Synthesis and bioactivity of pyrazole acyl thiourea derivatives

Article abstract of DOI:10.3390/molecules17055139

Sixteen novel pyrazole acyl thiourea derivatives 6 were synthesized from monomethylhydrazine (phenylhydrazine) and ethyl acetoacetate. The key 5-chloro-3- methyl_-1-substituted_-1H-pyrazole-4-carbonyl chloride intermediates 4 were first generated in four steps through cyclization, formylation, oxidation and acylation. Thess were then reacted with ammonium thiocyanate in the presence of PEG-400 to afford 5-chloro-3- methyl _-1-substituted_-1H-pyrazole-4-carbonyl isothiocyanates 5. Subsequent reaction with fluorinated aromatic amines resulted in the formation of the title compounds. The synthesized compound were unequivocally characterized by IR, ¹H-NMR, ¹³C-NMR and elemental analysis and some of the synthesized compounds displayed good antifungal activities against Gibberella zeae, Fusarium oxysporum, Cytospora mandshurica and anti-TMV activity in preliminary antifungal activity tests.

Full text of DOI:10.3390/molecules17055139

Molecules 2012, 17, 5139-5150; doi:10.3390/molecules17055139
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Bioactivity of Pyrazole Acyl Thiourea Derivatives
Jian Wu †, Qing Shi †, Zhuo Chen, Ming He, Linhong Jin and Deyu Hu *
State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering,
Key Laboratory of Green Pesticide and Agriculture Bioengineering, Ministry of Education,
Guizhou University, Guiyang 550025, China; E-Mails: jianwu2691@yahoo.com.cn (J.W.);
shiqing982816@163.com (Q.S.); fcc.zchen@gzu.edu.cn (Z.C.); hmher@126.com (M.H.);
fcc.jinlh@gzu.edu.cn (L.J.)
†
These authors contributed equally to this work.
* Author to whom correspondence should be addressed; E-Mail: fcc.dyhu@gzu.edu.cn;
Tel./Fax: +86-851-829-2170.
Received: 11 April 2012; in revised form: 19 April 2012 / Accepted: 19 April 2012 /
Published: 3 May 2012
Abstract: Sixteen novel pyrazole acyl thiourea derivatives 6 were synthesized from
monomethylhydrazine (phenylhydrazine) and ethyl acetoacetate. The key 5-chloro-3-
methyl-1-substituted-1H-pyrazole-4-carbonyl chloride intermediates 4 were first generated
in four steps through cyclization, formylation, oxidation and acylation. Thess were then
reacted with ammonium thiocyanate in the presence of PEG-400 to afford 5-chloro-3-
methyl-1-substituted-1H-pyrazole-4-carbonyl isothiocyanates 5. Subsequent reaction with
fluorinated aromatic amines resulted in the formation of the title compounds. The
1
13
synthesized compound were unequivocally characterized by IR, H-NMR, C-NMR and
elemental analysis and some of the synthesized compounds displayed good antifungal
activities against Gibberella zeae, Fusarium oxysporum, Cytospora mandshurica and
anti-TMV activity in preliminary antifungal activity tests.
Keywords: pyrazole acyl thiourea derivatives; synthesis; antifungal activity; antiviral activity
1. Introduction
Many acyl thiourea derivatives are well known to possess a wide range of bioactivities and are
often employed as fungicidal, antiviral and regulating activities for plant protection in agriculture [1–3].

Molecules 2012, 17
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Some of them are employed as commercial insecticides [4], herbicides [5,6] and fungicides [7,8]. Several
novel N'-(substituted pyrimidin-2-yl)-N-chrysanthemoylthiourea derivatives were synthesized by Xu’s
group in 2005, and preliminary biological indicated that compounds I (Figure 1) exhibited 90% and
80% activity on Bipolaris maydis and Powdery mildew at 100 µg/mL, respectively [9]. Compounds II
and III (Figure 1), arylthioureas containing 1,3,4-thiadiazole moieties, were found to show good plant
growth regulator activity at 10 µg/mL [10,11]; Wang et al. [12] reported that acyl thiourea derivatives
IV exhibited excellent selective herbicidal activity towards dicotyledons. In addition, some novel
N-[5-(2-chlorophenyl)-2-furamidothiocarbonyl]-L-α-amino acid ethyl esters (V) (see Figure 1) were
found to show good activity against K562 cells in vitro at 10 µg/mL [13]. Moreover, compound VI
(Figure 1) that can be prepared by a multi-step reaction sequence using 2-chloro-5-(chloromethyl)
pyridine, displayed 96.4% and 85.3% activities against rape root and barnyard root at 10 µg/mL,
respectively [14].
Figure 1. The structure of acyl thiourea derivatives I–VI.
Pyrazole derivatives play an important role in the development of pesticides and medicines, which
have become hot topics in recent years. Dozens of pyrazole products are available and widely used as
fungicides, antiviral agents, analgesic agents, insecticides and herbicides [15–17]. In order to discover
novel active compounds for use in agriculture, we sought to combine the active structures of thiourea
and pyrazole to design and synthesize a class of novel pyrazole acyl thiourea derivatives. The synthetic
route is shown in Scheme 1. All the compounds were unequivocally characterized by IR, NMR and
elemental analysis. Preliminary bioassay tests indicated that most of the compounds exhibited
antifungal activity against G. zeae, F. oxysporum and C. mandshurica and antiviral activity against
TMV in vivo to a certain extent. Especially, compounds 6b, 6h exhibited slightly superior or similar
activity on the corresponding fungi as compared to the commercial hymexazol.

Molecules 2012, 17
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Scheme 1. Synthetic route to compounds 6a–p.
CHO
Cl
H3C
H3C
DMF
EtOH
N
N
R1NHNH2
CH3COCH2COOC2H5
+
O
N
N
R1
1
POCl3
R1
2
CONCS
Cl
COCl
H3C
H3C
COOH
Cl
H3C
NH4SCN
PEG-400
KMnO4
SOCl2
N
N
Cl
N
H2O
N
N
N
R1
3
R1
R1
5a:CH3; 5b:C6H5
4
O
C
S
6a:R1=CH3, R2=2-Br-5-FC6H3; 6b:R1=CH3,R2=2-Br-4,6-diFC6H2;
2 6c: R1=CH3, R2=4-Br-2-CF3C6H3;6d: R1=CH3, R2=2-F-C6H4CH2;
6e: R1=CH3, R2=2-FC6H4;6f: R1=CH3, R2=2-F-5-CF3C6H3;
6g: R1=CH3, R2=2-F-3-CF3C6H3;6h: R1=CH3, R2=2-F-C6H4CH2CH2;
6i: R1=CH3, R2=3-F-C6H4CH2CH2;6j: R1=CH3, R2=2-F-5ClC6H3;
6k: R1=CH3, R2=4-CF3SC6H4;6l: R1=CH3, R2=4-Br-2-CF3C6H3;
6m: R1=C6H5, R2=2-F-C6H4CH2;6n: R1=C6H5, R2=2-F-5-CF3C6H3;
6o: R1=C6H5, R2=2-F-3-CF3C6H3;6p: R1=C6H5, R2=4-CF3SC6H4
H3C
C
R
N
N
R2NH2
H
H
N
Cl
N
R1
6a-6p
2. Results and Discussion
2.1. Chemistry
The synthetic route to title compounds 6a–p is depicted in Scheme 1. In present study, we have
conducted our reaction using PEG-400 as solid-liquid phase transfer catalyst (PTC). PEG-400 as a
phase transfer catalyst is indispensable for these reactions; it can easily react with NH4SCN to form a
[PEG-400-NH4+] SCN−complex, which make it possible for SCN− to readily react with 5-chloro-1,3-
dimethyl-1H-pyrazole-4-carbonyl chloride and lead to the formation of intermediates 5a. In the
preparation of 5-chloro-1,3-dimethyl-1H-pyrazole-4-carbonyl isothiocyanate 5a, phase transfer
catalysis (PEG-400) and a conventional method were employed (see Table 1). From Table 1 we can
see that the reaction in the presence of phase transfer catalysis (PEG-400) has many advantages such
as low reaction temperatures and high yields. The use of PTC make the heterogeneous system react
smoothly, the yield of the product be improved obviously [18,19]. The reaction of 5 with suitably
substituted fluorinated primary aromatic amines afforded the desired acyl thioureas 6a–p in
good yields.
Table 1. Phase transfer catalysis compared with conventional method for 6a.
Method
Agent
NH4SCN/PEG-400
NH4SCN
Reaction Temperature
Reaction Time
Yield
82.6%
51.2%
phase transfer catalysis
conventional method
25 °C
82 °C
3 h
3 h
The structures of the compounds 6a–p were established on the basis of their spectroscopic data. The
IR (KBr) spectra of the target compounds 6a–p showed characteristic N–H, C=O, and C=S
absorptions at 3,200–3,430 cm−1, 1,660–1,690 cm−1 and 1,150 cm−1 respectively. The significantly
reduced double bond character of the carbonyl group of the amide linkage accounts for the appearance

Molecules 2012, 17
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of its stretching frequency at a lower wave number compared to a non-conjugated carbonyl. The
1H-NMR (CDCl3) spectra of these compounds exhibited the expected multiplet near δ 7.10–8.50 ppm
due to the presence of aromatic protons. The two different N-H protons appeared as two broad singlets
at δ 9.07–9.58 ppm and δ 10.66–12.81 ppm respectively, whereas the pyrazole methyl protons showed
up near δ 2.50 ppm as a singlet. The characteristic chemical shifts for F/CF3 were also detected in the
19F-NMR (CDCl3) spectra of 6a–p.
2.2. Antifungal Activity Bioassay
The in vitro antifungal screening data of the pyrazole acyl thiourea derivatives are provided in
Table 2. It was observed that these synthesized compounds showed weak to good antifungal activities
against the tested fungi at 100 μg/mL. Compounds 6b, 6h and 6l were shown to inhibit the growth of
G. zeae at 24.7%, 48.6%, and 22.9%, respectively; compounds 6b and 6h exhibited good activities on
F. oxysporum at 50.3% and 57.9%, respectively, while compounds 6b and 6h inhibited the growth of
C. mandshurica at 48.7% and 57.9%, respectively. These figures were slightly lower than those of
hymexazol. Amongst the new products compound 6h exhibited similar activities as hymexozole on the
corresponding fungi. Although, a definite structure activity relationship could not be established with
the limited experimental data and available compounds, it appears that with the incorporation of
a 2-FC6H4CH2CH2- unit through R2 into thiourea in the resulting products 6 might have a positive
influence, enhancing the antifungal activity of the designed compounds, From the activity differences
between 6h and 6i, we can conclude that different fluorine atom (F) positions on benzene can result in
different activity: when F was substituted at the 2-position of benzene (6h), it showed much higher
activity than that of 6i (where F was substituted at the 3-position of benzene), this may be caused by a
steric effect on the internal hydrogen bond.
Table 2. Inhibition effects of pyrazole acyl thiourea derivatives 6 on phytopathogenic fungi.
Compd. (100 µg/mL)
G. zeae
2.84
24.7
8.24
12.8
9.57
−0.57
17.6
48.6
19.9
4.55
15.4
22.9
−1.14
5.85
7.18
3.46
69.7
F. oxysporum
1.46
C. mandshurica
−5.79
48.7
6a
6b
50.3
6c
6.92
1.98
6d
−3.21
−1.15
4.39
8.16
6e
8.47
6f
3.42
6g
−1.46
57.9
−3.68
59.7
6h
6i
4.09
6.57
6j
−3.80
11.8
−6.31
7.91
6k
6l
0.86
4.80
6m
6.73
3.42
6n
6o
−0.29
5.19
5.08
11.9
6p
9.22
4.24
hymexazol
67.1
69.5

Molecules 2012, 17
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2.3. Antiviral Activity Bioassay
The antiviral activities of compounds 6a–p against TMV were assayed by the reported method [20].
The results of the in vivo bioassay against TMV are given in Table 3. Ningnanmycin was used as a
reference antiviral agent. The data provided in Table 3 indicate that the title compounds 6a–p showed
curative rates ranging from 19.76%–41.23%.
Table 3. The curative effect of the compounds 6a–p against TMV in vivo.
Agent
Concentration (μg/mL)
Curative effect (%)
41.23
6a
500
500
500
500
500
500
500
500
500
500
500
500
500
500
500
500
500
6b
29.74
6c
35.17
6d
25.47
6e
35.44
6f
37.95
6g
32.71
6h
28.07
6i
23.45
6j
19.76
6k
27.84
6o
31.82
6m
32.39
6n
30.09
6o
6p
22.07
27.08
Ningnanmycin
51.92
3. Experimental
3.1. General
Unless otherwise stated, all the reagents and reactants were purchased from commercial suppliers.
Melting points were uncorrected and were determined on a XT-4 binocular microscope (Beijing Tech
1
13
Instrument Co., Beijing, China). The H-NMR and C-NMR spectra were recorded on a JEOL ECX
1
500 NMR spectrometer (Tokyo, Japan) at room temperature operating at 500 MHz for H-NMR,
125 MHz for 13C-NMR, and 470 MHz for 19F-NMR, using CDCl3 as solvents and TMS as an internal
standard; infrared spectra were recorded in KBr on a Bruker VECTOR 22 spectrometer (Ettlingen,
Germany); elemental analysis was performed on an Elemental Vario-III CHN analyzer (Hanau,
Germany). The course of the reactions was monitored by TLC; analytical TLC was performed on silica
gel GF254; column chromatographic purification was carried out using silica gel. The intermediates 1–3
were prepared according to the literature procedure [18].

Molecules 2012, 17
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3.2. Preparation of the Intermediate Acyl Isothiocyanates 5
The intermediates 3 (30 mmol) and thionyl chloride (15 mL) were placed in a dried round-bottomed
flask containing a magnetic stirrer bar. The reaction mixture was stirred at 80 °C for 4 h until evolution
of gas completely ceased. The excess thionyl chloride was carefully removed by rotary distillation to
give a white solid to which were added ammonium thiocyanate (36 mmol) in acetonitrile (60 mL) and
six drops of PEG-400. The solution was stirred at room temperature for 3 h, filtered and the solvent
was removed on a rotary evaporator to afford a crude yellow solid which was further purified by
column chromatography using a mixture of petroleum ether and ethyl acetate (v/v = 2:1) as developing
solvent to give pure acyl isothiocyanate 5. The appearance, melting points and yields of the
intermediates 5a, 5b are provided in Table 4.
Table 4. Appearance, melting points and yields of intermediates 5a–b.
Intermediate
R1
Appearance
Yellow solid
White solid
m.p./°C
51~52 °C
69~71 °C
Yield/％
82.6%
5a
5b
CH3
C6H5
81.7%
3.3. Preparation of Title Compounds 6a–p
To a well stirred solution of acyl isothiocyanate 5 (1.00 mmol) in THF (15 mL), a suitable
fluorinated aromatic amine (1.20 mmol) was added. The mixture was stirred at room temperature for
4 h and monitored by TLC. After completion of the reaction, the solid was filtered; the solvent was
evaporated and the crude product was purified by preparative TLC with a mixture of petroleum ether
and ethyl acetate (v/v = 3/1–1/3) to give title compounds 6a–p.
1-(2-Bromo-5-fluorophenyl)-3-(5-chloro-1,3-dimethyl-1H-pyrazole-4-carbonyl)thiourea (6a). White
1
solid, yield 73.9%; m.p. 148–150 °C; H-NMR (CDCl3): δ 12.65 (1H, brs, CSNH), 9.28 (1H, brs,
CONHCS), 8.33~6.88 (3H, m, Ph-H), 3.85 (3H, s, CH3), 2.50 (3H, s, CH3); 13C-NMR (CDCl3): δ
178.6, 160.9, 152.3, 137.7, 133.5, 127,6, 115.1, 114.9, 113.8, 113.6, 112.1, 108.7, 36.7, 14.8;
19F-NMR (CDCl3): δ −116.64; IR: ν 3396.6, 3145.9 (N–H), 1678.1 (C=O), 1159.2 (C=S) cm−1; Anal.
Calc. for C13H11BrClFN4OS: C 36.85%, H 2.38%, N 13.22%. Found: C 37.00%, H 2.14%, N 13.38%.
1-(2-Bromo-4,6-difluorophenyl)-3-(5-chloro-1,3-dimethyl-1H-pyrazole-4-carbonyl)thiourea
(6b).
White solid, yield 74.1%; m.p. 84–86 °C; 1H-NMR (CDCl3): δ 11.74 (1H, brs, CSNH), 9.46 (1H, brs,
CONHCS), 7.25~6.96 (2H, m, Ph-H), 3.84 (3H, s, CH3), 2.45 (3H, s, CH3); 13C-NMR (CDCl3): δ
181.3, 161.3, 152.1, 127.7, 124.0, 123.9, 122.8, 116.1, 115.9, 108.6, 104.7, 104.5, 104.3, 36.7, 14.7;
19F-NMR (CDCl3): δ −107.6, −109.1; IR: ν 3338.1, 3095.8 (N–H), 1668.4 (C=O), 1157.3 (C=S) cm−1;
Anal. Calc. for C13H10BrClF2N4OS: C 39.30%, H 2.73%, N 13.81%. Found: C 39.30%, H 2.54%,
N 13.98%.
1-(4-Bromo-2-(trifluoromethyl)phenyl)-3-(5-chloro-1,3-dimethyl-1H-pyrazole-4-carbonyl)thiourea (6c).
1
White solid, yield 71.9%; m.p. 112–114 °C; H-NMR (CDCl3): δ 12.43 (1H, brs, CSNH), 9.36 (1H,
brs, CONHCS), 7.89~7.73 (3H, m, Ph-H), 3.86 (3H, s, CH3), 2.49 (3H, s, CH3); 13C-NMR (CDCl3): δ
180.7, 161.3, 152.4, 135.3, 134.7, 131.4, 129.4, 127.6, 127.0, 126.8, 123.5, 121.3, 120.8, 108.7, 36.7,

Molecules 2012, 17
5145
19
14.8; F-NMR (CDCl3): δ −61.36; IR: ν 3373.5, 2993.5 (N–H), 1670.4 (C=O), 1126.4 (C=S) cm−1;
Anal. Calc. for C14H11BrClF3N4OS: C 36.90%, H 2.43%, N 12.30%. Found: C 37.41%, H 2.10%,
N 12.28%.
1-(2-Fluorobenzyl)-3-(5-chloro-1,3-dimethyl-1H-pyrazole-4-carbonyl)thiourea (6d). White solid,
1
yield 82.2%; m.p. 121–122 °C; H-NMR (CDCl3): δ 10.92 (1H, brs, CSNH), 9.13 (1H, brs,
CONHCS), 7.47~7.06 (4H, m, Ph-H), 4.96 (2H, d, J = 5.75 Hz CH2), 3.83 (3H, s, CH3), 2.44 (3H, s,
CH3); 13C-NMR (CDCl3): δ 178.3, 161.6, 152.0, 130.5, 130.5, 129.9, 129.8, 127.6, 124.4, 124.4,
19
115.7, 115.5, 109.0, 43.5, 14.7; F-NMR (CDCl3): δ −117.88; IR: ν 3398.6, 3228.8 (N–H), 1668.4
(C=O), 1166.9 (C=S) cm−1; Anal. Calc. for C14H14ClFN4OS: C 49.34%, H 4.14%, N 16.44%. Found:
C 49.57%, H 3.93%, N 16.93%.
1-(2-Fluorophenyl)-3-(5-chloro-1,3-dimethyl-1H-pyrazole-4-carbonyl)thiourea (6e). White solid,
1
yield 74.5%; m.p. 152–154 °C; H-NMR (CDCl3): δ 12.56 (1H, brs, CSNH), 9.27 (1H, brs,
13
CONHCS), 8.38~7.15 (4H, m, Ph-H), 3.87 (3H, s, CH3), 2.51 (3H, s, CH3); C-NMR: (CDCl3, 125
MHz): δ 178.8, 161.3, 152.3, 127.8, 127.7, 127.6, 126.0, 125.6, 124.0, 115.7, 115.6, 108.9, 36.7, 14.8;
19F-NMR (CDCl3, 500 MHZ): δ −124.43; IR (KBr): ν 3383.1, 2993.5 (N–H), 1666.5 (C=O), 1141.9
(C=S); Anal. Calcd for C13H12ClFN4OS: C 47.78%, H 3.70%, N 17.15%; Found: C 47.45%, H 3.83%,
N 17.21%.
1-(2-Fluoro-5-(trifluoromethyl)phenyl)-3-(5-chloro-1,3-dimethyl-1H-pyrazole-4-carbonyl)thiourea (6f).
White solid, yield 73.2%; m.p. 123–125 °C;1H-NMR (CDCl3): δ 12.80 (1H, brs, CSNH), 9.30 (1H,
brs, CONHCS), 8.95~7.27 (3H, m, Ph-H), 3.85 (3H, s, CH3), 2.49 (3H, s, CH3); 13C-NMR: (CDCl3): δ
178.7, 161.4, 152.3, 127.7, 126.9, 126.8, 126.7, 126.4, 124.5, 124.3, 122.3, 116.1, 116.0, 108.6, 36.6,
19
14.7; F-NMR (CDCl3): δ −61.1, −126.5; IR (KBr): ν 3373.5, 3145.9 (N–H), 1670.3 (C=O), 1163.1
(C=S); Anal. Calcd for C14H11ClF4N4OS: C 42.59%, H 2.81%, N 14.19%; Found: C 42.76%,
H 2.58%, N 14.34%.
1-(2-Fluoro-3-(trifluoromethyl)phenyl)-3-(5-chloro-1,3-dimethyl-1H-pyrazole-4-carbonyl)thiourea (6g).
White solid, yield 74.9%; m.p. 139–141 °C;1H-NMR (CDCl3): δ 12.80 (1H, brs, CSNH), 9.31 (1H,
brs, CONHCS), 8.75~7.26 (3H, m, Ph-H), 3.87 (3H, s, CH3), 2.51 (3H, s, CH3); 13C-NMR: (CDCl3): δ
180.1, 161.1, 151.8, 140.8, 140.8, 130.2, 130.1, 127.4, 124.4, 115.7, 115.6, 113.7, 113.5, 109.0, 36.7,
19
14.6; F-NMR (CDCl3): δ −62.0, −119.5; IR (KBr): ν 3387.0, 3145.9 (N–H), 1680.0 (C=O), 1143.8
(C=S); Anal. Calcd for C13H11BrClFN4OS: C 42.59%, H 2.81%, N 14.19%; Found: C 42.70%,
H 2.61%, N 14.29%.
1-(2-Fluorophenethyl)-3-(5-chloro-1,3-dimethyl-1H-pyrazole-4-carbonyl)thiourea (6h). White solid,
1
yield 79.3%; m.p. 97–99 °C; H-NMR (CDCl3): δ 10.66 (1H, brs, CSNH), 9.07 (1H, brs, CONHCS),
7.28~7.00 (4H, m, Ph-H), 3.92~3.89 (2H, q, CH2), 3.79 (3H, s, CH3), 3.06 (2H, t, J = 7.45 Hz, CH2),
2.40 (3H, s, CH3); 13C-NMR: (CDCl3): δ 180.0, 161.0, 151.6, 131.1, 128.5, 127.4, 125.2, 125.1, 124.3,
115.4, 115.3, 108.9, 45.5, 36.6, 27.8, 14.6; 19F-NMR (CDCl3): δ −118.20; IR (KBr): ν 3385.1, 3244.3
(N–H), 1666.5 (C=O), 1159.2 (C=S); Anal. Calcd for C15H16ClFN4OS: C 50.77%, H 4.55%,
N 15.79%; Found: C 50.90%, H 4.16%, N 15.79%.

Molecules 2012, 17
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1-(3-Fluorophenethyl)-3-(5-chloro-1,3-dimethyl-1H-pyrazole-4-carbonyl)thiourea (6i). White solid,
yield 78.8%; m.p. 117–119 °C; 1H-NMR (CDCl3): δ 10.68 (1H, brs, CSNH), 9.08 (1H, brs, CONHCS),
7.30~6.91 (4H, m, Ph-H), 3.93~3.89 (2H, q, CH2), 3.82 (3H, s, CH3), 3.03 (2H, t, J = 8.00 Hz, CH2), 2.43
(3H, s, CH3); 13C-NMR: (CDCl3): δ 180.1, 161.1, 151.8, 140.8, 130.2, 127.4, 124.4, 115.7, 115.6,
19
113.7, 113.5, 109.0, 46.6, 36.6, 34.1, 14.6; F-NMR (CDCl3): δ -112.97; IR (KBr): ν 3377.3, 3228.8
(N–H), 1672.3 (C=O), 1168.9 (C=S); Anal. Calcd for C15H16ClFN4OS: C 50.77%, H 4.55%,
N 15.79%; Found: C 50.55%, H 4.21%, N 15.99%.
1-(5-Chloro-2-fluorophenyl)-3-(5-chloro-1,3-dimethyl-1H-pyrazole-4-carbonyl)thiourea (6j). White
1
solid, yield 72.1%; m.p. 158–160 °C; H-NMR (CDCl3): δ 12.56 (1H, brs, CSNH), 9.27 (1H, brs,
CONHCS), 8.38~7.15 (4H, m, Ph-H), 3.87 (3H, s, CH3), 2.51 (3H, s, CH3); 13C-NMR: (CDCl3): δ
179.0, 161.5, 152.3, 134.2, 127.7, 126.5, 124.2, 121.3, 121.1, 116.7, 116.6, 108.7, 36.7, 14.8;
19F-NMR (CDCl3): δ −116.72; IR (KBr): ν 3375.4, 2991.6 (N–H), 1670.4 (C=O), 1138.0 (C=S); Anal.
Calcd for C13H11Cl2FN4OS: C 43.23%, H 3.07%, N 15.51%; Found: C 43.61%, H 2.96%, N 15.61%.
1-(4-(Trifluoromethylthio)phenyl)-3-(5-chloro-1,3-dimethyl-1H-pyrazole-4-carbonyl)thiourea
(6k).
1
White solid, yield 74.4%; m.p. 145–147 °C; H-NMR (CDCl3): δ 12.75 (1H, brs, CSNH), 9.21 (1H,
13
brs, CONHCS), 7.92~7.52 (4H, m, Ph-H), 3.86 (3H, s, CH3), 2.49 (3H, s, CH3); C-NMR: (CDCl3):
19
δ 178.2, 161.5, 152.3, 140.2, 137.0, 133.3, 127.7, 124.2, 121.6, 108.8, 36.7, 14.8; FNMR (CDCl3):
δ −42.66; IR (KBr): ν 3410.2, 3032.1 (N–H), 1666.5 (C=O), 1139.9 (C=S); Anal. Calcd for
C14H12ClF3N4OS2: C 41.13, H 2.96, N 13.70; Found: C 41.22, H 2.76, N 13.73.
1-(4-Bromo-2-(trifluoromethyl)phenyl)-3-(5-chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbonyl) thiourea
1
(6l): White solid, yield 75.9%; m.p. 142–144 °C; H-NMR (CDCl3): δ 12.43 (1H, brs, CSNH), 9.48
(1H, brs, CONHCS), 7.89~7.52 (8H, m, Ph-H), 2.60 (3H, s, CH3); 13C-NMR (CDCl3): δ 180.7, 161.5,
153.5, 136.9, 135.4, 134.7, 131.4, 129.7, 129.5, 127.4, 127.2, 126.9, 125.6, 120.9, 110.2, 15.0;
19F-NMR (CDCl3): δ −61.39; IR (KBr): ν 3373.5, 3119.2 (N–H), 1670.4 (C=O), 1149.6 (C=S); Anal.
Calcd for C19H13BrClF3N4OS: C 44.08%, H 2.53%, N 10.82%; Found: C 44.13%, H 2.12%,
N 10.72%.
1-(2-Fluorobenzyl)-3-(5-chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbonyl)thiourea (6m). White
1
solid, yield 82.9%; m.p. 106–108 °C; H-NMR (CDCl3): δ 10.93 (1H, brs, CSNH), 9.27 (1H, brs,
13
CONHCS), 7.54~7.07 (9H, m, Ph-H), 4.97 (2H, d, J = 5.75Hz, CH2), 2.53 (3H, s, CH3); C-NMR
(CDCl3): δ 180.1, 161.2, 152.9, 136.9, 130.5, 130.5, 129.9, 129.6, 129.4, 125.5, 124.4, 124.4, 115.7,
115.6, 110.6, 43.5, 14.8; 19F-NMR (CDCl3): δ −117.80; IR (KBr): ν 3394.7, 3248.1 (N–H), 1666.5
(C=O), 1165.0 (C=S); Anal. Calcd for C19H16ClFN4OS: C 56.64%, H 4.00%, N 13.91%; Found:
C 57.07%, H 4.46%, N 14.22%.
1-(2-Fluoro-5-(trifluoromethyl)phenyl)-3-(5-chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbonyl) thiourea
1
(6n). White solid, yield 75.1%; m.p. 178–180 °C; H-NMR (CDCl3): δ 12.77 (1H, brs, CSNH), 9.35
(1H, brs, CONHCS), 7.92~7.52 (9H, m, Ph-H), 2.60 (3H, s, CH3); 13C-NMR (CDCl3): δ 178.7, 161.5,
153.4, 136.9, 129.7, 129.5, 127.4, 126.8, 126.7, 125.6, 124.5, 122.6, 116.2, 116.1, 110.2, 15.0;
19F-NMR (CDCl3): δ −61.98, −119.33; IR (KBr): ν 3215.3, 3076.5 (N–H), 1685.8 (C=O), 1157.3

Molecules 2012, 17
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(C=S); Anal. Calcd for C19H13ClF4N4OS: C 49.95%, H 2.87%, N 12.26%; Found: C 50.07%,
H 2.73%, N 12.20%.
1-(2-Fluoro-3-(trifluoromethyl)phenyl)-3-(5-chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbonyl) thiourea
1
(6o). White solid, yield 76.2%; m.p. 146–148 °C; H-NMR (CDCl3): δ 12.81 (1H, brs, CSNH), 9.43
(1H, brs, CONHCS), 7.55~7.26 (8H, m, Ph-H), 2.61 (3H, s, CH3); 13C-NMR (CDCl3): δ 178.9, 161.5,
153.5, 136.9, 129.7, 129.5, 127.5, 125.5, 123.2, 117.5, 117.3, 110.2, 14.9; 19F-NMR (CDCl3): δ
−61.09, −126.50; IR (KBr): ν 3221.7, 3086.2 (N–H), 1678.4 (C=O), 1153.2 (C=S); Anal. Calcd for
C19H13ClF4N4OS: C 49.95%, H 2.87%, N 12.26%; Found: C 49.91%, H 2.77%, N 12.31%.
1-(4-(Trifluoromethylthio)phenyl)-3-(5-chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbonyl)thiourea (6p).
1
White solid, yield 75.7%; m.p. 109–111 °C; H-NMR (CDCl3): δ 12.77 (1H, brs, CSNH), 9.35 (1H,
brs, CONHCS), 7.92~7.52 (9H, m, Ph-H), 2.60 (3H, s, CH3); 13C-NMR: (CDCl3): δ 178.1, 161.6,
19
153.2, 140.2, 137.0, 136.9, 129.7, 129.5, 127.4, 125.5, 124.1, 121.8, 110.2, 15.0; F-NMR (CDCl3):
δ −42.64; IR (KBr): ν 3388.9, 3032.1 (N–H), 1674.2 (C=O), 1151.5 (C=S); Anal. Calcd for
C19H14ClF3N4OS2: C 48.46%, H 3.00%, N 11.90%; Found: C 48.36%, H 2.97%, N 11.87%.
3.4. Antifungal Bioassays
The antifungal activity of all synthesized compounds was tested against F. oxysporum, G. zeae, and
C. mandshurica by the poison plate technique [21]. All the compounds were dissolved in DMSO (10 mL)
before mixing with Potato Dextrose Agar (PDA, 90 mL). The final concentration of the compounds in
the medium was fixed at 100 μg/mL. The three kinds of fungi were incubated in PDA at 25 ± 1 °C for
5 days to get new mycelium for the antifungal assays, and then a mycelia disk of approximately
0.45 cm diameter cut from the culture medium was picked up with a sterilized inoculation needle and
inoculated in the center of PDA plate. The inoculated plates were incubated at 25 ± 1 °C for 5 days.
DMSO in sterilized distilled water served as control, while hymexazole was used as positive control
for each treatment with three replicates being conducted for each experiment. The radial growth of the
fungal colonies was measured on the sixth day and the data were statistically analyzed. The in vitro
inhibiting effects of the test compounds on the fungi were calculated by the formula CV = (A − B)/A,
where A represents the diameter of fungi growth on untreated PDA, B represents the diameter of fungi
on treated PDA, and CV represents the rate of inhibition.
3.5. Antiviral Activity Bioassay
Purification of tobacco mosaic virus. Using Gooding’s method [22], the upper leaves of
Nicotiana tabacum L. Inoculated with TMV were selected and ground in phosphate buffer, then
filtered through double layer pledget. The filtrate was centrifuged at 10,000 g, treated twice with PEG
and centrifuged again. The whole experiment was carried out 4 °C. Absorbance values were estimated
at 260 nm using an ultraviolet spectrophotometer:
0.1%,260nm
virus concn = (A260 × dilution ratio)/E1cm

Molecules 2012, 17
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Curative effect of compounds against TMV in vivo. Growing leaves of Nicotiana tabacum. L. of the
same ages were selected. The tobacco mosaic virus (concentration of 6 × 10−3 mg/mL) was dipped and
inoculated on the whole leaves. Then the leaves were washed with water and dried. The compound
solution was smeared on the left side and the solvent was on the right side for control. The local lesion
numbers were then recorded 3–4 days after inoculation [23]. For each compound, three repetitions
were conducted to ensure the reliability of the results, which were measured according to the following
formula:
av local lesion numbers of control (not treated with compound)  av local lesion numbers smeared with drugs
Inhibition rate (%) =
av local lesion numbers without drugs
4. Conclusions
In the present study, a mild and effective method for the preparation of sixteen novel pyrazole acyl
thiourea derivatives employing monomethylhydrazine (phenylhydrazine) and ethyl acetoacetate as the
starting materials is described. The synthesized compounds were characterized by spectral data
(1H-NMR, 13C-NMR, 19F-NMR, IR) and elemental analysis. The compounds were subjected to in vitro
fungicidal activity assays against G. zeae, F. oxysporum and C. mandshurica. The results showed that
the synthesized pyridazine compounds possessed weak to good antifungal activities against the tested
fungi, with compounds 6b, 6h displaying good activity. Preliminary bioassays indicated that some of
these compounds are also associated with good inhibitory activities against TMV at a concentration of
500 mg/L. Further studies are currently underway to establish a definite structure activity relationship.
Acknowledgements
We are grateful to the National Natural Science Foundation of China (No. 2010CB126105) and the
National Key Technologies R&D Program of China (No. 2011BAE06B05-6) and the National Natural
Science Foundation of China (No. 20962005) for supporting the project.
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Sample Availability: Samples of the intermediates 1–5 and title compounds 6a–p are available from
the authors.
© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).