Design, Synthesis, and Biological Evaluation of Pyridazinone-Containing Derivatives As Novel Protoporphyrinogen IX Oxidase Inhibitor.

Protoporphyrinogen IX oxidase (PPO, E.C. 1.3.3.4) plays a pivotal role in chlorophyll biosynthesis in plants, making it a prime target for herbicide development. In this study, we conducted an investigation aimed at discovering PPO-inhibiting herbicides. Through this endeavor, we successfully identified a series of novel compounds based on the pyridazinone scaffold. Following structural optimization and biological assessment, compound 10ae, known as ethyl 3-((6-fluoro-5-(6-oxo-4-(trifluoromethyl)pyridazin-1(6H)-yl)benzo[d]thiazol-2-yl)thio)propanoate, emerged as a standout performer. It exhibited robust activity against Nicotiana tabacum PPO (NtPPO) with an inhibition constant (Ki) value of 0.0338 µM. Concurrently, we employed molecular simulations to obtain further insight into the binding mechanism with NtPPO. Additionally, another compound, namely, ethyl 2-((6-fluoro-5-(5-methyl-6-oxo-4-(trifluoromethyl)pyridazin-1(6H)-yl)benzo[d]thiazol-2-yl)thio)propanoate (10bh), demonstrated broad-spectrum and highly effective herbicidal properties against all six tested weeds (Leaf mustard, Chickweed, Chenopodium serotinum, Alopecurus aequalis, Poa annua, and Polypogon fugax) at the dosage of 150 g a.i./ha through postemergence application in a greenhouse. This work identified a novel lead compound (10bh) that showed good activity in vitro and excellent herbicidal activity in vivo and had promising prospects as a new PPO-inhibiting herbicide lead.

Discovery and Development of 4-Hydroxyphenylpyruvate Dioxygenase as a Novel Crop Fungicide Target.

Plant pathogenic fungi pose a significant threat to crop yields and quality, and the emergence of fungicide resistance has further exacerbated the problem in agriculture. Therefore, there is an urgent need for efficient and environmentally friendly fungicides. In this study, we investigated the antifungal activity of (+)-Usnic acid and its inhibitory effect on crop pathogenic fungal 4-hydroxyphenylpyruvate dioxygenases (HPPDs) and determined the structure of Zymoseptoria tritici HPPD (ZtHPPD)-(+)-Usnic acid complex. Thus, the antifungal target of (+)-Usnic acid and its inhibitory basis toward HPPD were uncovered. Additionally, we discovered a potential lead fungicide possessing a novel scaffold that displayed remarkable antifungal activities. Furthermore, our molecular docking analysis revealed the unique binding mode of this compound with ZtHPPD, explaining its high inhibitory effect. We concluded that HPPD represents a promising target for the control of phytopathogenic fungi, and the new compound serves as a novel starting point for the development of fungicides and dual-purpose pesticides.

Structure-based discovery of pyrazole-benzothiadiazole hybrid as human HPPD inhibitors.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) has attracted increasing attention as a target for treating type I tyrosinemia and other diseases with defects in tyrosine catabolism. Only one commercial drug, 2-(2-nitro-4-trifluoromethylbenzoyl)-1, 3-cyclohexanedione (NTBC), clinically treat type I tyrosinemia, but show some severe side effects in clinical application. Here, we determined the structure of human HPPD-NTBC complex, and developed new pyrazole-benzothiadiazole 2,2-dioxide hybrids from the binding of NTBC. These compounds showed improved inhibition against human HPPD, among which compound a10 was the most active candidate. The Absorption Distribution Metabolism Excretion Toxicity (ADMET) predicted properties suggested that a10 had good druggability, and was with lower toxicity than NTBC. The structure comparison between inhibitor-bound and ligand-free form human HPPD showed a large conformational change of the C-terminal helix. Furthermore, the loop 1 and α7 helix were found adopting different conformations to assist the gating of the cavity, which explains the gating mechanism of human HPPD.

Synthesis and Biological Activity Evaluation of Benzoxazinone-Pyrimidinedione Hybrids as Potent Protoporphyrinogen IX Oxidase Inhibitor.

Protoporphyrinogen IX oxidase (PPO/Protox, E.C. 1.3.3.4) is recognized as one of the most important targets for herbicide discovery. In this study, we report our ongoing research efforts toward the discovery of novel PPO inhibitors. Specifically, we identified a highly potent new compound series containing a pyrimidinedione moiety and bearing a versatile building block-benzoxazinone scaffold. Systematic bioassays resulted in the discovery of compound 7af, ethyl 4-(7-fluoro-6-(3-methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-dihydropyrimidin-1(2H)-yl)-3-oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)butanoate, which exhibited broad-spectrum and excellent herbicidal activity at the dosage of 37.5 g a.i./ha through postemergence application. The inhibition constant (Ki) value of 7af to Nicotiana tabacum PPO (NtPPO) was 14 nM, while to human PPO (hPPO), it was 44.8 µM, indicating a selective factor of 3200, making it the most selective PPO inhibitor to date. Moreover, molecular simulations further demonstrated the selectivity and the binding mechanism of 7af to NtPPO and hPPO. This study not only identifies a candidate that showed excellent in vivo bioactivity and high safety toward humans but also provides a paradigm for discovering PPO inhibitors with improved performance through molecular simulation and structure-guided optimization.

Discovery of a Subnanomolar Inhibitor of Protoporphyrinogen IX Oxidase via Fragment-Based Virtual Screening.

Protoporphyrinogen IX oxidase (PPO, E.C. 1.3.3.4), a key functional enzyme existing in various organisms, is acknowledged to be one of the most important action targets in the development of herbicides due to its pivotal roles in chlorophyll and heme biosynthesis pathways. As our persistent research work on the discovery of novel PPO-inhibiting herbicides, a new compound methyl 2-((5-(3-chloro-4,5,6,7-tetrahydro-2H-indazol-2-yl)-6-fluorobenzo[d]thiazol-2-yl)thio)acetate (8aj, Ki = 16 nM) was screened out as a hit compound via a fragment-based virtual screening method performed in the Auto Core Fragment in silico Screening web server. Subsequently, through a fused process of "hit-to-lead" optimization guided by molecular simulation, a total of 30 3-chloro-4,5,6,7-tetrahydro-2H-indazol-benzo[d]thiazole derivatives were synthesized and characterized. The results of the enzymatic inhibition bioassay showed that more than half of the newly synthesized compounds displayed higher activity against Nicotiana tabacum PPO (NtPPO) than oxadiazon, a commercial PPO-inhibiting herbicide. In particular, compound 8ab, a subnanomolar inhibitor with a Ki value of 380 pM against NtPPO, was discovered, which showed to be 71-fold more active than the commercial control oxadiazon (Ki = 27 nM), and was proven to be the most potent PPO inhibitor so far. Furthermore, the greenhouse assay demonstrated that most of the synthetic compounds showed good herbicidal activity toward the tested weeds. Especially, compound 8ad (Ki = 670 pM) showed the most promising post-emergence herbicidal activity with a broad spectrum of weed control even at a concentration as low as 37.5 g a.i./ha and relatively safe to rice at a dosage of 150 g a.i./ha, indicating that 8ad has the greatest potential to be developed as a new herbicide for weed control in paddy fields. This work provides a paradigm for the rational design and discovery of a novel PPO-inhibiting herbicide guided by the fragment-based drug design.

Pyrazole-Isoindoline-1,3-dione Hybrid: A Promising Scaffold for 4-Hydroxyphenylpyruvate Dioxygenase Inhibitors.

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 µM to over 1 µM. 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.