key: cord-1012963-9p8ghblr authors: Chen, Weixiong; Feng, Bo; Han, Sheng; Wang, Peipei; Chen, Wuhong; Zang, Yi; Li, Jia; Hu, Youhong title: Discovery of highly potent SARS-CoV-2 M(pro) inhibitors based on benzoisothiazolone scaffold date: 2022-02-15 journal: Bioorg Med Chem Lett DOI: 10.1016/j.bmcl.2022.128526 sha: 422fa35db9bb07f3891412282857f1a662d93fd8 doc_id: 1012963 cord_uid: 9p8ghblr The COVID-19 pandemic has drastically impacted global economies and public health. Although vaccine development has been successful, it was not sufficient against more infectious mutant strains including the Delta variant indicating a need for alternative treatment strategies such as small molecular compound development. In this work, a series of SARS-CoV-2 main protease (M(pro)) inhibitors were designed and tested based on the active compound from high-throughput diverse compound library screens. The most efficacious compound (16b-3) displayed potent SARS-CoV-2 M(pro) inhibition with an IC(50) value of 116 nM and selectivity against SARS-CoV-2 M(pro) when compared to PL(pro) and RdRp. This new class of compounds could be used as potential leads for further optimization in anti COVID-19 drug discovery. sites of nsps4-11/nsps4-16 to release non-structural proteins (nsps). [12] [13] [14] [15] These non-structural proteins, including RNA-dependent RNA polymerase (nsp12) and helicase (nsp13), etc, participate in protein translation and viral genetic material synthesis, which collectively play important roles in the life cycle of coronaviruses. [16] [17] [18] Inhibiting the main protease could therefore block the coronaviruses replication cycle and prevent further viral infection. Various groups have previously reported protease inhibitors as potentially attractive targeted antiviral drug (Fig.1a) . [19] [20] [21] [22] [23] [24] [25] [26] The first crystal structure of SARS-CoV-2 main protease is covalently combined with ligand N3. 19 These covalent inhibitors typically contain an active warhead group that covalently binds to Cys145 of the main protease. For example, Pfizer's compound PF-007304814 is currently undergoing clinical research from a peptide mimic. 22 The development of diverse inhibitor is needed to prevent the various coronavirus for the future. Recently, researchers identified Ebsulfur, Ebselen and their derivatives as potent main protease inhibitors that combine through covalent interactions with M pro . 19, 27, 28 However, they did not explore the detailed structureactivity relationship of these compounds systematically. Through high-throughput screening a diverse compound library, we also identified active compound that is similar to Ebsulfur and Ebselen derivatives in their potency as inhibitors of the SARS-CoV-2 main protease. Herein, we report the structure-activity relationship of this series of compounds and provide a deeper understand of the key structural features that are responsible for their activities. As shown in the Fig. 1b , the structural optimization and structure-activity analysis were focused on three functional groups: the tail benzene ring (ring B), linker and the core benzoisothiazolone (ring A). Next, we optimized the linker. The inhibitory activities of these compounds are given in Table 2 . We first tested the inhibitory activities of intermediates 3a, 10a and 13a. The results showed that the inhibitory activity of the intermediates 3a and 13a was reduced dramatically, which illustrated the importance of the hydrophobic spatial pocket. Extending (16q, 16r) or shortening (21a) the linker also decreased inhibitory activity of the compounds. After the amide group was opposed, the activity of the compound 21b (IC 50 = 253 nM) remained at the same level as 16b. However, once the linker was replaced with an alkane chain, the inhibitory activity of compound 21c (IC 50 = 540 nM) dropped, which indicated that the acetamide group in the linker plays an important role in the inhibitory activity. (Table 3 ). These results indicate that the core structure of 16b binds with the main protease of SARS-CoV-2 covalently, which is supported by other research. 28 And then, we investigated the different substituents on the phenyl group (16b-2 ~ 16b-17) in Table 4 . The presence of the relative bulky groups at position 7 of phenyl ring (compound 16b-9, 16b-13, 16b-17) decreased the activity drastically. The steric hindrance might block the covalent attack of Cys145 in the main protease. Introducing substituents at other position on the phenyl ring maintained the activity. Compound 16b-3 contained an F substituent of the phenyl ring at position 4 yielded the good activity with an IC 50 value of 116 nM. Papain-like protease (PL pro ) is a cysteine protease, which hydrolyzes the polyproteins pp1a/pp1ab on the sites of nsps1-3 to release non-structural proteins. To verify whether these compounds act as covalent inhibitors of PL pro or RdRp, we evaluated the activities of the represented compounds against PL pro and RdRp. As shown in Table 5 , the compounds with a benzoisothiazolone core showed high selectivity against SARS-CoV-2 M pro comparing to PL pro and RdRp. Given the excellent potency and selectivity of this series compounds, the fast dilution experiment was performed to determine whether the inhibition of these compounds is reversible or not. The result of the fast dilution experiment (see Fig. S1 . and Table S1 .) indicated that compound 16b-3, just as GC-376, 32 is an irreversible inhibitor of SARS-CoV-2 M pro . In summary, we have identified a novel series of compounds that potently inhibited the SARS-CoV-2 main protease with high selectivity against SARS-CoV-2 M pro when compared to PL pro and RdRp. The most efficacious compound 16b-3 displayed IC 50 values of 116 nM against SARS-CoV-2 M pro , which was more potent than Ebsulfur (IC 50 = 490 nM). These new compounds could be a potential lead for further optimization in anti COVID-19 drug discovery. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 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