The discovery of new plant activators and scaffolds with potential induced systemic resistance: From jasmonic acid to pyrrolidone

Article abstract of DOI:10.1039/c6md00261g

Plants can develop multifaceted system resistance as a result of infection with pathogens, insects, and some specific microbes or after treatment with specific chemicals.¹ Among them, systemic acquired resistance (SAR) and induced systemic resi

Full text of DOI:10.1039/c6md00261g

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The Discovery of New Plant Activators and Scaffolds with
Potential Induced Systemic Resistance: From Jasmonic Acid to
Pyrrolidone†
Received 00th January 20xx,
Accepted 00th January 20xx
a,
c,
b
a
b
Kang Chang ‖, Yanxia Shi ‖, Jianqin Chen a, Zenghui He , Zheng Xu , Zhenjiang Zhao , Weiping
DOI: 10.1039/x0xx00000x
Zhu a, Honglin Lib, Yufang Xu a, * , BaoJu Li c,*, and Xuhong Qian a,*
www.rsc.org/
Plants can develop a multifaceted system resistance as a result of the infection of pathogen, insect, and some
specific microbes, or after treatment with specific chemicals1. Among them, systemic acquired resistance (SAR)
and induced systemic resistance (ISR)2 are the most important two types. To activate them, a large number of
chemical inducers have been exploited based on the SA pathway. However, the JA pathway was rarely investigated for
new plant activators. In this study, starting from JA and its analogs, based on molecular three-dimensional shape and
pharmacophore similarity comparison (SHAFTS), a new lead compound pyrrolidone as a new scaffold of plant activator
was predicted and a series of 1-phenyl-5-pyrrolidone-3-carboxylic acid derivatives were designed and synthesized. The
bioactivity was evaluated, most of them showed satisfying activity, especially compounds 7-11, showed almost no in vitro
activity but excellent efficacy against tested diseases, and the prediction of the virtual screening results was also
confirmed, which promoted the application of SHAFTS in the development of pesticide.
Pythium sp. etc fungal pathogens and the bacterial pathogen
Erwinia carotovora8-12 in ISR. As the most important pathway,
1. Introduction
Plants can develop a multifaceted system resistance as a result
of the infection of pathogen, insect, and some specific
microbes, or after treatment with specific chemicals1. Among
them, systemic acquired resistance (SAR) and induced systemic
resistance (ISR)2 are the most important two types. SAR is
induced by pathogen, along with the increased level of salicylic
acid (SA) and pathogen-related proteins (PR proteins). Early
experiments have demonstrated that SA accumulation and
signaling is crucial for the development of SAR3-7. In SA-
dependent SAR, plants exhibit enhanced diseases resistance
against a variety of pathogens including the fungal pathogens
Peronospora parasitica and Erisyphe sp., and the bacterial
pathogen Pseudomonas syringae etc8. In contrast to SA-
dependent SAR, ISR is induced by beneficial microorganisms,
and regulated by jasmonic acid (JA) and ethylene (ET)7, and the
JA-responsive genes, such as PLANT DEFENSIN1.2 (PDF1.2)
would be overexpressed in ISR9. Plants would show enhanced
resistance against Alternaria brassicicola, Botrytis cinerea, and
SA mediated SAR has been thoroughly investigated, and based
on its mode of action, a large number of exogenous inducers
were exploited, such as BTH13-15, INA16, probenazole17,
Tiadinil18, 19, and Isotianil. Among them, BTH is the most
successful one, and has been exploited for the control of
downy and powdery mildew13. Our group has also reported
two series of compounds, and all of which showed potent
bioactivity20, 21. Though more and more compounds have been
exploited as plant activators, most of them still work through
SA pathway, but JA mediated pathway was rarely investigated,
and the plant activators were rarely exploited from this view.
JA is the plant hormone essential for the immune response
against necrotrophic pathogens and herbivorous insects, its
analogue jasmonoyl-isoleucine (JA-Ile) was identified as the
biologically active signal1, 22-24, and coronatine (COR) which acts
as an agonist of JA-Ile has been found to strongly induce JA-
responsive genes1. Taking these into consideration, JA, JA-Ile
and COR were chosen as template molecules, with the three-
dimensional
shapes
and
pharmacophore
similarity
comparison, the SHAFTS was used for the virtual screening
(Figure 1)25, 26, SHAFTS is a hybrid 3D similarity calculation
algorithm considering both molecular shape and
pharmacophore features, which has been proved to robustly
improve the hit rate and increase the scaffold hopping ability
in virtual screening. Novel chemotypes were successfully
identified by SHAFTS targeting RSK2 as well as EGFR kinases
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respectively with potent promising inhibitory activities27, 28. In Considering the activity and synthesis, T3 was chosen as the
this study, to discover promising scaffold of plant activators, final lead compound and a series of 1-phenyl-5-pyrrolidone-3-
the MayBridge Database30, which contains more than 53,000 carboxylic acid derivatives was designed and synthesized
hit-like compounds, was searched by SHAFTS with the well- (Scheme 2), their activities as plant activators were evaluated
known plant hormones JA, JA-Ile and COR as query templates, both in vivo and in vitro against several plant diseases to
and six lead compounds were found (Scheme 1). Their confirm the prediction of the virtual screening results.
bioactivities were evaluated against three diseases (Table 1).
Figure 1. Virtual screening process
.
F
OH
O
O
O
O
O
N
N
S
N
OH
O
HO
JA
O
O
O
HO
T3
SHAFTS
T1
T2
O
Cl
Cl
O
N
O
O
S
O
NH
O
NH
HO
OH
OH
OH
S
HO
O
O
O
O
O
T6
T5
T4
COR
JA-Ile
Lead Compounds
Scheme 1. Structures of lead compounds
Scheme 3. All structures of compounds were characterized by
1H NMR, 13C NMR, and HRMS.
2. Results and discussion
2.1. Preparation of 1-phenyl-5-pyrrolidone-3-carboxylic acid
derivatives.
2.2. Bioactivity.
Firstly, the novel compounds inducing activities against seven
diseases were screened, and were shown in Table 2. Due to
the fact that positive control 50% procymidone (WP) showed
41.87% efficacy against B. cinerea, and in order to evaluate
and compare the efficacy of the novel compounds, so we
Compound 1-phenyl-5-pyrolidone-3-carboxylic acid scaffold
was synthesized via a simple solvent free approach. Compound
1-phenylpyrrolidone-3-carboxylate scaffold was synthesized
from acid by esterification with various alcohols, as shown in
2 | J. Name., 2012, 00, 1-3
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defined herein the efficacy above 40% as promising active
efficacy against the relevant disease.
To better understand the structure-activity relationship of the
activators in this chemical series, the 20 compounds were
aligned and scored by SHAFTS to the template molecule JA
(Figure 2B), the corresponding 3D molecular similarity scores
were listed in Table 3.
Table 1 In-vivo activities of the lead compounds
Efficacy (%)b
compda
C. cassiicola
C. cucumerinum
P.cubensis
42.95
74.58
64.93
67.57
44.83
41.89
60.55
64.06
41.97
44.05
48.33
34.73
45.41
52.01
70.98
80.36
6.54
2.22
0.00
0.00
3.97
0.00
28.47
66.67
T1
T2
Scheme 2. Design of target compounds
T3
T4
T5
T6
70% Mildothane(WP)
Scheme 3. Synthetic route of target conpounds
75%
chlorothalonil(WP)
50%
62.28
34.82
37.35
59.61
93.46
0
Figure 2A showed that lead T3 and template JA overlapped
well in terms of both shape and pharmacophore. Specifically,
besides the five-membered ring system, the phenyl group of
T3 also mimics the shape and hydrophobic features of the
pentenyl moiety of JA. The carbonyl group in T3 or JA on the
five- membered ring could act as hydrogen bond acceptor that
benefits the binding affinity. Moreover, the carboxyl group in
both JA and T3 may form a hydrophilic center, which is inclined
to generate salt bridge that strengthens the polar interactions.
Therefore, lead optimization was conducted by varying diverse
substituents at the hydrophobic as well as hydrophilic center
of lead T3 to obtain more potent plant activators.
dimethomorph(WP)
5% validamycin A(WP)
aThe concentration of compounds is 100 mg/L;
befficacy is the average of all repeated tests.
Table 2. In-vivo inducing activity of the target compounds
Efficacy (%)b
Number of Compounds a
MM
CC
PL
PI
RS
BC
FO
1
2
3
4
5
6
13.89
5.83
8.85
23.72
20.98
-8.2
55.07
26.42
37.03
32.08
18.63
26.95
21.38
60.69
31.03
15.66
-20.07
26.74
60.25
9.56
-7.90
-0.58
-2.08
-12.16
9.56
9.89
9.29
4.59
16.01
1.90
-3.47
13.33
8.06
25.52
46.95
-4.04
72.07
26.11
29.44
81.67
77.7
18.69
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7
100.00
98.06
48.33
100.00
79.72
6.67
75.74
89.59
52.13
83.61
41.64
-5.08
-1.64
90.49
-0.33
61.17
83.61
76.07
71.8
35.61
46.93
2.36
2.08
-3.45
3.05
2.36
-7.50
-6.16
-2.13
0.56
8
19.06
3.51
15.05
0.42
9
72.22
21.48
45.32
39.11
60.39
-2.86
3.05
10
11
12
13
14
15
16
17
18
19
26.77
28.54
25.71
19.52
39.33
-4.72
27.48
27.12
44.10
33.49
20.31
24.24
5.66
11.06
-5.90
2.85
47.59
-2.13
4.59
72.22
67.50
9.17
27.46
-14.53
78.20
2.80
2.24
-1.75
-12.39
-3.74
-14.88
-6.57
-5.74
-2.13
-2.13
26.76
-0.78
-6.16
-4.14
35.00
76.67
38.33
96.11
53.30
5.12
3.16
16.38
22.81
9.56
-1.08
20
93.61
88.06
78.69
-8.20
35.26
75.59
13.16
64.98
16.26
16.06
5.57
-6.16
BTH
46.80
19.37
50% kresoxim-methyl(WG)
98.43
75% chlorothalonil(WP)
20% bismerthlazol(WP)
50% dimethomorph(WP)
5% validamycin A(WP)
50% procymidone(WP)
70% Mildothane(WP)
90.16
67.80
96.78
83.38
41.87
75.14
aBTH represents S-methyl benzo[1,2,3]thiadiazole-7-carbothioate and was synthesized in our lab; The concentration of compounds is 100 mg/L;
bMicro-organisms used: M. melonis (MM), C. cassiicola (CC), P. syringae pv．Lachrymans (PL), P. infestans (PI), R. solani (RS), B. cinerea (BC), and F.
oxysporum (FO); efficacy is the average of all repeated tests.
In view of inducing activity against 7 diseases and SHAFTS
score of each compound, SAR (structure activity
relationship) of these 20 compounds was discussed as
follows.
Figure 2. (A): Key pharmacophore features of template JA and lead T3. (B): The superimposition of JA and 20 active compounds by SHAFTS. JA is shown
in blue sticks, while the other 20 activators are represented as gray sticks.
4 | J. Name., 2012, 00, 1-3
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Table 3. SHAFTS scores of the 20 target compounds
Whether can it achieve better efficacy if we introduced two
substituents to the meta and para position of benzene at the
same time? We compared two groups of bioactivity data:
compounds 18 vs 11, compounds 12 vs 10, both compounds in
each group had the same substituents in the para position of
benzene ring, the latter had another substituent in the meta
position of benzene ring. Compound 18 showed efficacy
against 2 diseases, comparatively, compound 11 showed
efficacy against 4 diseases. Compound 12 showed efficacy
against none of the diseases, comparatively, compound 10
O
R1
N
R2O
O
Compounds
R1
R2
SHAFTS Score
0.607
0.605
0.595
0.578
0.519
0.668
0.608
0.585
0.630
0.601
0.602
0.627
0.641
0.625
0.653
0.644
0.727
0.672
0.622
0.625
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
C6H5
H
H
4-FC6H4
4-FC6H4
4-FC6H4
CH3
CH2CH3
CH2CF3
H
H
H
H
H
showed efficacy against
2 diseases. It suggested that
4-FC6H4
2-CH3-5-FC6H3
3-F-4-CH3C6H3
4-CF3C6H4
4-CF3OC6H4
3-CF3-4-ClC6H3
3-CF3-4-BrC6H3
4-ClC6H4
introducing two proper substituents which could mimic the
methyl group of the pentenyl to the meta and para positions
of benzene at the same time could achieve a better efficacy.
In general, based on the molecular alignment by SHAFTS, the
pentenyl group of JA was similar to half of the benzene ring in
3D shape, and the terminal methyl of JA pentenyl was similar
to the substituent on the meta or para position of benzene
ring, and this provided constructive clues for lead modification.
R2 was unable to superimpose with the corresponding moiety
of JA, this was why R2 transformation had no effect. The
bioactivity assay had also confirmed the results of virtual
screening: when proper groups, which could mimic the methyl
moiety of JA , were introduced to the meta or para position of
H
H
H
3-ClC6H4
3-ClC6H4
CH2CF3
3,5-Cl2C6H3
3,5-Cl2C6H3
2-CH3-4-BrC6H3
4-BrC6H4
4-C6H4COOCH3
4-(CH3)3CC6H4
H
H
H
H
H
H
Firstly, we focused on the modification of R2. SHAFTS scores of
compounds 2, 3, 4 and were 0.605, 0.595, 0.578 and 0.519
benzene ring, the efficacy was excellent, such as
10, 11 (trifluoromethyl) and (trifluoromethoxy) all showed
potent efficacy especially against M. melonis and C. cassiicola
7 (methyl), 8,
5
9
respectively, significantly demonstrated a downward trend.
The experimental results of these compounds also showed low
efficacy, most of them only showed efficacy against one out
.
And some of them showed better efficacy than BTH and no
efficacy against C. cassiicola. Compound 11 showed good
efficacy against four diseases with broad-spectrum as BTH.
seven test diseases. Compound
3 even showed no activity. So
both SHAFTS scores and experiment data confirmed that
Although compound
8 showed efficacy against three pathogen
modification of R2 abated efficacy.
diseases, less than BTH, its efficacy against M. melonis and C.
cassiicola was better than BTH, also had the potential for
further application.
For R1, compounds 9, 13, 14, 16, 17, 18, 19 and 20 showed
efficacy against at least two diseases with SHAFTS scores
higher than 0.620 that significantly exceed other compounds.
Their common structure characteristic was that there was a
proper group substituted at the meta or para position of the
benzene group, which could simulate the terminal methyl
group of the pentenyl in JA. However, the score of compound
Table 4. In-vitro anti-microbial activity of the target
compounds
Efficacy (%)b
compda
PI
CC
MM
RS
2
was 0.605, which only showed efficacy against one disease.
8
4.30
8.94
25.17
10.69
-0.89
-0.30
2.13
6.78
That may because compared with methyl group, the fluorine
group was too small to occupy the position.
10
19
8.28
12
5.35
20
-0.89
4
3.29
48
BTH
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50%
80
3. Conclusions
dimethomorph(WP)
75%
21
In this paper, a series of 1-phenyl-5-pyrrolidone-3-carboxylic
acid derivatives has been designed and synthesized with the
help of virtual screening using SHAFTS, and their inducing
activities were evaluated by a broad-spectrum of pathogens,
including bacteria, fungi and oomycetes. In addition, the
quantitative Real Time PCR experiment showed that the
pyrrolidone derivatives could induce the overexpression of
defense gene PDF1.2 in JA-dependent ISR which met the
expectation of the original design. Among them, the
compounds 7-11 which were substituted with proper groups
at the meta and para position of the benzene were the most
potent candidates, especially 11, showed efficacy against four
diseases in our experiments, which was with great potential to
be exploited as an excellent plant activator. Besides, these
results were consistent with the results of virtual screening,
and confirmed the application of SHAFTS used in the
development of plant activators.
chlorothalonil(WP)
50% kresoxim-
methyl(WG)
5% validamycin
A(WP)
71
50
aBTH represents S-methyl benzo[1,2,3]thiadiazole-7-carbothioate and was
synthesized in our lab; The concentration of compounds is 100 mg/L;
bMicro-organisms used: M. melonis (MM), C. cassiicola (CC), P. infestans (PI),
R.solani (RS); efficacy is the average of all repeated tests.
Next, 8, 10 and 19 with good in vivo efficacy were selected to
screen their antimicrobial activity against four diseases in vitro.
The results can be seen in Table 4, as expected, all three
compounds had no direct antimicrobial activity against P.
infestans
,
C. cassiicola
,
M. melonis and R. solani.
Comparatively, the relative positive controls showed good
efficacy against those four diseases in vitro, respectively.
In order to directly reflect the bioactivity of pyrrolidone
derivatives, the expression of PR1 and PDF1.2 which
respectively are key defense genes in SAR and ISR were
determined by quantitative Real Time PCR after the treatment
of compound 11. The results were shown in Figure 3.
4. Experimental section
4.1. Discovery of lead compounds.
4.1.1. Confirmation of template molecules.
Reported plant hormones JA, JA-Ile and COR were selected as
template molecules for SHAFTS in the virtual screening
procedure. Due to the lack of bioactive conformational
information of those plant hormones, LigPrep31 module in
Maestro 9.0 was applied to obtain the lowest energy
conformation for each of the three template molecules.
4.1.2. Preparation of Database.
Commercially available compounds database MayBridge was
chosen for virtual screening by SHAFTS. According to the
characteristics of SHAFTS that utilize a semi-rigid strategy to
evaluate molecular flexibility, conformers of the compounds were
prepared in advance. Molecules with inorganic atoms were
removed. LigPrep module of Maestro 9.0 was then used to
generate the initial 3D conformation for every compound in the
database by adding hydrogen as well as predicting both the
ionization and the tautomerization states within the default pH
range of 7.0±2.0. Multiple conformers for each molecule in the
MayBridge database were generated by Cyndi29, which is an in
house molecular conformation generation program based on multi-
objective evolution algorithm, with default parameters.
Figure 3. The expression of PDF1.2 and PR1
.
According to the results, like MeJA, compound 11 could induce
the overexpression of PDF1.2 but have no inducing effect on
the expression of PR1. However, in contrary, BTH as an analog
of SA, could induce the overexpression of PR1 but did not
activate the expression of PDF1.2. From the results above, we
could conclude that like MeJA, the pyrrolidone derivatives
induced the disease resistance mainly through the JA-
dependent ISR, and BTH induced the disease resistance by SA-
dependent SAR, which was consistent with the literature
reported. This demonstrated that the pyrrolidone derivatives
indeed acted as plant activators and may participate in the JA
signal transduction or share the same signal pathway with JA.
The detailed mechanism of pyrrolidone derivatives will be
conducted in the next.
4.1.3. Virtual screening and scaffold hopping.
Using molecules JA, JA-Ile and COR as the query templates, virtual
screening was carried out against the prepared MayBridge
conformational library by SHAFTS. The top-ranked 500 compounds
were retained, and six molecules were selected for in vivo
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bioactivity assay after further visual inspection taking both similarity 36.89, 34.19, 17.31. HRMS (ESI) calcd for C12H11FNO3 [M-H]-:
scores and structural diversity into account. Through the analysis of 236.0723, found 236.0722.
the bioactivity as well as the synthesis of the six hits, T3 was chosen
1-(4-methyl-3-fluorophenyl)-5-pyrrolidone-3-carboxylic acid
(7). White crystal. Yield, 94%. 1H NMR (400 MHz, DMSO) δ
as the final lead for further optimization.
4.2. Synthesis of the compounds 1-2012
Compounds 1, 2, 6-13, 15-20 were synthesized via a solvent free
reaction. Compounds 3-5 and 14 were synthesized from and 13
.
( ppm ): 12.80 (s, 1H), 7.60 (d, J = 12.8 Hz, 1H), 7.32-7.25 (m,
2H), 4.06-3.93(m, 2H), 3.39-3.33 (m, 1H), 2.83-2.68 (m, 2H),
2.21 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ174.58, 172.47,
161.88, 159.49, 138.92, 138.81, 131.85, 131.79, 120.00, 119.83,
115.14, 106.72, 106.45, 50.37, 35.70, 35.44, 14.12, 14.09.
HRMS (ESI) calcd for C12H11FNO3 [M-H]-: 236.0723, found
236.0722.
2
via two steps (Scheme 3). General procedures for the synthesis of
compounds 1-20 were described as below:
The NMR spectra were recorded on a Brucker AM-400 (400 MHz)
spectrometer with DMSO-d6 or CDCl3 as the solvent and TMS as
internal standard. Chemical shifts are reported in δ (parts per
million) values. The MS spectra were measured with a HRMS
Micromass GCT CA 055 spectrometer. All solvents and chemicals
were of reagent grade and used without further purification.
1-(4-trifluoromethyl-phenyl)-5-pyrrolidone-3-carboxylic acid
1
(8). White crystal. Yield, 90%. H NMR (400 MHz, DMSO-d6) δ
( ppm ): 12.78 (s, 1H), 7.90 (d, J = 8.8 Hz, 2H), 7.73 (d, J = 8.4 Hz,
2H), 4.14-4.01 (m, 2H), 3.43-3.35 (m, 1H), 2.88-2.73 (m, 2H);
13C NMR (100 MHz, DMSO-d6): δ 174.49, 173.09, 142.92,
126.36, 126.32, 126.09, 124.51, 124.19, 123.39, 119.59, 50.21,
49.05, 35.73, 35.50; HRMS (ESI) calcd for C12H9F3NO3 [M-H]-:
272.0535, found 272.0535.
4.2.1. General procedure for the synthesis of compounds 1, 2, 6-
13, 15-20.
Itaconic acid (10 mmol) and corresponding substituted
arylamine (10 mmol) were added into a round bottomed flask.
The dry reactants were maintained at 100 oC. The mixture was
monitored by TLC to the end of reaction. After that, the mass
was cooled in ice-cold water and then dissolved in sodium
bicarbonate solution, filtered, and the filtrate was acidified
with ice-cold dilute hydrochloric acid. The liquid was filtered
and the residue was collected to give the corresponding
carboxylic acid. The data for compounds 1, 2, 6-13, 15-20 were
shown below.
1-(4-trifluoromethoxy-phenyl)-5-pyrrolidone-3-carboxylic
acid (9). White crystal. Yield, 95%. 1H NMR (400 MHz, DMSO-d6)
δ ( ppm ): 7.78 (d, J = 9.2 Hz, 2H), 7.38 (t, J = 8.8 Hz, 1H), 4.10-
3.97 (m, 2H), 3.41-3.35 (m, 1H), 2.84-2.69 (m, 2H); 13C NMR
(100 MHz, DMSO-d6): δ 174.55, 172.55, 144.59, 138.70, 121.97,
121.85, 121.31, 50.38, 35.55, 35.53. HRMS (ESI) calcd for
C12H9F3NO4 [M-H]-: 288.0484, found 288.0485.
1-(3-trifluoromethyl-4-chlorophenyl)-5-pyrrolidone-3-
carboxylic acid (10). White crystal. Yield, 89%. 1H NMR (400
MHz, DMSO-d6) δ ( ppm ): 12.84 (s, 1H), 8.30 (s, 1H), 7.85 (d, J
= 9.2 Hz, 1H), 7.73(t, J = 9.2 Hz, 1H), 4.14-4.01 (m, 2H), 3.42-
3.34 (m, 1H), 2.87-2.77 (m, 2H); 13C NMR (100 MHz, DMSO-d6):
δ 174.40, 173.16, 138.81, 132.46, 127.25, 126.95, 125.32,
125.31, 124.50, 124.44, 121.78, 118.58, 118.52, 118.47, 118.41,
50.17, 35.57, 35.44; HRMS (ESI) calcd for C12H10ClF3NO3 [M+H]+:
308.0301, found 308.0302.
1-phenyl-5-pyrrolidone-3-carboxylic acid (1). White crystal.
Yield, 90%.1H NMR (400 MHz, DMSO-d6): δ 7.65 (d, J = 8.0 Hz,
2H), 7.38 (t, J = 8.4 Hz, 2H), 7.15 (t, J1 = 7.2 Hz, 1H), 4.08-3.95
(m, 2H), 3.38-3.32(m, 1H), 2.83-2.67 (m, 2H); 13C NMR (100
MHz, DMSO-d6): δ 174.69, 172.28, 139.58, 129.15, 124.56,
119.91, 50.40, 35.69, 35.63. HRMS (ESI) calcd for C11H10NO3
[M-H]-: 204.0661, found 204.0662.
1-(4-fluorophenyl)-5-pyrrolidone-3-carboxylic acid (2). White
1
crystal. Yield, 91%. H NMR (400 MHz, DMSO-d6): δ 7.69-7.65
1-(3-trifluoromethyl-4-bromophenyl)-5-pyrrolidone-3-
carboxylic acid (11). White crystal. Yield, 91%. 1H NMR (400
MHz, DMSO-d6) δ ( ppm ): 12.83 (s, 1H), 8.29 (s, 1H), 7.88 (d, J
= 8.4 Hz, 1H), 7.71(d, J = 8.8 Hz, 1H), 4.14-4.01 (m, 2H), 3.42-
3.34 (m, 1H), 2.86-2.72 (m, 2H); 13C NMR (100 MHz, DMSO-d6):
δ 174.40, 173.19, 139.26, 135.86, 129.06, 128.75, 124.61,
124.48, 121.89, 118.81, 118.75, 118.69, 112.99, 50.13, 35.60,
35.43. HRMS (ESI) calcd for C12H8BrF3NO3 [M-H]-: 349.9640,
found 349.9643.
(m, 2H), 7.22 (t, J = 8.8 Hz, 2H), 4.07-3.94 (m, 2H), 3.38-
3.32(m,1H), 3.94-3.32 (m, 1H), 2.82-2.66 (m, 2H); 13C NMR
(100 MHz, DMSO-d6): δ 144.64, 172.19, 160.20, 157.80, 136.01,
122.02, 121.94, 115.89, 115.66, 50.60, 35.58, 35.48. HRMS (ESI)
calcd for C11H9FNO3 [M-H]-: 222.0566, found 222.0570.
1-(2-methyl-5-fluorophenyl)-5-pyrrolidone-3-carboxylic acid
1
(6). White crystal. Yield, 93%. H NMR (400 MHz, DMSO-d6) δ
( ppm ): 12.76 (s, 1H), 7.31 (t, J = 8.0 Hz, 1H), 7.16-7.08 (m,
2H), 3.93 (t, J = 9.2 Hz, 1H), 3.80 (q, J=5.2 Hz, 1H), 3.44-3.36 (m,
1H), 2.75-2.59 (m, 2H), 2.10 (s, 3H); 13C NMR (100 MHz, DMSO-
d6): δ 174.77, 171.88, 161.98, 159.57, 139.07, 138.98, 132.40,
132.31, 132.09, 132.06, 114.86, 114.66, 114.03, 113.81, 52.02,
1-(4-chloro-phenyl)-5-pyrrolidone-3-carboxylic acid (12).
White crystal. Yield, 96%. 1H NMR (400 MHz, DMSO-d6) δ
( ppm ): 7.70 (d, J = 8.8 Hz, 2H), 7.43 (d, J = 8.8 Hz, 2H), 4.07-
3.94 (m, 2H), 3.40-3.32 (m, 2H), 2.83-2.68 (m, 1H); 13C NMR
(100 MHz, DMSO-d6): δ 174.57, 172.49, 138.47, 129.02, 128.29,
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121.38, 50.31, 35.60, 35.49. HRMS (ESI) calcd for C11H11ClNO3 1-(4-tertiarybutylphenyl)-5-pyrrolidone-3-carboxylic acid (20).
[M+H]+: 240.0427, found 240.0425.
White crystal. Yield, 93%. 1H NMR (400 MHz, DMSO-d6) δ
( ppm ): 12.74 (s, 1H), 7.55 (d, J = 8.8 Hz, 2H), 7.39 (d, J = 8.4 Hz,
2H), 4.04 (t, J = 9.6 Hz, 1H), 4.06-3.93 (m, 1H), 3.39-3.31 (m,
1H), 2.91-2.65 (m, 2H), 1.28 (s, 9H); 13C NMR (100 MHz, DMSO-
d6): δ174.71, 171.99, 146.96, 137.07, 125.79, 119.77, 50.41,
35.66, 35.59, 34.52, 31.60. HRMS (ESI) calcd for C15H20NO3
[M+H]+: 262.1443, found 262.1445.
1-(3-chlorophenyl)-5-pyrrolidone-3-carboxylic
acid
(13).
White crystal. Yield, 95%. 1H NMR (400 MHz, DMSO-d6) δ
( ppm ): 7.87 (d, J = 6.4 Hz, 1H), 7.55 (t, J = 8.0 Hz, 1H), 7.41 (q,
J = 8 Hz, 1H), 7.20 (t, J = 7.2 Hz, 1H), 4.10-3.98 (m, 2H), 3.35 (s,
1H), 2.86-2.69 (m, 2H); 13C NMR (100 MHz, DMSO-d6): δ 174.54,
172.78, 140.94, 133.61, 130.86, 124.22, 119.40, 118.03, 50.32,
35.70, 35.52. HRMS (ESI) calcd for C11H11ClNO3 [M+H]+:
240.0427, found 240.0425.
4.2.2. General procedure for the synthesis of compounds 3-5 and
14.
1-(3,5-dichlorophenyl)-5-pyrrolidone-3-carboxylic acid (15).
White crystal. Yield, 94%. 1H NMR (400 MHz, DMSO-d6) δ
( ppm ): 7.77 (s, 2H), 7.37 (s, 1H), 4.10-3.96 (m, 2H), 3.38-
3.32(m, 1H), 2.86-2.70 (m, 2H); 13C NMR (100 MHz, DMSO-d6):
δ174.35, 173.24, 141.69, 134.63, 123.66, 117.88, 50.30, 35.70,
35.41. HRMS (ESI) calcd for C11H10Cl2NO3 [M+H]+: 274.0038,
found 274.0036.
To 100 mg (0.54 mmol) compound
2 suspended in 5 mL CH2Cl2
was added 0.5 mL (0.27 mmol) oxalyl chloride slowly. The
mixture was stirred at room temperature for 1 h. Solvent was
removed by vacuum distillation, the residue was dissolved in 2
mL CH2Cl2 and dropwise to a mixture containing 0.6 mmol
corresponding alcohol, 1.8 mmol triethylamine and 5 mL
CH2Cl2. The mixture was stirred at room temperature and
monitored by TLC to the end of reaction. Then, the solvent was
removed by vacuum distillation, the residue was recrystallized
from ethyl acetate and hexane to give the target compounds.
The data for compounds 3-5 and 14 were shown below.
1-(3,4-dichlorophenyl)-5-pyrrolidone-3-carboxylic acid (16).
White crystal. Yield, 94%. 1H NMR (400 MHz, DMSO-d6) δ
( ppm ): 12.82 (s, 1H), 8.03 (s, 1H), 7.63 (s, 2H), 4.09-3.96(m,
2H), 3.40-3.32 (m, 1H), 2.85-2.70 (m, 2H); 13C NMR (100 MHz,
DMSO-d6): δ174.45, 172.94, 139.50, 131.57, 130.97, 126.12,
121.08, 119.63, 50.25, 35.61, 35.42. HRMS (ESI) calcd for
C11H10Cl2NO3 [M+H]+: 274.0038, found 274.0037.
Methyl 1-(4-fluorophenyl)-5-pyrrolidone-3-carboxylate (3).
Yellow solid. Yield, 45%. 1H NMR (400 MHz
，DMSO-d6) δ
( ppm ): 7.58-7.55 (m, 2H), 7.08 (t, J = 8.4 Hz, 2H), 4.14-4.01 (m,
2H), 3.803(s,3H), 3.43-3.35 (m, 1H), 2.99-2.85 (m, 2H); 13C NMR
(100 MHz, DMSO-d6): δ 173.56, 171.94, 160.24, 135.91, 122.09,
122.01, 115.90, 115.68, 52.66, 50.40, 35.36, 35.28. HRMS (ESI)
calcd for C12H13FNO3 [M+H]+: 238.0879, found 238.0880.
1-(2-methyl-4-bromophenyl)-5-pyrrolidone-3-carboxylic acid
(17). White crystal. Yield, 89%. 1H NMR (400 MHz, DMSO-d6) δ
( ppm ): 12.74 (s, 1H), 7.62 (s, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.18
(d, J = 8.4 Hz, 1H), 3.90 (t, J = 8.8 Hz, 1H), 3.80-3.76 (m, 1H),
3.76-3.43 (m, 1H), 2.76-2.58 (m, 2H), 2.13 (s, 3H); 13C NMR
(100 MHz, DMSO-d6): δ174.83, 171.95, 138.84, 137.28, 133.63,
129.92, 129.17, 120.63, 52.09, 36.88, 34.21, 17.71. HRMS (ESI)
calcd for C12H13BrNO3 [M+H]+: 298.0079, found 298.0078.
Ethyl 1-(4-fluorophenyl)-5-pyrrolidone-3-carboxylate (4).
Yellow liquid. Yield, 20%. 1H NMR (400 MHz, DMSO-d6) δ
( ppm ): 7.55-7.51 (m, 2H), 7.03 (t, J = 8.8 Hz, 2H), 4.20 (q, J =
7.2 Hz, 2H), 4.08-3.96 (m, 2H), 3.37-3.29 (m, 1H), 2.93-2.79 (m,
2H), 1.28 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, DMSO-d6): δ
172.35, 171.48, 160.93, 158.50, 134.85, 134.82, 122.02, 121.94,
115.75, 115.53, 61.66, 50.60, 35.88, 35.30, 14.16. HRMS (ESI)
calcd for C13H15FNO3 [M+H]+: 252.1036, found 252.1037.
1-(4-bromophenyl)-5-pyrrolidone-3-carboxylic
acid (18).
White crystal. Yield, 92%. 1H NMR (400 MHz, DMSO-d6) δ
( ppm ): 12.81 (s, 1H), 7.64 (d, 2H), 7.55 (d, 2H), 4.06-3.94(m,
2H), 3.39-3.32 (m, 1H), 2.83-2.68 (m, 2H); 13C NMR (100 MHz,
DMSO-d6): δ 174.57, 172.52, 138.90, 131.94, 121.71, 116.39,
50.25, 35.64, 35.49. HRMS (ESI) calcd for C12H11BrNO3 [M+H]+:
283.9922, found 283.9926.
Trifluoroethyl 1-(4-fluorophenyl)-5-pyrrolidone-3-carboxylate
1
(5). Yellow liquid. Yield, 30%. H NMR (400 MHz
，DMSO-d6) δ
( ppm ): 7.54 (q, J = 4.8 Hz, 2H), 7.08 (t, J = 8.4 Hz, 2H), 4.58 (dd,
J1 = 2.8 Hz, J2 = 5.2 Hz, 2H), 4.09 (q, J = 2.8 Hz, 2H), 3.49 (t, J = 8
Hz, 1H), 2.94 (d, J = 8.4 Hz, 2H); 13C NMR (100 MHz, DMSO-d6):
δ 175.31, 171.82, 171.08, 170.88, 161.20, 158.76, 134.60,
134.40, 134.37, 124.00, 122.38, 122.30, 122.27, 122.19, 121.25,
115.92, 115.83, 115.69, 115.61, 61.60, 61.31, 61.24, 60.96,
60.87, 60.50, 50.68, 50.38, 35.60, 35.51, 35.17, 34.91. HRMS
(ESI) calcd for C13H10F4NO3 [M+H]+: 306.0753, found 306.0748.
1-(4-carboxylphenyl)-5-pyrrolidone-3-carboxylic acid (19).
White crystal. Yield, 89%. 1H NMR (400 MHz, DMSO-d6) δ
( ppm ): 12.75 (s, 1H), 7.79 (d, J = 7.6 Hz, 1H), 7.65 (t, J = 8.0 Hz,
1H), 7.44-7.38(m, 2H), 4.05 (t, J = 8.8 Hz, 1H), 3.96-3.92 (m, 1H),
3.74 (s, 3H), 3.45-3.37 (m, 1H), 2.66-2.63 (m, 2H); 13C NMR
(100 MHz, DMSO-d6): δ 174.50, 172.57, 166.67, 137.75, 133.36,
130.76, 128.36, 127.41, 126.87, 52.54, 52.22, 36.61, 34.38.
HRMS (ESI) calcd for C13H14NO5 [M+H]+: 264.0872, found
264.0869.
Trifluoroethyl 1-(3-chlorophenyl)-5-pyrrolidone-3-carboxylate
1
(14). Yellow liquid. Yield, 25%. H NMR (400 MHz, DMSO-d6) δ
( ppm ): 7.67 (d, J = 1.2 Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.31 (q,
8 | J. Name., 2012, 00, 1-3
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ARTICLE
J = 8.0 Hz, 1H), 7.17 (d, J = 8.0 Hz, 1H), 4.59 (t, J = 8.4 Hz, 2H), The disease index and efficacy were calculated according to
4.14-4.07 (m, 2H), 3.55-3.46 (m, 1H), 2.96 (d, J = 8.8 Hz, 2H); the equations (1) and (2) respectively.
13C NMR (100 MHz, DMSO-d6): δ175.70, 172.53, 171.71,
Ic − Ia
Ic
(Y×C)
170.76, 139.52, 139.34, 134.69, 134.62, 130.04, 129.99, 125.42,
125.25, 120.40, 120.37, 118.20, 61.63, 61.27, 60.90, 60.53,
50.40, 50.08, 35.54, 35.38, 35.30, 35.08. HRMS (ESI) calcd for
C13H12ClF3NO3 [M+H]+: 322.0458, found 322.0460.
∑
Efficacy =
I =
×100
N ×9
(1)
(2)
Where I is the disease index, Y is the leaf number of each level,
C is the level number of the corresponding lesion, N is the
number of the total investigated leaves. Ic is the disease index
of the control plants, and Ia is the disease index of the treated
plants.
4.3. Biological Assay.
The inducing activities of the target compounds were
evaluated according to the following procedures which
referred to the work we did before20, 21
.
4.3.2. In-vitro assay.
4.3.1. In-vivo screening.
During the in-vitro assay, M. melonis, C. cassiicola, P. infestans
and R. solani were used. All test compounds were dissolved in
N,N-dimethylformamide (DMF) to prepare a stock solution
with a concentration of 1000 mg/L. Then, 1 mL stock solution
and 9 mL potato dextrose agar (PDA) culture media were
mixed thoroughly in a petri dish to prepare a working solution
of 100 mg/L. The PDA medium was thoroughly mixed by
turning around the petri dish in the sterilized operation desk 5
times to scatter the compounds in PDA evenly. After
solidification, the micro-organism was inoculated on the plate
During the in-vivo screening of lead compounds, Corynespora
cassiicola, Cladosporium cucumerinum, Fusarium oxysporum,
and Pseudoperonospron cubensis were used on cucumber. For
the screening of the target compounds, Mycosphaerella
melonis, C. cassiicola, Pseudomonas syringae pv
．lachrymans,
Botrytis cinerea, and F. oxysporum were used on cucumber,
Phytophthora infestans was used on tomato, and Rhizoctonia
solani was used on rice. Among them, M. melonis, C. cassiicola,
P. syringae pv．lachrymans, P. infestans, and F. oxysporum
o
and incubated in the culture tank at 24-26 C, and solvent was
were inoculated by leaf spray, and B. cinerea, R. solani were
inoculated by irrigation. Additionally, commercialized 50%
kresoxim-methyl (WG), 50% Dimethomorph (WP), 75%
chlorothalonil (WP), 20% bismerthlazol (WP), 5% validamycin A
(WP), 50% procymidone (WP), and 70% Mildothane (WP) were
used as positive controls against M. melonis, P. infestans, C.
cassiicola, P. syringae pv. lachrymans, R. solani, B. cinerea and
F. oxysporum, respectively. All test compounds were dissolved
in acetone, and diluted with water to a concentration of 100
mg/L, solvent was used as blank control. During the test, the
agents were sprayed on the plant leaves, the first application
was conducted at proper life stage of plants, another two
applications were conducted every seven days. Then, the
inoculations were conducted three days after the third
application. During the screening, the plants were cultured in
used as blank control. After two days, the diameter of the
bacterial colony was measured, and the inhibition efficacy was
calculated according to the equation (3). Three replications
were conducted for each test.
Dc − Da
Dc
Effenciency =
(3)
Where Dc is the average diameter of the water control plants,
and Da is the average diameter of the treated plants.
4.3.3. Quantitative Real Time PCR.
Total RNAs were extracted from 3-week old Arabidopsis
thaliana after treatment of chemicals (compound 11, BTH,
MeJA and solvent) using Trizol reagent (Ambion). The
concentrations of all chemicals were all 100 mg/L. The RNAs
were quantitated using a NanoDrop 2000c spectrophotometer
(Thermo Fisher Scientific) and used to synthesize cDNAs using
ReverTra Ace qPCR RT Master Mix with gDNA Remover
(TOYOBO). Quantitative real-time PCR was performed using
SYBR Green (Takara) with CFX Real-Time System (Bio-Rad).
ACTIN2 mRNA was detected as an internal control for data
normalization. Experiments were repeated three times with
similar results.
o
green house maintaining the temperature between 24-26 C.
Each test was repeated four times, and in every replication,
five plants were used for the test. No other germicide or
insecticide was used during the screening. After the disease
conditions of controls became stable, the number of lesions on
test plant leaves were recorded, and classified into six levels as
below:
Level 0: no lesion;
Level 1: lesions occupied less than 5% of the entire leaf area;
Level 2: lesions occupied 6%-10% of the entire leaf area;
Level 5: lesions occupied 11%-25% of the entire leaf area;
Level 7: lesions occupied 26%-50% of the entire leaf area;
Level 9: lesions occupied more than 50% of the entire leaf area.
Acknowledgements
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This work was financially supported by the National Basic 25. X. Liu, H. Jiang and H. Li, Journal of Chemical Information and
Research Program of China (973 Program, 2010CB126100), the Modeling, 2011, 51, 2372-2385.
National High Technology Research and Development Program 26. W. Lu, X. Liu, X. Cao, M. Xue, K. Liu, Z. Zhao, X. Shen, H. Jiang,
of China (863 Program 2011AA10A207), and the Fundamental
Research Funds for the Central Universities.
Y. Xu, J. Huang and H. Li, J Med Chem, 2011, 54, 3564-3574.
27. W. Zhou, X. Liu, Z. Tu, L. Zhang, X. Ku, F. Bai, Z. Zhao, Y. Xu, K.
Ding and H. Li, J Med Chem, 2013, 56, 7821-7837.
28. S. Li, Y. Zhou, W. Lu, Y. Zhong, W. Song, K. Liu, J. Huang, Z.
Zhao, Y. Xu, X. Liu and H. Li, Journal of Chemical Information
and Modeling, 2011, 51, 2939-2947.
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10 | J. Name., 2012, 00, 1-3
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Graphical Abstract
The Discovery of New Plant Activators and Scaffolds with Potential
Induced Systemic Resistance: From Jasmonic Acid to Pyrrolidone
Kang Chang , Yanxia Shi, Jianqin Chen, Zenghui He, Zheng Xu , Zhenjiang Zhao, Weiping Zhu, Honglin Li,
Yufang Xu, BaoJu Li, and Xuhong Qian
A series of novel plant activators possessing pyrrolidone scaffold was developed with the help of
SHAFTS.