Exploring novel p-hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27, HPPD) inhibitors has become one of the most promising research directions in herbicide innovation. On the basis of our tremendous interest in exploiting more powerful HPPD inhibitors, we designed a family of benzyl-containing triketone-aminopyridines via a structure-based drug design (SBDD) strategy and then synthesized them. Among these prepared derivatives, the best active 3-hydroxy-2-(3,5,6-trichloro-4-((4-isopropylbenzyl)­amino)­picolinoyl)­cyclohex-2-en-1-one (23, IC50 = 0.047 μM) exhibited a 5.8-fold enhancement in inhibiting Arabidopsis thaliana (At) HPPD activity over that of commercial mesotrione (IC50 = 0.273 μM). The predicted docking models and calculated energy contributions of the key residues for small molecules suggested that an additional π–π stacking interaction with Phe-392 and hydrophobic contacts with Met-335 and Pro-384 were detected in AtHPPD upon the binding of the best active compound 23 compared with that of the reference mesotrione. Such a molecular mechanism and the resulting binding affinities coincide with the proposed design scheme and experimental values. It is noteworthy that inhibitors 16 (3-hydroxy-2-(3,5,6-trichloro-4-((4-chlorobenzyl)­amino)­picolinoyl)­cyclohex-2-en-1-one), 22 (3-hydroxy-2-(3,5,6-trichloro-4-((4-methylbenzyl)­amino)­picolinoyl)­cyclohex-2-en-1-one), and 23 displayed excellent greenhouse herbicidal effects at 150 g of active ingredient (ai)/ha after postemergence treatment. Furthermore, compound 16 showed superior weed-controlling efficacy against Setaria viridis (S. viridis) versus that of the positive control mesotrione at multiple test dosages (120, 60, and 30 g ai/ha). These findings imply that compound 16, as a novel lead of HPPD inhibitors, possesses great potential for application in specifically combating the malignant weed S. viridis.

4-Hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27) has been recognized as one of the most promising targets in the field of herbicide innovation considering the severity of weed resistance currently. In a persistent effort to develop effective HPPD-inhibiting herbicides, a structure-guided strategy was carried out to perform the structural optimization for triketone-quinazoline-2,4-diones, a novel HPPD inhibitor scaffold first discovered in our lab. Herein, starting from the crystal structure of Arabidopsis thaliana (At)­HPPD complexed with 6-(2-hydroxy-6-oxocyclohex-1-ene-1-carbonyl)-1,5-dimethyl-3-(o-tolyl)­quinazoline-2,4­(1H,3H)-dione (MBQ), three subseries of quinazoline-2,4-dione derivatives were designed and prepared by optimizing the hydrophobic interactions between the side chain of the core structure at the R1 position and the hydrophobic pocket at the active site entrance of AtHPPD. 6-(2-Hydroxy-6-oxocyclohex-1-ene-1-carbonyl)-1,5-dimethyl-3-(3-(trimethylsilyl)­prop-2-yn-1-yl)­quinazoline-2,4­(1H,3H)-dione (60) with the best inhibitory activity against AtHPPD was identified to be the first subnanomolar-range AtHPPD inhibitor (K i = 0.86 nM), which significantly outperformed that of the lead compound MBQ (K i = 8.2 nM). Further determination of the crystal structure of AtHPPD in complex with compound 60 (1.85 Å) and the binding energy calculation provided a molecular basis for the understanding of its high efficiency. Additionally, the greenhouse assay indicated that 6-(2-hydroxy-6-oxocyclohex-1-ene-1-carbonyl)-1,5-dimethyl-3-propylquinazoline-2,4­(1H,3H)-dione (28) and compound 60 showed acceptable crop safety against peanut and good herbicidal activity with a broad spectrum. Moreover, compound 28 also showed superior selectivity for wheat at the dosage of 120 g ai/ha and favorable herbicidal efficacy toward the gramineous weeds at the dosage of as low as 30 g ai/ha. We believe that compounds 28 and 60 have promising prospects as new herbicide candidates for wheat and peanut fields.

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4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a functional protein existing in almost all aerobic organisms. In the field of agricultural chemicals, HPPD is acknowledged to be one of the crucial targets for herbicides at present due to its unique bio-function in plants. In the Auto Core Fragment in silico Screening (ACFIS) web server, a potential HPPD inhibitor featuring 1,2,3-benzotriazine-4-one was screened out via a pharmacophore-linked fragment virtual screening (PFVS) method. Molecular simulation studies drove the process of “hit-to-lead” optimization, and a family of 1,2,3-benzotriazine-4-one derivatives was synthesized. Consequently, 6-(2-hydroxy-6-oxocyclohex-1-ene-1-carbonyl)-5-methyl-3-(2-methylbenzyl)­benzo­[d]­[1,2,3]­triazin-4­(3H)-one (15bu) was identified to be the best HPPD inhibitor (IC50 = 36 nM) among the 1,2,3-benzotriazine-4-one derivatives, which had over 8-fold improvement of enzyme inhibition compared with the positive control mesotrione (IC50 = 289 nM). Crystallography information for the AtHPPD–15bu complex revealed several important interactions of the ligand bound upon the target protein, i.e., the bidentate chelating interaction of the triketone motif with the metal ion of AtHPPD, a tight π–π stacking interaction consisting of the1,2,3-benzotriazine-4-one moiety and two benzene rings of Phe-424 and Phe-381, and the polydirectional hydrophobic contacts consisting of the ortho-CH3-benzyl group of the core scaffold and some hydrophobic residues. Furthermore, compound 15bu displayed 100% inhibition against the five species of target weeds at the tested dosage, which was comparable to the weed control of mesotrione. Collectively, the fused 1,2,3-benzotriazine-4-one-triketone hybrid is a promising chemical tool for the development of more potent HPPD inhibitors and provides a valuable lead compound 15bu for herbicide innovation.

Carboxylesterases (CEs) exist as multiple types of isomers in humans, and two major types are CE1 and CE2. They are widely distributed in human tissues and well-known for their important roles in drug metabolism and pathology of various diseases. Thus, the detection of CEs in living systems could provide efficient proof in disease diagnostics, as well as important information regarding chemotherapeutic effects of antitumor drugs and prognosis. To develop a specific probe to discriminate CEs from other hydrolases, especially cholinesterases, is quite challenging due to their structural similarities and substrate specificity. To date, almost all of the fluorescent probes developed for CEs have been constructed with an acetyl group as the recognition unit. Herein we proposed a new design strategy of probe–cavity matching, which led to the identification of a new fluorogenic substrate (termed as HBT-CE) with high specificity toward both CE isomers and improved sensitivity, considering the higher binding affinity and catalysis efficiency. The promising capability of HBT-CE was further demonstrated for endogenous CEs imaging in living cells, zebrafish, and nude mice. In addition, HBT-CE was successfully applied in kinetically monitoring drug-induced CE regulation in cancer cells. All of these findings suggest that HBT-CE is a valuable tool for tracking and imaging endogenous CEs in complex biological systems.