Pathogenic fungi are one of the major culprits in causing plant diseases, posing a serious threat to the production of food crops, economic crops, and horticultural crops worldwide [1,2]. Notably, M. oryzae and B. cinerea, as the pathogen of rice blast and grey mold diseases, respectively, are acknowledged as the two most significant plant pathogenic fungi due to the severe damage and diverse host range [3]. Traditionally, the use of chemical fungicides has been the most economical and effective manners for controlling plant fungal diseases [4]. However, owing to the limited targets and mechanism of action of existing fungicides, coupled with the long-term inappropriate application, resistance and environmental related issues have intensified, which presents substantial challenges for the management of plant fungal diseases [[5], [6], [7]]. For example, the resistance risk of broad-spectrum SDHI fungicides has been escalating over time, and the toxicity of benzimidazoles fungicides to aquatic organisms, particularly vertebrates, has attracted growing concern [8,9]. Consequently, the development of fungicides based on novel targets has gradually become a priority to cope with the issues associated with existing chemical fungicides.

Trehalose-6-phosphate synthase (TPS1 in fungi) is responsible for catalyzing the transformation of the glucose group from the donor substrate UDPG to the acceptor substrate G6P to synthesize T6P, being served as a precursor in trehalose biosynthesis in fungi [10,11]. Numerous studies have demonstrated that TPS1 is a crucial protein in regulating infection and colonization in plant pathogenic fungi. For M. oryzae, MoTPS1 has been proven to be closely participated in the growth and infection functions of M. oryzae by modulating carbon and nitrogen metabolism, conidia production, and the accumulation of turgor pressure within appressoria [[12], [13], [14]]. In B. cinerea, the mutant strain lacking the BcTPS1 encoding gene exhibited significantly reduced stress tolerance, and the normal sporulation process was disrupted as well [15]. Moreover, relative studies have highlighted the essential role of TPS1 in regulating various physiological processes, including the production of virulence factors, the response to environmental stress, sclerotium formation, and hyphal growth in Fusarium graminearum, Puccinia triticina, Rhizoctonia solani, Fusarium verticillioides, and Cytospora chrysosperma [[16], [17], [18], [19], [20], [21]]. In addition to these plant pathogenic fungi, the function of TPS1 in modulating the pathogenicity has also been well characterized in human pathogenic fungi, such as Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans [22]. Consequently, considering the essentiality of TPS1 function and its absence in vertebrates, TPS1 is considered as a potential target for the development of novel both fungicides and antifungal drugs [23]. Notably, with the advancement of structural biology research techniques, several crystal or cryo-EM structures of TPS1 in various plant pathogenic fungi and human pathogenic fungi including M. oryzae, C. albicans, and C. neoformans have been resolved, which provides an opportunity for the screening and rational designing of TPS1 inhibitors to be applied in pesticide or pharmaceutical fields [[24], [25], [26]].

In our previous study, an initial attempt to screen and develop inhibitors based on the structure of MoTPS1 for the control of M. oryzae has been made, through which isopropanolamine compound j11 with certain inhibitory activities against MoTPS1 and the pathogenicity of M. oryzae was discovered [27]. However, the structure of j11 still requires further optimization to enhance its bioactivity. Fragment replacement and merging are effective strategies for enhancing the bioactivity of lead compounds. Sulfonamide is a commonly used fragment in agrochemical fungicides, such as flumetylsulforim, amisulbrom, flusulfamide, and dichlofluanid (Fig. 1A), known for its structural stability and its ability to provide multiple hydrogen bond acceptors, making it a key fungicidal active fragment [28,29]. Triazole derivatives are among the most broad-spectrum and potent fungicides and antifungal agents including tebuconazole, hexaconazole, fluconazole, voriconazole and so on applied in both agriculture and medicine (Fig. 1B). The 1,2,4-triazole ring, a recurring fragment in the aforementioned derivatives, has gained significant attention in recent years for the design and development of novel fungicides and antifungal drugs [30,31].

Hence, in this study, the isopropanolamine compound j11 reported in our previous work was served as a lead, and a strategy combining fragment replacement with rational design was employed to perform a two-step structural optimization of j11. Specifically, the sulfonamide and 1,2,4-triazole fragments were sequentially used to substitute the groups linked to both sides of the isopropanolamine linker in j11, leading to the design and synthesis of thirty-two novel isopropanolamine-based compounds. The inhibitory activities of these novel isopropanolamine compounds against MoTPS1 and BcTPS1 were examined in vitro by applying the previously established IPC method. The interaction mechanism of isopropanolamine compounds with MoTPS1 was subsequently unveiled through molecular dynamics simulation and MM-GBSA techniques. Besides, the inhibitory activities of novel isopropanolamine compounds against the pathogenicity of M. oryzae and B. cinerea were assayed by means of barley leaf and cherry tomato fruit inoculations, respectively. Furthermore, the fungicidal mechanism of novel isopropanolamine compounds were preliminarily investigated through various biological experiments. The fungicidal activities of novel isopropanolamine compounds against different plant pathogenic fungi were also tested using mycelial growth inhibition assay to probe the possibility of isopropanolamine-based TPS1 inhibitors as broad-spectrum fungicide candidates. This study presented an example of discovering efficient TPS1 inhibitors through structural optimization for the control of plant diseases.