Synthesis and antiviral activities of pyrazole derivatives containing an oxime moiety

Article abstract of DOI:10.1021/jf802489e

Target compounds 4a-n were obtained by the reaction of 1 -substituted phenyl-3-methyl-5-substituted phenylthio-4-pyrazolaldoximes (3) with chloromethylated heterocyclic compounds (CICH₂-R₃) under reflux conditions in ethanol. Subsequ

Full text of DOI:10.1021/jf802489e

10160 J. Agric. Food Chem. 2008, 56, 10160–10167
Synthesis and Antiviral Activities of Pyrazole
Derivatives Containing an Oxime Moiety
GUIPING OUYANG, XUE-JIAN CAI, ZHUO CHEN, BAO-AN SONG,*
PINAKI S. BHADURY, SONG YANG, LIN-HONG JIN, WEI XUE, DE-YU HU, AND
SONG ZENG
Center for Research and Development of Fine Chemicals, Key Laboratory of Green Pesticide and
Agricultural Bioengineering, Ministry of Education, Guizhou University,
Guiyang 550025, People’s Republic of China
Target compounds 4a-n were obtained by the reaction of 1-substituted phenyl-3-methyl-5-substituted
phenylthio-4-pyrazolaldoximes (3) with chloromethylated heterocyclic compounds (ClCH2-R3) under
reflux conditions in ethanol. Subsequently, the oxidation of 4a-e with KMnO4 in HOAc at room
temperature afforded eight new compounds, 5a-h. The synthesized compounds were characterized
by physical constants, and the structures of the title compounds were confirmed by IR, 1H NMR, 13
C
NMR, and elemental analysis. The bioassay revealed that the compounds possessed antiviral
activities. It was found that title compounds 4a and 4g had the same inactivation effects against
TMV (EC50 ) 58.7 and 65.3 µg/mL) as the commercial product Ningnanmycin (EC50 ) 52.7 µg/mL).
To the best of our knowledge, this is the first report on the antiviral activity of pyrazole derivatives
containing an oxime moiety.
KEYWORDS: Pyrazole; oxime ethers moiety; antiviral activity; synthesis
INTRODUCTION
loss of tobacco, further research needs to be conducted in this
area for the development of a highly efficient, novel, environ-
mentally benign antiviral agent.
Pyrazole and its derivatives, a class of hererocyclic com-
pounds, occupy an important position in medicinal and pesticide
chemistry with a wide range of bioactivities. As medicines,
many of them display antibacterial (1), antimicrobial (2), anti-
cancer (3), antiinflammatory (4), and selective enzyme inhibitory
activities (5). As pesticides, they are used as insecticides (6),
fungicides (7, 8), and herbicides (9-12). As shown in Figure
1, for example, Pyrazophos, the first fungicide of this class to
be commercialized, was put on the market by Hoechst AG in
1974 to control powdery mildew in vegetables, and more than
10 pyrazole derivatives are now commercially available. The
advantages, such as a novel mode of action, wide spectrum,
low toxicity toward mammalian cells, and favorable profiles to
humans, have prompted chemists to design and synthesize novel
pyrazole derivatives. Recently, pyrazole compounds, such as
Penthiopyrad (Mitsui Toatsu Chemicals, 1995) and Pyraclos-
trobin (BASF, 2001), have been found to have potential
antifungal activities for the control of some plant diseases. With
growing applications on their synthesis and bioactivity, chemists
and biologists in recent years have directed considerable
attention to the research of pyrazole derivatives (13).
Modification of the structural profile by the change of
substituents at the 1-, 3-, or 4-position in pyrazole ring can bring
about significant change in bioactivity. However, pyrazolal-
doxime ethers bearing other heterocyclic moieties are scarcely
evaluated for their activities (14, 15). It has been demonstrated
that the pyridine and pyrazole groups are important pharma-
cophores of many fungicides. Linking the pyridine and pyrazole
group with a structurally diverse side chain is an effective way
to obtain new heterocyclic derivatives with high fungicidal
activities (13, 16). We assumed that if the pyridine and pyrazole
pharmacophores were introduced into the pyrazole scaffold, the
resulting compounds should have an interesting lead structure
for antiviral agent development (Figure 2), which will be
Tobacco mosaic virus (TMV) infection is very widely
distributed and can cause serious damage and large economic
loss. It has been found that in certain fields, 90-100% of the
plants show mosaic by harvest time. In view of the unsatisfac-
tory curatives (30-60%) cure rate obtained by common antiviral
agents (Ningnanmycin or Virus A) and widespread economic
Figure 1. The structures of commercial fungicides.
10.1021/jf802489e CCC: $40.75  2008 American Chemical Society
Published on Web 10/22/2008

Pyrazole Derivatives Containing an Oxime Moiety
J. Agric. Food Chem., Vol. 56, No. 21, 2008 10161
Figure 2. Design of the target compounds.
described (18, 19). Intermediates 1-aryl-3-methyl-5-chloro-1H-4-
pyrazolylformaldehydes 1-3 were prepared according to the reported
methods (20, 21).
expected to exhibit interesting features due to the coexistence
of two kinds of fungicidal pharmacophores with different action
mechanisms. In continuation of our research program of the
discovery of novel lead compounds (17), we describe herein
the synthesis and antiviral activity of a series of new pyrazola-
ldoxime ether analogues (3). Meanwhile, as a control, we also
synthesized and measured their antiviral activities in a series
of pyrazolaldoxime ether analogues (4). The synthetic route is
shown in Scheme 1. Starting from the key intermediate,
1-substitutedphenyl-3-methyl-5-substituted phenylthio-4-pyra-
zolaldoximes 3, the title compounds 4 and 5 are synthesized
by etherification with chloromethylated heterocyclic compound
followed by potassium permangante oxidation in acetic acid.
The structures of 4 and 5 were firmly established by well-defined
General Procedure for the Preparation of Title Compounds
4a-n. A 100 mL round-bottomed flask equipped with a magnetic
stirrer was charged with 3 (10 mmol), dimethylformamide (DMF)
(50 mL), and NaOH (12 mmol). The flask was stirred at room
temperature for 10 min, and then, R3CH2Cl (0.012mol) was added
dropwise over a period of 10 min and stirred at room temperature
for 4 h. The mixture was poured into ice water and filtered. The
white solid that resulted was washed with distilled water, dried under
vacuum, and recrystallized from ethanol to give the title compounds
4a-n in 54-87% yields.
Data for 1-Phenyl-3-methyl-5-(4-methylphenylthio)-4-pyrazolaldoxime-
(2-chloropyridine-5-ylmethyl) Ether (4a). White crystal; mp 95-96
°C; yield, 81%. IR (KBr, cm-1): ν 3063 (Ar-H), 2923, 2867 (CH3
+ CH2), 1593 (CdN), 1495, 1458 (Ar skeleton vibration), 1216
(CdN-N), 845 (p-disubstituded benzene), 655 (C-S-C) cm-1. 1H
NMR (DMSO-d6, 500 MHz, ppm): δ 8.42 (d, J ) 2.30 Hz, 1H,
Py-6-H), 8.25 (s, 1H, CH-H), 7.70 (dd, J ) 2.30 Hz, J ) 2.30 Hz,
1H, Py-4-H), 7.38-7.40 (m, 6H, 1-Ph-H, Py-3-H), 6.99 (d, J )
8.55 Hz, 2H, S-Ph-2, 6-H), 6.83 (d, J ) 8.60 Hz, 2H, S-Ph-3, 5-H),
5.12 (s, 2H, CH2-H), 2.52 (s, 3H, pyrazole-CH3-H), 2.26 (s, 3H,
Ph-CH3-H). 13C NMR (DMSO-d6, 125 MHz, ppm): δ 151.1, 149.9,
148.9, 144.0, 139.2, 138.9, 1136.9, 133.0, 132.4, 131.2, 130.2, 128.8,
128.4, 127.8, 125.6, 124.1, 118.7, 77.1, 21.0, 14.9. Anal. calcd for
C24H21ClN4OS (448.5): C, 64.24%; H, 4.66%; N, 12.46%. Found:
C, 64.32%; H, 4.38%; N, 12.60%.
1
IR, H NMR, 13C NMR, and elemental analysis. Preliminary
bioassay tests showed that some compounds possess a certain
degree of antiviral activity against TMV at 500 mg/L in vivo
as shown in Tables 1 and 2, however, with a degree of variation.
The bioassay results showed that title compounds 4a,g possess
high inactivation activities against TMV, and the EC50 values
range from 58.7 to 65.3 µg/mL.
MATERIAL AND METHODS
Instruments. The melting points of the products were determined
on a XT-4 binocular microscope (Beijing Tech Instrument Co., China)
and were not corrected. The IR spectra were recorded on a Bruker
VECTOR 22 spectrometer in KBr disk. 1H, 13C, and 31P NMR (solvent
DMSO-d6) spectra were performed on a JEOL-ECX 500 NMR
spectrometer at room temperature using tetramethylsilane as an internal
standard. Elemental analysis was performed on an Elementar Vario-
III CHN analyzer. Analytical thin-layer chromatography was performed
on silica gel GF254. Column chromatographic purification was carried
out using silica gel. All reagents were of analytical reagent grade or
chemically pure. All solvents were dried, deoxygenated, and redistilled
before use.
1-Phenyl-3-methyl-5-(4-methoxyphenylthio)-4-pyrazolaldoxime-(2-
chloropyridine-5-ylmethyl) Ether (4b). White crystal; mp 95-96 °C;
yield, 81%. IR (KBr, cm-1): ν 3094 (Ar-H), 2963, 2829 (CH3 +
CH2), 1593 (CdN), 1591, 1386 (Ar skeleton vibration), 1246
(CdN-N), 827 (p-disubstituded benzene), 665 (C-S-C). 1H NMR
(DMSO-d6, 500 MHz, ppm): δ 8.46 (d, J ) 2.30 Hz, 1H, Py-6-H),
8.23 (s, 1H, CH-H), 7.88 (dd, J ) 2.30 Hz, J ) 2.30 Hz, 1H, Py-
4-H), 7.44-7.53 (m, 6H, 1-Ph-H, Py-3-H), 6.91 (d, J ) 9.20 Hz,
2H, S-Ph-3,5-H), 6.81 (d, J ) 9.15 Hz, 2H, S-Ph-2, 6-H), 5.17 (s,
2H, CH2-H), 3.68 (s, 3H, O-CH3-H), 2.37 (s, 3H, CH3-H). 13C
NMR (DMSO-d6, 125 MHz, ppm): δ 169.3, 160.9, 150.4, 150.3,
143.6, 140.5, 138.9, 134.1, 133.3, 130.9, 129.4, 128.9, 126.1, 124.5,
117.7, 116.6, 115.7, 72.4, 55.7, 14.8. Anal. calcd for C24H21ClN4O2S
Synthetic Procedures. 1-Aryl-3-methyl-1H-4-pyrazoleylformal-
dehydes and 1-(4-chlorophenyl)-3-methyl-1H-4-pyrazoleylformal-
dehydes were prepared according to the literature method as

10162 J. Agric. Food Chem., Vol. 56, No. 21, 2008
Ouyang et al.
Scheme 1. Synthetic Route to Pyrazole Analogues 4 and 5 Containing Oxime
(464.5): C, 62.00%; H, 4.52%; N, 12.05%. Found: C, 61.89%; H,
4.20%; N, 11.90%.
1-(3-Chlorophenyl-3-methyl-5-(4-fluorophenylthio)-4-pyrazolaldoxime-
(2-chloro-pyridine-5-ylmethyl) Ether (4d). White crystal; mp 95-96
°C; yield, 81%. IR (KBr, cm-1): ν 3041 (Ar-H), 2855 (CH3
+
1-Phenyl-3-methyl-5-(4-fluorophenylthio)-4-pyrazolaldoxime-(2-chlo-
ropyridine-5-ylmethyl) Ether (4c). White crystal; mp 95-96 °C; yield,
81%. IR (KBr, cm-1): ν 3055 (Ar-H), 2925 (CH3), 2974 (CH3),
1591 (CdN), 1492 (Ar, skeleton vibration), 1225 (CdN-N), 821(p-
disubstituded benzene), 652 (C-S-C). 1H NMR (DMSO-d6, 500
MHz, ppm): δ 8.44 (d, J ) 2.30 Hz, 1H, Py-6-H), 8.26 (s, 1H,
CH-H), 7.70 (dd, J ) 2.30 Hz, J ) 2.30 Hz, 1H, Py-4-H), 7.27-7.39
(m, 6H, 1-Ph-H, Py-3-H), 6.90-6.84 (m, 4H, S-Ph-2, 3, 5, 6-H),
5.14 (s, 2H, CH2-H), 2.48 (s, 3H, CH3-H). 13C NMR (DMSO-d6,
125 MHz, ppm): δ 162.9, 160.9, 150.9, 149.9, 149.1, 143.6, 139.2,
132.3, 130.2, 130.1, 128.8, 128.5,125.6, 124.1, 118.5, 116.6, 116.4,
72.7,14.8. Anal. calcd for C23H18ClFN4OS (452.5): C, 60.95%; H,
3.96%; N, 12.38%. Found: C, 60.85%; H, 3.79%; N, 12.10%.
CH2), 16411 (CdN), 1589, 1493 (Ar, skeleton vibration), 1226
(CdN-N), 831(p-disubstituded benzene), 676 (C-S-C). 1H NMR
(DMSO-d6, 500 MHz, ppm): δ 8.43 (d, J ) 2.25 Hz, 1H, Py-6-H),
8.24 (s, 1H, CH-H), 7.69 (dd, J ) 2.85 Hz, J ) 2.30 Hz, 1H, Py-
4-H), 7.39 (s, 1H, 1-Ph-2-H), 7.26-7.36 (m, 5H, 1-Ph-H, Py-3-H),
6.88-6.70 (m, 4H, S-Ph-H), 5.12 (s, 2H, CH2-H), 2.49 (s, 3H,
pyrazole-CH3-H). 13C NMR (DMSO-d6, 125 MHz, ppm): δ 162.9,
161.0, 151.1, 149.9, 149.3, 143.4, 137.2, 134.2, 132.8, 132.2, 130.1,
129.1, 129.0, 124.1, 119.1, 118.5,116.7,116.5,72.8,14.8. Anal. calcd
for C23H17Cl2FN4OS(487.0): C, 56.65%; H, 3.45%; N, 11.46%.
Found: C, 56.47%; H, 3.60%; N, 11.28%.

Pyrazole Derivatives Containing an Oxime Moiety
J. Agric. Food Chem., Vol. 56, No. 21, 2008 10163
Table 1. Protection Effect, Inactivation Effect, and Curative Effect of the
New Compounds against TMV in Vivoa
°C; yield, 87%. IR (KBr, cm-1): ν 3041 (Ar-H), 2921, 2855 (CH3 +
CH2), 1611 (CdN), 1589, 1493 (Ar, skeleton vibration), 1226
(CdN-N), 831 (p-disubstituded benzene), 676 (C-S-C). H NMR
(DMSO-d6, 500 MHz, ppm): δ 8.43 (d, J ) 2.25 Hz, 1H, Py-6-H),
8.24 (s, 1H, CH-H), 7.69 (dd, J ) 2.85 Hz, J ) 2.30 Hz, 1H, Py-4-H),
7.26-7.36 (m, 5H, 1-Ph-H, Py-3-H), 6.88 (m, 4H, S-Ph-H), 5.12 (s,
2H, CH2-H), 2.49 (s, 3H, pyrazole-CH3-H). 13C NMR (DMSO-d6, 125
MHz, ppm): δ 162.9, 161.0, 151.1, 149.9, 149.3, 143.4, 137.2, 134.2,
132.8, 132.2, 130.1, 129.1, 129.0 124.1, 119.1, 116.7, 116.5, 72.8,14.8.
Anal. calcd for C23H17Cl2FN4OS (487.0): C, 56.67%; H, 3.49%; N,
11.50%. Found: C, 56.50%; H, 3.60%; N, 11.30%.
1
concentration
protection
effect (%)
inactivation
effect (%)
curative
agents
(µg/mL)
effect (%)
4a
4b
4c
4d
4e
4f
4g
4h
4i
500
500
500
500
500
500
500
500
500
500
500
500
500
500
500
500
500
500
500
500
500
500
500
31.3*
16.4
90.4**
71.2**
65.9*
61.7*
61.2*
34.5*
80.6**
56.6*
61.0*
43.0*
55.9*
51.2*
50.9**
36.7*
45.0*
30.1**
20.6
47.0*
26.6*
24.0*
34.3*
26.0*
12.9
37.3*
22.0
32.1*
31.9
35.6*
38.9*
22.0*
18.9
9.0a
15.6*
21.7
11.0
9.2
18.9*
28.6*
37.7*
22.0*
43.5*
31.0*
15.7
1-(4-Methylphenyl-3-methyl-5-(4-fluorophenylthio)-4-pyrazolaldoxime-
(2-chloropyridin-5-ylmethyl) Ether (4h). White crystal; mp 129-130
°C; yield, 78%. IR (KBr, cm-1): ν 3023 (Ar-H), 2935, 2867 (CH3 +
CH2), 1611 (CdN), 1598, 1497 (Ar, skeleton vibration), 1228
4j
4k
4l
19.0
32.0*
44.8*
41.0*
21.0
28.0*
29.8*
40.2*
12.9
31.3
16.4
42.2*
28.2
1
(CdN-N), 846 (p-disubstituded benzene), 658 (C-S-C). H NMR
4m
4n
5a
5b
5c
5d
5e
5f
(DMSO-d6, 500 MHz, ppm): δ 8.43 (d, J ) 2.25 Hz, 1H, Py-6-H),
8.16 (s, 1H, CH-H), 7.87 (dd, J ) 2.30 Hz, J ) 2.90 Hz, 1H, Py-4-H),
7.52 (d, J ) 8.00 Hz, 1H, Py-3-H), 7.30 (d, J ) 8.00 Hz, 2H, 1-Ph-2,
6-H), 7.25 (d, J ) 8.60 Hz, 2H, 1-Ph-3, 5-H), 6.98 (t, J ) 1.70 Hz, J
) 2.90 Hz, 2H, S-Ph-2, 6-H), 6.95 (dd, J ) 2.30 Hz, J ) 2.90 Hz, 2H,
S-Ph-3,5-H), 5.14 (s, 2H, CH2-H), 2.49 (s, 3H, pyrazole-CH3-H), 2.37
(s, 3H, Ph-CH3-H). 13C NMR (DMSO-d6, 125 MHz, ppm): δ 162.4,
161.0, 151.1, 149.9, 149.3, 143.4, 137.2, 134.2, 132.8, 132.2, 130.1,
129.1, 129.0, 124.5, 119.1, 117.9, 117.2, 72.1, 21.1, 14.7. Anal. calcd
for C24H20ClFN4OS (466.5): C, 61.74%; H, 4.27%; N, 12.00%. Found:
C, 61.55%; H, 4.21%; N, 11.85%.
30.0*
33.3
30.9
44.1*
11.0
98.0**
3.6
7.7*
22.0
54.8*
5g
5h
Ningnamycin
57.0*
a n ) 3 for all groups; *P < 0.05, and **P < 0.01.
1-(4-Methylphenyl-3-methyl-5-(4-methylphenylthio)-4-pyrazolaldoxime-
(2-chloropyridine-4-ylmethyl) Ether (4i). White crystal; mp 106-108
°C; yield, 81%. IR (KBr, cm-1): ν 3031 (Ar-H), 2937, 2868 (CH3 +
CH2), 1623 (CdN), 1587, 1487 (Ar, skeleton vibration), 1221
Table 2. Antiviral Activities in Vivo (%) of Compounds 4a,b,g
TMV
inactivation effect
1
(CdN-N), 843 (p-disubstituded benzene), 656 (C-S-C). H NMR
concentration
(µg mL-1
EC50
(µg mL-1
)
(DMSO-d6, 500 MHz, ppm): δ 8.42 (d, J ) 2.20 Hz, 1H, Py-6-H),
8.12 (s, 1H, CH-H), 7.86 (dd, J ) 2.30 Hz, J ) 2.85 Hz, 1H, Py-4-H),
7.51 (d, J ) 8.00 Hz, 1H, Py-3-H), 7.31 (d, J ) 8.60 Hz, 2H, 1-Ph-2,
6-H), 7.25 (d, J ) 8.60 Hz, 2H, 1-Ph-3, 5-H), 7.06 (d, J ) 8.05 Hz,
2H, S-Ph- 2, 6-H), 6.82 (d, J ) 8.60 Hz, 2H, S-Ph-3, 5-H), 5.14 (s,
2H, CH2-H), 2.37 (s, 3H, pyrazole-CH3-H), 2.33, 2.21 (2s, 6H, Ph-
CH3-H). 13C NMR (DMSO-d6, 125 MHz, ppm): δ 150.4, 148.2, 143.5,
140.5, 138.6, 136.5, 133.2, 132.6, 131.4, 130.7, 129.8, 127.5, 125.8,
124.5, 117.2, 72.3,21.1, 20.9, 14.8. Anal. calcd for C25H23ClN4OS
(462.5): C, 64.86%; H, 4.97%; N, 12.11%. Found: C, 64.70%; H,
4.85%; N, 11.99%.
)
500
250
125
62.5
31.8
4a
4b
4g
90.4
71.0
80.6
98.0
76.0
65.0
65.7
81.1
69.2
48.9
53.0
72.0
55.1
41.0
51.2
59.9
40.1
32.0
40.6
44.0
58.7
115.0
65.3
Ningnamycin
52.7
1-(4-Chlorophenyl-3-methyl-5-(4-methylphenylthio)-4-pyrazolaldoxime-
(2-chloropyridine-5-ylmethyl) Ether (4e). White crystal; mp 103-105
°C; yield, 87%. IR (KBr, cm-1): ν 3007 (Ar-H), 2978, 2887 (CH3 +
CH2), 1615 (CdN), 1579, 1489 (Ar, skeleton vibration), 1216
1-(4-Methylphenyl-3-methyl-5-(4-fluorophenylthio)-4-pyrazolaldoxime-
(5-chloro-3-methyl-1-(4-chlorophenyl)pyrazol-4-ylmethyl Ether (4j).
White crystal; mp 108-109 °C; yield, 58%. IR (KBr, cm-1): ν 3045
(Ar-H), 2924, 886 (CH3 + CH2), 1623 (CdN), 1599, 1499 (Ar,
skeleton vibration), 1221 (CdN-N), 874 (p-disubstituded benzene),
1
(CdN-N), 845 (p-disubstituded benzene), 653 (C-S-C). H NMR
(DMSO-d6, 500 MHz, ppm): δ 8.42 (d, J ) 2.30 Hz, 1H, Py-6-H),
8.24 (s, 1H, CH-H), 7.69 (dd, J ) 2.30 Hz, J ) 2.30 Hz, 1H, Py-4-H),
7.26-7.34 (m, 5H, 1-Ph-H, Py-3-H), 6.99 (d, J ) 8.55 Hz, 2H, S-Ph-
2, 6-H), 6.81 (d, J ) 8.60 Hz, 2H, S-Ph-3, 5-H), 5.12 (s, 2H, CH2-H),
2.47 (s, 3H, pyrazole-CH3-H), 2.27 (s, 3H, Ph-CH3-H). 13C NMR
(DMSO-d6, 125 MHz, ppm): δ 149.8, 149.2, 143.7, 139.1, 137.3, 137.0,
132.2, 130.8, 130.1, 128.9, 127.6, 126.6, 124.0, 127.6, 124.1, 119.8,
117.7, 116.7, 77.2, 72.6, 20.9, 14.8. Anal. calcd for C24H20Cl2N4OS
(483.0): C, 59.63%; H, 4.14%; N, 11.55%. Found: C, 59.45%; H,
4.02%; N, 11.48%.
1
660 (C-S-C). H NMR (DMSO-d6, 500 MHz, ppm): δ 8.24 (s, 1H,
CH-H), 7.50 (d, J ) 8.60 Hz, 2H, pyrazole-Ph-2, 6-H), 7.43 (d, J )
9.15 Hz, 2H, pyrazole-Ph-3, 5-H), 7.23 (d, J ) 8.00 Hz, 2H, 1-Ph-2,
6-H), 7.16 (d, J ) 8.60 Hz, 2H,1-Ph-3, 5-H), 6.92-6.85 (m, 4H, S-Ph-
H), 5.05 (s, 2H, CH2-H), 2.54 (s, 3H, pyrazole-CH3-H), 2.39 (s, 3H,
Ph-CH3-H), 2.36 (s, 3H, Cl-pyrazole-CH3-H). Anal. calcd for
C29H24Cl2FN5OS (580.0): C, 60.00%; H, 4.14%; N, 12.07%. Found:
C, 60.38%; H, 4.30%; N, 12.15%.
1-(4-Chlorophenyl-3-methyl-5-(4-methoxyphenylthio)-4-pyrazolal-
doxime-(2-chloropyridine-5-ylmethyl) Ether (4f). White crystal; mp
103-105 °C; yield, 87%. IR (KBr, cm-1): ν 3041 (Ar-H), 2921, 2855
(CH3 + CH2), 1611 (CdN), 1589, 1493 (Ar, skeleton vibration), 1226
1-Phenyl-3-methyl-5-(4-fluorophenylthio)-4-pyrazolaldoxime-(5-chloro-
3-methyl-1-(4-chlorophenyl)-pyrazol-4-ylmethyl Ether (4k). White crys-
tal; mp 105-106 °C; yield, 56%. IR (KBr, cm-1): ν 3045 (Ar-H),
2924, 886 (CH3 + CH2), 1599, 1499 (Ar, skeleton vibration), 874 (p-
disubstituded benzene), 660 (C-S-C). 1H NMR (DMSO-d6, 500 MHz,
ppm): δ 8.25 (s, 1H, CH-H), 7.50 (dd, J ) 2.30 Hz, J ) 1.75 Hz, 2H,
pyrazole-Ph-2, 6-H), 7.43 (dd, J ) 1.80 Hz, J ) 1.75 Hz, 2H, pyrazole-
Ph-3, 5-H), 7.33-7.48 (m, 5H, 1-Ph-H), 6.97 (d, J ) 8.00 Hz, 2H,
S-Ph-2, 6-H), 6.83 (d, J ) 8.60 Hz, 2H, S-Ph-3,5-H), 5.05 (s, 2H, CH2-
H), 2.55 (s, 3H, pyrazole-CH3-H), 2.38 (s, 3H, Ph-CH3-H), 2.24 (s,
3H, Cl-pyrazole-CH3-H). Anal. calcd for C28H22Cl2FN5OS (566.0): C,
59.36%; H, 3.89%; N, 12.37%. Found: C, 59.26%; H, 3.76%; N,
12.42%.
1
(CdN-N), 831 (p-disubstituded benzene), 676 (C-S-C). H NMR
(DMSO-d6, 500 MHz, ppm): δ 8.43 (d, J ) 2.25 Hz, 1H, Py-6-H),
8.24 (s, 1H, CH-H), 7.69 (dd, J ) 2.85 Hz, J ) 2.30 Hz, 1H, Py-4-H),
7.39 (s, 1H, 1-Ph-2-H), 7.26-7.36 (m, 4H, 1-Ph-3, 5, 6-H, Py-3-H),
6.88-6.70 (m, 4H, S-Ph-H), 5.12 (s, 2H, CH2-H), 3.68(s, 3H, O-CH3-
H), 2.49 (s, 3H, pyrazole-CH3-H). 13C NMR (DMSO-d6, 125 MHz,
ppm): δ 162.9, 161.0, 151.1, 149.9, 149.3, 143.4, 137.2, 134.2, 132.8,
132.2, 130.1, 129.1, 129.0 124.1, 119.1, 118.5, 116.7, 116.5, 72.8, 5.7,
14.8. Anal. calcd for C24H20Cl2N4O2S (499.0): C, 57.75%; H, 4.00%;
N, 11.22%. Found: C, 57.52%; H, 4.16%; N, 11.08%.
1-(4-Chlorophenyl-3-methyl-5-(4-fluorophenylthio)-4-pyrazolaldoxime-
(2-chloropyridine-5-ylmethyl) Ether (4g). White crystal; mp 103-105
1-(4-Methylphenyl-3-methyl-5-(4-methylphenylthio)-4-pyrazolaldoxime-
(5-chloro-3-methyl-1-(4-methylphenyl)pyrazol-4-ylmethyl Ether (4l).

10164 J. Agric. Food Chem., Vol. 56, No. 21, 2008
Ouyang et al.
White crystal; mp 115-117 °C; yield, 55%. IR (KBr, cm-1): ν 3035
(Ar-H), 2964, 730 (CH3 + CH2), 1609, 1488 (Ar, skeleton vibra-
tion),1213 (CdN-N), 834 (p-disubstituded benzene), 673 (C-S-C).
1H NMR (DMSO-d6, 500 MHz, ppm): δ 8.23 (s, 1H, CH-H), 7.40 (d,
J ) 8.60 Hz, 2H, pyrazole-Ph-2, 6-H), 7.25 (d, J ) 6.30 Hz, 4H,
pyrazole-Ph-3, 5-H, 1-Ph-2, 6-H), 7.15 (d, J ) 8.00 Hz, 2H, S-Ph-2,
6-H), 6.98 (d, J ) 8.00 Hz, 1-Ph-3, 5-H), 6.84 (d, J ) 8.55 Hz, 2H,
S-Ph-3, 5-H), 5.04 (s, 2H, CH2-H), 2.55 (s, 3H, pyrazole-CH3-H), 2.35,
2.37, 2.39 (3s, 9H, Ph-CH3-H), 2.24 (s, 3H, Cl-pyrazole-CH3-H). Anal.
calcd for C31H30ClN5OS (555.5): C, 66.96%; H, 5.40%; N, 12.60%.
Found: C, 67.21%; H, 5.68%; N, 12.86%.
1-(4-Methylphenyl-3-methyl-5-(4-fluorophenylthio)-4-pyrazolaldoxime-
(5-chloro-3-methyl-1-(4-methylphenyl)pyrazol-4-ylmethyl Ether (4m).
White crystal; mp 118-119 °C; yield, 54%. IR (KBr, cm-1): ν 3035
(Ar-H), 2925, 783 (CH3 + CH2), 1590, 1488 (Ar, skeleton vibra-
tion),1230 (CdN-N), 841 (p-disubstituded benzene), 670 (C-S-C).
1H NMR (DMSO-d6, 500 MHz, ppm): δ 8.24 (s, 1H, CH-H), 7.39 (d,
J ) 8.60 Hz, 2H, pyrazole-Ph-2, 6-H), 7.25-7.20 (m, 4H, pyrazole-
Ph-3, 5-H, 1-Ph-2, 6-H), 7.16 (d, J ) 8.05 Hz, 2H, S-Ph-2, 6-H),
6.92-6.85 (m, 4H, 1-Ph-3, 5-H, S-Ph-3, 5-H), 5.06 (s, 2H, CH2-H),
2.54 (s, 3H, pyrazole-CH3-H), 2.39, 2.40 (2s, 6H, Ph-CH3-H), 2.36 (s,
3H, Cl-pyrazole-CH3-H). Anal. calcd for C30H27ClFN5OS (559.5): C,
64.34%; H, 4.83%; N, 12.51%. Found: C, 64.54%; H, 5.00%; N,
12.63%.
1-(4-Chlorophenyl-3-methyl-5-(4-methylphenylthio)-4-pyrazolaldoxime-
(5-chloro-3-methyl-1-(4-methylphenyl)pyrazol-4-ylmethyl Ether (4n).
White crystal; mp 109-110 °C; yield, 66%. IR (KBr, cm-1): ν 3017
(Ar-H), 2923, 865 (CH3 + CH2), 1593, 1495 (Ar, skeleton vibration),
1235 (CdN-N), 834 (p-disubstituded benzene), 672 (C-S-C). 1H
NMR (DMSO-d6, 500 MHz, ppm): δ 8.24 (s, 1H, CH), 7.39 (d, J )
8.60 Hz, 2H, pyrazole-Ph-2, 6-H), 7.25-7.19 (m, 4H, pyrazole-Ph-
3,5-H, 1-Ph-2, 6-H), 7.16 (d, J ) 8.05 Hz, 2H, S-Ph-2, 6-H), 6.85-6.92
(m, 4H, 1-Ph-3, 5-H, S-Ph-3, 5-H), 5.06 (s, 2H, CH2-H), 2.54 (s, 3H,
pyrazole-CH3-H), 2.39, 2.40 (2s, 6H, Ph-CH3-H), 2.36 (s, 3H, Cl-
pyrazole-CH3-H). Anal. calcd for C30H27Cl2N5OS (576.0): C, 62.50%;
H, 4.69%; N, 12.15%. Found: C, 62.75%; H, 4.78%; N, 12.32%.
General Procedure for the Preparation of Title Compounds
5a-h. A 50 mL round-bottomed flask equipped with a magnetic stirrer
was charged with 4 (5 mmol) dissolved in ice HOAc (30 mL). The
flask was stirred at room temperature for 10 min, and then, potassium
permanganate (1.0 g) was added and stirred at room temperature for
2 h. Sodium bisulfite was then added to turn the mixture colorless again
and filtered. The white solid that resulted was washed with distilled
water, dried under vacuum, and recrystallized from ethanol to give the
title compounds 5a-h in 50-58% yields.
C24H21ClN4O4S (496.5): C, 58.00%; H, 4.22%; N, 11.28%. Found: C,
57.79%; H, 4.32%; N, 11.50%.
1-Phenyl-3-methyl-5-(4-fluorophenylsulfonyl)-4-pyrazolaldoxime-(2-
chloropyridine-5-ylmethyl) Ether (5c). White crystal; mp 125-126 °C;
yield, 50%. IR (KBr, cm-1): ν 3076 (Ar-H), 2929, 2873 (CH3 + CH2),
1615 (CdN), 1590, 1488 (Ar, skeleton vibration), 1343, 1149 (SO2
1
vibration), 1235 (CdN-N), 840 (p-disubstituded benzene). H NMR
(DMSO-d6, 500 MHz, ppm): δ 8.89 (s, 1H, CH-H), 8.45 (d, J ) 2.30
Hz, 1H, Py-6-H), 7.46-7.39 (m, 4H, Py-4-H + 1-Ph-3, 4, 5-H), 7.35
(dd, J ) 8.60 Hz, J ) 8.00 Hz, 2H, SO2-Ph-2, 6-H), 7.27 (d, J )
8.560 Hz, 2H, 1-Ph-2, 6-H), 7.14 (d, J ) 8.00 Hz, 1H, Py-3-H), 7.05
(t, J ) 8.55 Hz, J ) 8.60 Hz, 2H, SO2Ph-3, 5-H), 5.22 (s, 2H, CH2-
H), 2.41 (s, 3H, pyrazole-CH3-H). Anal. calcd for C23H18ClFN4O3S
(484.5): C, 56.96%; H, 3.71%; N, 11.56%. Found: C, 57.10%; H,
3.90%; N, 11.69%.
1-(3-Chlorophenyl-3-methyl-5-(4-fluorophenylsulfonyl)-4-pyrazola-
ldoxime-(2-chloropyridine-5-ylmethyl) Ether (5d). White crystal; mp
120-122 °C; yield, 58%. IR (KBr, cm-1): ν 3076 (Ar-H), 2929, 2873
(CH3 + CH2), 1590, 1488 (Ar, skeleton vibration), 1343, 1149 (SO2
1
vibration), 1235 (CdN-N), 840 (p-disubstituded benzene). H NMR
(DMSO-d6, 500 MHz, ppm): δ 8.89 (s, 1H, CH-H), 8.45 (d, J ) 2.30
Hz, 1H, Py-6-H), 7.46-7.51 (m, 1H, Py-4-H), 7.41-7.40 (m, 2H, 1-Ph-
4, 6-H), 7.35 (dd, J ) 8.60 Hz, J ) 8.00 Hz, 2H, S O2-Ph-2, 6-H),
7.27 (s, 1H, 1-Ph-2-H), 7.14 (d, J ) 7.90 Hz, 1H, Py-3-H), 7.05 (t, J
) 8.55 Hz, J ) 8.60 Hz, 2H, SO2-Ph-3, 5-H), 6.97 (t, J ) 2.3 Hz, J
) 1.75 Hz, 1H, 1-Ph-5-H), 5.22 (s, 2H, CH2-H), 2.41 (s, 3H, pyrazole-
CH3-H). Anal. calcd for C23H17Cl2FN4O3S (519.0): C, 53.18%; H,
3.28%; N, 10.79%. Found: C, 53.28%; H, 3.22%; N, 10.66%.
1-(4-Chlorophenyl-3-methyl-5-(4-methylphenylsulfonyl)-4-pyrazola-
ldoxime-(2-chloropyridine-5-ylmethyl) Ether (5e). White crystal; mp
138-140 °C; yield, 54%. IR (KBr, cm-1): ν 3045 (Ar-H), 2924, 2865
(CH3 + CH2), 1615 (CdN), 1592, 1459 (Ar, skeleton vibration), 1338,
1149 (SO2 vibration), 1235 (CdN-N), 840 (p-disubstituded benzene).
1H NMR (DMSO-d6, 500 MHz, ppm): δ 8.89 (s, 1H, CH-H), 8.46 (d,
J ) 2.30 Hz, 1H, Py-6-H), 7.75-7.34 (m, 1H, Py-4-H), 7.35 (d, J )
8.05 Hz, 1H, Py-3-H), 7.32 (d, J ) 8.55 Hz, 2H, SO2-Ph-2, 6-H), 7.26
(d, J ) 8.60 Hz, 2H, 1-Ph-2, 6-H), 7.14 (d, J ) 8.55 Hz, 2H, 1-Ph-3,
5-H), 7.07 (d, J ) 8.60 Hz, 2H, SO2-Ph-3, 5-H), 5.20 (s, 2H, CH2-H),
2.38 (s, 3H, pyrazole-CH3-H), 2,33, (s, H, Ph-CH3-H). Anal. calcd for
C24H20Cl2N4O3S (515.0): C, 55.92%; H, 3.88%; N, 10.87%. Found: C,
56.08%; H, 3.78%; N, 10.69%.
1-(4-Chlorophenyl)-3-methyl-5-(4-methoxylphenylsulfonyl)-4-pyra-
zolaldoxime-(2-chloropyridine-5-ylmethyl) Ether (5f). White crystal; mp
139-140 °C; yield, 58%. IR (KBr, cm-1): ν 3033 (Ar-H), 2933, 2852
(CH3 + CH2), 1615 (CdN), 1591, 1463 (Ar, skeleton vibration), 1315,
1148 (SO2 vibration), 1264 (CdN-N), 843 (p-disubstituded benzene),
Data for 1-Phenyl-3-methyl-5-(4-methylphenylsulfonyl)-4-pyrazola-
ldoxime-(2-chloropyridine-5-ylmethyl) Ether (5a). White crystal; mp
120-121 °C; yield, 56%. IR (KBr, cm-1): ν 3069 (Ar-H), 2926, 2877
(CH3 + CH2), 1605 (CdN), 1587, 1488 (Ar skeleton vibration), 1344,
1152 (SO2 vibration), 1230 (CdN-N), 845 (p-disubstituded benzene)
1
809, 771 (m-disubstituded benzene). H NMR (DMSO-d6, 500 MHz,
ppm): δ 8.89 (s, 1H, CH-H), 8.46 (d, J ) 2.30 Hz, 1H, Py-6-H),
7.75-7.39 (m, 1H, Py-4-H), 7.36 (d, J ) 8.05 Hz, 2H, SO2-Ph-2, 6-H),
7.32 (dd, J ) 5.30 Hz, J ) 1.75 Hz, 1H, Py-3-H), 7.29 (d, J ) 8.55
Hz, 2H, 1-Ph-3, 5-H), 7.08 (dd, J ) 2.25 Hz, J ) 2.30 Hz, 2H, 1-Ph-
2, 6-H), 6.80 (d, J ) 8.55 Hz, 2H, SO2-Ph-3, 5-H), 5.20 (s, 2H, CH2-
H), 3.84 (s, 3H, O-CH3-H), 2.39 (s, 3H, pyrazole-CH3-H). Anal. calcd
for C24H20Cl2N4O4S (531.0): C, 54.24%; H, 3.77%; N, 10.54%. Found:
C, 54.36%; H, 3.60%; N, 10.42%.
1-(4-Chlorophenyl)-3-methyl-5-(4-fluorophenylsulfonyl)-4-pyrazola-
ldoxime-(2-chloropyridine-5-ylmethyl) Ether (5g). White crystal; mp
144-145 °C; yield, 87%. IR (KBr, cm-1): ν 3041 (Ar-H), 2921, 2855
(CH3 + CH2), 1611 (CdN), 1589, 1493 (Ar, skeleton vibration), 1315,
1148 (SO2 vibration), 1226 (CdN-N), 831 (p-disubstituded benzene).
1H NMR (DMSO-d6, 500 MHz, ppm): δ 8.89 (s, 1H, CH-H), 8.43 (d,
J ) 2.30 Hz, 1H, Py-6-H), 8.24 (s, 1H, CH-H), 7.71 (d, J ) 2.30 Hz,
1H, Py-4-H), 7.32 (d, J ) 8.50 Hz, 2H, SO2-Ph-2, 6-H), 7.36 (d, J )
8.05 Hz, 1H, Py-3-H), 7.29 (d, J ) 8.60 Hz, 2H, 1-Ph-2, 6-H), 7.21
(d, J ) 8.50 Hz, 2H, 1-Ph-3, 5-H), 7.17 (d, J ) 8.60 Hz, 2H, SO2-
Ph-3, 5-H), 5.19 (s, 2H, CH2-H), 2.40 (s, 3H, pyrazole-CH3-H). Anal.
calcd for C23H17Cl2FN4O3S (519.0): C, 53.17%; H, 3.27%; N, 10.78%.
Found: C, 53.00%; H, 3.21%; N, 10.71%.
1
cm-1. H NMR (DMSO-d6, 500 MHz, ppm): δ 8.75 (s, 1H, CH-H),
8.46 (d, J ) 2.35 Hz, 1H, Py-6-H), 7.60 (d, J ) 2.30 Hz, 1H, Py-4-H),
7.48, ∼7.38 (m, 6H, 1-Ph-H, Py-3-H), 7.37 (dd, J ) 8.60 Hz, J )
8.00 Hz, 2H, SO2-Ph-2, 6-H), 7.10 (t, J ) 8.55 Hz, J ) 8.60 Hz, 2H,
SO2Ph-3, 5-H), 5.24 (s, 2H, CH2-H), 2.52 (s, 3H, pyrazole-CH3-H),
2.25 (s, 3H, Ph-CH3-H). Anal. calcd for C24H21ClN4O3S (480.5): C,
59.93%; H, 4.37%; N, 11.65%. Found: C, 59.92%; H, 4.39%; N,
11.50%.
1-Phenyl-3-methyl-5-(4-methoxyphenylsulfonyl)-4-pyrazolaldoxime-
(2-chloropyridine-5-ylmethyl) Ether (5b). White crystal; mp 125-126
°C; yield, 53%. IR (KBr, cm-1): ν 3098 (Ar-H), 2928, 2889 (CH3 +
CH2), 1613 (CdN), 1598, 1398 (Ar skeleton vibration), 1349, 1155
(SO2 vibration), 1250 (CdN-N), 847 (p-disubstituded benzene). 1H
NMR (DMSO-d6, 500 MHz, ppm): δ 8.46 (d, J ) 2.30 Hz, 1H, Py-
6-H), 8.23 (s, 1H, CH-H), 7.88 (dd, J ) 2.30 Hz, J ) 2.30 Hz, 1H,
Py-4-H), 7.44-7.53 (m, 6H, 1-Ph-H, Py-3-H), 7.37-7.28 (m, 5H, 1-Ph-
H), 6.91 (d, J ) 9.20 Hz, 2H, S-Ph-3,5-H), 6.81 (d, J ) 9.15 Hz, 2H,
S-Ph-2, 6-H), 5.17 (s, 2H, CH2-H), 3.68 (s, 3H, O-CH3-H), 2.37 (s,
3H, CH3-H). 13C NMR (DMSO-d6, 125 MHz, ppm): δ 169.3, 160.9,
150.4, 150.3, 143.6, 140.5, 138.9, 134.1, 133.3, 130.9, 129.4, 128.9,
126.1, 124.5, 117.7, 116.6, 115.7, 72.4, 55.7, 14.8. Anal. calcd for
1-(4-Methylphenyl-3-methyl-5-(4-fluorophenylsulfonyl)-4-pyrazola-
ldoxime-(2-chloropyridine-5-ylmethyl) Ether (5h). White crystal; mp
112-114 °C; yield, 52%. IR (KBr, cm-1): ν 3045 (Ar-H), 2924, 2865

Pyrazole Derivatives Containing an Oxime Moiety
J. Agric. Food Chem., Vol. 56, No. 21, 2008 10165
inoculated with the mixture of solvent and the virus for control. The
local lesion numbers were recorded 3-4 days after inoculation (23).
Three repetitions were conducted for each compound.
Curative Effect of Compounds against TMV in Vivo. Growing
leaves of N. tabacum L. of the same ages were selected. The TMV
(concentration of 6 × 10-3mg/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
smeared on the right side for control. The local lesion numbers were
then counted and recorded 3-4 days after inoculation (23). For each
compound, three repetitions were measured. The inhibition rate of the
compound was then calculated according to the following formula (“av”
means average).
inhibition rate (%) )
av local lesion numbers of control (not treated with compound) -
av local lesion numbers smeared with drugs
× 100%
av local lesion numbers of control (not treated with compound)
Figure 3. Molecular structure of 4b.
RESULTS AND DISCUSSION
(CH3 + CH2), 1615 (CdN), 1592, 1459 (Ar, skeleton vibration), 1338,
1149 (SO2 vibration), 1213 (CdN-N), 840 (p-disubstituded benzene).
1H NMR (DMSO-d6, 500 MHz, ppm): δ 8.90 (s, 1H, CH-H), 8.46 (d,
J ) 2.30 Hz, 1H, Py-6-H), 7.75-7.65 (m, 1H, Py-4-H), 7.35 (d, J )
8.05 Hz, 1H, Py-3-H), 7.32 (d, J ) 8.55 Hz, 2H, SO2-Ph-2, 6-H), 7.31
(d, J ) 8.60 Hz, 2H, 1-Ph-2, 6-H), 7.25 (d, J ) 8.60 Hz, 2H, 1-Ph-3,
5-H), 7.07 (d, J ) 8.60 Hz, 2H, SO2-Ph-3,5-H), 5.20 (s, 2H, CH2-H),
2.39 (s, 3H, pyrazole-CH3-H), 2.39 (s, 3H, Ph-CH3-H). Anal. calcd for
C24H20ClFN4O3S (498.5): C, 57.73%; H, 4.01%; N, 11.23%. Found:
C, 57.60%; H, 3.97%; N, 11.45%.
Synthesis. The synthetic route designed for the oxime ester
analogues 4 and 5 is summarized in Scheme 1. Starting from
1-substituted phenyl 3-methyl-pyrazolone derivative, the inter-
mediate 1, 1-substituted phenyl-3-methyl-4-formyl-5-chloropy-
razole was prepared by chlorination reaction with POCl3 in
DMF. Then, treatment of 1 with substituted thiophenol
afforded 1-substituted phenyl-3-methyl-5-substituted-phe-
nylthio-4-pyrazol-aldehyde 2. Reaction of 2 with hydroxy-
lamine hydrochloride gave 1-substituted phenyl-3-methyl-
5-substituted phenylthio-4-pyrazolaldoximes 3. The title
compounds, 1-substituted phenyl-3-methyl-5-substituted phe-
nylthio-4-pyrazolaldoxime ethers 4 were synthesized by the
etherification reaction of 3 with chloromethylated heterocyclic
compounds (ClCH2R3). 1-Substituted phenyl-3-methyl-5-
substituted phenyl- sulfonyl-4-pyrazolaldoxime ether deriva-
tives 5 were then obtained from the oxidation of 4 with
potassium permanganate in HOAc solution at room temper-
ature. While the compounds 4a-n were obtained in high
yields (54-87%), the compounds 5a-h were obtained in
good yields (50-58%). The X-ray single structure of typical
4b has been shown in Figure 3, and all compounds were
characterized unequivocally by spectroscopic data and el-
emental analysis as described in the Materials and Me-
thods.
X-ray Diffraction. Colorless blocks of 4b (0.20 mm × 0.19 mm ×
0.18 mm) were counted on a quartz fiber with protection oil. Cell
dimensions and intensities were measured at 293 K on a Bruker
SMART CCD area detector diffractometer with graphite monochro-
mated Mo KR radiation (λ ) 0.71073 Å), θmax ) 25.00, 19528
measured reflections, and 2498 independent reflections (Rint
)
0.1763) of which 4595 had I > 2δ(I). Data were corrected for
Lorentz and polarization effects and for absorption (Tmin ) 0.7680;
Tmax ) 0.8237). The structure was solved by direct methods using
SHELXS-97; all other calculations were performed with Bruker
SAINT System and Bruker SMART programs. Full-matrix least-
squares refinement based on F2 using the weight of 1/[σ2(F02) +
(0.0850P)2 + 0.0458P] gave final values of R ) 0.0517, ωR )
0.1631, and GOF(F) ) 1.011 for 453 variables and 4595 contributing
reflections. The maximum shift/error ) 0.001, and max/min residual
electron density ) 0.400/-0.322 e Å-3. Hydrogen atoms were
observed and refined with a fixed value of their isotropic displace-
ment parameter.
Antiviral Activity and Structure-Activity Relationship.
To make a judgment of the antiviral potency of the synthesized
compounds 4a-n and 5a-h, the commercially available plant
virucide Ningnanmycin (24), perhaps the most successful
registered antiplant viral agent available in China, was used as
the control. The antiviral bioassay against TMV is assayed by
the reported method (22, 23), and the antiviral results of all
of the compounds against TMV are listed in Table 1. The results
showed that most of our designed compounds had moderate
antiviral activities at 500 mg/L against TMV in vivo.
The title compounds 4a-n and 5a-h exhibited protection
activities of 15.7-44.8% at 500 mg/L. Compound 4g (R1 is
4-Cl, R2 is F, and R3 is 2-chloropyridine-5-ylmethyl), 4l [R1 is
4-Me, R2 is Me, and R3 is 5-chloro-3-methyl-1-(4-methylphe-
nyl)-pyrazol-4-ylmethyl], 4m [R1 is 4-Me, R2 is F, and R3 is
5-chloro-3-methyl-1-(4-methylphenyl)-pyrazol-4-ylmethyl], 5c
(R1 is H, R2 is F, and R3 is 2-chloropyridine-5-ylmethyl), and
5g (R1 is 4-Cl, R2 is F, and R3 is 2-chloropyridine-5-ylmethyl)
had moderate protection activities (43.5, 44.8, 41.0, 40.2, and
42.2, respectively), comparable to that that of the standard
reference (59.9%). In addition, the other compounds 4c-f,h-k
and 5a,b,d-f,h showed less than 40% protection activities at
Antiviral Biological Assay. Purification of TMV. Using Gooding’s
method (22), the upper leaves of Nicotiana tabacum L. inoculated with
TMV were selected, ground in phosphate buffer, and then filtered
through double layer pledget. The filtrate was centrifuged at 10000g,
treated twice with poly(ethylene glycol), and centrifuged again. The
whole experiment was carried out at 4 °C. Absorbance values were
estimated at 260 nm using an ultraviolet spectrophotometer.
0.1%,260nm
1cm
virus concn ) (A260 × dilution ratio) ⁄ E
Protective Effects of Compounds against TMV in Vivo. The
compound solution was smeared on the left side, while the solvent
served as the control on the right side of growing N. tabacum L. leaves
of the same ages. The leaves were then inoculated with the virus after
12 h. A brush was dipped in TMV of 6 × 10-3 mg/mL to inoculate
the leaves, which were previously scattered with silicon carbide. The
leaves were then washed with water and rubbed softly along the
nervature once or twice. The local lesion numbers appearing 3-4 days
after inoculation were counted (23). Three repetitions were conducted
for each compound.
Inactivation Effect of Compounds against TMV in Vivo. The virus
was inhibited by mixing with the compound solution at the same volume
for 30 min. The mixture was then inoculated on the left side of the
leaves of N. tabacum L., while the right side of the leaves was

10166 J. Agric. Food Chem., Vol. 56, No. 21, 2008
Ouyang et al.
oxime ester derivatives. Bioorg. Med. Chem. 2008, 16, 4075–
4082.
500 mg/L. From the data presented in Table 1, it can be
observed that the title compounds 4a-n possess potential
inactivation bioactivities, with values of 90.4, 71.2, 65.9, 61.7,
61.2, 34.5, 80.6, 56.6, 61.0, 43.0, 55.9, 51.2, 50.9, and 36.7%
at 500 µg/mL, respectively. Among these compounds, 4a and
4gare appreciably more active than the rest, with inactivation
rates of 90.4 and 80.6%, respectively, which are similar to
that of Ningnanmycin (98.0%) against TMV at 500 µg/mL.
The data also indicate that all compounds have a relatively
lower curative activity than that of Ningnanmycin. At this
moment, however, it is difficult to provide a rational account
of structure-activity relationships on the basis of steric,
electronic, and hydrophobic effects. The diversity of the
structures was limited by the availability of the reagents and
the ease with which para-substituted (for R1 and R2 groups)
products could be obtained as compared to those with ortho-
and meta-substituted ones. The electronic factor does not
seem to play a significant role as both the compounds 4a
(R1 as H and R2 Me) and 4g (R1 as Cl and R2 as F) with
electron-rich and electron-poor substituents, respectively,
were found to display good bioactivity. Comparison of
biological activities among 4a-n and 5a-h confirms that
the functional groups with alkylthio are potentially more
active than those with sulfonyl groups at the 5-position of
pyrazole, which is necessary for inactivation activity to occur.
In addition, as shown in Table 2, compounds 4a,b,g were
found to display good antiviral activities. These compounds were
bioassayed further to investigate their inactivation activities at
different concentrations with Ningnanmycin serving as the
commercial control. As shown in Table 2, the inactivation
effects against TMV of compounds 4a,b,g are significant. The
EC50 values were 58.7, 115.0, and 65.3 µg/mL, respectively.
Among these compounds, 4a displayed more potent antiviral
activity than the others, being similar to that of Ningnanmycin
(EC50) 52.7 µg/mL) against TMV.
In summary, a series of new pyrazole derivatives contain-
ing oxime moieties 4a-n were designed and synthesized by
the thioetherification reaction of 1-substituted phenyl-3-
methyl-5-substituted phenylthio-4-pyrazolaldoximes 3 with
chloromethylated heterocyclic compounds (ClCH2R3) and
NaOH in DMF. The in vivo tests indicated that compounds
4a,g exhibited a very similar inactivation bioactivity level
as that of Ningnanmycin against TMV. Therefore, the present
work demonstrates that the antiviral activity of pyrazole
derivatives was significantly improved via the introduction
of the oxime moiety. Although structure-activity relation-
ships could not be successfully established due to lack of
structural diversity, the present work paves the way toward
the synthesis and antiviral studies of new pyrazole derivatives
containing oxime moieties. Effects of steric, hydrophobic,
electronic, and electrostatic parameters on structure-activity
relationships and structural modification studies for identify-
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ACKNOWLEDGMENT
We thank the National Key Program for Basic Research
(2003CB114404) and the National Natural Science Foundation
of China (20672024 and 20762002) for the financial support.
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Received for review August 12, 2008. Revised manuscript received
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JF802489E