In Vitro Screening of an In-House Library of Structurally Distinct Chemotypes Towards the Identification of Novel SARS-CoV-2 Inhibitors

<b>Background/Objectives:</b> Four years after the COVID-19 pandemic, a very limited number of drugs has been marketed; thus, the search for new medications still represents a compelling need. In our previous work on antiviral, antiparasitic, and antiproliferative agents, we described several compounds (<b>1</b>-<b>13</b> and <b>16</b>-<b>20</b>) structurally related to clofazimine, chloroquine, and benzimidazole derivatives. Thus, we deemed it worthwhile to test them against the replication of SARS-CoV-2, together with a few other compounds (<b>14</b>, <b>15</b> and <b>21</b>-<b>25</b>), which showed some analogy to miscellaneous anti-coronavirus agents. <b>Methods</b>: Twenty-five structurally assorted compounds were evaluated in vitro for cytotoxicity against Vero E6 and for their ability to inhibit SARS-CoV-2 replication. <b>Results</b>: Several compounds (<b>2</b>, <b>3</b>, <b>10</b>, <b>11</b>, <b>13</b>-<b>15</b>, <b>18</b>-<b>20</b>) demonstrated antiviral activity (IC₅₀ range 1.5-28 µM) and six of them exhibited an interesting selectivity index in the range 4.5-20. The chloroquine analogs <b>10</b> and <b>11</b> were more potent than the reference chloroquine itself and doubled its SI value (20 versus 11). Also, the benzimidazole ring emerged as a valuable scaffold, originating several compounds (<b>13</b>-<b>15</b> and <b>18</b>-<b>20</b>) endowed with anti-SARS-CoV-2 activity. Despite the modest activity, the cytisine and the arylamino enone derivatives <b>23</b> and <b>25</b>, respectively, also deserve further consideration as model compounds. <b>Conclusions</b>: The investigated chemotypes may represent valuable hit compounds, deserving further in-depth biological studies to define their mechanisms of action. The derived information will guide the subsequent chemical optimization towards the development of more efficient anti-SARS-CoV-2 agents.