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

**Jing Wu 1,†, Bo Feng 2,3,†, Li-Xin Gao 1,3, Chun Zhang <sup>1</sup> , Jia Li 2,3,4, Da-Jun Xiang 5,\* , Yi Zang 3,\* and Wen-Long Wang 1,\***


**Abstract:** 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 8*H*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 IC<sup>50</sup> of 1.28 <sup>±</sup> 0.17 <sup>µ</sup>M 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 .

**Keywords:** COVID-19; Mpro inhibitors; drug design and synthesis; structure-activity relationships (SAR)

### **1. Introduction**

The global pandemic of coronavirus disease (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posted major challenges to public health systems and the economy worldwide [1–5]. 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 [9–11]. Therefore, it is urgent to develop effective drugs and specific treatments for people who are infected by COVID-19 with severe symptoms.

3CLpro (also called Mpro) 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 [12–14]. The development of 3CLpro 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 3CLpro inhibitors are

**Citation:** Wu, J.; Feng, B.; Gao, L.-X.; Zhang, C.; Li, J.; Xiang, D.-J.; Zang, Y.; Wang, W.-L. Synthesis and Biochemical Evaluation of 8*H*-Indeno[1,2-*d*]thiazole Derivatives as Novel SARS-CoV-2 3CL Protease Inhibitors. *Molecules* **2022**, *27*, 3359. https://doi.org/10.3390/ molecules27103359

Academic Editor: Joseph Sloop

Received: 19 April 2022 Accepted: 17 May 2022 Published: 23 May 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

peptidomimetic inhibitors containing a warhead of Michael acceptor, such as nirmatrelvir with nitrile [16], YH-53 with benzothiazolyl ketone [17], compound **1** with α-ketoamide [18], and compound **2** with aldehyde [19]. Others are nonpeptidic inhibitors including covalent and noncovalent inhibitors. Covalent inhibitors, such as Carmofur, Shikonin [20], and **3** [21], are identified by high-throughput screening. Noncovalent inhibitor CCF0058981 [22] and flavonoid analogs (baicalin, baicalein, and 40 -*O*-Methylscutellarein) [23,24] were obtained through structure-based optimization and from traditional Chinese medicines, respectively. trelvir with nitrile [16], YH-53 with benzothiazolyl ketone [17], compound **1** with α-ketoamide [18], and compound **2** with aldehyde [19]. Others are nonpeptidic inhibitors including covalent and noncovalent inhibitors. Covalent inhibitors, such as Carmofur, Shikonin [20], and **3** [21], are identified by high-throughput screening. Noncovalent inhibitor CCF0058981 [22] and flavonoid analogs (baicalin, baicalein, and 4′-*O-*Methylscutellarein) [23,24] were obtained through structure-based optimization and from traditional Chinese medicines, respectively. *Molecules* **2022**, *27*, x FOR PEER REVIEW 3 of 13

are peptidomimetic inhibitors containing a warhead of Michael acceptor, such as nirma-

*Molecules* **2022**, *27*, x FOR PEER REVIEW 2 of 11

**Figure 1.** SARS-CoV-2 3CLpro inhibitors. **Figure 1.** SARS-CoV-2 3CLpro inhibitors. **Figure 1.** SARS-CoV-2 3CLpro inhibitors.

In pursuit of novel 3CLpro inhibitors, we identified 8*H*-indeno[1,2-*d*]thiazole derivative **4** as a novel SARS-CoV-2 3CLpro 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 3CLpro. Herein, we designed and synthesized a series of 8*H*-indeno[1,2-*d*]thiazole derivatives, evaluated their inhibitory activities against SARS-CoV-2 3CLpro, and elucidated the SARs. Selected compound **7a** was subjected to molecular docking to predict the binding mode with SARS-CoV-2 3CLpro. In pursuit of novel 3CLpro inhibitors, we identified 8*H*-indeno[1,2-*d*]thiazole derivative **<sup>4</sup>** as a novel SARS-CoV-2 3CLpro inhibitor (IC<sup>50</sup> = 6.42 <sup>±</sup> 0.90 <sup>µ</sup>M) 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 3CLpro. Herein, we designed and synthesized a series of 8*H*-indeno[1,2-*d*]thiazole derivatives, evaluated their inhibitory activities against SARS-CoV-2 3CLpro, and elucidated the SARs. Selected compound **7a** was subjected to molecular docking to predict the binding mode with SARS-CoV-2 3CLpro . In pursuit of novel 3CLpro inhibitors, we identified 8*H*-indeno[1,2-*d*]thiazole derivative **4** as a novel SARS-CoV-2 3CLpro inhibitor (IC<sup>50</sup> = 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 3CLpro . Herein, we designed and synthesized a series of 8*H*-indeno[1,2-*d*]thiazole derivatives, evaluated their inhibitory activities against SARS-CoV-2 3CLpro, and elucidated the SARs. Selected compound **7a** was subjected to molecular docking to predict the binding mode with SARS-CoV-2 3CLpro .

Based on the structure of compound **4**, 14 new 8*H*-indeno[1,2-*d*]thiazole derivatives (compounds **7a–7l**, and **10a–10b**) (shown in Scheme 1 and Scheme 2) were designed and

[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

**Figure 2.** Structure of 8*H*-indeno[1,2-*d*]thiazole derivatives. **Figure 2.** Structure of 8*H*-indeno[1,2-*d*]thiazole derivatives. **Figure 2.** Structure of 8*H*-indeno[1,2-*d*]thiazole derivatives.

**2. Results and Discussion**

*2.1. Design and Synthesis of 8H-Indeno[1,2-d]thiazole Derivatives*

#### **2. Results and Discussion** 2.1. Design and Synthesis of 8H-Indeno[1,2-d]thiazole Derivatives 2. Results and Discussion

2. Results and Discussion

#### *2.1. Design and Synthesis of 8H-Indeno[1,2-d]thiazole Derivatives* Based on the structure of compound 4, 14 new 8H-indeno[1,2-d]thiazole derivatives

Molecules 2022, 27, x FOR PEER REVIEW 3 of 11

Based on the structure of compound **4**, 14 new 8*H*-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 [25–28]. 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 4-chlorobenzamido, 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 3CLpro . (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 [25–28]. 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 4 chlorobenzamido, 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 3CLpro . 2.1. Design and Synthesis of 8H-Indeno[1,2-d]thiazole Derivatives 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 [25–28]. 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 4 chlorobenzamido, compounds 7f–7k were obtained. To evaluate the effect of ring expansion, compound 7l was synthesized. Finally, ring opening analogues 10a and 10b were

Scheme 1. (a) thiourea, bromine, ethanol, 100 °C, 5–6 h; (b) aromatic acid, HATU, DIPEA, DMF, r t, 2–3 h, 25–50%. **Scheme 1.** (**a**) thiourea, bromine, ethanol, 100 ◦C, 5–6 h; (**b**) aromatic acid, HATU, DIPEA, DMF, r t, 2–3 h, 25–50%. Scheme 1. (a) thiourea, bromine, ethanol, 100 °C, 5–6 h; (b) aromatic acid, HATU, DIPEA, DMF, r t, 2–3 h, 25–50%.

Scheme 2. (a) thiourea, iodine, 110 °C, 10 h; (b) aromatic acid, HATU, DIPEA, DMF, r t, 2–3 h, 35– **Scheme 2.** (**a**) thiourea, iodine, 110 ◦C, 10 h; (**b**) aromatic acid, HATU, DIPEA, DMF, r t, 2–3 h, 35–40%.

#### Scheme 2. (a) thiourea, iodine, 110 °C, 10 h; (b) aromatic acid, HATU, DIPEA, DMF, r t, 2–3 h, 35– 40%. *2.2. SARS-CoV-2 3CLpro Inhibitory Activities and Structure-Activity Relationships*

40%. 2.2. SARS-CoV-2 3CLpro Inhibitory Activities and Structure-Activity Relationships All synthesized compounds were evaluated for inhibitory activity against SARS-CoV-2 3CLpro using PF-07321332 as positive control [29–31], 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 3CLpro 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 signifi-2.2. SARS-CoV-2 3CLpro Inhibitory Activities and Structure-Activity Relationships All synthesized compounds were evaluated for inhibitory activity against SARS-CoV-2 3CLpro using PF-07321332 as positive control [29–31], 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 3CLpro 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 3CLpro. 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 All synthesized compounds were evaluated for inhibitory activity against SARS-CoV-2 3CLpro using PF-07321332 as positive control [29–31], 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 3CLpro 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 3CLpro. 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

cantly affected inhibitory activities against SARS-CoV-2 3CLpro. 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

abolished. These results demonstrated that the effect of steric hindrance at this position was detrimental to inhibitory activities. The SAR of **R <sup>3</sup>** 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 8*H*-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 3CLpro . 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 3CLpro . demonstrated that the effect of steric hindrance at this position was detrimental to inhibitory activities. The SAR of R<sup>3</sup> 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 3CLpro. 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 3CLpro . demonstrated that the effect of steric hindrance at this position was detrimental to inhibitory activities. The SAR of R<sup>3</sup> 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 3CLpro. 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 3CLpro . demonstrated that the effect of steric hindrance at this position was detrimental to inhibitory activities. The SAR of R<sup>3</sup> 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 3CLpro. 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 3CLpro . demonstrated that the effect of steric hindrance at this position was detrimental to inhibitory activities. The SAR of R<sup>3</sup> 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 3CLpro. 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 3CLpro . demonstrated that the effect of steric hindrance at this position was detrimental to inhibitory activities. The SAR of R<sup>3</sup> 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 3CLpro. 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 demonstrated that the effect of steric hindrance at this position was detrimental to inhibitory activities. The SAR of R<sup>3</sup> 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 3CLpro. 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 demonstrated that the effect of steric hindrance at this position was detrimental to inhibitory activities. The SAR of R<sup>3</sup> 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 3CLpro. 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 demonstrated that the effect of steric hindrance at this position was detrimental to inhibitory activities. The SAR of R<sup>3</sup> 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 3CLpro. 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

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**Table 1.** Inhibitory activities of target compounds against SARS-CoV-2 3CLpro . Table 1. Inhibitory activities of target compounds against SARS-CoV-2 3CLpro . Table 1. Inhibitory activities of target compounds against SARS-CoV-2 3CLpro Table 1. Inhibitory activities of target compounds against SARS-CoV-2 3CLpro Table 1. Inhibitory activities of target compounds against SARS-CoV-2 3CLpro Table 1. Inhibitory activities of target compounds against SARS-CoV-2 3CLpro is important for the inhibitory activity against SARS-CoV-2 3CLpro .

Molecules 2022, 27, x FOR PEER REVIEW 4 of 11

is important for the inhibitory activity against SARS-CoV-2 3CLpro

is important for the inhibitory activity against SARS-CoV-2 3CLpro

is important for the inhibitory activity against SARS-CoV-2 3CLpro

Table 1. Inhibitory activities of target compounds against SARS-CoV-2 3CLpro

Table 1. Inhibitory activities of target compounds against SARS-CoV-2 3CLpro

Table 1. Inhibitory activities of target compounds against SARS-CoV-2 3CLpro


7i methoxy 1 20.3 ± 4.7 >20

7i methoxy 1 20.3 ± 4.7 >20

7h methoxy 1 72.5 ± 6.1 2.86 ± 0.11

7h methoxy 1 72.5 ± 6.1 2.86 ± 0.11

7h methoxy 1 72.5 ± 6.1 2.86 ± 0.11

7i methoxy 1 20.3 ± 4.7 >20

7i methoxy 1 20.3 ± 4.7 >20

7i methoxy 1 20.3 ± 4.7 >20

7i methoxy 1 20.3 ± 4.7 >20

7i methoxy 1 20.3 ± 4.7 >20

7i methoxy 1 20.3 ± 4.7 >20

Compd. R<sup>1</sup>

Compd. R<sup>1</sup>


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is important for the inhibitory activity against SARS-CoV-2 3CLpro

is important for the inhibitory activity against SARS-CoV-2 3CLpro

Table 1. Inhibitory activities of target compounds against SARS-CoV-2 3CLpro

Table 1. Inhibitory activities of target compounds against SARS-CoV-2 3CLpro

Molecules 2022, 27, x FOR PEER REVIEW 4 of 11

Molecules 2022, 27, x FOR PEER REVIEW 4 of 11

demonstrated that the effect of steric hindrance at this position was detrimental to inhib-

demonstrated that the effect of steric hindrance at this position was detrimental to inhib-

zamido 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 3CLpro. 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

zamido 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 3CLpro. 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

was explored next. Replacement of the 3,5-dimethoxyben-

was explored next. Replacement of the 3,5-dimethoxyben-

is important for the inhibitory activity against SARS-CoV-2 3CLpro

7d methyl 1 21.7 ± 2.2 >20

7a methoxy 1 89.5 ± 2.0 1.28 ± 0.17

7a methoxy 1 89.5 ± 2.0 1.28 ± 0.17

7b butoxy 1 0.5 ± 4.9 >20

7b butoxy 1 0.5 ± 4.9 >20

7c isobutoxy 1 −3.1 ± 1.7 >20

7c isobutoxy 1 −3.1 ± 1.7 >20

demonstrated that the effect of steric hindrance at this position was detrimental to inhib-

zamido 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 3CLpro. 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

was explored next. Replacement of the 3,5-dimethoxyben-

.

.

.

.

SARS-CoV-2 3CLpro Inhibition (%) at 20 μM IC50 (μM)

SARS-CoV-2 3CLpro Inhibition (%) at 20 μM IC50 (μM)

.

.

**Table 1.** *Cont.* Table 1. Inhibitory activities of target compounds against SARS-CoV-2 3CLpro

itory activities. The SAR of R<sup>3</sup>

R<sup>3</sup> n

R<sup>3</sup> n

itory activities. The SAR of R<sup>3</sup>

itory activities. The SAR of R<sup>3</sup>

#### 2.3. Predicting Binding Mode of 4b with 3CLpro 2.3. Predicting Binding Mode of 4b with 3CLpro 2.3. Predicting Binding Mode of 4b with 3CLpro 2.3. Predicting Binding Mode of 4b with 3CLpro 2.3. Predicting Binding Mode of 4b with 3CLpro 2.3. Predicting Binding Mode of 4b with 3CLpro *2.3. Predicting Binding Mode of* **7a** *with 3CLpro*

To explore the interaction mode between small molecule 7a and 3CLpro (PDB code: 6M2N) [23], we carried out molecular docking by applying AutoDock 4.2 program [31– 34]. Figure 3a showed that 7a docked well into the binding pockets S1 and S2 of 3CLpro, 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 3CLpro (PDB code: 6M2N) [23], we carried out molecular docking by applying AutoDock 4.2 program [31– 34]. Figure 3a showed that 7a docked well into the binding pockets S1 and S2 of 3CLpro, 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 3CLpro (PDB code: 6M2N) [23], we carried out molecular docking by applying AutoDock 4.2 program [31– 34]. Figure 3a showed that 7a docked well into the binding pockets S1 and S2 of 3CLpro, 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 3CLpro (PDB code: 6M2N) [23], we carried out molecular docking by applying AutoDock 4.2 program [31– 34]. Figure 3a showed that 7a docked well into the binding pockets S1 and S2 of 3CLpro, 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 3CLpro (PDB code: 6M2N) [23], we carried out molecular docking by applying AutoDock 4.2 program [31– 34]. Figure 3a showed that 7a docked well into the binding pockets S1 and S2 of 3CLpro, 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 3CLpro (PDB code: 6M2N) [23], we carried out molecular docking by applying AutoDock 4.2 program [31– 34]. Figure 3a showed that 7a docked well into the binding pockets S1 and S2 of 3CLpro, 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 3CLpro (PDB code: 6M2N) [23], we carried out molecular docking by applying AutoDock 4.2 program [31–34]. Figure 3a showed that **7a** docked well into the binding pockets S1 and S2 of 3CLpro , 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.

(a) (b) Figure 3. (a) surf representation of the compound 7a (bonds representation) in the 3CLpro S1 (red), S2 (blue), S1′ (green), S4 (orange) binding pocket; (b) the docking results of 7a and 3CLpro (PDB

(a) (b) Figure 3. (a) surf representation of the compound 7a (bonds representation) in the 3CLpro S1 (red), S2 (blue), S1′ (green), S4 (orange) binding pocket; (b) the docking results of 7a and 3CLpro (PDB

(a) (b) Figure 3. (a) surf representation of the compound 7a (bonds representation) in the 3CLpro S1 (red), S2 (blue), S1′ (green), S4 (orange) binding pocket; (b) the docking results of 7a and 3CLpro (PDB

(a) (b) Figure 3. (a) surf representation of the compound 7a (bonds representation) in the 3CLpro S1 (red), S2 (blue), S1′ (green), S4 (orange) binding pocket; (b) the docking results of 7a and 3CLpro (PDB

(a) (b) Figure 3. (a) surf representation of the compound 7a (bonds representation) in the 3CLpro S1 (red), S2 (blue), S1′ (green), S4 (orange) binding pocket; (b) the docking results of 7a and 3CLpro (PDB

(a) (b) Figure 3. (a) surf representation of the compound 7a (bonds representation) in the 3CLpro S1 (red), S2 (blue), S1′ (green), S4 (orange) binding pocket; (b) the docking results of 7a and 3CLpro (PDB

code: 6M2N, active residues in 3.0 Å range around 7a).

code: 6M2N, active residues in 3.0 Å range around 7a).

code: 6M2N, active residues in 3.0 Å range around 7a).

code: 6M2N, active residues in 3.0 Å range around 7a).

code: 6M2N, active residues in 3.0 Å range around 7a).

code: 6M2N, active residues in 3.0 Å range around 7a).

**Figure 3.** (**a**) surf representation of the compound **7a** (bonds representation**)** in the 3CLpro S1 (red), S2 (blue), S1′ (green), S4 (orange) binding pocket; (**b**) the docking results of **7a** and 3CLpro (PDB code: 6M2N, active residues in 3.0 Å range around **7a**). **Figure 3.** (**a**) surf representation of the compound **7a** (bonds representation**)** in the 3CLpro S1 (red), S2 (blue), S10 (green), S4 (orange) binding pocket; (**b**) the docking results of **7a** and 3CLpro (PDB code: 6M2N, active residues in 3.0 Å range around **7a**).

#### **3. Materials and Methods**

### *3.1. Chemistry*

All chemical reagents are reagent grade and used as purchased. <sup>1</sup>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 (1H NMR) and the 39.52 signal of DMSO (13C 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 Na2SO4. 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%). <sup>1</sup>H NMR (400 MHz, DMSO-*d*6) δ 12.81 (s, 1H), 7.46 (d, *J* = 8.4 Hz, 1H), 7.33 (d, *J* = 2.0 Hz, 2H), 7.22 (d, *J* = 2.0 Hz, 1H), 6.94 (dd, *J* = 8.0, 2.4 Hz, 1H), 6.74 (t, *J* = 2.0 Hz, 1H), 3.87 (s, 2H), 3.84 (s, 6H), 3.80 (s, 3H) ppm. <sup>13</sup>C NMR (100 MHz, DMSO-*d*6) δ 164.22, 162.12, 160.52, 157.76, 155.03, 147.98, 133.82, 130.05, 128.39, 118.28, 112.37, 111.83, 105.74, 105.08, 55.60, 55.36, 32.43 ppm. MS (ESI): *m*/*z* calcd for C20H19N2O4S [M + H]<sup>+</sup> 383.11, found 383.20.

*N-(6-butoxy-8H-indeno[1,2-d]thiazol-2-yl)-3,5-dimethoxybenzamide* (**7b**), eluting with DCM, yield = 32%; <sup>1</sup>H NMR (400 MHz, DMSO-*d*6) δ 12.81 (s, 1H), 7.43 (d, *J* = 8.0 Hz, 1H), 7.33 (d, *J* = 2.4 Hz, 2H), 7.18 (s, 1H), 6.91 (dd, *J* = 8.4, 2.4 Hz, 1H), 6.73 (d, *J* = 2.4 Hz, 1H), 3.99 (t, *J* = 6.4 Hz, 2H), 3.84 (s, 2H), 3.83 (s, 6H), 1.74–1.67 (m, 2H), 1.49–1.40 (m, 2H), 0.95–0.91 (m, 3H) ppm. <sup>13</sup>C NMR (100 MHz, DMSO-*d*6) δ 164.25, 162.15, 160.54, 157.20, 155.04, 147.96, 133.85, 129.95, 128.32, 118.29, 112.97, 112.39, 105.76, 105.10, 67.48, 55.62, 32.43, 30.89, 18.84, 13.76 ppm. MS (ESI): *m*/*z* calcd for C23H25N2O4S [M + H]<sup>+</sup> 425.15, found 425.10.

*N-(6-isobutoxy-8H-indeno[1,2-d]thiazol-2-yl)-3,5-dimethoxybenzamide* (**7c**), eluting with DCM, yield = 40%; <sup>1</sup>H NMR (400 MHz, DMSO-*d*6) δ 8.27 (s, 1H), 7.43 (dd, *J* = 8.4, 3.2 Hz, 1H), 7.36–7.32 (m, 2H), 7.19 (d, *J* = 2.8 Hz, 1H), 6.92 (dd, *J* = 8.4, 3.2 Hz, 1H), 6.73 (t, *J* = 2.4 Hz, 1H), 3.85 (s, 2H), 3.83 (s, 6H), 3.78 (d, *J* = 6.4 Hz, 2H), 2.06–2.00 (m, 1H), 0.99 (d, *J* = 7.2 Hz, 6H) ppm. <sup>13</sup>C NMR (100 MHz, DMSO-*d*6) δ 164.25, 162.17, 160.50, 157.26, 154.99, 147.94, 133.86, 129.95, 128.31, 118.26, 112.99, 112.45, 105.74, 105.06, 74.11, 55.59, 32.41, 27.78, 19.11 ppm. MS (ESI): *m*/*z* calcd for C23H25N2O4S [M + H]<sup>+</sup> 425.15, found 425.20.

*3,5-dimethoxy-N-(6-methyl-8H-indeno[1,2-d]thiazol-2-yl)benzamide* (**7d**), eluting with DCM, yield = 47%; <sup>1</sup>H NMR (400 MHz, DMSO-*d*6) δ 12.81 (s, 1H), 7.45 (d, *J* = 7.6 Hz, 1H), 7.39 (s, 1H), 7.34 (d, *J* = 2.4 Hz, 2H), 7.18 (d, *J* = 7.6 Hz, 1H), 6.74 (t, *J* = 2.0 Hz, 1H), 3.87 (s, 2H), 3.84 (s, 6H), 2.38 (s, 3H) ppm; <sup>13</sup>C NMR (100 MHz, DMSO-*d*6) δ 164.26, 162.14, 160.50, 155.22, 146.31, 134.42, 134.30, 133.78, 129.84, 127.40, 126.00, 117.56, 105.73, 105.09, 55.58, 32.16, 21.13 ppm. MS (ESI): *m*/*z* calcd for C20H19N2O3S [M + H]<sup>+</sup> 367.11, found 366.95.

*N-(6-chloro-8H-indeno[1,2-d]thiazol-2-yl)-3,5-dimethoxybenzamide* (**7e**), eluting with DCM, yield = 39%; <sup>1</sup>H NMR (400 MHz, DMSO-*d*6) δ 12.85 (s, 1H), 7.64 (d, *J* = 1.6 Hz, 1H), 7.54 (d, *J* = 8.0 Hz, 1H), 7.42 (dd, *J* = 8.0, 2.0 Hz, 1H), 7.33 (d, *J* = 2.0 Hz, 2H), 6.74 (t, *J* = 2.0 Hz, 1H), 3.94 (s, 2H), 3.84 (s, 6H) ppm. <sup>13</sup>C NMR (100 MHz, DMSO-d6) δ 164.36, 162.60, 160.48, 154.14, 148.13, 135.82, 133.64, 131.60, 129.72, 126.87, 125.43, 118.83, 105.75, 105.13, 55.57, 32.49 ppm. MS (ESI): *m*/*z* calcd for C19H16ClN2O3S [M + H]<sup>+</sup> 387.06, found 387.15.

*3,5-dimethoxy-N-(5-methoxy-8H-indeno[1,2-d]thiazol-2-yl)benzamide* (**4**), eluting with DCM, yield = 50%; <sup>1</sup>H NMR (400 MHz, DMSO-*d*6) δ 12.80 (s, 1H), 7.44 (d, *J* = 8.4 Hz, 1H), 7.34 (d, *J* = 2.4 Hz, 2H), 7.07 (d, *J* = 2.4 Hz, 1H), 6.81 (dd, *J* = 8.4, 2.4 Hz, 1H), 6.73 (t, *J* = 2.4 Hz, 1H), 3.83 (s, 6H), 3.82 (s, 2H), 3.81 (s, 3H) ppm. <sup>13</sup>C NMR (100 MHz, DMSO-*d*6) δ 164.32, 162.22, 160.50, 158.81, 138.17, 137.75, 133.77, 132.04, 125.68, 110.29, 105.74, 105.10, 104.65, 103.93, 55.58, 55.20, 31.64 ppm. MS (ESI): *m*/*z* calcd for C20H19N2O4S [M + H]<sup>+</sup> 383.11, found 383.15.

*3,4,5-trimethoxy-N-(6-methoxy-8H-indeno[1,2-d]thiazol-2-yl)benzamide* (**7f**), eluting with DCM, yield = 44%; <sup>1</sup>H NMR (400 MHz, DMSO-*d*6) δ 12.76 (s, 1H), 7.52 (s, 2H), 7.45 (d, *J* = 8.4 Hz, 1H), 7.21 (d, *J* = 2.4 Hz, 1H), 6.93 (dd, *J* = 8.4, 2.4 Hz, 1H), 3.89 (s, 6H), 3.86 (s, 2H), 3.80 (s, 3H), 3.75 (s, 3H) ppm. <sup>13</sup>C NMR (100 MHz, DMSO-*d*6) δ 163.94, 162.29, 157.73, 154.98, 152.79, 147.95, 141.00, 130.07, 128.23, 126.73, 118.20, 112.35, 111.81, 105.61, 60.14, 56.11, 55.35, 32.41 ppm. MS (ESI): *m*/*z* calcd for C21H21N2O5S [M + H]<sup>+</sup> 413.12, found 413.15.

*5-((6-methoxy-8H-indeno[1,2-d]thiazol-2-yl)carbamoyl)-1,3-phenylene diacetate* (**7g**), eluting with DCM, yield = 25%; <sup>1</sup>H NMR (400 MHz, DMSO-*d*6) δ 12.91 (s, 1H), 7.86 (d, *J* = 2.0 Hz, 2H), 7.46 (d, *J* = 8.4 Hz, 1H), 7.33 (t, *J* = 2.0 Hz, 1H), 7.22 (d, *J* = 2.0 Hz, 1H), 6.94 (dd, *J* = 8.4, 2.0 Hz, 1H), 3.88 (s, 2H), 3.80 (s, 3H), 2.33 (s, 6H) ppm. <sup>13</sup>C NMR (100 MHz, DMSO-*d*6) δ 172.06, 169.05, 164.01, 158.37, 157.74, 151.38, 147.94, 134.01, 130.00, 128.36,119.23, 118.29, 113.15, 112.35, 111.83, 55.35, 32.42, 20.86 ppm. MS (ESI): *m*/*z* calcd for C22H19N2O6S [M + H]<sup>+</sup> 439.10, found 439.05

*3-methoxy-N-(6-methoxy-8H-indeno[1,2-d]thiazol-2-yl)benzamide* (**7h**), eluting with DCM, yield = 47%; <sup>1</sup>H NMR (400 MHz, DMSO-*d*6) δ 12.81 (s, 1H), 7.72–7.70 (m, 2H), 7.48–7.44 (m, 2H), 7.22–7.18 (m, 2H), 6.94 (dd, *J* = 8.0, 2.4 Hz, 1H), 3.88 (s, 2H), 3.86 (s, 3H), 3.80 (s, 3H) ppm. <sup>13</sup>C NMR (100 MHz, DMSO-*d*6) δ 164.43, 162.14, 159.31, 157.73, 155.00, 147.95, 133.25, 130.05, 129.78, 128.32, 120.45, 119.02, 118.26, 112.58, 112.34, 111.80, 55.41, 55.34, 32.40 ppm. MS (ESI): *m*/*z* calcd for C19H17N2O3S [M + H]<sup>+</sup> 353.10, found 353.15.

*3-fluoro-N-(6-methoxy-8H-indeno[1,2-d]thiazol-2-yl)benzamide* (**7i**), eluting with DCM, yield = 34%; <sup>1</sup>H NMR (400 MHz, DMSO-*d*6) δ 12.92 (s, 1H), 8.00–7.94 (m, 2H), 7.65–7.59 (m, 1H), 7.52 (dd, *J* =8.4, 2.4 Hz, 1H), 7.47 (d, *J* = 8.0 Hz, 1H), 7.22 (d, *J* = 2.4 Hz, 1H), 6.94 (dd, *J* = 8.0, 2.4 Hz, 1H), 3.88 (s, 2H), 3.80 (s, 3H) ppm. <sup>13</sup>C NMR (100 MHz, DMSO-*d*6) δ 163.43,163.21, 161.90, 160.77, 157.77, 147.93, 134.26, 130.84 (d, *J* = 8.0 Hz), 129.95, 128.48, 124.36 (d, *J* = 3.0 Hz), 119.52 (d, *J* = 21.0 Hz), 118.30, 114.91 (d, *J* = 23.0 Hz), 112.36, 111.80, 55.34, 32.42 ppm. MS (ESI): *m*/*z* calcd for C18H14FN2O2S [M + H]<sup>+</sup> 341.08, found 341.05.

*N-(6-methoxy-8H-indeno[1,2-d]thiazol-2-yl)thiophene-2-carboxamide* (**7j**), eluting with DCM, yield = 30%; <sup>1</sup>H NMR (400 MHz, DMSO-*d*6) δ 12.92 (s, 1H), 8.28 (d, *J* = 8.0 Hz, 1H), 7.98 (d,

*J* = 4.8 Hz, 1H), 7.45 (d, *J* = 8.0 Hz, 1H), 7.27 (t, *J* = 4.8 Hz, 1H), 7.22 (d, *J* = 2.4 Hz, 1H), 6.94 (dd, *J* = 8.0, 2.4 Hz, 1H), 3.87 (s, 2H), 3.80 (s, 3H) ppm. <sup>13</sup>C NMR (100 MHz, DMSO-*d*6) δ 161.76, 159.35, 157.74, 154.97, 147.91, 137.30, 133.61, 130.69, 129.96, 128.64, 128.29, 118.24, 112.34, 111.78, 55.33, 32.43 ppm. MS (ESI): *m*/*z* calcd for C16H13N2O2S<sup>2</sup> [M + H]<sup>+</sup> 329.04, found 329.10.

*4-chloro-N-(6-methoxy-8H-indeno[1,2-d]thiazol-2-yl)benzamide* (**7k**), eluting with DCM, yield = 38%; <sup>1</sup>H NMR (400 MHz, DMSO-*d*6) δ 12.90 (s, 1H), 8.14 (dt, *J* = 8.8, 2.0 Hz, 2H), 7.63 (dt, *J* = 8.4, 2.0 Hz, 2H), 7.47 (d, *J* = 8.4 Hz, 1H), 7.22 (d, *J* = 2.0 Hz, 1H), 6.94 (dd, *J* = 8.4, 2.4 Hz, 1H), 3.88 (s, 2H), 3.80 (s, 3H) ppm. <sup>13</sup>C NMR (100 MHz, DMSO-*d*6) δ 163.77, 162.03, 157.74, 154.90, 147.93, 137.48, 130.82, 130.03, 129.97, 128.73, 128.38, 118.28, 112.34, 111.79, 55.34, 32.41 ppm. MS (ESI): *m*/*z* calcd for C18H14ClN2O2S [M + H]<sup>+</sup> 357.05, found 356.90.

#### 3.1.2. Procedure for the Synthesis of Compound **7l**

To a solution of **5f** (528.2 mg, 3.0 mmol) in dry ethanol (10 mL) were added thiourea (456.7 mg, 6.0 mmol) and bromine (0.2 mL, 3.3 mmol) 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 **6f** was collected without purification for the next step. The mixture of 6f (255.2 mg, 1.1 mmol), 3,5-dimethoxybenzoic acid (182.1 mg, 1.0 mmol), HATU (380.2 mg, 1.0 mmol), and DIPEA (0.5 mL 3.0 mmol) in DMF (6 mL) was stirred at room temperature for 2 h. The reaction mixture was quenched with water. The aqueous layer was extracted with EtOAc (20 mL × 2). The combined organic layers were dried over Na2SO4. The residue was purified by column chromatography on silica gel (eluting with DCM) to afford compound **7l** (103.0 mg, yield 26%) as a white solid.

<sup>1</sup>H NMR (400 MHz, DMSO-*d*6) δ 12.66 (s, 1H), 7.66 (dd, *J* = 8.4, 2.0 Hz, 1H), 7.32 (t, *J* = 2.0 Hz, 2H), 6.88 (s, 1H), 6.85 (dd, *J* = 8.4, 2.4 Hz, 1H), 6.73 (d, *J* = 2.4 Hz, 1H), 3.83 (s, 6H), 3.77 (s, 3H), 3.00–2.91 (m, 4H) ppm. <sup>13</sup>C NMR (100 MHz, DMSO-*d*6) δ 164.31, 160.45, 158.42, 156.44, 143.66, 136.68, 133.90, 124.23, 123.33, 121.55, 114.08, 111.82, 105.75, 105.06, 55.59, 55.09, 28.65, 20.74 ppm. MS (ESI): *m*/*z* calcd for C21H21N2O4S [M + H]<sup>+</sup> 397.12, found 396.95.

#### 3.1.3. General Procedure of Synthesis of **10a**–**10b** (Exemplified by **10a**)

A mixture of **8a** (10.0 mmol, 1.0 equiv), thiourea (20.0 mmol, 2.0 equiv), and iodine (10.0 mmol, 1.0 equiv) was stirred at 110 ◦C for 10 h. After the reaction was completed, the residue was triturated with MTBE and adjusted to pH 9–10 with 25% ammonium hydroxide. The precipitated solid was collected and washed with EtOAc (30 mL × 2) and NaHCO<sup>3</sup> (15 mL × 2) aqueous solution. The separated organic layer dried over Na2SO<sup>4</sup> and evaporated to dryness to afford crude product **9a**. The mixture of **9a** (3.3 mmol, 1.1 equiv), aromatic acid (3.0 mmol, 1.0 equiv), HATU (3.0 mmol, 1.0 equiv), and DIPEA (9.0 mmol, 3.0 equiv) in DMF (20 mL) was stirred at room temperature for 2 h. Then the reaction mixture was quenched with water. The aqueous layer was extracted with EtOAc (30 mL × 2). The combined organic layers were dried over Na2SO4. The residue was purified by column chromatography on silica gel (eluting with DCM) to afford compound **10a** as a white solid (406.7 mg, yield 35%). <sup>1</sup>H NMR (400 MHz, DMSO-*d*6) δ 12.67 (s, 1H), 7.57 (d, *J* = 8.4 Hz, 1H), 7.32 (d, *J* = 2.0 Hz, 2H), 7.21 (s, 1H), 6.86 (d, *J* = 2.4 Hz, 1H), 6.83 (dd, *J* =8.4, 2.8 Hz, 1H), 6.74 (t, *J* = 2.4 Hz, 1H), 3.83 (s, 6H), 3.77 (s, 3H), 2.43 (s, 3H) ppm. <sup>13</sup>C NMR (100 MHz, DMSO-*d*6) δ 164.74, 160.45, 158.60, 157.76, 149.01, 136.97, 134.22, 130.74, 127.31, 115.96, 111.22, 110.14, 105.80, 104.90, 55.58, 55.04, 21.26 ppm. MS (ESI): *m*/*z* calcd for C20H21N2O4S [M + H]<sup>+</sup> 385.12, found 385.20.

*3,5-dimethoxy-N-(4-(4-methoxy-3-methylphenyl)thiazol-2-yl)benzamide* (**10b**), eluting with DCM, yield = 40%; <sup>1</sup>H NMR (400 MHz, DMSO-*d*6) δ 12.70 (s, 1H), 7.77 (d, *J* = 2.4 Hz, 1H), 7.75 (s, 1H), 7.49 (s, 1H), 7.33 (d, *J* = 2.4 Hz, 2H), 6.99 (d, *J* = 8.8 Hz, 1H), 6.74 (t, *J* = 2.4 Hz, 1H), 3.84 (s, 6H), 3.82 (s, 3H), 2.20 (s, 3H) ppm. <sup>13</sup>C NMR (100 MHz, DMSO-*d*6) δ 164.59, 160.46, 158.26, 157.15, 149.27, 133.86, 128.07, 126.72, 125.68, 124.62, 110.36, 106.38, 105.79, 105.09, 55.59, 55.31, 16.21 ppm. MS (ESI): *m*/*z* calcd for C20H21N2O4S [M + H]<sup>+</sup> 385.12, found 385.25.

#### *3.2. Molecule Docking*

The protease structure, SARS-CoV-2 3CLpro enzyme (PDB code: 6M2N) with 2.2 Å, was obtained from the the Protein Data Bank at the RCSB site (http://www.rcsb.org (accessed on 6 March 2022)). The molecule docking used the Lamarckian genetic algorithm local search method and the semiempirical free energy calculation method in the AutoDock 4.2 program. Additionally, the charge was added by Kollman in AutoDock 4.2, The docking methold was employed on rigid receptor conformation, all the rotatable torsional bonds of compound **7a** were set free, the size of grid box was set at to 10.4 nm × 12.6 nm × 11.0 nm points with a 0.0375 nm spacing and grid center (−33.798 −46.566 39.065), and the other parameters were maintained at their default settings.

### *3.3. Enzymatic Activity and Inhibition Assays*

The enzyme activity and inhibition assays of SARS-CoV-2 3CLpro have been described previously [20,36]. Briefly, the recombinant SARS-CoV-2 3CLpro (40 nM at a final concentration) was mixed with each compound in 50 µL of assay buffer (20 mM Tris, pH 7.3, 150 mM NaCl, 1% Glycerol, 0.01% Tween-20) and incubated for 10 min. The reaction was initiated by adding the fluorogenic substrate MCA-AVLQSGFRK (DNP) K (GL Biochem, Shanghai, China), with a final concentration of 40 µM. After that, the fluorescence signal at 320 nm (excitation)/405 nm (emission) was immediately measured by continuous 10 points for 5 min with an EnVision multimode plate reader (Perkin Elmer, Waltham, MA, USA). The initial velocity was measured when the protease reaction was proceeding in a linear fashion; plots of fluorescence units versus time were fitted with linear regression to determine initial velocity. Plots of initial velocity versus inhibitor concentration were fitted using a four-parameter concentration–response model in GraphPad Prism 8 to calculate the IC<sup>50</sup> values. All data are shown as mean ± SD, *n* = 3 biological replicates.

#### **4. Conclusions**

In summary, we synthesized a series of 8*H*-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 IC<sup>50</sup> of 1.28 ± 0.17 µM against SARS-CoV-2 3CLpro. Molecular docking elucidated that **7a** was well-docked into the binding pockets S1 and S2 of 3CLpro. These preliminary results could provide a possible opportunity for the development of novel inhibitors against SARS-CoV-2 3CLpro with optimal potency and improved pharmacological properties.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules27103359/s1, copies of the <sup>1</sup>H NMR and <sup>13</sup>C NMR spectra for compounds **4**, **7a**–**7l**, **10a**–**10b** and Figure S1. surf representation of the compound **4** (bonds representation) in the 3CLpro S1 (red), S2 (blue), S1<sup>0</sup> (green), S4 (orange) binding pockets, Figure S2. surf representation of the compound **7h** (bonds representation) in the 3CLpro S1 (red), S2 (blue), S1<sup>0</sup> (green), S4 (orange) binding pockets.

**Author Contributions:** Investigation, J.W. (synthesis); B.F. and L.-X.G. (bioassay); C.Z. (molecule docking); Conceptualization, J.L., D.-J.X., Y.Z. and W.-L.W.; writing—original draft preparation, J.W., B.F. and D.-J.X.; writing—review and editing, Y.Z. and W.-L.W.; supervision, Y.Z. and W.-L.W.; project administration, D.-J.X. and W.-L.W.; funding acquisition. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was financially supported by the science and technology development foundation of Wuxi (N2020X016) and the Natural Science Foundation of Jiangsu Province (BK20190608).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data are available upon request to the Corresponding Authors.

**Acknowledgments:** The authors express their gratitude to the BioDuro-Sundia in Wuxi for NMR spectral data and mass spectral data.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Sample Availability:** Some of the compounds may be available in mg quantities upon request from the corresponding authors.

### **References**

