Synthesis and antiviral activity of novel pyrazole derivatives containing oxime esters group

Article abstract of DOI:10.1016/j.bmc.2008.09.070

Fourteen title compounds, 1-substituted-5-substitutedphenylthio-4-pyrazolaldoxime ester derivatives 4a-4n, were synthesized from the starting material 1-substitutedphenyl-3-methyl-5-substitutedphenylthio-4-pyrazolaldoximes 3 by treatment with acyl chlorid

Full text of DOI:10.1016/j.bmc.2008.09.070

Bioorganic & Medicinal Chemistry 16 (2008) 9699–9707
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antiviral activity of novel pyrazole derivatives containing
oxime esters group
*
Guiping Ouyang, Zhuo Chen, Xue-Jian Cai, Bao-An Song , Pinaki S. Bhadury, Song Yang, Lin-Hong Jin,
Wei Xue, De-Yu Hu, Song Zeng
Center for Research and Development of Fine Chemicals, Key Laboratory of Green Pesticide and Bioengineering, Ministry of Education, Guizhou University, Guiyang 550025, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Fourteen title compounds, 1-substituted-5-substitutedphenylthio-4-pyrazolaldoxime ester derivatives
4a–4n, were synthesized from the starting material 1-substitutedphenyl-3-methyl-5-substitutedphenyl-
thio-4-pyrazolaldoximes 3 by treatment with acyl chloride. The synthesized compounds were character-
ized by physical constants, and the structures of the title compounds were further confirmed by IR, 1H
NMR, 13C NMR and elemental analysis. The bioassay results showed that title compounds possessed
weak to good anti-TMV bioactivity with 4l showing significant enhancement of disease resistance in
tobacco leaves with high affinity for TMV CP.
Received 20 August 2008
Revised 27 September 2008
Accepted 30 September 2008
Available online 2 October 2008
Keywords:
Pyrazole
Oxime ester moiety
Synthesis
Ó 2008 Elsevier Ltd. All rights reserved.
Antiviral bioactivity
Mechanism of action
1. Introduction
of this, we turned our attention to evaluate the modification of
the biological profile induced by the change of the substituents
Pyrazole and their derivatives exhibit a broad spectrum of biolog-
ical activities such as antimicrobial,1 herbicidal,2,3 antitumor,4,5
anti-inflammatory activities.6–8 Among them, some are used as
insecticides, herbicides and fungicides, such as fripronil (Colliot
et al., 1992), topramezon (BASF, 2006), pyraelostrobin (BASF,
2001), and so on. With growing application on their synthesis and
bioactivity, chemists and biologists in recent years have directed
considerable attention on the research of pyrazole derivatives.7,8
TMV infection is very widely distributed, and can cause serious
damage and large economic loss. It was found that in certain fields
90–100% of the plants show mosaic by harvesting time. Due to the
unsatisfactory curatives (30–60%) cure rate by common antiviral
agent Ningnanmycin (or Virus A) and economic loss of tobacco, a
wide range of chemicals have been synthesized and tested for anti-
viral effects during screening for biological activity. However, since
there are only a few reported economically viable antiviral chem-
icals available for practical application in agriculture,9 there still
lies a great deal of scope for further research in this field. In view
at the 1or 3 or 4 and 5-position in pyrazole ring.
Pyrazolaldoxime esters at 4 position are scarcely evaluated for
their antiviral activities. Due to their known fungicidal activities,
we reasoned that if oxime esters are linked at 4 position in the pyr-
azole ring, the resulting compounds will have better bioactivities.
Therefore, in our pursuit to develop new class of agrochemicals,
we synthesized the title compounds and studied their antiviral
activities. The synthetic route is shown in Scheme 1. Starting from
the key intermediate 1-substitutedphenyl-3-methyl-5-substitut-
edphenylthio-4-pyrazolaldoximes 3, the title compounds 4 are
synthesized in one step. Esterification reaction of 3 with acyl chlo-
ride affords appropriate oxime ester. The structures of 4 were
firmly established by well-defined IR, 1H NMR, 13C NMR and ele-
mental analysis. Preliminary bioassay tests showed that some
compounds possess certain degree of antiviral activity against
TMV at 500 mg/L in vivo as shown in Table 1, however with a de-
gree of variation. The title compounds 4l and 4m had high anti-
TMV activities, and the inhibitory effect for the two treated meth-
ods ranged from 60.0% to 85.6%. The preliminary studies on action
mechanism of compound 4l against TMV revealed that it was capa-
ble to enhance the defense enzyme activity within a certain period
and up-regulate PR-1a gene expression. Further studies on the
relationship among compound 4l and TMV CP and TMV RNA
showed special affinity of 4l to CP, but not to RNA. Therefore, we
inferred that the action target of compound 4l against anti-TMV
Abbreviations: 1H NMR, 1H nuclear magnetic resonance; 13C NMR, 13C nuclear
magnetic; TMV, tobacco mosaic virus; PAL, L-phenylalanine ammonia-lyase; SOD,
superoxide dismutase; POD, peroxidase; SA, salicylic acid; SAR, systemic acquired
resistance; PR, pathogenesis-related proteins; PCR, polymerase chain reaction;
BLAST, basic local alignment search tool; CP, coat protein.
* Corresponding author. Tel.: +86 851 362 0521; fax: +86 851 362 2211.
E-mail address: songbaoan22@yahoo.com (B.-A. Song).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.09.070

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G. Ouyang et al. / Bioorg. Med. Chem. 16 (2008) 9699–9707
H3C
H3C
H3C
CHO
CHO
S
N
N
N
O
Cl
R2
R2
SH
N
N
N
POCl3
DMF
NH2OH·HCl
R1
R1
R1
2
1
H3C
H3C
NOCOR3
CH NOH
S
CH
S
N
N
R2
R2
N
ClCOR3
N
r.t , DMF, NaOH
R1
R1
3
4
Compd. R1
R2
Me
F
R3
Compd. R1
R2
R3
4a
4c
4e
4g
4i
H
H
Me
Ph
Ph
Ph
4b
4d
4f
H
F
F
F
F
F
F
F
Me
3-Cl
4-Me
4-Me
Me
Ph
4-Cl MeO
Ph
4-Cl
H
F
F
4h
4j
Me-CH=CH-CH=CH
Me-(CH2)4
Me-(CH2)4
n-Pr
Me-(CH2)4
4k
4m
4-Cl Me
4-Cl
Me-CH=CH-CH=CH 4l
Me-CH=CH-CH=CH 4n
4-Cl
H
F
Scheme 1.
was related to up-regulation of the PR gene and promotion of de-
fense enzymes activity to acquire SAR, as well as to limit transfer
and proliferation of TMV with highly affinity to CP.
(1.453 Å) are shorter than normal C–C (1.54 Å), C(11)–N(3)
(1.269 Å) and C(3)–N(1) (1.444 Å) bonds are shorter than the nor-
mal C–N single bond (1.49 Å), suggesting the existence of an elec-
tron density delocalization among O(1)–N(3)–C(11)–C(8)–C(7)–
N(1), C(9) and S(1).
2. Chemistry
2.1. Synthesis of novel pyrazole derivatives
3. Antiviral activity
The synthetic route designed for the oxime ester analogues 4 is
summarized in Scheme 1. Starting from 1-substitutedphenyl-3-
methyl-4-pyrazolone derivatives, the intermediate 1, 1-substi-
tuted phenyl-3-methyl-4-formyl-5-chloropyrazole was prepared
by chlorination reaction with POCl3 in DMF. Then, treatment of 1
with substituted thiophenol afforded 1-substitutedphenyl-3-
methyl-5-substitutedphenylthio-4-pyrazolaldehyde 2. Reaction of
2 with hydroxylamine hydrochloride gave 1-substitutedphenyl-
3-methyl-5-substitutedphenylthio-4-pyrazolaldoximes 3. The
title compounds, 1-substituted-5-substitutedphenylthio-4-pyra-
zolaldoxime ester derivatives 4 were synthesized by the esterifica-
tion reaction of 3 with acyl chloride (ClCOR3).
3.1. Preliminary antiviral activity assay
The commercially available plant virucide Ningnanmycin, per-
haps the most successful registered anti-plant viral agent available
in China, was used as the control. The antiviral bioassay against
TMV is assayed by the reported method and the antiviral results
of all the compounds against TMV are listed in Table 1. The results
showed that most of our designed compounds had moderate to
good antiviral activities at 500 mg/L against TMV in vivo.
The title compounds 4a–4n exhibited good curative activities of
30.0–62.0% at 500 mg/L. Compound 4l (R1 was 4-Cl, R2 was F, and
R3 was Me-(CH2)4), 4m (R1 was 4-Cl, R2 was F and R3 was Me–
CH@CH–CH@CH) had higher activities (62.0% and 60.0%, respec-
tively) than that of the standard reference (56.0%). In addition,
compounds 4a, 4b, 4f, 4g, 4h and 4i showed 40.8–50.0% curative
activities at 500 mg/L, with values of 47.8%, 40.4%, 50.0%, 47.7%,
46.7%, and 40.0%, respectively. From the data presented in Table
1, it can be observed that the title compounds 4a–4n possess po-
tential inactivation bioactivities, with values of 68.3%, 81.4%,
55.0%, 55.0%, 56.8%, 69.2%, 63.8%, 80.2%, 67.8%, 61.5%, 68.9%,
2.2. Crystal structure analysis
It could be seen from the X-ray single-crystal analysis that title
compound 4n maintains a planar structure. The structure of target
compound 4n was established by X-ray diffraction. The bond
lengths of C(7)–C(8) (1.389 Å) and C(8)–C(9) (1.410 Å) are longer
than normal C@C (1.34 Å), C(12)–O(1) (1.365 Å) is shorter than
normal single C–O (1.44 Å), C(8)–C(11) (1.458 Å) and C(12)–C(13)
85.6%, 82.9% and 79.9% at 500 lg/mL, respectively. Among these

G. Ouyang et al. / Bioorg. Med. Chem. 16 (2008) 9699–9707
9701
Table 1
and R3 was Ph), 4j (R1 was Me, R2 was F and R3 was Me-(CH2)4])
and 4k (R1 was 4-Cl, R2 was Me and R3 was Me-CH@CH–CH@CH)
had moderate protection activities against TMV of up to 41.0%,
48.4%, 40.9% and 50.0%, respectively, at 500 lg/mL. The other com-
pounds had a relatively lower curative activity than those of4d, 4f,
The protection, inactivation and curative effect of the title compounds 4a–4n against
TMV in vivo.
Agents
Concentration
(mg/L)
Protection
effect(%)
Inactivation
effect (%)
Curative
effect (%)
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
500
500
500
500
500
500
500
500
500
500
500
500
500
500
33.8 1*
36.7 4*
28.8 3*
41.0 8*
27.6 5*
48.4 7*
35.0 5*
26.0 6*
28.9 5*
40.9 5*
50.0 7*
31.0 6*
28.3 5*
25.9 6*
68.0 5*
68.3 3*
81.4 4**
55.0 4*
55.0 6*
56.8 5*
69.2 7*
63.8 9*
80.2 4**
67.8 4*
61.5 3*
68.9 6*
85.6 4**
82.9 5**
79.9 4**
97.0 6**
47.8 2*
40.4 3*
34.9 6*
30.2 7*
35.0 6*
50.0 9*
47.7 9*
46.4 3*
40.2 6*
30.0 5*
38.9 4*
62.0 8**
60.0 8*
34.0 3*
56.0 4*
4j, and 4k.
3.2. Effect of 3l treatment on PAL, POD and SOD activity in
tobacco
As shown in Figure 3, using enzyme activity method, PAL, POD,
SOD contents in 4l-treated tobacco showed some definite trend
within a certain period of time. POD and SOD levels in 4l-treated
tobacco increased by the end of the first day after the inoculation
and reached up to 2.4 EU/mg and 40 EU/mg on the 7th day, respec-
tively, four times greater than the first day after inoculation in en-
zyme levels. In contrast, in control and TMV, no significant increase
was measured. We found that PAL activity in 4l-treated tobacco
decreased from 1st day to 7th day after inoculation, similar trend
was not observed on control and TMV.
Ningnanmycin 500
All results are expressed as means SD; n = 3 for all groups.
*
P < 0.05.
P < 0.01.
**
3.3. Gene expression analysis of PR-1a and PR-5 in 4l-treated
tobacco leaf
For the results of product of PCR in PR-1a and PR-5 and their
sequence identification, see Supporting information. In vitro syn-
thesized single-stranded cDNA from RNA samples was isolated
from leaf in Ningnanmycin-treated tobacco and the TMV-treated
tobacco and the 4l and TMV-treated tobacco. The differential
expression analysis of the PR-1a and PR-5 gene was determined
by the relative quantification PCR and real-time PCR analysis. As
shown in Figure 4, the mRNAs of PR-1a gene accumulated to
detectable levels in all-treated tobacco leaf, although, the gene
up-regulation in 4l-treated was found to be higher than the rest
from 3rd day to 7th day. The gene expression ratio reached up to
26.6 on the 5th day, the corresponding values for Ningnanmycin-
treated and TMV-treated reached only up to 13.85 and 7.64,
respectively. The values of compound 4l were almost twice as large
as compared to Ningnanmycin-treated tobacco.
Figure 1. The molecular structure of compound 4n.
As shown in Figure 5, it could be seen that the mRNA content of
4l-treated tobacco leaf for PR-5 gene slowly decreased from 1st day
to the start of the 3rd day, then gradually increased and reached a
peak on the 5th day after the inoculation before showing a gradual
decreasing trend again. Meanwhile, similar trends were also ob-
served in TMV-treated, although, for Ningnanmycin-treated tobac-
co leaf, significant increase in the levels of gene expression ratio
was noticed from 3rd day to 5th day, the value reaching up to
17.95. From above results, we found that compound 4l could in-
duce gene up-regulation of PR-1a without any effect in the regula-
tion of PR-5 gene.
3.4. The primary spectroscopic study of 4l for TMV CP
3.4.1. The ultraviolet–vis spectroscopic study of 4l for TMV CP
For isolation of TMV CP, see Supporting information. The
intrinsic UV–vis absorbance peak of CP appeared mainly due to
the presence of tryptophan and tyrosine in the CP peptide. As
shown in Figure 6, blue line and red line correspond to the UV–
vis spectra for TMV CP-treated and TMV CP-4l-treated, respec-
tively. It could be seen from the red line inFigure 6A that the absor-
bance peak values were 2.726 and 0.195 at 207.0 nm and
281.0 nm, respectively, for 4S CP. The absorption peak values of
4S CP shifted toward the longer wavelength side as the compound
4l was added and the maximum absorption peak increased from
2.726 to 3.136. In addition, the relatively smaller absorption peak
Figure 2. Packing diagram of the unit cell of compound 4n.
compounds, 4b, 4h, 4l, 4m and 4n were appreciably more active
than the rest, with the inactivation rates of 81.4%, 80.2%, 85.6%,
82.9% and 79.9%, respectively, which were similar to that of Nin-
gnanmycin (97%) against TMV at 500 lg/mL. The data also indi-
cated that a change in the substituent might also affect the
protection activity of title compounds 4a–4n. Compound 4d (R1
was 3-Cl, R2 was F and R3 was Ph), 4f (R1 was 4-Me, R2 was F

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G. Ouyang et al. / Bioorg. Med. Chem. 16 (2008) 9699–9707
Figure 3. Effect of 4l treatment on PAL and SOD and POD activity in Nicotiana tabacum. Nicotiana tabacum inoculated by TMV was treated with 4l at 500 lg/mL for 1, 3, 5 and
7 day. Leaf sample extracts using homogenized and centrifuged methods were used to determine the PAL and SOD and POD activities as described in Section 6. Water
treatment and TMV treatment were used for negative and positive controls. Data were expressed as means SD (n = 3) of three individual experiments. One-way ANOVA
revealed significant difference.
Figure 4. Real-time PCR analysis of RNA isolated from tobacco leaf against PR-1a gene. Letters A in the figures indicate that real-time log view of PR-1a gene. Letters B in the
figures indicate that the melting curve of real-time PCR against PR-1a gene. The temperature of the melting curve was 85 °C, the results show the PCR products were purified
relatively and had not other non-specific products. Letters C in the figures indicate that the results of PR-1a gene expression ratio through CT value formula DDCT
(test) ꢀ CT (calibrator). Relative values of real-time PCR between target gene and b-actin gene were calculated, each value represents the mean SD (n = 4) and each
experiment was performed in triplicate sets. The data was analyzed by one-way ANOVA.
= DCT
D

G. Ouyang et al. / Bioorg. Med. Chem. 16 (2008) 9699–9707
9703
Figure 5. Real-time PCR analysis of RNA isolated from tobacco leaf against PR-5 gene. Letters A in the figures indicate that real-time log view of PR-5 gene. Letters B in the
figures indicate that the melting curve of real-time PCR against PR-5 gene. The temperature of the melting curve was 83 °C, the results show the PCR products were purified
relatively and had not other non-specific products. Letters C in the figures indicate that the results of PR-5 gene expression ratio through CT value formula DDCT
(test) ꢀ CT (calibrator). Relative values of real-time PCR between target gene and b-actin gene were calculated, each value represents the mean SD (n = 4) and each
experiment was performed in triplicate sets. The data was analyzed by one-way ANOVA.
= DCT
D
Figure 6. The ultraviolet spectroscopy of TMV CP 4S (A) or 20S (B) protein and 4l. TMV CP concentration was 0.65 mg/L and concentration of 4l was varied from 0% to 0.01%.
value shifted slightly toward the longer wavelength side as shown
by the blue line in Figure 6A. Similar phenomenon could be ob-
served for TMV CP 20S fromFigure 6B. To conclude, as evident from
Figure 6, a change in UV–vis spectrum was noticed in the range
190–400 nm as compound 4l was added, red-shift-phenomenon
occurred and peak value increased sharply around 207 and
280 nm. The results indicated that the compound 4l could associ-
ate with CP.
3.4.2. Fluorescence spectroscopic study of 4l for TMV CP
For studying the relationship of compound 4l with TMV CP,
fluorescence spectroscopic assay was used. At 278 nm excitation
wavelength, emission wavelength of TMV CP revealed a maximum
of emission at 325 nm. As shown in Figure 7A, the excitation wave-
lengths of TMV CP 4S shifted from 320 to 331 nm as compound 4l
was added, and the fluorescence intensity (a.u.) of TMV CP de-
creased from 175 to 87. Meanwhile, from Figure 7B, it could be

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G. Ouyang et al. / Bioorg. Med. Chem. 16 (2008) 9699–9707
Figure 7. The fluorescence quenching spectra of TMV CP 20S or 4S protein as 4l is added. 1. 4l = 0
4l = 79.808 5. 4l = 159.616 6. 4l = 203 7. 4l = 319.232 8. 4l = 464
mol Lꢀ1 mol Lꢀ1 mol Lꢀ1 mol Lꢀ1
CP = 1.32
mol Lꢀ1. The data was recorded on Fluorescence Spectrophotometer (VARIAN CARY Eclipse) at 250 °C. The excitation wavelength and emission wavelength of
l
mol Lꢀ1; 2. 4l = 19.952
l
mol Lꢀ1; 3. 4 l = 39.904
l
mol Lꢀ1; 4.
l
;
l
;
l
;
l
;
l
mol Lꢀ1
;
9. 4l = 638
l
mol Lꢀ1
,
10. Curves 1–10: TMV
l
TMV CP are 278 nm and 325 nm, respectively. The EX. Slit and EM. Slit and smoothing factor are 5 nm, 5 nm and 10 nm, respectively.
Figure 8. The Stern-Volmer curves (A part) and the double-reciprocal curves (B part) of TMV CP 4S and 20S protein.
Table 2
Regression equations and correlation coefficient.
Regression equations
R2
Figure 7A 4S
Figure 7A 20S
Figure 7B 4S
Figure 7B 20S
F0/F = 1.0231 + 0.0031 ꢁ 104[4l]
0.986
F0/F = 0.9608 + 0.0016 ꢁ 104[4l]
0.9898
0.9997
0.9728
(F0 ꢀ F)ꢀ1 = 0.0028 + 2.1681 ꢁ 10ꢀ7[4l]ꢀ1
(F0 ꢀ F)ꢀ1 = 0.011 + 3.9027 ꢁ 10ꢀ7[4l]ꢀ1
seen that the excitation wavelengths of TMV CP 20S shifted from
321.07 to 330 nm as compound 4l was added, and the fluorescence
intensity (a.u.) of TMV CP 20S decreased from 168.14 to 87.35.
From above results, red-shift of wavelengths and fluorescence
quenching phenomenon for CP 4S and 20S were noticed when
compound 4l was added. These results also showed that the com-
pound 4l exhibited a higher affinity for TMV CP.
3.4.3. The determination of quenching constants
The intrinsic fluorescence of TMV CP can be quenched by com-
pound 4l at 321 and 330 nm. As shown in Figure 8A, F0/F presented
a linear relationship against compound 4l concentration for TMV
CP 4S and 20S protein. From the plot 8A of F0/F against compound
4l concentration, the dynamic quenching constants, Ksv, were eas-
ily calculated from the slope of the straight line, the values were
0.0031 ꢁ 104 and 0.0016 ꢁ 104 for TMV CP 4S and 20S protein,
respectively (Table 2). According to the equation of the constants
of collisional quenching rates (Kq), the Kq values were 31 ꢁ 108
and 16 ꢁ 108 for the above protein. The value was lower than the
value of Kq, which is 2.0 ꢁ 1010 L molꢀ1 Sꢀ1 for all the fluorescence
Figure 9. The Fluorescence quenching spectra of TMV RNA as 4l and cyanine dye
are added. 1. Cyanine dye by treated only; 2. TMV RNA by treated only; 3. cyanine
dye and TMV RNA by treated; 4–9. the resolution of cyanine dye and TMV RNA as 4l
was added. The concentration of 4l was increased from 19.952
l
mol Lꢀ1 to
159.616 The data was recorded on Fluorescence Spectrophotometer
l
mol Lꢀ1
.
(VARIAN CARY Eclipse) at 250 °C. The excitation wavelength and emission
wavelength of TMV CP are 495 nm and 525 nm, respectively. The EX. Slit and EM.
Slit and smoothing factor are 5 nm, 5 nm and 15 nm, respectively.
quenchers of biomacromolecule. Meanwhile, as shown in plot 8B,
the static quenching constants (KLB), were 2.1681 ꢁ 10ꢀ7 and

G. Ouyang et al. / Bioorg. Med. Chem. 16 (2008) 9699–9707
9705
3.9027 ꢁ 10ꢀ7 for the above protein (Table 2). Above results sug-
gested a probable binding of compound 4l with TMV CP mainly
through dynamic quenching. The relation between 4l and TMV
CP is worthy of further investigation.
specific action location of the CP for compound 4l was made
through the single-crystal diffraction and simulation methods to
further verification.
5. Conclusion
3.5. Fluorescence spectroscopic study of 4l for TMV RNA
In conclusion, a series of novel pyrazole derivatives were syn-
thesized by the treatment of 1-substitutedphenyl-3-methyl-5-
substituted phenylthio-4-pyrazolaldoximes 3 with acyl chloride.
Their structures were verified by spectroscopic data. The results
of bioassay showed that these title compounds exhibited weak to
good anti-TMV bioactivity. Title compounds 4l, 4m showed better
biological activity than their structurally related analogues 4a–4k
and 4n. Preliminary studies showed that treatment by compound
4l can significantly enhance disease resistance of tobacco and also
show that the compound 4l is structurally related to TMV by
exhibiting a higher affinity for TMV CP. So, the action mechanism
of curative effect by the compound 4l was mainly attributed to
the induced disease resistance against tobacco while the action
mechanism of inactivation effect was caused by its affinity towards
TMV CP. More detailed studies on mechanistic aspects are cur-
rently underway.
For isolation of TMV RNA, see Supporting information. As
shown in Figure 9, at 495 nm excitation wavelength, emission
wavelength of TMV RNA was 525 nm, the fluorescence intensity
of TMV RNA was relatively moderate, and reached up to
52.30 a.u. The fluorescence intensity was lower for cyanine dye,
the value being 3.73 a.u. which was close to 0 a.u. When the TMV
RNA was mixed with cyanine dye, the fluorescence intensity of
the system was increased suddenly, reached to 857.2 a.u. at
528.95 nm for emission wavelength. When compound 4l was
added to the system of TMV RNA and cyanine dye, the fluorescence
intensity was not significantly changed but the fluorescence inten-
sity of the system containing compound 4l was noticed to be com-
paratively lower. As the differences of the fluorescence intensity
were not observed in every concentration group, it was believed
that compound 4l had no effect on TMV RNA.
4. Discussion
6. Experimental
With the help of bio-chemistry and molecular biology, signifi-
cant progress has been made in the antiviral action mechanism
studies. At present, we have great understanding about pathogenic
mechanism of plant virus and disease resistance mechanism from
plant host and pharmacological mechanism of some commercially
employed antiviral reagents. Some studies have revealed that the
plant defense enzymes10–12 and some signal transduction path-
ways mediated by SA and PR can correlate SAR and induce expres-
sion by antiviral agents thereby playing an important role in
antiviral action mechanism.13–17 Other studies showed that small
natural molecules possessed inhibition activity of plant virus pro-
liferation by specially binding with sub-components of virus
against virus nucleic acid or protein.18 Our results indicate that
compound 4l induced up-regulation of PR-1a gene, the down-
stream molecule of SA-mediated signal transduction pathway,
from 3rd day to 5th day, the corresponding ratio in 4l-treated to-
bacco leaf was four times as large as that in TMV-treated tobacco
leaf on the 5th day after inoculation. Meanwhile, during 1st day
to 7th day, the activity increase of POD and SOD was noticed in
compound 4l-treated, without CK-treated and TMV-treated. So
we assumed that compound 4l possessed the capacity to induce
disease resistance. Moreover, using ultraviolet–vis spectroscopic
and fluorescence spectroscopic methods, we found that compound
4l had affinity to TMV CP 4S and 20S protein by induction to red-
shift and fluorescence quenching phenomenon, but not to TMV
RNA. As TMV CP possessed the ability to protect TMV RNA from
digestion by ribonuclease and then help the formation of normal
virus particle, antiviral activity of compound 4l was also thought
to be associated with affinity towards TMV CP. So we infer that
the antiviral action mechanism might be through a multiply action
mode for compound 4l. Future research would be focused on the
study of gene regulation by Northern blot and real-time PCR meth-
ods such as pathogenesis-related gene family member, determina-
tion of SA content by isolation, extraction and HPLC methods,
studies on various molecular levels in SA signaling pathway by
gene knock-out and gene knock-in technology, and ascertaining
of molecular target sites. Meanwhile, capillary electrophoresistion
mode between compound 4l and TMV CP, the site-directed muta-
genesis technology was used to construct the CP peptide contain-
ing anticipated amino acids in advance, and analysis of the
6.1. Analysis and 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 VEC-
TOR22 spectrometer in KBr disks. 1H and 13C NMR (solvent DMSO-
d6) spectra were recorded on a JEOL-ECX 500 NMR spectrometer at
room temperature using TMS as an internal standard. Elemental
analysis was performed on an Elementar Vario-III CHN analyzer.
The reagents were all of analytical grade or chemically pure. Ana-
lytical TLC was performed on silica gel GF254. Column chromato-
graphic purification was carried out using silica gel. 1-aryl-3-
methyl-1H-4-pyrazoleylformaldehydes and 1-(4-chlorophenyl)-3-
methyl-1H-4-pyrazolyl-form-aldehydes were prepared according
to literature method as described.19–21 Intermediates 1-aryl-3-
methyl-5-chloro-1H-4-pyrazolyl-formaldehydes 1, 2 and 3 were
prepared according to the reported methods.22
6.2. Preparation of pyrazolaldoxime ester derivatives (4a–4n)
A 100 mL round-bottomed flask equipped with a magnetic stir-
rer was charged with 3 (1.5 mmol) and dichloromethane (50 mL)
and pyridine (0.012 mol). The flask was stirred at room tempera-
ture for 10 min, and then was added drop wise acyl chloride
R3COCl (0.012 mol) dissolved in 20 mL dichloromethane over a
period of 0.5 h and refluxed for 4 h. The mixture was cooled and
washed with 5% Na2CO3 solution and distilled water, dried over
anhydrous MgSO4. The solvent was removed and the crude product
was purified by chromatography (petroleum ether/ethyl acetate,
2:1) and recrystallized from ethanol to give compound 4 (see the
Supporting Information).
6.2.1. 1-Phenyl-3-methyl-5-(4-methylphenylthio)-4-
pyrazolaldoxime acetate (4a)
White crystal, mp 90–91 °C, yield, 81%; IR (KBr, cmꢀ1
) m: 3019
(Ar-H), 2975, 923 (CH3 + CH2), 1775 (C@O), 1616 (C@N), 1499 (Ar
skeleton vibration), 1194 (C@N–N), 801 (p-disubstituted benzene),
662 (C–S–C); 1H NMR (DMSO-d6, 500 MHz, ppm) d: 8.45 (s, 1H,
CH–H), 7.44–7.49 (m, 5H, Ph-H), 7.06 (d, J = 8.05 Hz, 2H, S–Ph-
3,5-H), 6.88 (q, J = 8.00 Hz, 2H, S–Ph-2,6-H), 6.87 (t, J = 8.05 Hz,

9706
G. Ouyang et al. / Bioorg. Med. Chem. 16 (2008) 9699–9707
J = 9.16 Hz 2H, S–Ph-3,5-H); 3.38 (s, 3H, O@C–CH3–H), 2.2 (s, 3H,
pyrazole-CH3–H), 2.1 (s, 3H, Ph–CH3–H); 13C NMR (DMSO-d6,
125 MHz, ppm) d: 168.9, 149.8, 149.2, 138.7, 137.3, 135.2, 130.7,
130.5, 129.4, 129.2, 128.1, 126.1, 116.9, 20.9, 19.9, 14.9; Anal. Calcd
for C20H19N3O2S (365.0): C 65.47%, H 5.20%, N 11.50%; found: C
66.55%, H 5.12%, N 10.54%.
plate in PCR. The primers for PCR amplification are shown in Table
3. The PCR was performed in Tris–HCl buffer (10 mM, pH 8.3), KCl
(50 mM), MgCl2 (1.8–2.0 lL), dNTPs (0.02 lM), primers (0.04 lM),
DNA polymerase (1 U). PCR amplification steps consisted of a pre-
liminary denaturation step at 94 °C for 1 min, followed by 35 cycles
at 94 °C for 40 s, at 58 °C for 40 s and at 72 °C for 50 s on icycle of
BioRad. PCR products were separated on 1.5% agarose gel in 0.5ꢁ
TBE buffer and visualized under UV light after staining with ethi-
dium bromide.32
6.3. Crystal structure determination
In order to study the single-crystal structure (Figs. 1 and 2), X-ray
intensity data were recorded on a Rigaku Raxis-IV diffraction meter
6.7. Semi-quantity PCR for expression of gene
using graphite monochromatic MoKa radiation (k = 0.71073 Å). In
the range 1.88° 6 h 6 27.44°, 2498 independent reflections were ob-
tained. Intensities were corrected for Lorentz and polarization ef-
fects and empirical absorption, and all data were corrected using
SADABS program.23 The structure was solved by direct methods
SHELXS-97 program.24 All the non-hydrogen atoms were refined
on F2 anisotropically by full-matrix least squares method. The
hydrogen atoms were located from the difference Fourier map, but
theirpositionswerenotrefined. Thecontributionsof thesehydrogen
atoms were included in structure-factor calculations. The final least-
In order to assess relative expression levels of target gene in
water-treated tobacco and 4l and TMV-treated tobacco and tobac-
co by inoculated TMV only, semi-quantity PCR consisting of 20 cy-
cles (within the logarithmic range of amplification of gene) with
putting primer of b-actin serving as an internal reference gene
was employed for the study. The amplified products were analyzed
on a 1.5% agarose gel by the method of Mohamed.33
6.8. The relative quantification real-time PCR for expression of
the target gene
square cycle gave wR = 0.1358, R = 0.0517 for 4595 reflection with
2
I > 2
r
(I); the weighting scheme, w ¼ 1=½r2ðF02Þ þ ð0:00805PÞ þ
0:0458Pꢂ, where P ¼ ½ðF20Þ þ 2F2c ꢂ=3. The max and min difference
The relative quantification real-time PCR was carried out with
iCycler IQ according to manufacturer’s protocol with primer of b-
actin serving as an internal reference gene. Precautions were taken
to ascertain reliable quantitative results: log-linear dilution curves
were performed with primers for the target gene as well as with
primers for the b-actin. Reactions performed without reverse
transcriptase or without template did not result in any product.
By following PCR, 110 steps for melt curve analysis were com-
pleted in 10 s at temperature ranging from 40 to 95 °C. The ampli-
fication efficiency was 95–99% for PR-1a and PR-5 gene,
respectively (the standard curve figures are not shown). Each tar-
get gene peak was assigned an arbitrary quantitative value corre-
lated to the b-actin gene peak, according to the formula
peaks and holes were 0.537 and ꢀ0.204 e Aꢀ3
, respectively.
s = 1.101. Atomic scattering factors and anomalous dispersion cor-
rections were taken from International Table for X-ray Crystallogra-
phy.25 Crystallographic data (excluding structure factors) for the
structure have been deposited with the Cambridge Crystallographic
Data Center as supplementary publication No. CCDC-696918.
6.4. Protection and inactivation and cure effect of compound
against TMV in vivo
Purification of TMV was assessed by Gooding’s method, as de-
scribed in the Supporting Information.26 The bioactivity assay for
protection and inactivation and cure effect was assessed according
to Li’s method (see the Supporting Information).27
DDCT =
D
CT (test)
ꢀ
DCT (calibrator), CT being the cycle threshold. Rates
of stimulation of RNA expression were calculated from the
DCT val-
ues at various time points.34
6.5. Determination of PAL and POD and SOD activity
6.9. The primary spectroscopic study of compound 4l for TMV
CP
The leaf samples were homogenized in the 2-mercaptoethanol-
boric acid buffer (5 mM, pH 8.8) on the ice bath and centrifuged.
The supernatant was used for experiment. PAL and POD and SOD
activities were determined by He’s method,28 Polle’s method,29
Beauchamp’s method,30 respectively (see the Supporting
Information).
Owing to the presence of tryptophan and tyrosine, protein can
emit fluorescence. Therefore, the fluorescence method has some-
times been employed to study the interaction between protein and
small drug molecules, the latter being known as fluorescence
quencher35,36 as it can quench the intrinsic fluorescence from the
protein. It could be seen that TMV CP containing 3 tryptophan and
5 tyrosine is constituted from 155 amino acids from NCBI protein
AAD20291. TMV CP was isolated by acetic acid methods.37 TMV CP
was dissolved in 10 mM phosphate potassium buffer, pH 7.1,
50 mM NaCl. At the concentration of 0.01% of 4l and 0.65 mg/mL of
TMV CP, the interaction between the 4l and TMV CP ceased to exist
and a full spectrum scan was made for the samples in the range
200–600 nm wavelength with ultraviolet spectroscopy. Using fluo-
rescence spectroscopy method, at the resolution of TMV CP 4S and
6.6. RT-PCR assay
Trizol kit was used according to the standard protocol for total
RNA isolation. Prior to RT-PCR, the total RNA samples were treated
with DNase I for 10 min and quantified by spectrophotometer and
identified by agarose gel electrophoresis.31 cDNA was synthesized
with Oligo (dT)18 at the 30 end of mRNA as a primer. Total RNA
(1 lg) was used for temple of first-strand cDNA synthesis using ex-
tend reverse transcriptase. Reverse transcription was carried out at
37 °C for 1 h. The single-stranded DNA mixture was used as tem-
20S protein at concentration of 1.32 lmol/L, a full spectrum scan
to the samples was made in 280–400 nm of wavelength range at
278 nm excitation wavelength.
Table 3
Sequences of gene-specific primers used in RT-PCR analysis.
Gene
family
Accession 50 primer
No.
30 primer
6.10. The determination of quenching constants
b-Actin U60495
50-gacatgaaggaggagcttgc-30
50-atcatggatggctggaagag-30
The course of fluorescence quenching was divided into the dy-
namic quenching and the static quenching.35 The dynamic quench-
ing involves the interaction between fluorescence quencher and
PR-1a
PR-5
X12737
X03913
50-caatacggcgaaaacctagctga -30 50-cctagcacatccaacacgaa-30
50-gcttccccttttatgccttc-30
50-cctgggttcacgttaatgct-30

G. Ouyang et al. / Bioorg. Med. Chem. 16 (2008) 9699–9707
9707
the excited molecular state of the substance. The action mecha-
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Acknowledgments
We are grateful for the financial support from the National Key
Project for Basic Research (2003CB114404), Key Technologies R&D
Program (2006BAE01A01-15) and the National Natural Science
Foundation of China (20562003).
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A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.09.070.