Hyperuricemia (HUA) is a chronic disease caused by abnormal metabolism of purine nucleotides and has been confirmed to be associated with many diseases, such as gout, debate, and cardiovascular diseases [1,2]. In recent years, the incidence of HUA and gout is rapidly increasing in the worldwide [3]. Xanthine oxidase (XO) is a key enzyme that catalyzes the conversion of hypoxanthine to xanthine and then to uric acid. XO inhibitors (XOIs) that can reduce uric acid levels by blocking uric acid production in the body have a good effect on the treatment of HUA, gout, and other related diseases [[4], [5], [6]].

Classical XOIs clinically used as anti-gout drugs, mainly including allopurinol, febuxostat, and topiroxostat, as shown in Fig. 1. Allopurinol has been approved by the FDA as a purine XOI for the treatment of HUA since 1966 [7]. Regrettably, given its similar backbone to purine, allopurinol can cause many severe and life-threatening side effects including renal failure, fulminant hepatitis, and Stevens–Johnson syndrome [8]. Furthermore, non-purine XOIs have been proven to exhibit fewer side effects in reducing the level of uric acid in the body and have drawn worldwide researchers' attention. Currently, two non-purine XOIs, febuxostat (approved in the United States in 2009) and topiroxostat (approved in Japan in 2013), have been approved for the treatment of HUA [9,10]. In addition, in recent years, different febuxostat analogues with excellent inhibitory potencies against XO have been reported, including pyrazole [[11], [12], [13]], imidazoles [14,15], isoxazoles [16], selenazoles [17], pyrimidines [[18], [19], [20], [21]], thiazoles [[22], [23], [24]], benzofurans [25], 1,2,3-triazole [[26], [27], [28]], 1,2,4-beoxadiazol-5(4H)-ones [29,30], and some natural products [31,32]. However, febuxostat has been proven to show potential cardiovascular side effects, and the FDA issued a black box warning in 2019, suggesting that the drug may increase the risk associated with cardiac death [33]. Furthermore, the clinical studies of topiroxostat indicated that it could increase the incidence of gouty arthritis in the treatment of HUA [34]. There is an urgent need to develop novel non-purine XOIs with new chemical scaffolds for the treatment of HUA or gout.

Isoxazole derivatives have been identified to exhibit various biological activities such as anti-inflammatory, antibacterial, analgesic, and antituberculosis. The isoxazole derivatives have certain immunomodulatory effects and potential antiarthritic efficacy. In recent years, they have attracted much attention in the field of drug design research [35]. In a previous study, a series of 5-phenylisoxazole-3-carboxylic acid derivatives with remarkable inhibitory potencies against XO have been reported [16]. The molecular docking analysis indicated that the isoxazole fragment of these XOIs could form hydrogen bonds with key amino acid residues, including Glu802 and Arg880 in the active pocket of XO. Thus, the isoxazole moiety was a reliable and effective fragment to interact with XO.

Indoles are widely distributed in a variety of synthetic and natural products that usually possess various biological activities, including anti-inflammatory, antidiabetic, antiviral, and antitumor effects. In 2015, Song [22] synthesized a series of 2-(indol-5-yl)thiazole derivatives by expanding the benzene ring of febuxostat to the indole ring. The molecular docking results indicated that the 3-cyano indole moiety could form hydrogen bonds with the key amino acid residue Asn768 in the active pocket of XO, which greatly contributed to its remarkable inhibitory potency. Thus, the 3-cyano indole moiety could be a key pharmacophore for the design of non-purine XOIs. In this study, a series of novel non-purine XOIs, N-5-(1H-indol-5-yl)isoxazole-3-carboxylic acid derivatives, were designed according to the isosteric/bioisosteric strategy by replacing the benzene rings of febuxostat with indole and replacing the thiazole rings of febuxostat with isoxazole, as shown in Fig. 2. The designed target compounds (6a-6v, 8, and 9) were then synthesized effectively, and their inhibitory activities against XO as well as the structure-activity relationships were further investigated. Additionally, molecular modeling studies and enzyme kinetic assays were also used to analyze the inhibitory behaviors of the representative compounds 6c.