Synthesis and Biochemical Evaluation of 8H-Indeno[1,2-d]thiazole Derivatives as Novel SARS-CoV-2 3CL Protease Inhibitors

The COVID-19 pandemic caused by SARS-CoV-2 is a global burden on human health and economy. The 3-Chymotrypsin-like cysteine protease (3CLpro) becomes an attractive target for SARS-CoV-2 due to its important role in viral replication. We synthesized a series of 8H-indeno[1,2-d]thiazole derivatives and evaluated their biochemical activities against SARS-CoV-2 3CLpro. Among them, the representative compound 7a displayed inhibitory activity with an IC50 of 1.28 ± 0.17 μM against SARS-CoV-2 3CLpro. Molecular docking of 7a against 3CLpro was performed and the binding mode was rationalized. These preliminary results provide a unique prototype for the development of novel inhibitors against SARS-CoV-2 3CLpro.


Introduction
The global pandemic of coronavirus disease  caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posted major challenges to public health systems and the economy worldwide [1][2][3][4][5]. There have been 434 million confirmed cases of COVID-19 worldwide as of the end of February 2022, and almost 6 million deaths have been reported [6]. Although multiple effective vaccines against COVID-19 are available, reinfections and breakthrough infections are frequently reported [7,8]. In addition, the virus is continuing to evolve, and a new variant named Omicron enables the virus to evade the immune protective barrier due to a large number of mutations in the receptor binding sites [9][10][11]. Therefore, it is urgent to develop effective drugs and specific treatments for people who are infected by COVID-19 with severe symptoms.
3CL pro (also called M pro ) plays an essential role during replication and transcription of SARS-CoV-2 and has been regarded as an attractive target for treating COVID-19 and other coronavirus-caused diseases [12][13][14]. The development of 3CL pro inhibitors has attracted much attention from medicinal chemists and the pharmaceutical industry. The collective efforts culminated in the recent approval of Paxlovid (nirmatrelvir) by FDA for the treatment of SARS-CoV-2 [15]. As shown in Figure 1, Most known 3CL pro inhibitors are In pursuit of novel 3CL pro inhibitors, we identified 8H-indeno[1,2-d]thiazole derivative 4 as a novel SARS-CoV-2 3CL pro inhibitor (IC50 = 6.42 ± 0.90 μM) through highthroughput screening of our compound collection ( Figure 2). This result provided us with an opportunity to explore novel small molecule inhibitors against SARS-CoV-2 3CL pro . Herein, we designed and synthesized a series of 8H-indeno[1,2-d]thiazole derivatives, evaluated their inhibitory activities against SARS-CoV-2 3CL pro , and elucidated the SARs. Selected compound 7a was subjected to molecular docking to predict the binding mode with SARS-CoV-2 3CL pro .  In pursuit of novel 3CL pro inhibitors, we identified 8H-indeno[1,2-d]thiazole derivative 4 as a novel SARS-CoV-2 3CL pro inhibitor (IC 50 = 6.42 ± 0.90 µM) through high-throughput screening of our compound collection ( Figure 2). This result provided us with an opportunity to explore novel small molecule inhibitors against SARS-CoV-2 3CL pro . Herein, we designed and synthesized a series of 8H-indeno[1,2-d]thiazole derivatives, evaluated their inhibitory activities against SARS-CoV-2 3CL pro , and elucidated the SARs. Selected compound 7a was subjected to molecular docking to predict the binding mode with SARS-CoV-2 3CL pro . In pursuit of novel 3CL pro inhibitors, we identified 8H-indeno[1,2-d]thiazole derivative 4 as a novel SARS-CoV-2 3CL pro inhibitor (IC50 = 6.42 ± 0.90 μM) through highthroughput screening of our compound collection ( Figure 2). This result provided us with an opportunity to explore novel small molecule inhibitors against SARS-CoV-2 3CL pro . Herein, we designed and synthesized a series of 8H-indeno[1,2-d]thiazole derivatives, evaluated their inhibitory activities against SARS-CoV-2 3CL pro , and elucidated the SARs. Selected compound 7a was subjected to molecular docking to predict the binding mode with SARS-CoV-2 3CL pro .

SARS-CoV-2 3CL pro Inhibitory Activities and Structure-Activity Relationships
All synthesized compounds were evaluated for inhibitory activity against SARS-CoV-2 3CL pro using PF-07321332 as positive control [29][30][31], and the results were detailed in Table 1. We initially prepared 7a from the commercially available compound 5a by the route outlined in Scheme 1. We noticed that compound 7a with 6-methoxy group on the phenyl ring exhibited inhibitory activity against SARS-CoV-2 3CL pro with 1.28 ± 0.17 μM, about five times more potent than compound 4 with 5-methoxy group on the phenyl ring. The result indicated that the position of the methoxy group on the phenyl ring significantly affected inhibitory activities against SARS-CoV-2 3CL pro . To explore the SAR of this seemingly important position, methoxy group on compound 7a was replaced by butoxy (7b), isobutoxy (7c), methyl groups (7d), and chlorine atom (7e); the inhibitory activities of the corresponding compounds 7b-7e were completely abolished. These results  Based on the structure of compound 4, 14 new 8H-indeno[1,2-d]thiazole derivatives (compounds 7a-7l, and 10a-10b) (shown in Schemes 1 and 2) were designed and synthesized through a two-step synthesis from the appropriate ketone and thiourea [25][26][27][28]. Adjusting the methoxy group of compound 4 from position 5 to position 6 afforded compound 7a. Considering the effects of steric hindrance and electron withdrawing, compounds 7b-7e were synthesized by substitution of the methoxy group for the butoxy, isobutoxy, and methyl groups and for the chlorine atom. After replacing the 3,5-dimethoxybenzamido moiety in compound 7a with 3,4,5-trimethoxybenzamido, 3,5-diacetoxybenzamido, 3-methoxybenzamido, 3-fluorobenzamido, thiophene-2-carboxamido, and 4chlorobenzamido, compounds 7f-7k were obtained. To evaluate the effect of ring expansion, compound 7l was synthesized. Finally, ring opening analogues 10a and 10b were synthesized to elucidate the effect of the central ring on the inhibition of 3CL pro .

SARS-CoV-2 3CL pro Inhibitory Activities and Structure-Activity Relationships
All synthesized compounds were evaluated for inhibitory activity against SARS-CoV-2 3CL pro using PF-07321332 as positive control [29][30][31], and the results were detailed in Table 1. We initially prepared 7a from the commercially available compound 5a by the route outlined in Scheme 1. We noticed that compound 7a with 6-methoxy group on the phenyl ring exhibited inhibitory activity against SARS-CoV-2 3CL pro with 1.28 ± 0.17 μM, about five times more potent than compound 4 with 5-methoxy group on the phenyl ring. The result indicated that the position of the methoxy group on the phenyl ring significantly affected inhibitory activities against SARS-CoV-2 3CL pro . To explore the SAR of this seemingly important position, methoxy group on compound 7a was replaced by butoxy (7b), isobutoxy (7c), methyl groups (7d), and chlorine atom (7e); the inhibitory activities of the corresponding compounds 7b-7e were completely abolished. These results

SARS-CoV-2 3CL pro Inhibitory Activities and Structure-Activity Relationships
All synthesized compounds were evaluated for inhibitory activity against SARS-CoV-2 3CL pro using PF-07321332 as positive control [29][30][31], and the results were detailed in Table 1. We initially prepared 7a from the commercially available compound 5a by the route outlined in Scheme 1. We noticed that compound 7a with 6-methoxy group on the phenyl ring exhibited inhibitory activity against SARS-CoV-2 3CL pro with 1.28 ± 0.17 µM, about five times more potent than compound 4 with 5-methoxy group on the phenyl ring. The result indicated that the position of the methoxy group on the phenyl ring significantly affected inhibitory activities against SARS-CoV-2 3CL pro . To explore the SAR of this seemingly important position, methoxy group on compound 7a was replaced by butoxy (7b), isobutoxy (7c), methyl groups (7d), and chlorine atom (7e); the inhibitory activities of the corresponding compounds 7b-7e were completely abolished. These results demonstrated that the effect of steric hindrance at this position was detrimental to inhibitory activities. The SAR of R 3 was explored next. Replacement of the 3,5-dimethoxybenzamido moiety with 3,4,5-trimethoxybenzamido moiety, 3,5-diacetoxybenzamido moiety, 3-methoxybenzamido moiety, 3-fluorobenzamido moiety, thiophene-2-carboxamido moiety, and 4-chlorobenzamido moiety led to compounds 7f, 7g, 7h, 7i, 7j, and 7k, respectively. The inhibitory activity of compounds 7f and 7g dropped significantly, while compound 7h almost maintained its inhibitory activities. These results indicated that the extra steric hindrance had negative impact on the inhibitory activities. Compared to compound 7h, the inhibitory activities of compounds 7i-7k diminished; these results indicated that introduction of an electron-withdrawing group or heterocyclic ring on the scaffold of 8H-indeno[1,2-d]thiazole took negative roles for inhibitory activities. Expanding the five-membered ring on compound 7a to a six-membered ring led to compound 7l, which unfortunately did not show any inhibitory activity against SARS-CoV-2 3CL pro . Opening the five-membered ring on compound 7a resulted in compounds 10a and 10b, which also lost inhibitory activities. These results indicated that the five-membered ring on compound 7a is important for the inhibitory activity against SARS-CoV-2 3CL pro . demonstrated that the effect of steric hindrance at this position was detrimental to inhibitory activities. The SAR of R 3 was explored next. Replacement of the 3,5-dimethoxybenzamido moiety with 3,4,5-trimethoxybenzamido moiety, 3,5-diacetoxybenzamido moiety, 3-methoxybenzamido moiety, 3-fluorobenzamido moiety, thiophene-2-carboxamido moiety, and 4-chlorobenzamido moiety led to compounds 7f, 7g, 7h, 7i, 7j, and 7k, respectively. The inhibitory activity of compounds 7f and 7g dropped significantly, while compound 7h almost maintained its inhibitory activities. These results indicated that the extra steric hindrance had negative impact on the inhibitory activities. Compared to compound 7h, the inhibitory activities of compounds 7i-7k diminished; these results indicated that introduction of an electron-withdrawing group or heterocyclic ring on the scaffold of 8Hindeno[1,2-d]thiazole took negative roles for inhibitory activities. Expanding the fivemembered ring on compound 7a to a six-membered ring led to compound 7l, which unfortunately did not show any inhibitory activity against SARS-CoV-2 3CL pro . Opening the five-membered ring on compound 7a resulted in compounds 10a and 10b, which also lost inhibitory activities. These results indicated that the five-membered ring on compound 7a is important for the inhibitory activity against SARS-CoV-2 3CL pro . demonstrated that the effect of steric hindrance at this position was detrimental to inhibitory activities. The SAR of R 3 was explored next. Replacement of the 3,5-dimethoxybenzamido moiety with 3,4,5-trimethoxybenzamido moiety, 3,5-diacetoxybenzamido moiety, 3-methoxybenzamido moiety, 3-fluorobenzamido moiety, thiophene-2-carboxamido moiety, and 4-chlorobenzamido moiety led to compounds 7f, 7g, 7h, 7i, 7j, and 7k, respectively. The inhibitory activity of compounds 7f and 7g dropped significantly, while compound 7h almost maintained its inhibitory activities. These results indicated that the extra steric hindrance had negative impact on the inhibitory activities. Compared to compound 7h, the inhibitory activities of compounds 7i-7k diminished; these results indicated that introduction of an electron-withdrawing group or heterocyclic ring on the scaffold of 8Hindeno[1,2-d]thiazole took negative roles for inhibitory activities. Expanding the fivemembered ring on compound 7a to a six-membered ring led to compound 7l, which unfortunately did not show any inhibitory activity against SARS-CoV-2 3CL pro . Opening the five-membered ring on compound 7a resulted in compounds 10a and 10b, which also lost inhibitory activities. These results indicated that the five-membered ring on compound 7a is important for the inhibitory activity against SARS-CoV-2 3CL pro . demonstrated that the effect of steric hindrance at this position was detrimental to inhibitory activities. The SAR of R 3 was explored next. Replacement of the 3,5-dimethoxybenzamido moiety with 3,4,5-trimethoxybenzamido moiety, 3,5-diacetoxybenzamido moiety, 3-methoxybenzamido moiety, 3-fluorobenzamido moiety, thiophene-2-carboxamido moiety, and 4-chlorobenzamido moiety led to compounds 7f, 7g, 7h, 7i, 7j, and 7k, respectively. The inhibitory activity of compounds 7f and 7g dropped significantly, while compound 7h almost maintained its inhibitory activities. These results indicated that the extra steric hindrance had negative impact on the inhibitory activities. Compared to compound 7h, the inhibitory activities of compounds 7i-7k diminished; these results indicated that introduction of an electron-withdrawing group or heterocyclic ring on the scaffold of 8Hindeno[1,2-d]thiazole took negative roles for inhibitory activities. Expanding the fivemembered ring on compound 7a to a six-membered ring led to compound 7l, which unfortunately did not show any inhibitory activity against SARS-CoV-2 3CL pro . Opening the five-membered ring on compound 7a resulted in compounds 10a and 10b, which also lost inhibitory activities. These results indicated that the five-membered ring on compound 7a is important for the inhibitory activity against SARS-CoV-2 3CL pro . demonstrated that the effect of steric hindrance at this position was detrimental to inhibitory activities. The SAR of R 3 was explored next. Replacement of the 3,5-dimethoxybenzamido moiety with 3,4,5-trimethoxybenzamido moiety, 3,5-diacetoxybenzamido moiety, 3-methoxybenzamido moiety, 3-fluorobenzamido moiety, thiophene-2-carboxamido moiety, and 4-chlorobenzamido moiety led to compounds 7f, 7g, 7h, 7i, 7j, and 7k, respectively. The inhibitory activity of compounds 7f and 7g dropped significantly, while compound 7h almost maintained its inhibitory activities. These results indicated that the extra steric hindrance had negative impact on the inhibitory activities. Compared to compound 7h, the inhibitory activities of compounds 7i-7k diminished; these results indicated that introduction of an electron-withdrawing group or heterocyclic ring on the scaffold of 8Hindeno[1,2-d]thiazole took negative roles for inhibitory activities. Expanding the fivemembered ring on compound 7a to a six-membered ring led to compound 7l, which unfortunately did not show any inhibitory activity against SARS-CoV-2 3CL pro . Opening the five-membered ring on compound 7a resulted in compounds 10a and 10b, which also lost inhibitory activities. These results indicated that the five-membered ring on compound 7a is important for the inhibitory activity against SARS-CoV-2 3CL pro . demonstrated that the effect of steric hindrance at this position was detrimental to inhibitory activities. The SAR of R 3 was explored next. Replacement of the 3,5-dimethoxybenzamido moiety with 3,4,5-trimethoxybenzamido moiety, 3,5-diacetoxybenzamido moiety, 3-methoxybenzamido moiety, 3-fluorobenzamido moiety, thiophene-2-carboxamido moiety, and 4-chlorobenzamido moiety led to compounds 7f, 7g, 7h, 7i, 7j, and 7k, respectively. The inhibitory activity of compounds 7f and 7g dropped significantly, while compound 7h almost maintained its inhibitory activities. These results indicated that the extra steric hindrance had negative impact on the inhibitory activities. Compared to compound 7h, the inhibitory activities of compounds 7i-7k diminished; these results indicated that introduction of an electron-withdrawing group or heterocyclic ring on the scaffold of 8Hindeno[1,2-d]thiazole took negative roles for inhibitory activities. Expanding the fivemembered ring on compound 7a to a six-membered ring led to compound 7l, which unfortunately did not show any inhibitory activity against SARS-CoV-2 3CL pro . Opening the five-membered ring on compound 7a resulted in compounds 10a and 10b, which also lost inhibitory activities. These results indicated that the five-membered ring on compound 7a is important for the inhibitory activity against SARS-CoV-2 3CL pro . demonstrated that the effect of steric hindrance at this position was detrimental to inhibitory activities. The SAR of R 3 was explored next. Replacement of the 3,5-dimethoxybenzamido moiety with 3,4,5-trimethoxybenzamido moiety, 3,5-diacetoxybenzamido moiety, 3-methoxybenzamido moiety, 3-fluorobenzamido moiety, thiophene-2-carboxamido moiety, and 4-chlorobenzamido moiety led to compounds 7f, 7g, 7h, 7i, 7j, and 7k, respectively. The inhibitory activity of compounds 7f and 7g dropped significantly, while compound 7h almost maintained its inhibitory activities. These results indicated that the extra steric hindrance had negative impact on the inhibitory activities. Compared to compound 7h, the inhibitory activities of compounds 7i-7k diminished; these results indicated that introduction of an electron-withdrawing group or heterocyclic ring on the scaffold of 8Hindeno[1,2-d]thiazole took negative roles for inhibitory activities. Expanding the fivemembered ring on compound 7a to a six-membered ring led to compound 7l, which unfortunately did not show any inhibitory activity against SARS-CoV-2 3CL pro . Opening the five-membered ring on compound 7a resulted in compounds 10a and 10b, which also lost inhibitory activities. These results indicated that the five-membered ring on compound 7a is important for the inhibitory activity against SARS-CoV-2 3CL pro . demonstrated that the effect of steric hindrance at this position was detrimental to inhibitory activities. The SAR of R 3 was explored next. Replacement of the 3,5-dimethoxybenzamido moiety with 3,4,5-trimethoxybenzamido moiety, 3,5-diacetoxybenzamido moiety, 3-methoxybenzamido moiety, 3-fluorobenzamido moiety, thiophene-2-carboxamido moiety, and 4-chlorobenzamido moiety led to compounds 7f, 7g, 7h, 7i, 7j, and 7k, respectively. The inhibitory activity of compounds 7f and 7g dropped significantly, while compound 7h almost maintained its inhibitory activities. These results indicated that the extra steric hindrance had negative impact on the inhibitory activities. Compared to compound 7h, the inhibitory activities of compounds 7i-7k diminished; these results indicated that introduction of an electron-withdrawing group or heterocyclic ring on the scaffold of 8Hindeno[1,2-d]thiazole took negative roles for inhibitory activities. Expanding the fivemembered ring on compound 7a to a six-membered ring led to compound 7l, which unfortunately did not show any inhibitory activity against SARS-CoV-2 3CL pro . Opening the five-membered ring on compound 7a resulted in compounds 10a and 10b, which also lost inhibitory activities. These results indicated that the five-membered ring on compound 7a is important for the inhibitory activity against SARS-CoV-2 3CL pro . demonstrated that the effect of steric hindrance at this position was detrimental to inhibitory activities. The SAR of R 3 was explored next. Replacement of the 3,5-dimethoxybenzamido moiety with 3,4,5-trimethoxybenzamido moiety, 3,5-diacetoxybenzamido moiety, 3-methoxybenzamido moiety, 3-fluorobenzamido moiety, thiophene-2-carboxamido moiety, and 4-chlorobenzamido moiety led to compounds 7f, 7g, 7h, 7i, 7j, and 7k, respectively. The inhibitory activity of compounds 7f and 7g dropped significantly, while compound 7h almost maintained its inhibitory activities. These results indicated that the extra steric hindrance had negative impact on the inhibitory activities. Compared to compound 7h, the inhibitory activities of compounds 7i-7k diminished; these results indicated that introduction of an electron-withdrawing group or heterocyclic ring on the scaffold of 8Hindeno[1,2-d]thiazole took negative roles for inhibitory activities. Expanding the fivemembered ring on compound 7a to a six-membered ring led to compound 7l, which unfortunately did not show any inhibitory activity against SARS-CoV-2 3CL pro . Opening the five-membered ring on compound 7a resulted in compounds 10a and 10b, which also lost inhibitory activities. These results indicated that the five-membered ring on compound 7a is important for the inhibitory activity against SARS-CoV-2 3CL pro . introduction of an electron-withdrawing group or heterocyclic ring on the scaffold of 8Hindeno[1,2-d]thiazole took negative roles for inhibitory activities. Expanding the fivemembered ring on compound 7a to a six-membered ring led to compound 7l, which unfortunately did not show any inhibitory activity against SARS-CoV-2 3CL pro . Opening the five-membered ring on compound 7a resulted in compounds 10a and 10b, which also lost inhibitory activities. These results indicated that the five-membered ring on compound 7a is important for the inhibitory activity against SARS-CoV-2 3CL pro . To explore the interaction mode between small molecule 7a and 3CL pro (PDB code: 6M2N) [23], we carried out molecular docking by applying AutoDock 4.2 program [31][32][33][34]. Figure 3a showed that 7a docked well into the binding pockets S1 and S2 of 3CL pro , in which the S1, S2 sites play a key role in substrate recognition [35]. As illustrated in Figure  3b, the indene moiety of compound 7a buried deeply into the hydrophobic S2 subsite with π-electrons with Arg188 and hydrophobic interaction with Met165; the 3,5-dimethoxybenzamido moiety of compound 7a formed strong H-bonds with Asn142, Glu166 on S1 subsite, while compounds 4 and 7h escaped from S1 subsite, as shown in Supplementary Materials Figures S1 and S2. To explore the interaction mode between small molecule 7a and 3CL pro (PDB code: 6M2N) [23], we carried out molecular docking by applying AutoDock 4.2 program [31][32][33][34]. Figure 3a showed that 7a docked well into the binding pockets S1 and S2 of 3CL pro , in which the S1, S2 sites play a key role in substrate recognition [35]. As illustrated in Figure  3b, the indene moiety of compound 7a buried deeply into the hydrophobic S2 subsite with π-electrons with Arg188 and hydrophobic interaction with Met165; the 3,5-dimethoxybenzamido moiety of compound 7a formed strong H-bonds with Asn142, Glu166 on S1 subsite, while compounds 4 and 7h escaped from S1 subsite, as shown in Supplementary Materials Figures S1 and S2. To explore the interaction mode between small molecule 7a and 3CL pro (PDB code: 6M2N) [23], we carried out molecular docking by applying AutoDock 4.2 program [31][32][33][34]. Figure 3a showed that 7a docked well into the binding pockets S1 and S2 of 3CL pro , in which the S1, S2 sites play a key role in substrate recognition [35]. As illustrated in Figure  3b, the indene moiety of compound 7a buried deeply into the hydrophobic S2 subsite with π-electrons with Arg188 and hydrophobic interaction with Met165; the 3,5-dimethoxybenzamido moiety of compound 7a formed strong H-bonds with Asn142, Glu166 on S1 subsite, while compounds 4 and 7h escaped from S1 subsite, as shown in Supplementary Materials Figures S1 and S2. To explore the interaction mode between small molecule 7a and 3CL pro (PDB code: 6M2N) [23], we carried out molecular docking by applying AutoDock 4.2 program [31][32][33][34]. Figure 3a showed that 7a docked well into the binding pockets S1 and S2 of 3CL pro , in which the S1, S2 sites play a key role in substrate recognition [35]. As illustrated in Figure  3b, the indene moiety of compound 7a buried deeply into the hydrophobic S2 subsite with π-electrons with Arg188 and hydrophobic interaction with Met165; the 3,5-dimethoxybenzamido moiety of compound 7a formed strong H-bonds with Asn142, Glu166 on S1 subsite, while compounds 4 and 7h escaped from S1 subsite, as shown in Supplementary Materials Figures S1 and S2 To explore the interaction mode between small molecule 7a and 3CL pro (PDB code: 6M2N) [23], we carried out molecular docking by applying AutoDock 4.2 program [31][32][33][34]. Figure 3a showed that 7a docked well into the binding pockets S1 and S2 of 3CL pro , in which the S1, S2 sites play a key role in substrate recognition [35]. As illustrated in Figure  3b, the indene moiety of compound 7a buried deeply into the hydrophobic S2 subsite with π-electrons with Arg188 and hydrophobic interaction with Met165; the 3,5-dimethoxybenzamido moiety of compound 7a formed strong H-bonds with Asn142, Glu166 on S1 subsite, while compounds 4 and 7h escaped from S1 subsite, as shown in Supplementary Materials Figures S1 and S2 To explore the interaction mode between small molecule 7a and 3CL pro (PDB code: 6M2N) [23], we carried out molecular docking by applying AutoDock 4.2 program [31][32][33][34]. Figure 3a showed that 7a docked well into the binding pockets S1 and S2 of 3CL pro , in which the S1, S2 sites play a key role in substrate recognition [35]. As illustrated in Figure  3b, the indene moiety of compound 7a buried deeply into the hydrophobic S2 subsite with π-electrons with Arg188 and hydrophobic interaction with Met165; the 3,5-dimethoxybenzamido moiety of compound 7a formed strong H-bonds with Asn142, Glu166 on S1 subsite, while compounds 4 and 7h escaped from S1 subsite, as shown in Supplementary Materials Figures S1 and S2. 99.5 ± 0.1 0.012 ± 0.001

Predicting Binding Mode of 7a with 3CL pro
To explore the interaction mode between small molecule 7a and 3CL pro (PDB code: 6M2N) [23], we carried out molecular docking by applying AutoDock 4.2 program [31][32][33][34]. Figure 3a showed that 7a docked well into the binding pockets S1 and S2 of 3CL pro , in which the S1, S2 sites play a key role in substrate recognition [35]. As illustrated in Figure 3b, the indene moiety of compound 7a buried deeply into the hydrophobic S2 subsite with π-electrons with Arg188 and hydrophobic interaction with Met165; the 3,5-dimethoxybenzamido moiety of compound 7a formed strong H-bonds with Asn142, Glu166 on S1 subsite, while compounds 4 and 7h escaped from S1 subsite, as shown in Supplementary Materials Figures S1 and S2.

Chemistry
All chemical reagents are reagent grade and used as purchased. 1 H NMR (400 MHz) spectra were recorded on a Bruker AVIII 400 MHz spectrometer (Bruker, Billerica, MA, USA). The chemical shifts were reported in parts per million (ppm) using the 2.50 signal of DMSO ( 1 H NMR) and the 39.52 signal of DMSO ( 13 C NMR) as internal standards. ESI Mass spectra (MS) were obtained on a SHIMADZU 2020 Liquid Chromatograph Mass Spectrometer (SHIMADZU, Kyoto, Japan).
3.1.1. General Procedure for the Synthesis of Compounds 7a-7k (Exemplified by 7a) To a solution of 5a (6.2 mmol, 1.0 equiv) in dry ethanol (25 mL) were added thiourea (12.4 mmol, 2.0 equiv) and bromine (6.8 mmol, 1.1 equiv) at room temperature. The reaction solution was stirred at 100 • C for 5-6 h, At the end of the reaction, the solvent was evaporated, and aqueous ammonium hydroxide (25%) was added to the residue. The precipitated solid was collected without purification for the next step. The mixture of 6a (2.2 mmol, 1.1 equiv), aromatic acid (2.0 mmol, 1.0 equiv), HATU (2.0 mmol, 1.0 equiv), and DIPEA (6.0 mmol, 3.0 equiv) in DMF (15 mL) was stirred at room temperature for 2 h. The reaction mixture was quenched with water. The aqueous layer was extracted with EtOAc (30 mL × 2). The combined organic layers were dried over Na 2 SO 4 . The residue was purified by column chromatography on silica gel (eluting with DCM) to afford compound 7a as a yellow solid (280.0 mg, yield 37%). 1 13   ppm. 13