Discovery of Potent Succinate-Ubiquinone Oxidoreductase Inhibitors via Pharmacophore-linked Fragment Virtual Screening Approach

Article abstract of DOI:10.1021/acs.jafc.6b00325

Succinate-ubiquinone oxidoreductase (SQR) is an attractive target for fungicide discovery. Herein, we report the discovery of novel SQR inhibitors using a pharmacophore-linked fragment virtual screening approach, a new drug design method developed in our laboratory. Among newly designed compounds, compound 9s was identified as the most potent inhibitor with a K_i value of 34 nM against porcine SQR, displaying approximately 10-fold higher potency than that of the commercial control penthiopyrad. Further inhibitory kinetics studies revealed that compound 9s is a noncompetitive inhibitor with respect to the substrate cytochrome c and DCIP. Interestingly, compounds 8a, 9h, 9j, and 9k exhibited good in vivo preventive effects against Rhizoctonia solani. The results obtained from molecular modeling showed that the orientation of the R² group had a significant effect on binding with the protein.

Full text of DOI:10.1021/acs.jafc.6b00325

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Article
Discovery of Potent Succinate-ubiquinone Oxidoreductase Inhibitors
via Pharmacophore-linked Fragment Virtual Screening (PFVS) Approach
Li Xiong, Xiao-Lei Zhu, Hua-Wei Gao, Yu Fu, Sheng-
Qua Hu, Li-Na Jiang, Wen-Chao Yang, and Guang-Fu Yang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00325 • Publication Date (Web): 26 May 2016
Downloaded from http://pubs.acs.org on May 29, 2016
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Page 1 of 33
Journal of Agricultural and Food Chemistry
Discovery of Potent Succinate-ubiquinone Oxidoreductase Inhibitors via
Pharmacophore-linked Fragment Virtual Screening (PFVS) Approach
1
1*
1
1
1
1
Li Xiong, XiaoꢀLei Zhu, HuaꢀWei Gao, Yu Fu, ShengꢀQuan Hu, LiꢀNa Jiang, WenꢀChao
1
1,2*
Yang, GuangꢀFu Yang
1
Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, College of
Chemistry, Central China Normal University, Wuhan 430079, P.R. China
2
Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 30071,
P.R.China
Correspondence:
GuangꢀFu Yang, Ph.D. & Professor
College of Chemistry
Central China Normal University
Luoyu Road 152, Wuhan 430079
P. R. China
TEL: 86ꢀ27ꢀ67867800
FAX: 86ꢀ27ꢀ67867141
Eꢀmail: gfyang@mail.ccnu.edu.cn
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Abstract:
Succinateꢀubiquinone oxidoreductase (SQR) is an attractive target for fungicide discovery.
Herein we report the discovery of novel SQR inhibitors using pharmacophoreꢀlinked
fragment virtual screening approach, a new drug design method developed in our
laboratory. Among newly designed compounds, compound 9s was identified as the most
potent inhibitor with the K value of 34 nM against porcine SQR, displaying about 10ꢀfold
i
higher potency than the commercial control penthiopyrad. The further inhibitory kinetics
studies revealed that compound 9s is a nonꢀcompetitive inhibitor with respect to the
substrate cytochrome c and DCIP. Interestingly, compounds 8a, 9h, 9j, and 9k exhibited
good in vivo preventive effects against Rhizoctonia solani. The results obtained from
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molecular modeling showed that the orientation of R group displayed a significant effect
on the binding with the protein.
Key Words: Succinateꢀubiquinone oxidoreductase, complex II, pharmacophoreꢀlinked
fragment virtual screening, molecular modeling, structureꢀbased design
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Introduction
Pesticides play a key role in the control of diseases in crops, a vital part of ensuring
global food security. However, the unrestricted use of highly toxic pesticides over several
decades has resulted in negative effects on the environment and poisoning of nontargeted
species. To reduce the negative impacts of the pesticides, new compounds with high
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efficacy and selectivity against target species are desirable. Among the known pesticides
classes, fungicides targeting succinateꢀubiquinone oxidoreductase (SQR, EC 1.3.5.1, also
known as succinate dehydrogenase or complex II) can offer such solutions. SQR is an
important membrane complex in the tricarboxylic acid cycle (Kreb cycle) that catalyzes the
oxidation of succinate to fumarate in the mitochondrial matrix as succinate dehydrogenase.
Succinate oxidation is coupled to the reduction of ubiquinone to ubiquinol at the
mitochondrial inner membrane as one part of the electron transfer chain. Electrons are
transferred from succinate to ubiquinone through the burried prosthetic groups
flavinꢀadenine dinucleotid (FAD), [2Feꢀ2S], [4Feꢀ4S], [3Feꢀ3S] clusters and heme, which
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form an integral part of SQR. Due to its critical roles in life processes,
SQR has been
identified as a promising action target for agricultural fungicides. SQRꢀinhibiting
fungicides have many advantages, such as broadꢀspectrum fungi control (including
resistance to other fungicides), excellent crop selectivity, benign environmental effects, low
application rate, and low toxicity.
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Since the launch of carboxin as the first commercial SQR fungicide in 1966, about
18 commercial SQR fungicides have been successfully developed, which could be
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classified into eight categories: pyrazoleꢀ4ꢀcarboxamides, thiazole carboxamides, pyridine
carboxamides, phenylꢀoxoꢀethyl thiophene amide, oxathiin carboxamides, pyridinylꢀethyl
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benzamides, furan carboxamides, and phenylꢀbenzamides. Our enzyme inhibition kinetics
results showed that these carboxamide fungicides should bind into the ubiquinoneꢀbinding
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site (Qꢀsite) of SQR. The acyl moiety of carboxamide fungicides formed vanꢀderꢀWaals
(VDW) interactions with residues C_R46, C_S42, B_I218, and B_P169 of Qꢀsite, and the
carbonyl oxygen atom of the carboxamide formed Hꢀbonds with residues B_W173 and
D_Y91. These key interactions are highly conserved among different carboxamide
fungicides. On the contrary, the hydrophobic subꢀpocket surrounded by residues, C_W35,
C_I43, and C_I30, extending to the mouth of the Qꢀsite, can accommodate
structurallyꢀdiverse and bulky amine groups. Therefore, the acyl moiety and amide bond
could be regarded as the pharmacophore of the carboxamide fungicides. Modification of
the amine moiety should be an effective way to obtain new inhibitors with improved
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potency.
Recently, fragmentꢀbased drug discovery (FBDD) has been rapidly developed as an
effective method of hit identification. Although FBDD has several significant advantages,
such as higher ligand efficiency (LE), higher hit rate and more opportunities of structure
optimization, it also poses great challenges. First, FBDD often relies on sensitive
biophysical methods to detect the binding of fragments. Secondly, FBDD can only screen
fragments with excellent solubility due to their much weaker binding affinity. Furthermore,
FBDD requires large amounts of purified protein (>10 mg), which are very difficult to
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achieve in most cases, especially for membrane proteins. In order to overcome the
lowꢀthroughput nature of FBDD and meet these challenges, we developed a new approach
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named pharmacophoreꢀlinked fragment virtual screening (PFVS). Using this new
approach, we successfully discovered a series of new picomolarꢀrange Q site inhibitors of
the cytochrome bc complex, an important membrane protein for drug and fungicide
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discovery. As a continuation of our computational discovery of new inhibitors of membrane
protein, we extended the PFVS method to the SQR system with the pyrazoleꢀcarboxamide
moiety as pharmacophore and designed a series of new potent SQR inhibitors. Herein, we
report
the
discovery
and
structural
optimization
of
indoleꢀcontaining
pyrazoleꢀcarboxamides, one of the hits identified by PFVS method. Very fortunately, one
nanomolar SQR inhibitor was successfully obtained, with significantly improved potency
compared with the commercial product penthiopyrad. In addition, some compounds
displayed good preventive effects in vivo against Rhizoctonia solani (R.solani).
Materials and Methods
Chemicals
Unless otherwise noted, all chemical reagents were commercially purchased and treated
with standard methods before use. Solvents were dried in a routine way and redistilled.
Rhizoctonia solani, Botrytis cinerea, Pseudoperonospora cubensis, Sphaerotheca fuliginea
were provided through the courtesy of the Center for Bioassay, Zhejiang Chemical Industry
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Research Institute. H NMR spectra were recorded on a MercuryꢀPlus 400/600
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spectrometer in CDCl
3
or DMSO with TMS as the internal reference. Mass spectral data
were obtained on a MocroMass platform by electrospray ionization (ESIꢀMS). Elementary
analyses were performed on a Vario EL III elementary analysis instrument. Melting points
were taken on a Buchi Bꢀ545 melting point apparatus and uncorrected. The starting
materials 2ꢀ(2ꢀnitrophenyl) hydrazinꢀ1ꢀium
chloride,
ethyl
2ꢀoxopropanoate,
3ꢀ(difluoromethyl)ꢀ1ꢀ methylꢀ1Hꢀpyrazoleꢀ4ꢀcarboxylic acid were commercially available.
Computational protocol
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Based on the modification and combination of AutoGrow and the Amber 8.0
programs, the PFVS method was designed to discovered new type SQR inhibitor. Depicted
in Figure 1 is the workflow of PFVS calculations.
Synthetic Chemistry
The
preparation
of
ethyl
2ꢀmethylꢀ3ꢀ[2ꢀ(2ꢀnitrophenyl)hydrazono]propanoate,
ethylꢀ7ꢀnitroꢀ1Hꢀindoleꢀ2ꢀcarboxylate, 7ꢀnitroꢀ1Hꢀindoleꢀ2ꢀcarboxylic acid, 7ꢀnitroꢀ1Hꢀ
indole, Nꢀsubstitutedꢀ7ꢀnitroꢀ1Hꢀindole, Nꢀsubstitutedꢀ1Hꢀindolꢀ7ꢀamine and the title
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compounds 8ꢀ9 were performed according to the reported methods.
The detailed
procedure and the characterization data were supplied in the Supporting Information.
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Enzymatic Kinetics
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The preparation of succinateꢀcytochrome c reductase (SCR, mixture of respiratory SQR
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01 and complex III) from porcine heart was essentially as reported . The enzymatic activities
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02 of SCR, SQR and complex III were analyzed in respective reaction mixtures as reported
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03 previously
. Kinetic analyses for the inhibition mechanism were performed as
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04 previously described .
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06 Data analysis
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According to the measured substance’s extinction coefficient, the change of absorbance
08 was converted to product variation, then making linear fitting of time, the slope is the
09 enzymatic reaction velocity. Compared with the control sample, the inhibition rates of
10 inhibitors were calculated.
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12 Fungicidal Activity Assay
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The fungicidal activities of compounds against R. solani, B. cinerea, P. cubensis, and S.
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14 fuliginea were evaluated according to a previously published method . The results are
15 summarized in Table 4.
16
17 Results and Discussion
18 Hit identification through PFVS
19
It may need to be pointed out that the PVFS is picking a new starting point for
20 optimization, getting away from the "local minimum" of the current scaffold which has
21 already been optimized, and ideally picking a starting point that can be optimized further
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22 than the current scaffold.
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The workflow of hit identification via PFVS method is shown in Figure 1. The first key
24 step of PFVS method is to define the pharmacophore. According to our previous study on
25 enzyme inhibition kinetics of ten commercial carboxamideꢀtype fungicides, we carried out
26 systematic computational simulations of the interaction between SQR and
27 carboxamideꢀtype inhibitors. The results indicated that the acyl moiety and carboxamide
28 group have relative conserved binding conformation (Supporting information, Figure 1S),
29 and made great contribution to the inhibitor binding. Therefore, we defined the
30 pyrazoleꢀ4ꢀcarboxamide moiety as the pharmacophore, the structure of the
31 pharmacophoreꢀbound SQR was prepared based on the computationally simulated
32 penthiopyradꢀSQR binding mode. Then, a modified library of 1735 fragments derived from
33 commercial pesticides was screened and each fragment was set to link with the nitrogen
34 atom of the pharmacophore. After performing a threeꢀstep computational screening (see
35 Figure 1), 8 hits with the most favorable binding free energies (Supporting information,
36 Figure 2S) were identified. Considering the synthetic feasibility, six hits (3, 4, 5, 6, 7, and
37 8) were finally selected for further syntheses and structural optimization.
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These six hit compounds were successfully synthesized and characterized using H NMR
39 and MS spectral data. However, hits 4, 6 and 7 were eventually abandoned due to their too
40 low inhibition activity (IC > 100 ꢁM) towards porcine SQR. Fortunately, the IC values
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41 against porcine SCR of compounds 3, 5, and 8a were found to be 24.39 ꢁM, 19.79 ꢁM, and
42 51.87 ꢁM, respectively. Although compound 3 showed high inhibition effect against
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43 porcine SQR, it was also eventually abandoned because further optimization of compound
44 3 did not result in significant improvement of the potency. Finally, compounds 5 and 8a
45 were allowed to progress to hitꢀtoꢀlead chemistry. In this work, we only focused on the
46 structural optimization of hit 8, the structural optimization of hit 5 will be reported
47 elsewhere.
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49 Optimization of Compound 8
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It is known that most of the commercial pyrazoleꢀ4ꢀcarboxamide fungicides have a CHF
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51 group on the R position of pyrazole ring. Previously, we have also found that the ꢀCHF
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52 compound displayed higher potency than the corresponding ꢀCF
3
compound. Therefore, it
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53 is not surprising that compound 8a was first synthesized with ꢀCHF group on the R
54 position (Table 1). The binding mode of compound 8a with SQR was shown in Figure 3A,
55 conservatively forming hydrogen bonds with B_W173 and D_Y91. The pyrazole ring of 8a
56 inserted into the Qꢀsite and formed cationꢀπ interaction with C_R46, while the indole ring
57 is located in the entrance of the Qꢀsite and formed hydrophobic interaction with C_I43,
58 similar to the binding mode of carboxin in 2WQY. The IC50 value of 8a was 51.87 ꢁM
59 against porcine SCR, still showing lower activity than the commercial control penthiopyrad
60 (IC50 = 1.32 ꢁM). As we noted, the value of ꢂEvdw energy of 8a was ꢀ39.60 kcal/mol, also
61 less favorable than that of penthiopyrad (ꢂEvdw = ꢀ41.91 kcal/mol, Table 2). Considering
62 the characteristic of Qꢀsite, optimizing the hydrophobic interactions between the indole
63 ring and some important residues while keeping the key hydrogen bonds should be an
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64 effective way to improve the potency. Bearing this consideration in mind, the ꢀCH
3
or
65 ꢀCH
66 these two compounds almost showed no inhibition activity against porcine SCR (IC50
67 100ꢁM). When introducing group ꢀCH CH CH , the resulted compound 8d (IC50
2 3
CH group was respectively introduced to result in 8b and 8c (Table 1). However,
>
=
2
2
3
68 60.59ꢁM) displayed the same level of activity as compound 8a. When introducing
69 ꢀCH CH CH CH group, the resulted compound 8e (IC = 4.02 ꢁM) showed about 13ꢀfold
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70 improved potency than 8a. As shown in Figure 3B, the ꢀCH CH CH CH group stayed
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71 nearly the same plane as the indole ring and its hydrophobic interaction with C_I43 and
72 C_I30 was significantly improved. However, when introducing cyclopropylmethyl (8f),
73 isobutyl (8g), cyclopentyl (8h), allyl (8i), propꢀ2ꢀynꢀ1ꢀyl (8j), the resulting compounds
74 displayed lower activity, even complete loss of activity.
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These results led us to carefully analyze the interaction between 8 and the protein. As
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76 shown in Table 2, the value of ∆Evdw became more favorable with increasing the size of R
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2
77 group, such as ꢀ39.60 kcal/mol for 8a (R = H), ꢀ41.43 kcal/mol for 8d (R = ꢀCH
3
), ꢀ44.28
2
2
78 kcal/mol for 8e (R = ꢀCH
2
CH
2
CH
2
CH
3
). These results indicated that the substitute in R
79 position did increase the hydrophobic interaction between ligand and protein. However,
80 compounds 8b-c and 8f-h displayed no inhibition towards porcine SCR, indicating that too
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81 small or too large group in R position was unsuitable for the binding. In addition, when R
82 group changed from saturated alkyl to unsaturated alkyl, although the ꢂEele energies
83 contribution were increased, the less favorable entropy contributions (ꢀTꢂS) and polar
84 solvation energies (ꢂGpol) were produced (e.g 8i and 8j), which accounted reasonably for
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85 the lower activity of compounds 8f-j.
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Then, we attempted to introduce groups to the R position linked directly with the N
87 atom of the indole ring with the aim to increase the VDW interaction with some key
88 residues. As we know, the entrance of the Qꢀsite has some hydrophobic residues, such as
89 D_Y91, C_W35, C_I43, and C_I30. How to modulate the conformation of indole ring to
90 increase the VDW interaction with these residues was our primary concern. It has been
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91 well established that the arylꢀaryl interaction energy is stronger than alkylꢀaryl. So, we
92 introduced a hydrophobic benzyl group to link with the N atom of the indole ring,
93 producing compound 9a with just slightly increased activity (IC50 = 1.89 ꢁM) as indicated
94 by 8a. As shown in Figure 3C, the indole ring of 9a took about 180° turn, and then resulted
95 in the benzyl group forming edgeꢀtoꢀedge πꢀπ interaction with D_Y91. As a result, the
96 ꢂEvdw energy was increased to ꢀ46.35 kcal/mol. These results confirmed the reasonability
97 of our hypothesis, and promoted us to design more compounds along with the same
98 strategy.
99
In order to probe the structureꢀactivity relationship of benzyl substitute, a number of
00 benzyl substituted indoles containing pyrazoleꢀcarboxamide with diverse properties were
01 synthesized (Table 1). A series of simple paraꢀsubstituted benzyl groups such as 4ꢀF (9d,
02 IC50 = 0.44 ꢁM), 4ꢀCl (9g, IC50 = 0.75 ꢁM), 4ꢀBr (9i, IC50 = 0.58 ꢁM), and 4ꢀOCH
03 IC50 = 0.80 ꢁM) did produce a significant increase in activity over 9a, but 4ꢀCF (9l, IC50
04 100 ꢁM) and 4ꢀOCF (9n, IC > 100 ꢁM) almost complete loss of activity. Meanwhile,
3
(9o,
3
>
3
50
3
05 orthoꢀsubstituted benzyl groups resulted in less or almost no activity, such as 9j (2ꢀCH ,
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06 IC50 = 8.45 ꢁM), 2ꢀF (9b, IC50 = 22.06 ꢁM), 2ꢀCl (9e, IC50 > 100 ꢁM), and 2ꢀBr (9h, IC50 >
07 100 ꢁM). For the derivatives bearing metaꢀsubstituted benzyl groups, compound 9c (3ꢀF,
08 IC50 = 2.76 ꢁM) and 9m (3ꢀOCH , IC50 = 9.89 ꢁM) showed a little decreased potency,
09 whereas 9f (3ꢀCl, IC50 = 0.45 ꢁM) and 9k (3ꢀCH , IC50 = 0.50 ꢁM) showed about 4ꢀfold
3
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10 improved potency compared with 9a. Interestingly, when introducing diꢀsubstituted benzyl
11 groups onto the N atom of the indole ring, the opposite results were obtained. As shown in
12 Table 1, 9q (2,4ꢀdichlorobenzyl, IC > 100 ꢁM) exhibited almost no inhibition effects
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13 towards porcine SCR, and 9r (3,4ꢀdimethylbenzyl, IC50 = 0.49 ꢁM) exhibited strong
14 inhibition effects, but 9s (3,5ꢀdimethylbenzyl, IC50 = 0.09 ꢁM) exhibited significant
15 inhibition effects with a IC50 of 0.09 ꢁM, about 576ꢀfold and 15ꢀfold improvement,
16 respectively, over compound 8a and the commercial control penthiopyrad (IC50 = 1.32 ꢁM).
17 In Figure 3D, the carbonyl oxygen atom of 9s formed hydrogen bonds with B_W173 and
18 D_Y91, the indole ring formed a cationꢀπ interaction with C_R46, and the benzyl ring
19 formed an edgeꢀtoꢀedge πꢀπ interaction with D_Y91. As a result, compound 9s harvested
20 the largest ꢂEvdw and ꢂGcal energy with values of ꢀ51.08 kcal/mol and ꢀ17.38 kcal/mol,
21 respectively.
22
From the energy component analysis, we found that 9 analogues displayed relative larger
23 ∆Evdw energy than 8 analogues. These results indicated that the hydrophobic interaction
24 between arylꢀaryl were indeed stronger than alkylꢀaryl. As shown in Table 2, both of the
25 electronꢀdonating and electronꢀwithdrawing groups attached on the benzyl always
26 increased the hydrophobic interaction. For example, the ꢂEvdw energies were ꢀ47.24
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27 kcal/mol for 9b (R = 2ꢀF), ꢀ48.86 kcal/mol for 9k (R = 3ꢀCH
3
), and ꢀ51.06 kcal/mol for
2
3
28 9m (R = 3ꢀOCH ).
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To further understand the mechanism of inhibition of newly designed compounds, the
30 binding mode of these compounds was superimposed. As shown in Figure 4, analogous to
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31 our previously study, the pyrazol ring of the title compounds deeply inserted into the
32 Qꢀsite. The extremely interesting thing was that two directions were found when different
33 substituted groups linked to indole ring. As for 8 derivatives, the alkyl group would mainly
34 point to the left side from the perspective of Figure 4. On the contrary, the benzyl group in
35 9 derivatives primarily pointed to the right side, forming πꢀπ interaction with D_Y91
36 (Figure 3C and 3D), perhaps correlating with higher activity compared to 8 derivatives. For
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37 example, compound 9k (R = 3ꢀCH
3
, IC50 = 0.50 ꢁM) showed higher potency than 8b (R =
38 ꢀCH , IC50 >100 ꢁM). It should be noted that the optimization of compound 8 was based on
3
39 the aboveꢀmentioned computational strategy. The experimental and calculated binding free
40 energies (ꢂG) of compounds summarized in Table 2 showed a good linear correlation with
2
41 the correlation coefficient of R = 0.92, further confirming the reliability of this
42 computational strategy (Supporting Information Figure 3S).
43
44 Inhibitory Kinetics of 9s and penthipyrad
45
In order to understand the mechanism by which these compounds inhibit this electron
46 transport complex, compound 9s was selected as an example to carry out further inhibitory
47 kinetics against porcine SCR and SQR. As shown in Figure 5A, doubleꢀreciprocal plots
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48 revealed the nonꢀcompetitive inhibition of 9s with respect to cytochrome c. Furthermore,
49 we examined the effect of 9s on the reactions of the SCR pathway catalyzed by SQR. We
50 determined the kinetics of SQR activity with respect to the substrate DCIP in the absence
51 and presence of 9s. As shown in Figure 5B, 9s was a nonꢀcompetitive inhibitor against
52 SQR with respect to the substrate DCIP. These results indicate that 9s should bind to the
2
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53 Qꢀsite of SQR, which is consistent with our previous study. The commercial fungicide
54 penthiopyrad also showed nearly the same chracteristics, nonꢀcompetitive with respect to
3
7
55 both cytochrome c and DCIP.
Next, we compared the inhibitory activities of both compounds against porcine SCR and
57 SQR (Table 3 and Figure 5). As indicated by the K values, Compound 9s and penthiopyrad
56
i
58 exhibited about 1.44ꢀfold and 4.25ꢀfold higher inhibition against the porcine SQR than
59 SCR, respectively. We also found that compound 9s and penthiopyrad nearly did not show
60 any inhibitory effects against complex III at the concentration of 20ꢁM (data not show).
61 Furthermore, we also measured the IC50 values (Table 3) of 9s and penthiopyrad against
62 porcine SQR, showing about 2.3ꢀfold and 2.5ꢀfold more potent than against porcine SCR
63 (Supporting Information Figure 4S). All these results indicated that the portion of complex
64 III in SCR system did not display any effect upon the SQR assay. In addition, 9s showed
65 the same action mechanism as the commercial fungicide. Therefore, we can conclude that
i
66 compound 9s (K = 0.034 ꢁM against porcine SQR) was indeed a potent SQR inhibitor,
67 displaying about 10ꢀfold higher potency than penthiopyrad (K = 0.33 ꢁM).
i
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69 In vivo test
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70
The antifungal activities of all of the titled compounds were evaluated against R. solani,
71 B. cinerea, P. cubensis, and S. fuliginea in the greenhouse environment. Thifluzamide was
72 used as the positive control, and the results were shown in Table 4. Thifluzamide did not
73 exhibit fungicidal activity against B. cinerea and P. cubensis at a concentration of 100
74 mg/L, although it exhibited a total inhibition effect against R.solani and S. filiginea. As
75 shown in Table 4, although showing lower fungicidal activity against R.solani and S.
76 filiginea than Thifluzamide, some compounds still displayed inhibition effects to some
77 extent against R. solani (9i: 54%, 9h: 72%; 9k: 73%, 9j: 62%) and S. filiginea (9n: 60%
78 and 9l: 50%). In addition, all compounds along with the control thifluzamide displayed no
79 inhibition against B. cinerea and P. cubensis. One of the possible reason of low in vivo
80 activity of the newly designed compounds was due to the high reactivity of 3ꢀposition of
81 indole ring, which resulted in the rapid metabolization of the title compounds in the plant.
82 Researche has established that the indole derivatives were easily metabolized in wheat, pea,
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9ꢀ40
83 and rat liver tissues.
Therefore, all these results indicate that indoleꢀcontaining
84 pyrazoleꢀcarboxamide could be regarded as a new type of SQR inhibitor and further
85 structural optimization should be carried out in the future.
86
87 Chemistry
88
The Figure 2 gives the steps necessary to synthesize 8a-j and 9a-s based on
89 nucleophilic substitution reaction. Nꢀsubstituted indolꢀ7ꢀamine was prepared by a multiple
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90 step
synthetic
route
using
2ꢀ(2ꢀnitrophenyl)ꢀhydrazinꢀ1ꢀium
chloride
and
91 3ꢀsubstitutedꢀ1ꢀmethylꢀ1Hꢀpyrazoleꢀ4ꢀcarboxylic acid as starting materials. Finally,
92 indolꢀ7ꢀamine reacted with 3ꢀ(difluoromethyl)ꢀ1ꢀmethylꢀ1Hꢀpyrazoleꢀ4ꢀ carbonyl chloride
93 to produce the title compound 8-9 smoothly. The synthesis routes for compound 4~7 was
94 described in the Supporting information. The structure of all intermediates and designed
1
95 compounds were confirmed by elemental analyses, by H NMR and ESIꢀMS spectral data.
96
97
In summary, a series of novel indoleꢀcontaining pyrazoleꢀcarboxamides 8-9 were
98 rationally designed and synthesized based on the PFVS method. Among these compounds,
99 compound 9s, 3ꢀ(difluoromethyl)ꢀNꢀ[1ꢀ(3,5ꢀdimethylbenzyl)ꢀ1Hꢀindolꢀ7ꢀyl]ꢀ1ꢀmethylꢀ
i
00 1Hꢀpyrazoleꢀ4ꢀcarboxamide, displayed the most potent activity with the value of K 34 nM
01 against porcine SQR. Further inhibitory kinetic assays indicated that compound 9s was a
02 nonꢀcompetitive inhibitor with respect to the substrate cytochrome c and DCIP. The in vivo
03 tests indicated that compounds 8a, 9h, 9j, and 9k exhibited well preventive effects against
04 R.solani at the concentration of 100mg/L. The computational simulation results strongly
05 indicated that the Nꢀbenzyl indole ring was favorable for the binding into the Qꢀsite of
06 SQR. The hydrogen bonds with D_Y91 and B_W173 and the cationꢀπ interaction with
07 C_R46 make great contribution to the binding free energy. In addition, the orientation of
2
08 the group in R position of the title compound has a significant effect on the potency.
09
10 SUPPORTING INFORMATION
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11
Supplemental Figure 1S describes the chemical structure of ten commercial fungicides,
12 Figure 2S shows the chemical structures of 8 top candidates, Figure 3S shows the
13 coorelation between the calculated and experiment binding free energy, and Figure 4S
14 describes the inhibition of 9s against SCR and SQR, followed by the synthetic route of hit
15 3, 4, 5, 6, and 7. These materials are available free of charge via the Internet at
16 http://pubs.acs.org.
17
18 FUNDING
19
This research was supported by the Special Fund for Agroꢀscientific Research in the
20 Public Interest (No. 201203022) and the National Natural Science Foundation of China
21 (No. 21332004 and 21272092).
22
23 AUTHOR INFORMATION
24
25
26
Corresponding Author
*Eꢀmail: (XLZ) xlzhu@mail.ccnu.edu.cn; (GFY) gfyang@mail.ccnu.edu.cn
27 NOTES
28
29
The authors declare no competing financial interest.
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06 28. Zhao, P. L.; Wang, L.; Zhu, X. L.; Huang, X.; Zhan, C. G.; Wu, J. W.; Yang, G. F.
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FIGURE CAPTIONS
Figure 1. The protocl of PFVS.
Figure 2. General Synthesis of Compound 8-9.
Figure 3. (A) The binding mode of 8a. (B) The binding mode of 8e. (C) The binding mode of
9a. (D) The binding mode of 9s.
Figure 4. The binding mode overlay of 8 (red stick) and 9 (blue stick) analogues in the Qꢀsite
of SQR.
Figure 5. Kinetic analysis of inhibition by 9s (A for SCR and B for SQR) and penthiopyrad
(
C for SCR and D for SQR, obtained from ref 35) against porcine SCR and SQR. The
inhibition of porcine SCR by (A) 9s (1, 0 nM; 2, 10nM; 3, 30nM; 4, 50 nM and 5, 80 nM),
C) penthiopyrad (1, 0 nM; 2, 300nM; 3, 500 nM; 4, 1000 nM and 5, 2000 nM). Each reaction
mixture contains 100 mM PBS (pH 7.4), 0.3 mM EDTA, 20 mM succinate, 0.1 nM enzyme,
(
1
.16ꢀ16.0 ꢁM cytochrome c and the indicated penthiopyrad or 9s. K was estimated to be
i
0.049±0.002 µM for 9s and 1.393±0.087 µM for penthiopyrad by assuming nonꢀcompetitive
inhibition. The inhibition of porcine SQR by (B) 9s (1, 0 nM; 2, 5 nM; 3, 10 nM; 4, 30 nM
and 5, 50 nM), (D)penthiopyrad (1, 0 nM; 2,100 nM; 3, 200 nM; 4, 300 nM and 5, 500 nM).
Each reaction mixture contains 100 mM PBS (pH 7.4), 0.3 mM EDTA, 20 mM succinate,
i
2nM SCR, 1.18ꢀ41.22 ꢁM DCIP and the indicated amount of penthiopyrad or 9s. K was
estimated to be 0.034±0.002 µM for 9s and 0.327±0.009 µM for penthiopyrad by assuming
nonꢀcompetitive inhibition.
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Table 1. Structures of compound 8-9 derivatives and their IC50 value against porcine SCR.
1
2
1
2
No.
R
R
IC （ꢁM） No.
R
R
IC （ꢁM）
50
5
0
8
a
CHF
CHF
CHF
CHF
CHF
CHF
CHF
CHF
CHF
CHF
CHF
CHF
CHF
CHF
CHF
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
H
51.87±1.22 9f CHF
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3ꢀClꢀPhCH
2
2
0.45±0.01
0.75±0.12
>100
8b
CH
CH
CH
CH
3
3
3
3
>100 9g CHF
4ꢀClꢀPhCH
8
c
CH
CH
CH
2
>100 9h CHF
2ꢀBrꢀPhCH
4ꢀBrꢀPhCH
2
2
8d
2
2
CH
2
2
60.59±3.46 9i CHF
4.02±1.04 9j CHF
>100 9k CHF
0.58±0.12
8.45±1.14
0.50±0.13
>100
8
e
CH
CH
2
2ꢀCH
3ꢀCH
3
3
ꢀPhCH
2
2
8
f
(CH
(CH
(CH
2
3
2
)
)
)
2
2
5
CHCH
CHCH
CH
2
ꢀPhCH
8
8
g
h
2
>100 9l CHF
3 2
4ꢀCF ꢀPhCH
>100 9m CHF
3ꢀOCH
3
ꢀPhCH
ꢀPhCH
ꢀPhCH
2
9.89±1.07
>100
8
i
j
CH
CH≡CCH
PhCH
2
=CHCH
2
74.49±4.34 9n CHF
51.83±1.40 9o CHF
1.89±1.13 9p CHF
22.06±1.24 9q CHF
2.76±1.15 9r CHF
0.44±0.07 9s CHF
4ꢀOCF
4ꢀOCH
3
2
8
2
3
2
0.80±0.11
21.57±1.66
>100
9
a
2
2,4ꢀF
2
ꢀPhCH
2
9b
2ꢀFꢀPhCH
3ꢀFꢀPhCH
4ꢀFꢀPhCH
2
2
2
2,4ꢀCl
2
ꢀPhCH
2
9
c
3,4ꢀ(CH
3
3
)
2
2
ꢀPhCH
ꢀPhCH
2
2
0.49±0.13
0.09±0.01
1.32±0.11
9d
3,5ꢀ(CH
)
9
e
2ꢀClꢀPhCH
2
>100 Penthiopyrad
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Table 2. The individual energy terms of the calculated free energy of title compounds
(kcal/mol).
a
No.
ꢂEele
ꢂEvdw ꢂGnp ꢂGpol
ꢂH
ꢀ23.33 ꢀ39.60 ꢀ4.47 46.95 ꢀ20.46 10.16 ꢀ10.30 ꢀ5.82
ꢀ18.48 ꢀ41.43 ꢀ4.86 44.42 ꢀ20.36 10.40 ꢀ9.96 ꢀ5.73
ꢀ14.66 ꢀ44.28 ꢀ5.39 42.29 ꢀ22.04 10.05 ꢀ11.99 ꢀ7.33
ꢀ20.38 ꢀ42.8 ꢀ4.84 47.31 ꢀ20.05 11.58 ꢀ8.47 ꢀ5.61
ꢀTꢂS ꢂGcal ꢂGexp
8
a
8d
8
e
8
8
i
j
ꢀ21.33 ꢀ41.99 ꢀ4.91 45.88 ꢀ22.35 11.74 ꢀ10.61 ꢀ5.82
ꢀ25.54 ꢀ46.35 ꢀ5.25 53.48 ꢀ23.66 11.71 ꢀ11.95 ꢀ7.78
ꢀ18.89 ꢀ47.24 ꢀ5.34 46.97 ꢀ24.50 8.25 ꢀ11.25 ꢀ6.33
ꢀ20.36 ꢀ47.70 ꢀ5.37 50.84 ꢀ22.60 10.22 ꢀ12.38 ꢀ7.55
ꢀ21.33 ꢀ47.50 ꢀ5.25 48.10 ꢀ25.98 10.23 ꢀ15.75 ꢀ8.64
ꢀ25.24 ꢀ48.44 ꢀ5.41 52.62 ꢀ26.47 10.85 ꢀ15.62 ꢀ8.63
ꢀ21.15 ꢀ48.39 ꢀ5.29 50.33 ꢀ24.50 10.01 ꢀ14.49 ꢀ8.32
ꢀ19.35 ꢀ48.93 ꢀ5.48 50.01 ꢀ23.76 8.41 ꢀ15.35 ꢀ8.48
ꢀ24.8 ꢀ47.43 ꢀ5.41 53.80 ꢀ23.16 10.82 ꢀ12.34 ꢀ6.89
ꢀ22.14 ꢀ48.86 ꢀ5.40 51.28 ꢀ25.11 9.94 ꢀ15.17 ꢀ8.56
ꢀ25.79 ꢀ51.06 ꢀ5.52 57.60 ꢀ24.77 12.74 ꢀ12.03 ꢀ6.80
ꢀ29.85 ꢀ46.73 ꢀ5.36 56.22 ꢀ25.72 11.25 ꢀ14.47 ꢀ8.29
ꢀ19.53 ꢀ46.15 ꢀ5.36 48.17 ꢀ22.87 11.45 ꢀ11.42 ꢀ6.34
ꢀ16.24 ꢀ45.70 ꢀ5.37 42.12 ꢀ25.19 9.55 ꢀ15.64 ꢀ8.57
ꢀ23.14 ꢀ51.08 ꢀ5.61 52.18 ꢀ27.66 10.28 ꢀ17.38 ꢀ9.59
9
a
9b
9
c
9d
9
f
9
g
9
9
i
j
9
k
9
m
9o
9p
9
9
r
s
Penthiopyrad ꢀ13.84 ꢀ41.91 ꢀ5.10 34.28 ꢀ26.57 13.54 ꢀ13.03 ꢀ7.99
a
ꢂGexp = ꢀRTLnIC50
2
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Journal of Agricultural and Food Chemistry
Page 26 of 33
Table 3. The inhibitory effect of some inhibitors against porcine SCR and SQR.
SCR (succinateꢀcyt.c system 23°C)
SQR (DCIPꢀsystem 23°C)
Inhibition type
Inhibitor
Inhibition type
IC (ꢁM)
K (ꢁM)
i
IC (ꢁM)
50
K (ꢁM)
i
5
0
(
with cyt. c)
(with DCIP)
nonꢀ
nonꢀ
9
s
0.090±0.01
1.32±0.110
0.049±0.002 0.040±0.004
1.39±0.087 0.53±0.111
0.034±0.002
0.33±0.009
competitive
nonꢀ
competitive
nonꢀ
a
penthiopyrad
competitive
competitive
a
The data were taken from ref 37.
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Journal of Agricultural and Food Chemistry
Table 4. The titled compounds fungicidal activities in vivo.
concentration (mg/L) R. solani B.cinerea P.cubensis S. filiginea
No.
8
a
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
68
44
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
20
0
8b
8
c
0
8d
0
0
8
e
31
0
0
8
f
0
8
8
g
h
0
0
22
0
0
8
8
i
j
0
0
0
9
a
0
0
9b
0
0
9
c
0
0
9d
0
0
9
e
0
0
9
f
18
18
72
54
62
73
0
0
9
9
g
h
0
0
9
9
i
j
0
0
9
k
0
9
l
50
0
9
m
0
9
n
o
p
q
0
60
0
9
0
9
9
0
0
11
0
0
9
r
0
9
s
0
0
Thifluzamid
e
100
100
0
0
100
2
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Page 28 of 33
Figure 1.
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Journal of Agricultural and Food Chemistry
Figure 2.
NO2
NO2
NO2
H
O
H
NHNH2 HCl
N
a
N
b
N
COOEt
COOEt
+
COOEt
NO2
NO2
NO2
R2
NH2
R2
N
H
H
N
N
N
f
c
d
e
COOH
R1
N
NH2
R2
N
COCl
R1
O
g
+
N
N
H
N
2N
R
N
8
and 9
(
(
a) NaOAc, MeOH, rt. (b) PPA,110 (c) NaOH/H O, 45 ; HCl, 40 (d) CuO, quinoline, 194
2
2
e) NaH/DMF, R X (f) EtOH/H O, Fe, NH Cl, reflux (g) (CH CH ) N, CH Cl , 0
2
4
3
2 3
2
2
2
9
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