Using bioisosterism as a medicinal chemistry tool, 16 3,5-diaryl-isoxazole analogues of the tetrahydrofuran neolignans veraguensin, grandisin and machilin G were synthesized via 1,3-dipolar cycloaddition reactions, with yields from 43% to 90%. Antitrypanosomatid activities were evaluated against Trypanosoma cruzi, Leishmania (L.) amazonensis and Leishmania (V.) braziliensis. All compounds were selective for the Leishmania genus and inactive against T. cruzi. Isoxazole analogues showed a standard activity on both promastigotes of L. amazonensis and L. braziliensis. The most active compounds were 15, 16 and 19 with IC 50 values of 2.0, 3.3 and 9.5 μM against L. amazonensis and IC 50 values of 1.2, 2.1 and 6.4 μM on L. braziliensis, respectively. All compounds were noncytotoxic, showing lower cytotoxicity (>250 μM) than pentamidine (78.9 μM). Regarding the structure-activity relationship (SAR), the methylenedioxy group was essential to antileishmanial activity against promastigotes. Replacement of the tetrahydrofuran nucleus by an isoxazole core improved the antileishmanial activity. K E Y W O R D S bioisosterism, cycloaddition [3+2], isoxazole, neolignans 314 | TREFZGER ET al.

Isoxazole analogues derived from the neolignans veraguensin, grandisin, and machilin G were previously synthesized with different substitution patterns through the bioisosterism strategy. These compounds were tested on intracellular amastigotes of Leishmania (Leishmania) amazonensis; the derivatives proved to be active against intracellular amastigotes, with IC50 values ranging from 0.4 to 25 μM. The most active analogues were 4′, 14′, 15′, and 18′, with IC50 values of 0.9, 0.4, 0.7, and 1.4 μM, respectively, showing high selectivity indexes (SI = 277.0; 625.0; 178.5 and 357.1). Overall, the isoxazole analogues did not induce nitric oxide (NO) production by infected cells; there was no evidence that NO influences the antileishmanial mechanism of action, except for compound 4′. Trimethoxy groups as substituents seemed to be critical for antileishmanial activity. The SAR study demonstrated that the isoxazole compounds were more active than 1,2,3‐triazole compounds with the same substitution pattterns, demonstrating the importance of the bioisosterism strategy in drug design.

Chagas disease affects 6–8 million people worldwide, remaining a public health concern. Toxicity, several adverse effects and inefficiency in the chronic stage of the disease are the major challenges regarding the available treatment protocols. This work involved the synthesis of twenty‐two 1,4‐disubstituted‐1,2,3‐triazole analogues of benznidazole (BZN), by using a click chemistry strategy. Analogues were obtained in moderate to good yields (40‐97 %). Antitrypanosomal activity was evaluated against the amastigote forms of Trypanosoma cruzi. Compound 8 a (4‐(2‐nitro‐1H‐imidazol‐1‐yl)methyl)‐1‐phenyl‐1H‐1,2,3‐triazole) without substituents on phenyl ring showed similar biological activity to BZN (IC50=3.0 μM, SI>65.3), with an IC50=3.1 μM and SI>64.5. Compound 8 o (3,4‐di‐OCH3−Ph) with IC50 = 0.65 μM was five‐fold more active than BZN, and showed an excellent selectivity index (SI>307.7). Compound 8 v (3‐NO2, 4‐CH3−Ph) with IC50=1.2 μM and relevant SI>166.7, also exhibited higher activity than BZN. SAR analysis exhibited a pattern regarding antitrypanosomal activity relative to BZN, in compounds with electron‐withdrawing groups (Hammett σ+) at position 3, and electron‐donating groups (Hammett σ‐) at position 4, as observed in 8 o and 8 v. Further research might explore in vivo antitrypanosomal activity of promising analogues 8 a, 8 o, and 8 v. Overall, this study indicates that approaches such as the bioisosteric replacement of amide group by 1,2,3‐triazole ring, the use of click chemistry as a synthesis strategy, and design tools like Craig‐plot and Topliss tree are promising alternatives to drug discovery.

Nineteen 3,5-disubstituted-isoxazole analogs were synthesized based on nitrofuran scaffolds, by a [3 + 2] cycloaddition reaction between terminal acetylenes and 5-nitrofuran chloro-oxime. The compounds were obtained in moderate to very good yields (45-91%). The antileishmanial activity was assayed against the promastigote and amastigote forms of Leishmania (Leishmania) amazonensis. Alkylchlorinated compounds 14p-r were active on both the promastigote and amastigote forms, with emphasis on compound 14p, which showed strong activity against the amastigote form (IC 50 = 0.6 μM and selectivity index [SI] = 5.2). In the alkyl series, compound 14o stands out with an IC 50 = 8.5 μM and SI = 8.0 on the amastigote form. In the aromatic series, the most active compounds were those containing electron-donor groups, such as trimethoxy isoxazole 14g (IC 50 = 1.2 μM and SI = 20.2); compound 14h, with IC 50 = 7.0 μM and SI = 6.1; and compound 14j containing the 4-SCH 3 group, with IC 50 = 5.7 μM and SI = 10.2. In addition, the antifungal activity of 19 nitrofuran isoxazoles was evaluated against five strains of Candida (C. albicans, C. parapsilosis, C. krusei, C. tropicalis, and C. glabrata). Eleven isoxazole derivatives were active against C. parapsilosis, and compound 14o was found to be the most active (minimal inhibitory concentration [MIC] = 3.4 μM) for this strain. Compound 14p was active against all the strains tested, with an MIC = 17.5 μM for C. glabrata, lower than that of the fluconazole used as the reference drug. K E Y W O R D S5-nitrofuran scaffolds, antifungal activity, antileishmanial activity, drug design, isoxazole core *Ozildéia S. Trefzger, Natália V. Barbosa, and Renata L. Scapolatempo contributed equally to this work.

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Chagas disease (CD) is a neglected disease caused by the protozoan Trypanosoma cruzi. The two drugs used in the treatment schedules exhibit adverse effects and severe toxicity. Thus, searching for new antitrypanosomal agents is urgent to provide improved treatments to those affected by this disease. 5-Nitrofuran-isoxazole analogs were synthesized by cycloaddition reactions [3+2] between chloro-oximes and acetylenes in satisfactory yields. We analyzed the structure-activity relationship of the analogs based on Hammett's and Hansch's parameters. The 5-nitrofuran-isoxazole analogs exhibited relevant in vitro antitrypanosomal activity against the amastigote forms of T. cruzi. Analog 7s was the trending hit of the series, showing an IC 50 value of 40 nM and a selectivity index of 132.50. A possible explanation for this result may be the presence of an electrophile near the isoxazole core. Moreover, the most active analogs proved to act as an in vitro substrate of type I nitroreductase rather than the cruzain, enzymes commonly investigated in molecular target studies of CD drug discovery. These findings suggest that 5-nitrofuran-isoxazole analogs are promising in the studies of agents for CD treatment.

New treatment approaches targeting cutaneous leishmaniasis (CL) are required since conventional drugs exhibit limitations due to their several adverse effects and toxicity. In this study, we aimed to evaluate the in vivo intralesional treatment efficacy of five isoxazole derivatives previously synthesized and effective in vitro against intracellular amastigote forms of Leishmania (L.) amazonensis. Among the tested analogues, 7 exhibited relevant in vivo therapeutic effects. The in silico predictions provided interesting information about the toxicity, suggesting the safety of analogue 7. Experiments performed with Salmonella typhimurium strains (TA98, TA100, and TA102) showed a non-mutagenicity profile of 7. The treatment of Leishmania-infected BALB/c mice with isoxazole 7 showed remarkably smaller CL lesions and decreased the parasitism (by 98.4%) compared to the control group. Hence, analogue 7 is a promising drug candidate and alternative treatment for CL caused by L. amazonensis.