Pyrazole-isoindoline-1,3-dione hybrid: A promising scaffold for 4-hydroxyphenylpyruvate dioxygenase inhibitors

Article abstract of DOI:10.1021/acs.jafc.9b04917

The discovery of 4-hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27) inhibitors has been an active area of research due to their great potential as herbicides for weed control. Starting from the binding mode of known inhibitors of HPPD, a series of HPPD inhibitors with new molecular scaffolds were designed and synthesized by hybridizing 2-benzoylethen-1-ol and isoindoline-1,3-dione fragments. The results of the in vitro tests indicated that the newly synthesized compounds showed good HPPD inhibitory activity with IC50 values against the recombinant Arabidopsis thaliana HPPD (AtHPPD) ranging from 0.0039 ?M to over 1 ?M. Most promisingly, compound 4ae, 2-benzyl-5-(5-hydroxy-1,3-dimethyl-1H-pyrazole-4-carbonyl)isoindoline-1,3-dione, showed the highest AtHPPD inhibitory activity with a Ki value of 3.92 nM, making it approximately 10 times more potent than pyrasulfotole (Ki = 44 nM) and slightly more potent than mesotrione (Ki = 4.56 nM). In addition, the cocrystal structure of the AtHPPD-4ae complex was successfully resolved at a resolution of 1.8 ?. The X-ray diffraction analysis indicated that the two carbonyl groups of 2-benzoylethen-1-ol formed a bidentate chelating interaction with the metal ion, while the isoindoline-1,3-dione moiety formed pronounced π-π stacking interactions with Phe381 and Phe424. Moreover, water-mediated hydrogen bonding interactions were observed between Asn282 and the nitrogen atoms of the pyrazole ring of 4ae. The above results showed that the pyrazole-isoindoline-1,3-dione hybrid is a promising scaffold for developing HPPD inhibitors.

Full text of DOI:10.1021/acs.jafc.9b04917

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Agricultural and Environmental Chemistry
Pyrazole-Isoindoline-1,3-dione Hybrid: A Promising Scaffold
for 4-Hydroxyphenylpyruvate Dioxygenase Inhibitors
Bo He, Jin Dong, Hong-Yan Lin, Meng-yao Wang, Xian-Kai Li, Bai-Feng
Zheng, Qiong Chen, Ge-Fei Hao, Wen-Chao Yang, and Guang-Fu Yang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b04917 • Publication Date (Web): 17 Sep 2019
Downloaded from pubs.acs.org on September 17, 2019
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Pyrazole-Isoindoline-1,3-dione Hybrid: A Promising Scaffold for
4-Hydroxyphenylpyruvate Dioxygenase Inhibitors
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Bo He,† Jin Dong, † Hong-Yan Lin, † Meng-Yao Wang, † Xian-Kai Li, † Bai-Feng Zheng, †
Qiong Chen,† Ge-Fei Hao,† Wen-Chao Yang,†* Guang-Fu Yang†#*
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†Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, International Joint
Research Center for Intelligent Biosensor Technology and Health, and Chemical Biology
Center, College of Chemistry, Central China Normal University, Wuhan 430079, P.R. China
#Collaborative Innovation Center of Chemical Science and Engineering, Nankai University,
Tianjin 300071, P.R. China
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ORCID:
Guang-Fu Yang: 0000-0003-4384-2593
—————————
* Wen-Chao Yang, e-mail: tomyang@mail.ccnu.edu.cn; Tel: 86-27-67867706; Fax:
86-27-67867141; Guang-Fu Yang, e-mail: gfyang@mail.ccnu.edu.cn; Tel: 86-27-67867800;
Fax: 86-27-67867141.
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ABSTRACT:
The discovery of 4-hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27) inhibitors has
been an active area of research due to their great potential as herbicides for weed control.
Starting from the binding mode of known inhibitors of HPPD, a series of HPPD inhibitors with
new molecular scaffolds were designed and synthesized by hybridizing 2-benzoylethen-1-ol
and isoindoline-1,3-dione fragments. The results of the in vitro tests indicated that the newly
synthesized compounds showed good HPPD inhibitory activity with IC50 values against the
recombinant Arabidopsis thaliana HPPD (AtHPPD) ranging from 0.0039 M to over 1 M.
Most promisingly, compound 4ae, 2-benzyl-5-(5-hydroxy-1,3-dimethyl-1H-pyrazole-4-
carbonyl)isoindoline-1,3-dione, showed the highest AtHPPD inhibitory activity with a Ki value
of 3.92 nM, making it approximately 10 times more potent than pyrasulfotole (Ki = 44 nM) and
slightly more potent than mesotrione (Ki = 4.56 nM). In addition, the cocrystal structure of the
AtHPPD-4ae complex was successfully resolved at a resolution of 1.8 Å. The X-ray diffraction
analysis indicated that the two carbonyl groups of 2-benzoylethen-1-ol formed a bidentate
chelating interaction with the metal ion, while the isoindoline-1,3-dione moiety formed
pronounced π-π stacking interactions with Phe381 and Phe424. Moreover, water-mediated
hydrogen bonding interactions were observed between Asn282 and the nitrogen atoms of the
pyrazole ring of 4ae. The above results showed that the pyrazole-isoindoline-1,3-dione hybrid
is a promising scaffold for developing HPPD inhibitors.
KEYWORDS: 4-Hydroxyphenylpyruvate Dioxygenase, Pyrazole, Isoindoline-1,3-dione,
Inhibition Kinetics, X-ray Crystallography
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INTRODUCTION
Currently, crops suffer severely from infestations of various weeds, so the control of
weeds in fields is still a great challenge in agricultural development.1, 2 In this context, the
development of green herbicides that are highly efficient and environmentally friendly while
showing low toxicity and high crop selectivity is becoming increasingly important.3 As an
important target of herbicides, 4-hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27, HPPD)
is widely distributed in various aerobic organisms, and it is involved in the catabolism of
tyrosine and plays a pivotal role in the biosynthesis of many biologically relevant compounds.4,
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In plants, HPPD catalyzes the conversion of p-hydroxyphenylpyruvic acid (HPPA) to
homogentisic acid (HGA) under aerobic conditions.6-9 HGA can be further transformed into
tocopherol and plastoquinone, which are crucial for the normal growth of plants.10, 11 Hence,
inhibiting the activity of HPPD will disrupt photosynthesis, leading to the death of weeds
through bleaching.5, 12, 13
Recently, IHARA Chemical Industry Co., Ltd. of Japan launched a new HPPD-inhibiting
herbicide, fenquinotrione, which contained a bicyclic core skeleton not seen in previously
reported HPPD herbicides (Figure 1). Over the past 30 years, more than ten
HPPD-inhibition-based herbicides containing monocyclic skeletons have been developed and
widely applied in the field. However, fenquinotrione, the first inhibitor containing a bicyclic
core, was recently developed as a commercial HPPD-inhibiting herbicide. Fenquinotrione,
bearing a benzopyrazinone ring, showed high safety for rice at a dose of 125-250 g ai/ha and
effectively controlled a variety of broadleaf and grass weeds. Therefore, we were inspired to
explore new HPPD inhibitors with a bicyclic core as promising leads for green herbicide
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development.
The pharmacophore was first put forward by Paul Ehrlich.14, 15 The International Union of
Pure and Applied Chemistry (IUPAC) summarized the concept as follows: ‘‘A pharmacophore
is the ensemble of steric and electronic features that is necessary to ensure the optimal
supramolecular interactions with a specific biological target and to trigger (or block) its
biological response.’’16 Analysis of all the commercialized HPPD inhibitors showed that there
was a shared fragment (Figure 1), a 2-benzoylethen-1-ol or 2-heteroaroylethen-1-ol. To date,
studies on HPPD herbicides have mainly focused on the modification of the 2- and 4- positions
of the aromatic ring of the pharmacophore. Modifications of the substituents (especially the
electron-withdrawing group) could afford inhibitors with excellent herbicidal activity (Figure 1,
pyrasulfotole, bicyclopyone, and mesotrione).4, 17, 18 At the same time, certain herbicidal
activity can be achieved by introducing different alkoxy chains or heterocyclic fragments at the
3 position of the aromatic ring (Figure 1, tolpyralate and ZJ10361).19, 20 We previously
reported several series of nitrogen-containing compounds (quinoline, quinazoline-2,4-dione,
and benzimidazolone derivatives) and identified a candidate compound (named quinotrione,
Supporting Information S1) that is now in the development and registration stage.21-23 To obtain
a new bioactive compound with a novel and diverse scaffold, a molecular skeleton with an
isoindoline-1,3-dione fragment attracted our attention due to the extensive biological activity of
this fragment in the fields of pharmaceuticals and agrochemicals. For example, derivatives of
isoindoline-1,3-dione (thalidomide) exhibit extensive histone deacetylase-inhibitory activity,
and thalidomide was widely used as sedative or hypnotic drugs in the 1960s.24 In the field of
agrochemicals, inhibitors containing an isoindoline-1,3-dione moiety were used to control an
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exceptionally wide variety of plants by inhibiting the activity of the enzymes acetohydroxyacid
synthase or protoporphyrinogen oxidase.25-27 Therefore, isoindoline-1,3-dione would be a
promising bicyclic core skeleton to integrate into the pyrazole pharmacophore. Herein, we
designed a novel molecular skeleton (Figure 2, 4aa) by hybridizing 2-benzoylethen-1-ol and
isoindoline-1,3-dione fragments. Accordingly, we comprehensively investigated the synthetic
chemistry, the inhibition activity towards HPPD and the structure-activity relationships (SAR)
of these compounds. Finally, compound 4ae, 2-benzyl-5-(5-hydroxy-1,3-dimethyl-1H-
pyrazole-4-carbonyl)-isoindoline-1,3-dione, was identified as the most potent compound,
showing the highest AtHPPD inhibition activity with a Ki value of 3.92 ± 0.71 nM, making it
approximately 10 times more potent than pyrasulfotole (Ki = 44 nM) and slightly more potent
than mesotrione (Ki = 4.56 nM). Furthermore, to elucidate the mechanism of action of 4ae at
the atomic level, the cocrystal structure of the AtHPPD-4ae complex was successfully resolved
at a resolution of 1.8 Å.
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MATERIALS AND METHODS
Chemicals and Instruments. All the purchased reagents and solvents were analytically or
chemically pure and were used as received. The non-aqueous reactions were conducted under a
N2 atmosphere in flame-dried glassware. The reaction progress was monitored using thin-layer
chromatography (TLC) on silica gel-coated glass plates (Yantai Jiangyou Silica Gel
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Development Co., Ltd., Yantai, China). Usually, the H NMR and 13C NMR spectra were
acquired in DMSO-d6 or CDCl3 as solvent using tetramethylsilane (TMS) as the internal
reference on a Varian Mercury-Plus 600/400 spectrometer (Varian Inc., Palo Alto, CA).
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Chemical shift values (δ) are listed in parts per million (ppm). The melting point of each
synthesized compound was tested on a melting point instrument B-545 (Büchi, Flawil,
Switzerland). High-resolution mass spectra (HRMS) were obtained on a model 6224
time-of-flight liquid chromatograph-mass spectrometer (Agilent Technologies, Santa Clara,
CA).
Molecular Docking. The crystal structure of AtHPPD (PDB ID: 1TFZ) was obtained from
the RCSB Protein Data Bank. The 3D structures of our compounds were prepared using
SYBYL 7.3 and GOLD 3.0, which were used to search the binding conformations of the
compounds with AtHPPD. The Fe2+ center was used to define the binding pocket, and the
radius of the active site was set to 10 Å. Other parameters were set to the defaults. A total of
100 runs were performed using a genetic algorithm (GA).28 The binding mode of DAS645 with
AtHPPD (PDB ID: 1TG5) was used as a reference to help us determine the final binding
conformation.29 Finally, we selected the top-ranked conformation as the binding conformation,
and for every compound studied, the binding mode was similar to that of DAS645.
AtHPPD Enzymatic Assay and Kinetic Analysis of Slow-Binding Inhibition. We
selected AtHPPD as the target enzyme to assess the biological activity of target compounds
4aa−4bc in vitro. Recombinant AtHPPD was obtained and purified by the method shown in
Supporting Information S3. With purified AtHPPD in hand, the evaluation of the activity of the
AtHPPD inhibitors was carried out in vitro using a coupled enzyme assay as we reported
previously (S3).30-32 The half-maximal inhibitory concentration (IC50) values were determined,
and the values are shown in Table 1.
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The kinetic analysis method was established by John F. Morrison.33 We used an
optimized experimental protocol to determine the kinetic constant of compound 4ae using a
spectrophotometer (Perkin-Elmer Lambda 45, Waltham, MA, USA) at a constant temperature
of 25 ℃ and measuring the absorbance change at 318 nm.34, 35 The kinetic constants (compound
4ae, mesotrione and pyrasulfotole) were shown in Table 1. Details of the experimental
procedures and data processing methods are shown in Supporting Information S4.
Crystallization and Structure Determination of AtHPPD in Complex with 4ae. We
adopted the same method as mentioned above to express and purify AtHPPD. CoCl2 (0.2 mM)
was added to purified AtHPPD (6-10 mg/mL) and the mixture was incubated for approximately
one hour. Then, compound 4ae (0.075 mM) was added, and the mixture was kept for two hours
on ice before crystallization. The complex of AtHPPD with the inhibitor (compound 4ae)
crystallized well and grew via hanging drop diffusion under constant temperature conditions
(18 °C). During crystallization, the AtHPPD-4ae complex (1.5 μL) was mixed with an equal
volume of the reservoir solution (0.1 M Tris/Bicine pH 8.5, 13% (v/v) MPD, 13% (w/v)
PEG1000, 13% (w/v) PEG3350, 0.03 M NaBr, 0.03 M NaF, and 0.03 M NaI). After 10-20 days
of crystallization, the crystal was rapidly frozen in liquid nitrogen to facilitate data collection.
Diffraction data were collected at a wavelength of 0.97774 Å and over 225° in 1° increments at
beamline 18U at Shanghai Synchrotron Radiation Facility (SSRF) and processed with
HKL2000.34
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RESULTS AND DISCUSSION
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Chemistry. 1,3-Dioxo-1,3-dihydroisobenzofuran-5-carboxylic acid (compound 1) was
selected as the starting material, and the synthetic route to the title compounds is shown in
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Figure 3. Compounds 4aa-bc were synthesized by the continuous multistep synthesis method.
First, the N-substituted isoindoline-1,3-dione derivatives (2aa−bc) were obtained in high yields
by reacting starting material 1 with the appropriate primary amine in anhydrous acetic acid.24, 36
Then, 3aa−bc were synthesized from 2aa−bc using 2-chloro-1-methylpyridinium iodide
(CMPI) as a condensation reagent in dichloromethane in high yield, and these compounds were
further transformed into target compounds 4aa−bc in 47−89% yields through a Fries
rearrangement at room temperature. The final products (4ad and 4af) are racemic. We used the
racemic for subsequent determination of vivo activity test. The chemical structures of all the
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synthesized compounds (4aa−bc) were identified by H NMR and C NMR spectroscopy and
ESI-HRMS analyses. Furthermore, a suitable single crystal of compound 4au was obtained
directly from CDCl3 in the NMR tube (Supporting Information S2), allowing the structure of
this compound to be confirmed by single-crystal X-ray diffraction (Figure 4).
AtHPPD Inhibition Assay and Structure-Activity Relationships (SAR). Based on the
docking results of 4aa and 4ae (Figure 5A and 5B), all the inhibitors coordinated to Fe2+ in a
bidentate manner and formed π−π interactions with adjacent amino acid residues (Phe 381 and
Phe 424), which is quite similar to the binding mode of mesotrione in AtHPPD (-7.681 kcal/mol,
Figure 5D). In addition to the above interaction, the terminal benzene rings of both 4aa and
4ae formed hydrophobic interactions with the surrounding residues (Leu368 and Lue427) in the
simulated binding modes. It is worth mentioning that the distances between the terminal
benzene ring and the hydrophobic residues are different for 4aa and 4ae. As expected, the
binding energy of compound 4ae (-6.322 kcal/mol, Figure 5B) is significantly lower than that
of compound 4aa (-3.758 kcal/mol, Figure 5A), which means 4ae would show stronger
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biological activity. In addition, we evaluated the effects of the five-membered heterocycle (a
furan ring) and benzene ring on the activity by molecular docking. The calculated binding
energies showed that compound 4ae binds more strongly than 4ai (-5.903 kcal/mol, Figure 5C),
which suggested that compound 4ae may be a better candidate.
To verify the molecular simulation results, we synthesized compounds 4aa-4aj and
evaluated their activities against AtHPPD in vitro. Mesotrione and pyrasulfotole, commercial
HPPD-inhibiting herbicides, were chosen as the positive controls. As shown in Table 1, the
half-maximal inhibitory concentration (IC50) of compound 4aa (IC50 = 0.607 μM) was higher
than that of mesotrione and better than that of pyrasulfotole. 4ab and 4ac were synthesized by
replacing the benzene ring with a pyridine or a pyrazole, respectively. However, 4ab and 4ac
showed weak inhibitory activities against AtHPPD (IC50 > 1 μM), which may be caused by the
poor solubility of 4ab and 4ac. Interestingly, when a 2,3-dihydro-1H-inden-2-yl moiety (4ad)
was introduced, the inhibitory potency against AtHPPD improved significantly to IC50 = 0.161
μM, which is approximately three times better than that of compound 4aa and seven times
better than that of pyrasulfotole. The above results indicated that the inhibitory activities of the
title compounds against AtHPPD could be enhanced by the addition of a hydrophobic alkyl
group between the benzene ring and isoindoline-1,3-dione (Figure 2).
In this context, we tried to install alkyl chains of different lengths between the terminal
benzene ring and the isoindoline-1,3-dione. Importantly, the IC50 value of compound 4ae (R =
benzyl) against AtHPPD reached 90 nM. Compounds containing two or three methylene units,
4ag (R = 2-phenylethyl, IC50 = 0.165 μM) and 4ah (R =3-phenylpropyl, IC50 = 0.135 μM), also
showed significantly increased potencies against AtHPPD compared with that of 4aa. The
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activity of compound 4af (IC50 = 0.208 μM) containing a 1-phenylethyl group showed
significantly lower inhibitory potency relative to that of 4ae. The overall trend in the in vitro
activities of these compounds is benzyl (4ae) > 3-phenylpropyl (4ah) > 2-phenylethyl (4ag) >
1-phenylethyl (4af) > phenyl (4aa). Apart from the above structures, we also introduced
five-membered heterocycles into our scaffold. Compounds 4ai (R = furan-2-ylmethyl, IC50 =
0.276 μM) and 4aj (R = thiophen-2-ylmethyl, IC50 = 0.148 μM) also showed better inhibitory
activity than compound 4aa. However, the inhibitory activities of those compounds were lower
than those of compound 4ae. The trend in the IC50 values of these compounds can be
summarized as follows: benzyl (4ae) < thiophen-2-ylmethyl (4aj) < furan-2-ylmethyl (4ai). In
general, the results of the in vitro activity assay were consistent with the results of the
molecular docking study. Therefore, compound 4ae could be regarded as a lead compound for
further optimization.
Based on the above study, we tried to further optimize the substituents around the benzyl
moiety of 4ae to improve the activity. We synthesized compounds 4ak−bc by introducing a
wide range of substituents on the benzyl ring of compound 4ae (Table 1). First, we considered
the effect of the electronic properties of the substituents on the biological activities of the
compounds. These compounds also showed a general trend through the analysis of
structure-activity relationships. The AtHPPD inhibitory activities of these compounds increased
as the substituents increase the electron donating capacity. On the contrary, the biological
activity of these compounds decreased with the enhancement of the electron-withdrawing
ability of the substituent. i.e., 2-OCH3-benzyl (4am, IC50 = 0.160 μM) > 2-Me-benzyl (4ak,
IC50 = 0.220 μM), 3-OCH3-benzyl (4an, IC50 = 0.105 μM) > 3-OCF3-benzyl (4av, IC50 = 0.172
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μM), and 4-Cl-benzyl (4at, IC50 = 0.116 μM) > 4-F-benzyl (4aq, IC50 = 0.135 μM) >
4-SO2CH3-benzyl (4ao, IC50 = 0.274 μM). When different halogens were introduced on the
terminal benzene ring, monohalogens 4ap-4at, the derivatives showed higher inhibitory
activities than that of mesotrione. We also found that chloro-substituted compounds exhibited
better activity than fluoro-substituted compounds and higher activity than trifluoromethyl or
trifluoromethoxy substituted compounds. The activities of the monochloro-compounds were
similar to that of compound 4ae, i.e., 3-Cl-benzyl (4as, IC50 = 0.097 μM) > 4-F-benzyl (4aq,
IC50 = 0.135 μM)> 3-CF3-benzyl (4au, IC50 = 0.152 μM) > 3-OCF3-benzyl (4av, IC50 = 0.172
μM)> mesotrione. We further studied the influence of the steric effects on the terminal benzene
ring on the potency of the title compounds. Ortho-substituted compound 4aw (2-Ph-benzyl,
IC50 = 0.211 μM) showed better inhibitory activity than para-substituted compound 4ax
(4-Ph-benzyl) against AtHPPD and showed lower inhibitory activity than compound 4ae.
Therefore, introducing large steric hindrance did not improve the in vitro biological activity. In
addition, we also investigated the effect of multiple substituents on the benzyl group on the
inhibitory activity against AtHPPD. Compounds 4ay−bc were all superior to the controls.
Compounds with fluoro and bromo substituents (4bb) showed higher inhibitory activity against
AtHPPD than did the di-fluoro and di-chloro-substituted compounds (4ay, 4az, and 4ba). In
general, except for compounds 4ao and 4ax, other compounds with substituents on the terminal
benzene ring of 4ae showed better inhibitory activity against AtHPPD than the control agents.
Enzymatic Kinetics of Compound 4ae. In vitro, we used the coupled enzyme method to
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measure the kinetics following a known procedure.22,
To avoid systematic errors, the
inhibitory activity of the inhibitor against recombinant homogentisate 1,2-dioxygenase (HGD)
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alone was tested prior to starting the study of the kinetic constants of 4ae. In the end, 4ae did
not inhibit HGD. Similar to other slow-binding inhibitors, the curve of HPPD-catalyzed product
formation as a function of time was also curved in the presence of the inhibitor. As the reaction
progressed, the reaction process curve changed from a straight line to a curve and finally
showed a simple linear increase (Figure 6A). The curve could be fitted with equation (I)
(Supporting Information S4) by nonlinear regression analysis, and this equation yielded the
following kinetic parameters: ⱽ0 (initial velocity), vs (steady-state velocity) and kobs (the
observed first-order rate constant). Figure 6B shows the plot of kobs against the concentration of
4ae. The experimental results clearly revealed that kobs and the inhibitor concentration were
positively correlated. Using equations (II) (Supporting Information S4), the apparent rate
constants, A (slope) and B (intercept), were obtained using the straight lines of the kobs values
against the inhibitor concentrations at the indicated HPPA concentrations. We further evaluated
the dependence of constants A and B on the HPPA concentrations. Figure 6C and 6D show that
constant A (Supporting Information S4) decreased as the concentration of HPPA increased,
while constant B (Supporting Information S4) was unaffected by the concentration of HPPA.
The association and dissociation rate constants of 4ae, k+0 = 24.3072 ± 1.7513 μM-1min-1 and
k-0 = 0.0952 ± 0.0172 min-1, were measured by fitted equations (III) and (IV) (Supporting
Information S4), and the inhibition constant, Ki = k-0/k+0, was 3.92 ± 0.71 nM. Compound 4ae
exhibited better AtHPPD inhibition activity with a Ki value of 3.92 ± 0.71 nM, making it
approximately 10 times more potent than pyrasulfotole (Ki = 44.0 ±3.3 nM) and slightly more
potent than mesotrione (Ki = 4.56 ± 0.82 nM).
The Crystal Structure of AtHPPD-4ae Complex (PDB ID: 6JX9). To study the binding
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mode of the inhibitor, we obtained the crystal structure of the AtHPPD-compound 4ae complex.
After extensive screening and optimizing of the conditions, a suitable crystal was obtained
using the hanging drop vapor diffusion method. The crystal of the AtHPPD-compound 4ae
complex contained one molecule per asymmetric unit and belonged to space group C2. Using
Phaser MR with the AtHPPD-mesotrione complex (PDB: 5YWG) as the search model, the
structure was subsequently analyzed by molecular replacement.37 The standard refinement was
performed with the programs PHENIX and Coot (the final statistics for the structure are given
in Supporting Information S6 Table 1).38, 39 All structural representations in this paper were
prepared with PYMOL (http://www.pymol.org).
A clear and complete crystal structure (Supporting Information S5, the resolution of the
complex is 1.8 Å) could be obtained by processing and analyzing the original data. One chain
contained a clear electron density map of the ligand in the asymmetric unit (Figure 7B). The
metal ion (Fe2+) was modeled in the active site of each chain. As shown in Figure 7A, on one
hand, the Fe2+ could chelate with two carbonyl groups of inhibitor 4ae. On the other hand, the
Fe2+ was involved in a facial triad consisting of His226, His308, Glu394, and a free water
molecule. In addition, the isoindoline-1,3-dione fragment of the inhibitor formed pronounced
π-π stacking interactions with Phe381 and Phe424. Moreover, Asn282 interacts with 4ae
through water-mediated hydrogen bonds. Phe392, Met335, Leu368, and Leu427 form van der
Waals interactions with 4ae (Figure 7C). When compared with other inhibitors, such as
mesotrione, this inhibitor has a similar binding mode in AtHPPD (Figure 7D) in one respect.
They used the same carbonyl groups to coordinate with the metal ion and formed π-π stacking
interactions with Phe381 and Phe424. The binding mode of the inhibitor with HPPD was
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generally consistent with the binding mode determined by molecular docking. The crystal
structure of the complex further confirmed that the target of the inhibitor was HPPD.
In summary, a series of novel pyrazole−isoindoline-1,3-dione hybrids 4aa-4bc as
promising HPPD inhibitors were designed by hybridizing two fragments, 2-benzoylethen-1-ol
and isoindoline-1,3-dione, that are often present in the molecular structure of drugs and
pesticides. An efficient and convenient synthetic method was successfully established to
prepare the target compounds. Based on the inhibition effect assay against AtHPPD, the
structure-activity relationships of compounds 4aa-4bc were systematically investigated. The
results indicated that electron-donating groups (e.g., 4ak−an) on the benzene ring of the
N-linked benzyl moiety improved the AtHPPD inhibitory activity relative to what was seen
with electron-withdrawing groups (4ao, 4-SO2CH3-benzyl). In contrast, a sterically bulky group,
such as phenyl, on the benzene ring of N-linked benzyl decreased the enzymatic inhibition
activity. Importantly, compound 4ae was identified as the most potent inhibitor with a Ki value
of 3.92 nM, making it approximately 10 times more potent than pyrasulfotole (Ki = 44 nM) and
slightly more potent than mesotrione (Ki = 4.56 nM). Finally, the successful determination of
the cocrystal structure of the AtHPPD-4ae complex at a resolution of 1.8 Å confirmed the
mechanism of action at the atomic level, which will provide useful information for the further
design of novel HPPD inhibitors.
Supporting Information
Supporting information includes S1 The structure of the candidate quinoline compound, S2
X-ray diffraction data of compound 4au, S3 Protein expression and purification, S4 Kinetic
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analysis method for slow-binding inhibition, S5 A clear and complete crystal structure of
AtHPPD-4ae, S6 Data collection and refinement statistics of AtHPPD-4ae, S7 General
synthetic procedure and physical data of intermediates 2aa-bc, S8 General synthetic procedure
and physical data of intermediates 3aa-bc, S9 General synthetic procedure and physical data of
intermediates 4aa-bc, and S10 Spectra of representative compounds. These materials are
available free of charge via the internet at http://pubs.acs.org.
Acknowledgment
This work was supported by the National Key Research and Development Program of China
(2017YFD0200507) and the National Natural Science Foundation of China (Nos. 21837001
and 21672079).
Notes
The authors declare no competing financial interest.
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Figure captions:
Figure 1. The partially commercialized HPPD inhibitors.
Figure 2. The design strategy of compounds 4aa and 4ae.
Figure 3. Reagents and conditions: (a) AcOH, R-NH2, reflux 3-4 h; (b) Et3N, DCM,
2-chloro-1-methylpyridin-1-ium iodide (CMPI), room temperature, 1 h; (c) Et3N,
1,3-dimethyl-1H-pyrazol-5-ol, room temperature, 8-10 h; (d) Acetone cyanohydrin, Et3N,
acetonitrile, room temperature, 8-10 h; (e) 1 N HCl solution.
Figure 4. X-ray crystal structure of compound 4au.
Figure 5. Binding models of compounds 4aa (A), 4ae (B), 4ai (C) and the commercial
herbicide mesotrione (D) with AtHPPD (PDB ID: 1TFZ). The key residues surrounding the
active site are shown as green sticks, and the structures of representative compounds 4aa, 4ae,
4ai, and mesotrione are shown in light blue. The binding energies (△H) of compounds 4aa, 4ae,
4ai and mesotrione are -3.758 kcal/mol, -6.322 kcal/mol, -5.903 kcal/mol and -7.681 kcal/mol,
respectively. The red dotted line indicates the distance from the adjacent amino acid to the
center of the terminal benzene ring or the furan. The yellow dotted line indicates chelation
between the inhibitor and Fe2+ in the catalytic center.
Figure 6. Inhibition kinetics of HPPD by 4ae. (A) Progress curves in the presence of varying
concentrations of inhibitor 4ae (1, 0 µM; 2, 0.075µM; 3, 0.1µM; and 4, 0.13µM) for a reaction
initiated by the addition of AtHPPD to a mixture containing HPPA (20µM). (B) Plot of kobs
against the inhibitor concentration. (C) The plot of the apparent rate constant, A, against the
HPPA concentration. Insets: Plots of 1/A against the HPPA concentration. (D) Plots of the
apparent rate constant, B, against HPPA the concentration.
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501
Figure 7. Crystal structure of AtHPPD-4ae (green) and the structural comparison with
AtHPPD-mesotrione (pink). (A) Close-up view of the active site and the binding mode of
compound 4ae. (B) 2Fo-Fc map (contoured at 1.0 σ) of compound 4ae. (C) Hydrophobic
interactions between inhibitor 4ae and AtHPPD. (D) The superposition of 4ae and mesotrione
in AtHPPD. Inhibitors and their key surrounding residues are highlighted as sticks. The
chelation interactions are indicated with blue dashed lines.
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O
CH3
O
O
O
CH3
O
OH
O
O
O
O
O O S CH3
HO
2
2
1
1
CH3
O
N3
3
H3C
H3C
H3C
N
N
O
CH3
6
O
6
CH3
4
CF3
N
N
4
CF3
5
5
SO2Me
Pyrasulfotole
(2-benzoylethen-1-ol)
Bicyclopyone
(2-heteroaroylethen-1-ol)
Tolpyralate
O
CH3
O
OH
O
O
O
CH3
OH
O
O
NO2
CH3
O
CH3
O
O
N
O
CH3
N
O
H3C
N
S
O
N
O
SO2Me
ZJ10361
CH3
Cl
Fenquinotrione
Mesotrione
502
503
Figure 1
504
505
506
507
508
509
510
511
512
513
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517
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H2
O
HO
O
O
HO
HO
O
2
5
C
O
1
3
4
Cyclization With
Bioactivity Fragment
H3C
N
N
H3C
N
N
H3C
N
N
N
N
6
CH3
Optimization
CH3
CH3
O
The pharmacophore
(2-benzoylethen-1-ol)
O
4aa
4ae
518
519
Figure 2
520
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
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538
O
O
O
O
a
b, c
HO
HO
H2N
+
R
O
N R
O
O
2aa-bc
1
2aa-aj, R = phenyl, substituted pyridyl or
pyrazolyl or alkyl, benzyl
2ak-bc, R = substituted benzyl
H3C
N
O
HO
O
O
O
d, e
H3C
N
N
N
O
N R
N R
H3C
CH3
O
O
3aa-bc
4aa-bc
4aa-aj, R = phenyl, substituted pyridyl or
pyrazolyl or alkyl, benzyl
4ak-bc, R = substituted benzyl
3aa-aj, R = phenyl, substituted pyridyl or
pyrazolyl or alkyl, benzyl
3ak-bc, R = substituted benzyl
539
540
Figure 3
541
542
543
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551
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Figure 4
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Figure 5
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569
Figure 6
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