molecular docking were used to verify the rationality of our design.
1. The introduction of thiazole or oxazole ring does make our target compounds have
very good antifungal activities.
2. The replacing of the bioisostere (phenyl thiazole) makes the three-dimensional
structure of the leading compound (insecticide) has changed greatly, which is
necessary for the transformation from insecticidal activity to antifungal activity.
3. The introduction of phenyl thiazole makes the target compound A4 has some
special properties on the theory of frontier orbit, which are conducive to explain the
antifungal activity of the target compounds.
4. Mulliken atomic charges and molecular docking verify the interaction between
small molecule and protein. This point is very significant to succinodehydrogenase
inhibitors (SDHIS).
These findings indicate that the incorporation of the thiazole or oxazole fragment
improves the antifungal activities as expected. In this way, we supposed that we can
transform an insecticide (tebufenpyrad) into a fungicide. Therefore, further studies on
structural optimization and structure-activity relationships of these pyrazole amide
derivatives are in progress.
3. Experimental Section
3.1 Materials and methods
All of the original raw materials and reagents were commercially available and used
without further purification. Melting points were determined on WPS-1B melting point
1
apparatus made in shanghai physical optics instrument plant. H NMR spectra were
recorded in CDCl3 with a Varian INOVA-300, using TMS as the internal standard; high

resolution mass spectra were acquired using an Agilent 5973-6890 gas
chromatography-mass spectrometer (GC-MS) and an Agilent 1100 series Liquid
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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.
3.2 Chemical synthesis
The target compounds were synthesized as shown in Scheme 1 and most of the
intermediates were synthesized immediately for the next step without further
purification. The general procedures for the intermediates are as follows:
3.2.1. General procedure for the preparation of compounds 2
A solution of starting material 1 (20 mmol), thionyl chloride (SOCl2) (40 mmol) and
dichloromethane (30 mL) was stirred under reflux for 3 hours, then the reaction
mixture was concentrated under reduced pressure, the residue was added to the aqueous
ammonia (20 mL) at room temperature or below. Upon reaction completion (as
monitored by TLC), the reaction was terminated with water, followed by extraction
with ethyl acetate (3×10 mL), dried over anhydrous sodium sulphite and then filtered to
obtain a crude product and recrystallized to get compounds 2.
3.2.2 General procedure for the preparation of compounds 3
Benzamides 2 (6mmol) was dissolved in dry THF (20 mL) and thereto was added
P2S5 (6mmol) and heated at 65 for 3 hours, after which the reaction mixture was
cooled, poured in NaHCO3 (10%, 100mL), stirred for half an hour, then the resulting
solid was filtered, dried and recrystallized from ethyl acetate to get compounds 3.
3.2.3 General procedure for the preparation of compounds 4
A mixture of 2 (6 mmol) and 1,3-dichloroacetone (12 mmol) was heated at 130°C
for 1 hour. After the mixture was cooled to room temperature, water (20 mL) was
added, and the mixture was extracted with CH2Cl2 (3×20 mL). The organic layer was
dried over anhydrous sodium sulphite, filtered, and concentrated under reduced
pressure, and then the crude product was recrystallized with petroleum ether and ethyl
acetate.
3.2.4 General procedure for the preparation of compounds 4’
Thiobenzamides 3 (3 mmol) was dissolved in a mixture ethyl alcohol (14 mL) and
THF (6 mL), and the solution was heated to 65°C. 1,3-dichloroacetone (3.3 mmol) was
added, and the mixture was heated to reflux for 6 hours. The solvents were evaporated,
and the residue was taken up with ethyl acetate, and washed with an aqueous solution

of NaHCO3. The organic phase was dried, filtered and evaporated to dryness for the
next step without further purification.
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3.2.5 General procedure for the preparation of compounds 5a-h (5’a-f)
A mixture 4 (4’) (2 mmol) and potassium phthalimide (3 mmol) in DMF (15 mL)
was heated at 80 °C for 3 hour. The reaction was diluted with ethyl acetate and washed
with water. The aqueous phase was extracted with ethyl acetate (2×20 mL). The
combined organic phases were washed with brine, filtered through Na2SO4 and
concentrated. The intermediate was dissolved in ethyl alcohol (15 mL), treated with
hydrazine monohydrate (80%) (15 mmol) and stirred overnight. The reaction mixture
was quenched with water and diluted with ethyl acetate. The aqueous and organic
phases were separated and the aqueous phase extracted with ethyl acetate (2×20 mL).
The combined organic phases were washed with brine, filtered over Na2SO4,
concentrated and the residue was purified by silica gel column chromatography.
3.2.6 General procedure for the preparation of compounds 7
Take compound 7a (R3=CH3, R4=Ph) for example and the others of 7 were
synthesized in the same way. Phenylhydrazine (38 mmol) was added slowly into ethyl
2,4-dioxovalerate (45.6 mmol) in anhydrous ethanol below -5 . Then the solution was
stirred acutely for 2 hours. The solvent was removed under reduced pressure and the
aqueous phase was extracted with ethyl acetate. The organic layer was washed twice
with 30 mL water and dried over anhydrous sodium sulfate, filtered and evaporated to
dryness for the next step without further purification (column chromatography was
also used when compounds 7-isomers were present).
3.2.6 General procedure for the preparation of compounds 9a
7a (15 mmol) was dissolved in tetrahydrofuran (20 mL) and 36% hydrochloric acid
(16 mL) solution. The reaction was kept at reflux and monitored by TLC. After the
evaporation of tetrahydrofuran, water (24 mL)/ethanol (6 mL) was added to the residue.
The solution was brought to pH 10 with 50% sodium hydroxide solution and then
heated to reflux for 4 hours. After cooling to room temperature, the resulting mixture
was brought to pH 1 with 36% hydrochloric acid solution. Precipitated solid was
filtered and the filter cake was recrystallized from ethanol to obtain white solid 9a.
The syntheses of other intermediates were similar.
3.2.7 General procedure for the preparation of target compounds
Pyrazole acid 9a-k (3 mmol) and thionyl chloride (7.5mmol) were successively

added to 20 mL 1,2-dichloroethane, the mixture were stirred under reflux for 3 hours
and then concentrated under reduced pressure, the crude products were added in drops
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into a solution of 5'a-f (5a-h) (3 mmol) and trimethylamine (6 mmol) in THF (15
mL) .The reaction mixture was stirred under the 0 for the night. After reaction the
mixture was poured in Na2CO3 solution and aqueous phase was extracted with ethyl
acetate (2×20 mL). The combined organic phase was washed with saturated salt water
(2×20 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure
and the residue was purified by silica gel column chromatography.
3-Methyl-1-phenyl-N-((2-phenylthiazol-4-yl)methyl)-1H-pyrazole-5-carboxamide
1
(A1).Yield, 88%;White solid; mp 149.7~149.8 ; H NMR (CDCl3): δ2.337 (s, 3H,
CH3), 4.615 (dd, J=5.4, 0.3 Hz, 2H, CH2), 6.580 (s, 1H, Pyrazole H), 6.626 (bs, 1H,
NH), 7.105 (s, 1H, Thiazole H), 7.274-7.474 (m, 8H, Ph H), 7.865-7.915 (m, 2H, Ph
H); GC-MS M+=374. Anal.calcd. for C21H18N4OS: C, 67.36; H, 4.85; N, 14.96; Found:
C, 67.42; H, 4.88; N, 14.86.
3-Ethyl-1-methyl-N-((2-phenylthiazol-4-yl)methyl)-1H-pyrazole-5-carboxamide
(A2).Yield,93%; White solid; mp 98.9~99.9 ; 1H NMR (CDCl3): δ 1.235 (d, J=6.9 Hz,
6H, 2CH3), 2.937-2.984 (m, 1H, CH), 4.130 (s, 3H, CH3), 4.716 (dd, J=5.4, 0.6 Hz, 2H,
CH2), 6.387 (s, 1H, Pyrazole H), 6.828 (bs, 1H, NH), 7.196 (s, 1H, Thiazole H),
7.463-7.476 (m, 3H, Ph H), 7.939-7.971 (m, 2H, Ph H). GC-MS M+=340; Anal.calcd.
for C18H20N4OS: C, 63.50; H, 5.92; N, 16.46; Found: C, 63.44; H, 5.98; N, 16.53.
3-Cyclopropyl-1-methyl-N-((2-phenylthiazol-4-yl)methyl)-1H-pyrazole-5-carboxa
1
mide(A3). Yield,95%; Brown oil; H NMR (CDCl3) : δ 0.873-0.937 (m, 4H, 2CH2),
1.865-1.921 (m, 1H, CH), 4.101 (s, 3H, CH3), 4.694 ( dd, J=5.4, 0.9 Hz, 2H, CH2),
6.227 (s, 1H, Pyrazole H), 6.767 (bs, 1H, NH), 7.210 (s, 1H, Thiazole H), 7.434-7.485
(m, 3H, Ph H), 7.928-7.964 (m, 2H, Ph H); GC-MS M+=338. Anal.calcd. for
C18H18N4OS: C, 63.88; H, 5.36; N, 16.56; Found: C, 63.92; H, 5.44; N, 16.58.
3-Ethyl-1-methyl-N-((2-phenylthiazol-4-yl)methyl)-1H-pyrazole-5-carboxamide
(A4).Yield, 96%; Brown oil; 1H NMR (CDCl3) : δ 1.196 (t, J=7.5 Hz, 3H, CH3), 2.582
(q, J=7.5H, 2H, CH2), 4.120 (s, 3H, CH3), 4.691 ( dd, J=5.4, 0.6 Hz, 2H, CH2), 6.376 (s,
1H, Pyrazole H), 6.906 (bs, 1H, NH), 7.206 (t, J=0.9 H, 1H, Thiazole H), 7.434-7.467
(m, 3H, Ph H), 7.905-7.938 (m, 2H, Ph H); HPLC-MS [M+1]+=326. Anal.calcd. for

C17H18N4OS: C, 62.55; H, 5.56; N, 17.16; Found: C, 62.56; H, 5.58; N, 17.14.
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4-Chloro-3-ethyl-1-methyl-N-((2-(p-tolyl)thiazol-4-yl)methyl)-1H-pyrazole-5-carb
1
oxamide (A5).Yield, 95%; Pale yellow solid; mp 130.5~131.0 ; H NMR (CDCl3): δ
1.196 (t, J=5.1 Hz, 3H, CH3), 2.443 (s, 3H, CH3), 2.586 (q, J=7.5 Hz, 2H, CH2), 4.075
(s, 3H, CH3), 4.936 (d, J=6.3 Hz, 2H, CH2), 7.347 (d, J=8.4 Hz, 2H, Ph H), 7.467 (s,
1H, Thiazole H), 8.097 (d, J=8.4 Hz, 2H, Ph H), 8.271 (bs, 1H, NH). GC-MS M+=374;
Anal.calcd. for C18H19ClN4OS：C, 57.67; H, 5.11; N, 14.95；Found: C, 57.68; H, 5.11;
N, 14.94.
1,3-Dimethyl-N-((2-(p-tolyl)thiazol-4-yl)methyl)-1H-pyrazole-5-carboxamide
1
(A6).Yield, 95%; Pale yellow solid; mp 127.3~129.3℃; H NMR (CDCl3): δ 2.257 (s,
3H, CH3), 2.416 (s, 3H, CH3), 4.116 (s, 3H, CH3), 4.714 (d, J=5.7 Hz, 2H, CH2), 6.412
(s, 1H, Pyrazole H), 7.219 (s, 1H, Thiazole H), 7.262 (d, J=10.2 Hz, 2H, Ph H), 7.871
(d, J=8.1 Hz, 2H, Ph H); GC-MS M+=326. Anal.calcd. for C17H18N4OS: C, 62.55; H,
5.56; N, 17.16; Found: C, 62.54; H, 5.58; N, 17.18.
4-Chloro-1,3-dimethyl-N-((2-(p-tolyl)thiazol-4-yl)methyl)-1H-pyrazole-5-carboxa
mide(A7). Yield, 92%; White solid; mp 142.1~143.5℃; 1H NMR (CDCl3) : δ 2.241 (s,
3H, CH3), 2.416 (s, 3H, CH3), 4.107 (s, 3H, CH3), 4.820 (t, J=2.85 Hz, 2H, CH2), 7.274
(s, 1H, Thiazole H), 7.276 (d, J=7.8 Hz, 2H, Ph H), 7.800 (s, 1H, NH), 7.913 (d, J=8.4
Hz, 2H, Ph H); GC-MS M+=360. Anal.calcd for C17H17ClN4OS: C, 56.58; H, 4.75; N,
15.53; Found: C, 56.60; H, 4.78; N, 15.56.
4-Chloro-N-((2-(2-chlorophenyl)thiazol-4-yl)methyl)-3-ethyl-1-methyl-1H-pyrazol
1
e-5-carboxamide(A8). Yield, 91%; Pale yellow solid; mp 136.2~136.4℃; H NMR
(CDCl3): δ 1.222 (t, J=7.5 Hz, 3H, CH3), 2.610 (q, J=6.4 Hz, 2H, CH2), 4.153 (s, 3H,
CH3), 4.819 (dd, J=5.4, 0.6 Hz, 2H, CH2), 7.345 (s, 1H, thiazole H), 7.362-7.427(m,
2H, Ph H). 7.498-7.529 (m, 1H, Ph H), 7.572 (bs, 1H, NH), 8.227-8.259 (m, 1H, Ph H);
GC-MS M+=394. Anal.calcd. for C17H16Cl2N4OS: C, 51.65; H, 4.08; N, 14.17; Found:
C, 51.60; H, 4.10; N, 14.18.
4-Chloro-N-((2-(2-chlorophenyl)
thiazol-4-yl)
methyl)-1,3-dimethyl-1H-pyrazole-5-carboxamide(A9). Yield, 91%; White solid; mp
1
136.6~137.6℃; H NMR (CDCl3): δ 2.248 (s, 3H, CH3), 4.140 (s, 3H, CH3), 4.838 (d,

J=5.4 Hz, 2H, CH2), 7.378-7.419 (m, 3H, Ph H), 7.514 (s, 1H, Thiazole H), 7.641 (bs,
1H, NH), 8.307 (d, J=9.2 Hz, 1H, Ph H); HPLC-MS Pos [M+1]+=381. Anal.calcd. for
ACCEPTED MANUSCRIPT
C16H14Cl2N4OS: C, 50.40; H, 3.70; N, 14.69; Found: C, 50.42; H, 3.72; N, 14.70.
N-((2-(2-Chlorophenyl)
thiazol-4-yl)
methyl)-3-methyl-1-phenyl-1H-pyrazole-5-carboxamide(A10). Yield,85%; Pale
yellow solid; mp 120.0~125.0 ; 1H NMR (CDCl3): δ 2.351 (s, 3H, CH3), 4.817 (dd, J=
6.0, 0.6 Hz, 2H, CH2), 6.767 (d, J=0.9 Hz, 1H, Pyrazole H), 7.324-7,387 (m, 3H, Ph H),
7.417 (s, 1H, Thiazole H), 7.447-7.507 (m, 5H, Ph H), 7.559 (bs, 1H, NH), 8.202-8.234
(m, 1H, Ph H). HPLC-MS Pos [M+1]+=409; Anal.calcd. for C21H17ClN4OS: C, 61.68;
H, 4.19; N, 13.70; Found: C, 61.70; H, 4.18; N, 13.71.
N-((2-(2-Chlorophenyl)thiazol-4-yl)methyl)-3-ethyl-1-phenyl-1H-pyrazole-5-
carboxamide (A11).Yield, 90%; White solid; mp 142.5~143.0 ; 1H NMR (CDCl3) : δ
1.259 (t, J=7.5 Hz, 3H, CH3), 2.678 (q, J=7.5 Hz, 2H, CH2), 4.618 (dd, J=6.0, 0.6 Hz,
2H, CH2), 6.641 (s, 1H, Pyrazole H), 6.684 (bs, 1H, NH), 7.088 (s, 1H, Thiazole H),
7.088-7.258 (m, 1H, Ph H), 7.268-7.296 (m, 1H, Ph H), 7.305-7.340 (m, 1H, Ph H),
7.355-7.388 (m, 2H, Ph H), 7.407-7.449 (m, 2H, Ph H), 7.778-7.813 (m, 2H, Ph H);
HPLC-MS Pos [M+1]+=423 ; Anal.calcd. for C22H19ClN4OS: C, 62.48; H, 4.53; N,
13.25; Found: C, 62.52; H, 4.50; N, 13.20.
N-((2-(4-Chlorophenyl)thiazol-4-yl)methyl)-1,3,4-trimethyl-1H-pyrazole-5-carbox
1
amide(A12). Yield, 91%; White solid; mp 165.5~165.9 ; H NMR (CDCl3) : δ 2.199
(s, 3H, CH3), 2.210 (s, 3H, CH3), 4.036 (s, 3H, CH3), 4.744 (dd, J=5.4, 0.9Hz,, 2H,
CH2), 6.691 (bs, 1H, NH), 7.228 (s, 1H, Thiazole H), 7.407-7.453 (m, 2H, Ph H),
7.853-7.898 (m,, 2H, Ph H); GC-MS M+=360. Anal.calcd. for C17H17ClN4OS: C, 56.58;
H, 4.75; N, 15.53; Found: C, 56.54; H, 4.68; N, 15.59.
4-Chloro-N-((2-(4-chlorophenyl)thiazol-4-yl)methyl)-3-ethyl-1-methyl-1H-pyrazol
e-5-carboxamide(A13). Yield, 92%; Pale yellow solid; mp 136.8~141.3 ; 1H NMR
(CDCl3) : δ 1.221-1.278 (m, 3H, CH3), 2.609-2.686 (s, 2H, CH2), 4.147 (s, 3H, CH3),
4.772-4.790 (m, 2H, CH2), 7.227 (s, 1H, Thiazole H), 7.411-7.441 (m, 2H, Ph H),7.544
(bs, 1H, NH), 7.871 (d, J=8.7 Hz, 2H, Ph H); GC-MS M+=394. Anal.calcd. for
C17H16Cl2N4OS: C, 51.65; H, 4.08; N, 14.17; Found: C, 51.68; H, 4.09; N, 14.16.
N-((2-(4-Chlorophenyl)thiazol-4-yl)methyl)-3-ethyl-1-methyl-1H-pyrazole-5-carbo

xamide（A14）.Yield, 93%; White solid; mp 118.6~119.6℃；1H NMR (CDCl3) : δ
1.210-1.262 (m, 3H, CH3), 2.597 (q, J=7.7 Hz, 2H, CH2), 4.130 (s, 3H, CH3), 4.705 (dd,
ACCEPTED MANUSCRIPT
J=6.0, 0.9 Hz, 2H, CH2), 6.378 (s, 1H, Pyrazole H), 6.753 (bs, 1H, NH), 7.242 (s, 1H,
Thiazole H), 7.422 (d, J=8.7 Hz, 2H, Ph H), 7.880 (d, J=8.7 Hz, 2H, Ph H); GC-MS
M+=361. Anal.calcd. for C17H17ClN4OS: C, 56.58; H, 4.75; N, 15.53; Found: C, 56.55;
H, 4.69; N, 15.63.
N-((2-(4-Chlorophenyl)thiazol-4-yl)methyl)-1,3-dimethyl-1H-pyrazole-5-carboxam
1
ide(A15).Yield, 90%; Pale yellow solid; mp 123.1~124.0℃; H NMR (CDCl3) : δ
2.262 (s, 3H, CH3), 4.121 (s, 3H, CH3), 4.703 (d, J=5.7 Hz, 2H, CH2), 6.361 (s, 1H,
Pyrazole H), 6.792 (bs, 1H, NH), 7.240 (s, 1H, Thiazole H), 7.415-7.460 (m, 2H, Ph H),
7.874-7.920 (m,, 2H, Ph H); GC-MS M+=346. Anal.calcd. for C16H15ClN4OS: C, 55.41;
H, 4.36; N, 16.15; Found: C, 55.46; H, 4.29; N, 16.20.
N-((2-(4-Chlorophenyl)thiazol-4-yl)methyl)-3-methyl-1-phenyl-1H-pyrazole-5-car
boxamide(A16). Yield, 84%; Pale yellow solid; mp 147.1~147.9℃; 1H NMR (CDCl3):
δ 2.342-2.359 (m, 3H, CH3), 4.765-4.789 (m, 2H, CH2), 6.764 (d, J=0.9, 1H, Pyrazole
H), 7.222(s, 1H, Thiazole H), 7.384-7.399(m, 2H, Ph H), 7.473-7.503(m, 5H, Ph H),
+
7.544(bs, 1H, NH), 7,851-7.880 (m, 2H, Ph H). HPLC-MS Pos [M+1] =409;
Anal.calcd. for C21H17ClN4OS: C, 61.68; H, 4.19; N, 13.70; Found: C, 61.70; H, 4.22;
N, 13.73.
4-Chloro-N-((2-(2-methoxyphenyl)thiazol-4-yl)methyl)-1,3-dimethyl-1H-pyrazole-
1
5-carboxamide(A17).Yield, 89%; White solid; mp 155.4~155.8℃; H NMR (CDCl3):
δ 2.232 (s, 3H, CH3), 4.074 (s, 3H, CH3), 4.098 (s, 3H, CH3), 4.894 (d, J=5.4 Hz, 2H,
CH2), 7.065 (d, J=8.4 Hz, 1H, Ph H), 7.159-7.242 (m, 1H, Ph H), 7.402 (s, 1H,
Thiazole H), 7.485-7.609(m, 1H, Ph H), 8.052 (bs, 1H, NH), 8.707(s, 1H, Ph H).
HPLC-MS Pos [M+1] +=376; Anal.calcd. for C17H17ClN4O2S: C, 54.18; H, 4.55; N,
14.87; Found: C, 54.22; H, 4.49; N, 14.83.
N-((2-(4-Methoxyphenyl)thiazol-4-yl)methyl)-1,3-dimethyl-1H-pyrazole-5-carboxa
1
mide(A18).Yield,93%;Pale yellow solid; mp 139.6~140.1℃; H NMR (CDCl3): δ
2.258 (s, 3H, CH3), 3.876 (s, 3H, CH3), 4.117 (s, 3H, CH3), 4.690 (d, J=5.7 Hz, 2H,
CH2), 6.386 (s, 1H, Pyrazole H), 6.964 (d, J=8.7 Hz, 2H, Ph H), 7.115 (bs, 1H, NH)，
7.261 (s, 1H, Thiazole H), 7.905 (d, J=9.0 Hz, 2H, Ph H); GC-MS M+=342. Anal.calcd.

for C17H18N4O2S: C, 59.63; H, 5.30; N, 16.36; Found: C, 59.59; H, 5.36; N, 16.43.
ACCEPTED MANUSCRIPT
3-Ethyl-N-((2-(4-methoxyphenyl)thiazol-4-yl)methyl)-1-methyl-1H-pyrazole-5-car
1
boxamide(A19). Yield,93%; Pale yellow solid; mp 102.6~103.1 ; H NMR (CDCl3):
δ 1.200 (t, J=7.5 Hz, 3H, CH3), 2.587 (q, J=7.6 Hz, 2H, CH2), 3.864 (s, 3H, CH3),
4.124 (s, 3H, CH3), 4.672 (dd, J=6.3, 2.1 Hz, 2H, CH2), 6.369 (s, 1H, Pyrazole H),
6.830 (bs, 1H, NH), 6.934-6.983 (m, 2H, Ph H), 7.126 (s, 1H, Thiazole H), 7.841-7.890
(m, 2H, Ph H); GC-MS M+=356. Anal.calcd. for C18H20N4O2S: C, 60.65; H, 5.66; N,
15.72; Found: C, 60.71; H, 5.58; N, 15.70.
4-Chloro-3-ethyl-N-((2-(4-methoxyphenyl)thiazol-4-yl)methyl)-1-methyl-1H-pyraz
1
ole-5-carboxamide(A20).Yield, 91%; White solid; mp 136.0~136.2 ; H NMR
(CDCl3): δ 1.222 (t, J=7.5Hz, 3H, CH3), 2.609 (q, J=7.6 Hz, 2H, CH2), 3.867 (s, 3H,
CH3), 4.148 (s, 3H, CH3), 4.751(dd, J=5.7, 1.2 Hz, 2H, CH2), 6.946-6.986 (m, 2H, Ph
H), 7.125 (s, 1H, Thiazole H), 7.550 (bs, 1H, NH), 7.859-7.899 (m, 2H, Ph H); GC-MS
M+=390. Anal.calcd. for C18H19ClN4O2S: C, 55.31; H, 4.90; N, 14.33; Found: C, 55.36;
H, 4.86; N, 14.41.
3-Ethyl-1-methyl-N-((2-phenyloxazol-4-yl)methyl)-1H-pyrazole-5-carboxamide
(B1). Yield,94%; White solid; mp 98.4~99.1 ; 1H NMR (CDCl3): δ 1.200 (t, J=7.8Hz,
3H, CH3), 2.587 (dd, J=15.6, 7.8Hz, 2H, CH2), 4.121 (s, 3H, CH3), 4.536 (q, J=0.6Hz,
2H, CH2), 6.364 (s, 1H, Pyrazole H), 6.643 (bs, 1H, Oxazole H), 7.455-7.498 (m, 3H,
Ph H), 7.692 (s, 1H, NH), 8.011-8.052 (m, 2H, Ph H); GC-MS M+=310. Anal.calcd. for
C17H18N4O2: C, 65.79; H, 5.85; N, 18.05; Found: C, 65.80; H, 5.88; N, 18.07.
4-Chloro-3-methyl-N-((2-phenyloxazol-4-yl)methyl)-1H-pyrazole-5-carboxamide
1
(B2).Yield,97%; Pale yellow solid; mp 192.1~195.4 ; H NMR (CDCl3): δ 2.304 (s,
3H, CH3), 4.526 (d, J=6.3 Hz, 2H, CH2), 7.423-7.495 (m, 4H, Oxazole / Ph H), 7.845
(s, 1H, NH), 7.945-7.977 (m, 2H, Ph H), 9.045 (bs, 1H, Pyrazole H); GC-MS M+=316.
Anal.calcd. for C15H13ClN4O2: C, 56.88; H, 4.14; N, 17.69; Found: C, 56.91; H, 4.16;
N, 17.68.
4-Chloro-1,3-dimethyl-N-((2-phenyloxazol-4-yl)methyl)-1H-pyrazole-5-carboxami
1
de(B3). Yield, 96%; White solid; mp 114.1~116.3 ；H NMR (CDCl3) : δ 2.242 (s, 3H,
CH3), 4.132 (s, 3H, CH3), 4.610 (dd, J=0.9, 5.4 Hz, 2H, CH2), 7.340 (d, J=0.9 Hz, 1H,

NH), 7.452-7.500 (m, 3H, Ph H), 7.708 (s, 1H, Oxazole H), 8.010-8.064 (m, 2H, Ph H);
GC-MS M+=330. Anal.calcd. for C16H15ClN4O2：C, 58.10; H, 4.57; N, 16.94; Found: C,
ACCEPTED MANUSCRIPT
58.12; H, 4.54; N, 16.90.
1,3-dimethyl-N-((2-(o-tolyl)oxazol-4-yl)methyl)-1H-pyrazole-5-carboxamide
1
(B4).Yield,97%; White solid; mp 91.4~92.0℃；H NMR (CDCl3): δ 2.254 (s, 3H, CH3),
2.670 (s, 3H, CH3), 4.120 (s, 3H, CH3), 4.554 (q, J=0.9Hz, 2H, CH2), 6.337 (s, 1H,
Pyrazole H), 6.690 (s, 1H, Oxazole H),7.276-7.398 (m, 3H, Ph H), 7.716 (s, 1H, NH),
7.954-7.986 (m, 1H, Ph H); GC-MS M+=310. Anal.calcd. for C17H18N4O2: C, 65.79; H,
5.85; N, 18.05; Found: C, 65.76; H, 5.84; N, 18.06.
3-Ethyl-1,4-dimethyl-N-((2-(o-tolyl)oxazol-4-yl)methyl)-1H-pyrazole-5-carboxami
1
de(B5).Yield,92%;White solid; mp 78.2~80.0℃; H NMR (CDCl3) : δ 1.181 (t, J=7.8
Hz, 3H, CH3), 2.203 (s, 3H, CH3), 2.550 (q, J=7.5 Hz 2H, CH2), 2.679 (s, 3H, CH3),
4.045 (s, 3H, CH3),4.604 (d, J=5.1 Hz 2H, CH2), 6.501 (s, 1H, NH), 7.292-7.388 (m,
3H, Ph H) 7.716 (m, 1H, Oxazole H), 7.973 (d, J=1.8 Hz 1H, Ph H); GC-MS
M+=338. Anal.calcd. for C19H22N4O2: C, 67.44; H, 6.55; N, 16.56; Found: C, 67.48; H,
6.56; N, 16.52.
4-Chloro-1,3-dimethyl-N-((2-(p-tolyl)oxazol-4-yl)methyl)-1H-pyrazole-5-carboxa
mide(B6).Yield,91%;White solid; mp 138.7~138.8℃; 1H NMR (CDCl3): δ 2.239 (s, 3H,
CH3), 2.412 (s, 3H, CH3), 4.128 (s, 3H, CH3), 4.598 (t, J=1.2 Hz, 2H, CH2),
7.262-7.288 (m, 2H, Ph H), 7.326 (bs, 1H, Oxazole H), 7.675 (s, 1H, NH), 7.904-7.938
(m, 2H, Ph H); GC-MS M+=344. Anal.calcd. for C17H17ClN4O2: C, 59.22; H, 4.97; N,
16.25; Found: C, 59.24; H, 4.99; N, 16.24.
N-((2-(2-Chlorophenyl)
oxazol-4-yl)
methyl)-3-ethyl-1,4-dimethyl-1H-pyrazole-5-carboxamide(B7). Yield,90%; White
solid; mp 98.3~99.1℃; 1H NMR (CDCl3) : δ 1.171 (t, J=7.8 Hz, 3H, CH3), 2.194 (s, 3H,
CH3), 2.536 (q, J=7.5 Hz, 2H, CH2), 4.025 (s, 3H, CH3), 4.612 (dd, J=1.2, 5.4 Hz, 2H,
CH2), 6.557 (s, 1H, NH),7.337-7.442 (m, 2H, Ph H) 7.484-7.532 (m, 1H, Ph H), 7.774
(t, J=0.9 Hz 1H, Oxazole H), 7.913-7.960 (m, 1H, Ph H); HPLC-MS Pos [M+1]+=359.
Anal.calcd. for C18H19ClN4O2: C, 60.25; H, 5.34; N, 15.61; Found: C, 60.28; H, 5.35;
N, 15.58.

4-Chloro-1,3-dimethyl-N-((2-(2-(trifluoromethyl)
phenyl)
oxazol-4-yl)
methyl)-1H-pyrazole-5-carboxamide (B8). Yield, 88%; White solid; mp 69.4~72.1 ;
1
ACCEPTED MANUSCRIPT
H NMR (CDCl3): δ 2.245 (s, 3H, CH3), 4.138(s, 3H, CH3), 4.633 (t, J=1.2 Hz, 2H,
CH2), 7.320 (bs, 1H, Oxazole H), 7.585-7.694 (m, 2H, Ph H), 7.791 (s, 1H, NH),
7.813-7.843 (m, 1H, Ph H), 7.980-8.004 (m, 1H, Ph H); GC-MS M+=398. Anal.calcd.
for C17H14ClF3N4O2: C, 51.20; H, 3.54; N, 14.05; Found: C, 51.22; H, 3.56; N, 14.03.
3-Ethyl-1-methyl-N-((2-(2-(trifluoromethyl)phenyl)oxazol-4-yl)methyl)-1H-pyrazo
1
le-5-carboxamide(B9). Yield, 95%; Brown oil; H NMR (CDCl3) : δ 1.235 (q, J=5.4
Hz, 3H, CH3), 2.602 (dd, J=15.0, 7.5 Hz, 2H, CH2), 4.138 (s, 3H, CH3), 4.566 (t, J=0.9
Hz, 2H, CH2), 6.369 (s, 1H, Pyrazole H), 6.671 (bs, 1H, Oxazole H), 7.589-7.700 (m,
2H, Ph H), 7.787 (s, 1H, NH), 7.819-7.850 (m, 1H, Ph H), 7.968-7.998 (m, 1H, Ph H);
GC-MS M+=378. Anal.calcd. for C18H17F3N4O2: C, 57.14; H, 4.53; N, 14.81; Found: C,
57.13; H, 4.56; N, 14.79.
N-((2-(2,6-difluorophenyl)oxazol-4-yl)methyl)-1,3-dimethyl-1H-pyrazole-5-carbox
amide(B10).Yield,92%; Yellow solid; mp 145.0~146.4 ; 1H NMR (CDCl3): δ 2.247 (s,
3H, CH3), 4.113 (s, 3H, CH3), 4.572 (q, J=0.9Hz, 2H, CH2), 6.311 (s, 1H, Pyrazole H),
7.957 (s, 1H, Oxazole H), 7.023-7.078 (m, 2H, Ph H), 7.418-7.475 (m, 1H, Ph H),
7.818 (s, 1H, NH); GC-MS M+=332. Anal.calcd. for C16H14F2N4O2: C, 57.83; H, 4.25;
N, 16.86; Found: C, 57.82; H, 4.27; N, 16.88.
4-Chloro-N-((2-(2,6-difluorophenyl)oxazol-4-yl)methyl)-1,3-dimethyl-1H-pyrazole
1
-5-Carboxamide(B11). Yield, 92%; Off-white solid; mp 116.3~118.8 ; H NMR
(CDCl3): δ 2.237 (s, 3H, CH3), 4.129 (s, 3H, CH3), 4.648 (q, J=0.9Hz, 2H, CH2),
7.010-7.089 (m, 2H, Ph H), 7.337 (bs, 1H, Oxazole H), 7.393-7.490 (m, 1H, Ph H),
7.826 (s, 1H, NH); GC-MS M+=366. Anal.calcd. for C16H13ClF2N4O2: C, 52.40; H,
3.57; N, 15.28; Found: C, 52.38; H, 3.59; N, 15.25.
4-Chloro-N-((2-(2,6-difluorophenyl)oxazol-4-yl)methyl)-3-ethyl-1-methyl-1H-pyra
zole-5-carboxamide(B12). Yield, 89%; White solid; mp 116.3~117.0 . 1H
NMR(CDCl3): δ 1.212 (t, J=7.8Hz, 3H, CH3), 2.598 (dd, J=15, 7.5Hz, 2H, CH2), 4.137
(s, 3H, CH3), 4.650 (q, J=0.9Hz, 2H, CH2), 7.021-7.078 (m, 2H, Ph H), 7.413 (bs, 1H,
Oxazole H), 7.441-7.470 (m, 1H, Ph H), 7.826 (s, 1H, NH); GC-MS M+=380.
Anal.calcd. for C17H15ClF2N4O2: C, 53.62; H, 3.97; N, 14.71; Found: C, 53.65; H, 3.98;

N, 14.74.
ACCEPTED MANUSCRIPT
N-((2-(2,4-dichlorophenyl)oxazol-4-yl)methyl)-1,3-dimethyl-1H-pyrazole-5-carbox
1
amide (B13). Yield, 92%; White solid; mp 122.1~124.8 ; H NMR (CDCl3): δ 2.276
(s, 3H, CH3), 4.136 (s, 3H, CH3), 4.572 (s, 2H, CH2), 6.339 (s, 1H, Pyrazole H), 6.633
(s, 1H, NH),7.347 (dd, J=2.1, 8.4 Hz, 1H, Ph H),7.541 (d, J=2.1 Hz, 1H, Ph H) 7.778 (s,
1H, Oxazole H), 7.907 (d, J=8.4 Hz, 1H, Ph H); GC-MS M+=364. Anal.calcd. for
C16H14Cl2N4O2: C, 52.62; H, 3.86; N, 15.34; Found: C, 52.65; H, 3.84; N, 15.36.
N-((2-(2,4-dichlorophenyl)oxazol-4-yl)methyl)-3-ethyl-1-methyl-1H-pyrazole-5-car
1
boxamide(B14).Yield,91%; White solid; mp 123.5~124.5 ; H NMR (CDCl3): δ
1.216 (t, J=7.5 Hz, 3H, CH3), 2.617 (dd, J=14.7, 7.2Hz, 2H, CH2), 4.152 (s, 3H, CH3),
4.568 (d, J=3.9Hz, 2H, CH2), 6.383 (s,1H, Pyrazole H), 6.742 (s, 1H, Oxazole H),
7.348-7.383 (m, 1H, Ph H), 7.541-7.548 (m, 1H, Ph H), 7.782 (s, 1H, NH), 7.904-7.933
(m, 1H, Ph H); GC-MS M+=378. Anal.calcd. for C17H16Cl2N4O2: C, 53.84; H, 4.25; N,
14.77; Found: C, 53.87; H, 4.28; N, 14.74.
N-((2-(2,4-dichlorophenyl)oxazol-4-yl)methyl)-1,3-diethyl-1H-pyrazole-5-carboxa
1
mide(B15).Yield,91%; White solid; mp 101.3~105.5 ; H NMR (CDCl3): δ 1.218 (t,
J=7.8 Hz, 3H, CH3), 1.421 (t, J=7.2Hz, 3H, CH3),2.630 (dd, J=15.3, 7.8Hz, 2H, CH2),
4.539-4.611 (m, 4H, 2CH2), 6.360 (s, 1H, Pyrazole H), 6.649 (bs, 1H, Oxazole H),
7.345-7.381 (m, 1H, Ph H), 7.540-7.546 (m, 1H, Ph H), 7.778 (s, 1H, NH), 7.907-7.935
(m, 1H, Ph H); GC-MS M+=392. Anal.calcd. for C18H18Cl2N4O2: C, 54.97; H, 4.61; N,
14.25; Found: C, 54.99; H, 4.58; N, 14.26.
4-Chloro-N-((2-(2,6-dichlorophenyl)oxazol-4-yl)methyl)-3-methyl-1H-pyrazole-5-c
1
arboxamide(B16).Yield,94%; White solid; mp 201.7~204.4 ; H NMR (CDCl3): δ
2.226 (s, 3H, CH3), 4.389 (d, J=6.0Hz, 2H, CH2), 7.606-7.704 (m, 3H, Ph H), 8.105 (s,
1H, Oxazole H), 8.614 (s, 1H, NH), 13.393 (s, 1H, Pyrazole H); HPLC-MS Pos [M+1]
+=386. Anal.calcd. for C15H11Cl3N4O2: C, 46.72; H, 2.88; N, 14.53; Found: C, 46.74; H,
2.91; N, 14.50.
3.3 Biological assay
Fungicidal activity of compounds A1~A22 and B1~B16 were tested in vitro against
G. zeae, P. capsici and S. sclerotiorum and which were tested in vivo against E.

graminis and P. sorghi using the mycelium growth rate method [36,37]. The
commercially available agricultural fungicide, thifluzamide and boscalid were used as
ACCEPTED MANUSCRIPT
the positive control, whereas water (with 0.2% DMF and 0.2% Tween80) served as the
negative control. The compounds 0.0111g were dissolved in 0.5 mL DMF, and then
Tween80 and sterile water were added to 22.2 mL to prepare 500 mg/L solution and
then 2 mL of the solution was taken out to mix with 38 mL potato dextrose agar (PDA)，
thus 25 mg/L final concentration was got for the following antifungal test. G. zeae, P.
capsici and S. sclerotiorum were incubated in PDA at 27 ± 1 °C for 4 days to get new
mycelium for antifungal assay. Then mycelia dishes of approximately 6 mm diameter
were cut from culture medium and one of them was picked up with a sterilized
inoculation needle and inoculated in the center of PDA plate aseptically. The inoculated
plates were incubated at 27 ± 1℃for at least 72 h and when the control colony diameter
reached 70 mm, the diameter of each stain was measured. Water (with 0.2%DMF and
0.2% Tween80) served as the negative control while thifluzamide and boscalid severed
as the positive control.The growth inhibition rates were calculated by the following
equation: I = [(C - T)/C] × 100%, where I is the growth inhibition rate (%), C is the
control settlement radius (mm) and T is the treatment group fungus settlement radius
(mm).
Wheat powdery mildew was caused by Erysiphe graminis f. sp tritici and the E.
graminis f.sp. tritici was cultured on wheat seedlings, cultivar Qingnong No.142
(Variety protection number: CNA20060353E), in a temperature-controlled room at
18 °C. Take the plant pots with a diameter of 15cm, and seed the wheat with 20
granules per pot. Seven days later, when the third true leaf comes out, it can be used for
testing. Mildew spores collected from the infected culture plants were sprayed onto
seven-day-old wheat seedlings that had been sprayed with the fungicide solution and
the dose of all the tests in vivo is 5 mL per pot. The inoculated seedlings were kept in a
controlled temperature room at 18 °C with subirrigation. The percentage disease
control was rated 7 days after the inoculation. Corn rust was caused by Puccinia
polysora Unedrw. and the maize leaves were cut off and washed with 0.05% Tween80
sterile aqueous solution, and then filtered with 2 to 4 layers of gauze. The suspension
was adjusted to 1×105 spores/mL with 0.05% Tween80 sterile aqueous solution. Take
the plant pots with a diameter of 15cm, and seed the corn with 10~12 granules per pot.
When the corn sprouts break out into the third leaf, the above solution (500 mg/L) was
sprayed onto the plants and 24 hours later the plants were inoculated with fungal spores.
After inoculation, it was moved to the moisture storage cabinet (relative humidity

above 95%, temperature 20 ~ 22 , darkness) for 12 h and then it was cultivated in
normal environment (Shine for 12 hours and keep darkness for 12 hours a day，
ACCEPTED MANUSCRIPT
temperature 20 ~ 22℃, light intensity 5000 Lux to 10000 Lux) until the negative
control was found obviously that the test began. The results are the percentage disease
control compared with the untreated check, wherein 100 is rated as complete disease
control and 0 as no disease control. The compounds that displayed high activity
(control effects more than 95%) against E. graminis were evaluated further at
concentration of 200,100, 50, 25, 10 and 0 mg/L. The EC50 (mg/L) values were
estimated statistically by probit analysis using SPSS on a personal computer.
Acknowledgements
The authors extend their appreciation to the support of the National Natural Science
Foundation of China (Grant 21572050, 21672057).
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Legents
Table 1. Chemical structures and antifungal activities of the target compounds
Table 2. EC50 values of some compounds with excellent activity against E. graminis.
Table 3. The molecular total energy and frontier orbital energy of A4 and some
commercial compounds
Table 4. Mulliken atomic charges except for H atoms (e)
Figure 1. Bioisosterism in the application of pesticide design of pyrazole amide.
Figure 2. Design strategy of the target compounds.
Figure 3. Geometry optimization and the transformation of configuration and activity.
Figure 4. Frontier molecular orbitals of lead compound, A4, carboxin and thifluzamid.
Figure 5. (a) Connolly surface of complex with carboxin shown as a stick model. (b)
Hydrogen-bond interaction of carboxin and amion acid residues near the ligands. (c) Transparent
surface of complex with compound A4 as a stick model. (d) Main interaction of compound A4 and
amion acid residues near the ligands.

ACCEPTED MANUSCRIPT
Table 1. Chemical structures and antifungal activities of the target compounds
R2
O
N
R3
N
H
R1
N
N
Z
R4
Inhibition rate (control effects) (%)
No.
Z
S
R1
R2
H
R3
R4
Ph
Gzα
Pcα
Ssα
Egβ
Psβ
A1
H
H
CH3
37.9
10.8
95.2
70
94.5
A2
A3
S
S
H
H
CH3
CH3
31.8
40.4
13.3
18.8
91.8
20.5
100
85
95.6
_
H
A4
A5
S
S
H
4-CH3
4-CH3
4-CH3
2-Cl
H
Cl
H
CH3CH2
CH3CH2
CH3
CH3
CH3
CH3
CH3
CH3
CH3
Ph
29.5
31.1
34.1
45.5
31.1
41.4
34.5
12.8
42.2
31.1
44.4
62.1
41.4
34.1
51.7
42.2
28.9
11.4
6.8
41.4
35.9
42.7
6.3
41.7
24.4
0
100
74
90
90
60
90
70
0
95.0
80.3
75.9
91.8
44.7
13.5
0
A6
S
A7
S
Cl
Cl
Cl
H
CH3
0
A8
S
CH3CH2
CH3
46.2
13.8
20
35.6
52.4
81
A9
S
2-Cl
A10
A11
A12
A13
A14
A15
A16
A17
A18
A19
A20
B1
S
2-Cl
CH3
S
2-Cl
H
CH3CH2
CH3
Ph
20.5
46.2
5.1
45.3
44.4
55.6
46.7
57.1
90.5
0
43.7
40.2
83
S
4-Cl
CH3
Cl
H
CH3
CH3
CH3
CH3
Ph
91
91
90
95
90
70
95
90
91
95
10
10
80
40
92
20
65
S
4-Cl
CH3CH2
CH3CH2
CH3
S
4-Cl
59
90.9
44.1
11.9
69.1
70.1
97.3
91.2
90
S
4-Cl
H
26.2
41.5
51.2
10.8
38.5
38.5
43.2
22.7
0
S
4-Cl
H
CH3
S
2-OCH3
4-OCH3
4-OCH3
4-OCH3
H
Cl
H
CH3
CH3
CH3
CH3
CH3
CH3
H
S
CH3
57.1
53.3
44.4
10.3
20.5
0
S
H
CH3CH2
CH3CH2
CH3CH2
CH3
S
Cl
H
O
O
O
O
O
O
O
O
B2
H
Cl
Cl
H
0
B3
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
42.9
30.6
31
90
B4
2- CH3
2- CH3
4- CH3
2- Cl
2- CF3
CH3
27.3
-12.8
34.1
12.8
43.2
43.6
40
90
B5
CH3 CH3CH2
Cl CH3
CH3 CH3CH2
Cl CH3
70
B6
31.8
42.9
43.2
43.6
2.9
20
B7
60
B8
20.5
85

ACCEPTED MANUSCRIPT
B9
B10
O
O
O
O
O
O
O
O
2- CF3
2,6-di F
2,6-di F
2,6-di F
2,6-di F
2,4-di Cl
2,4-di Cl
2,6-di Cl
H
H
CH3CH2
CH3
CH3
CH3
CH3
CH3
CH3
CH3
34.1
27.3
22.7
34.1
40.5
15.9
11.4
0
38.6
36.4
27.3
38.6
38.5
29.5
38.6
0
30.8
30.8
43.6
38.5
17.1
56.4
61.5
53.8
36.9
100
90
92
90
50
10
60
85
90
20
B11
Cl
Cl
H
CH3
15
B12
CH3CH2
CH3
80
B13
85
B14
H
CH3CH2
95
B15
H
CH3CH2 CH3CH2
CH3
25
B16
Cl
H
5
90
_
Thifluzamide
Boscalid
14.0
37.0
63.0
5.3
85.0
100
_
“_” no test
β
αIn vitro (25mg/L), Gz, Gibberella zeae; Pc, Phytophythora capsici; Ss, Sclerotonia sclerotiorum; In
vivo (500mg/L), Eg, Erysiphe graminis, white powder of wheat germs preserves spores in wheat
seedlings for test; Ps, Puccinia sorghi, from corn seedling living save.

ACCEPTED MANUSCRIPT
NO.
MW
340
326
310
379
528
EC50
A2
A4
12.6 µM
9.3 µM
13.8 µM
11.9 µM
8.6 µM
4.28 mg/L
3.04 mg/L
4.29 mg/L
4.52 mg/L
4.52 mg/L
B1
B14
Thifluzamide

ACCEPTED MANUSCRIPT
Lead compound
DFT
A4
Carboxin
Thifluzamide
(tebufenpyrad)
-1400.08912
-0.23632
Etotal /Hartreeb
EHOMO / Hartree
ELUMO /Hartree
∆Ea / Hartree
-1351.10001
-0.22358
-0.05786
0.16572
-1068.50757
-0.20192
-0.02962
0.17230
-6899.61428
-0.25765
-0.07817
0.17948
-0.02804
0.20828
a∆E =ELUMO-EHOMO ; b1 Hartree= 4.35974417×10-18J=27.2113845eV.

ACCEPTED MANUSCRIPT
Charge
Atom
DFT
Charge
DFT
Atom
1C
2C
0.113158
-0.132108
-0.094250
-0.077969
-0.089464
-0.094587
0.032820
-0.483247
0.290934
-0.331041
0.241499
-0.088683
13N
14C
15C
16O
17C
18C
19N
20N
21C
22C
23C
-0.569898
0.583698
0.299249
-0.516211
-0.208388
0.286149
-0.340361
-0.353665
-0.234326
-0.313502
-0.195919
3C
4C
5C
6C
7C
8N
9C
10C
11S
12C

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT