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The biological evaluation results showed that compound 5k was the most active inhibitor, exhibiting moderate-to-excellent potency against HIV-1 wild-type (WT) and a panel of NNRTI-resistant strains, with EC50 values ranging from 0.042 \u03bcM to 7.530 \u03bcM. Of special note, 5k exhibited the most potent activity against single-mutant strains (K103N and E138K), with EC50 values of 0.031 \u03bcM and 0.094 \u03bcM, being about 4.3-fold superior to EFV (EC50 = 0.132 \u03bcM) and 1.9-fold superior to NVP (EC50 = 0.181 \u03bcM), respectively. In addition, 5k demonstrated lower cytotoxicity (CC50 = 27.9 \u03bcM) and higher selectivity index values. The HIV-1 reverse transcriptase (RT) inhibition assay was further performed to confirm their binding target. Moreover, preliminary structure\u2013activity relationships (SARs) and molecular docking studies were also discussed in order to provide valuable insights for further structural optimizations. In summary, 5k turned out to be a promising NNRTI lead compound for further investigations of treatments for HIV-1 infections.<\/jats:p>","DOI":"10.3390\/ijms25169028","type":"journal-article","created":{"date-parts":[[2024,8,20]],"date-time":"2024-08-20T09:13:48Z","timestamp":1724145228000},"page":"9028","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Discovery of Novel Amino Acids (Analogues)-Substituted Thiophene[3,2-d]pyrimidine Derivatives as Potent HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitors: Design, Synthesis, and Biological Evaluation"],"prefix":"10.3390","volume":"25","author":[{"given":"Zongji","family":"Zhuo","sequence":"first","affiliation":[{"name":"Key Laboratory of Chemical Biology (Ministry of Education), Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, 44 West Culture Road, Jinan 250012, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhao","family":"Wang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Chemical Biology (Ministry of Education), Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, 44 West Culture Road, Jinan 250012, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lanlan","family":"Jing","sequence":"additional","affiliation":[{"name":"Key Laboratory of Chemical Biology (Ministry of Education), Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, 44 West Culture Road, Jinan 250012, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tao","family":"Zhang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Chemical Biology (Ministry of Education), Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, 44 West Culture Road, Jinan 250012, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anchao","family":"Ge","sequence":"additional","affiliation":[{"name":"Key Laboratory of Chemical Biology (Ministry of Education), Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, 44 West Culture Road, Jinan 250012, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhenzhen","family":"Zhou","sequence":"additional","affiliation":[{"name":"Key Laboratory of Chemical Biology (Ministry of Education), Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, 44 West Culture Road, Jinan 250012, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ying","family":"Liu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Chemical Biology (Ministry of Education), Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, 44 West Culture Road, Jinan 250012, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xin","family":"Li","sequence":"additional","affiliation":[{"name":"Key Laboratory of Chemical Biology (Ministry of Education), Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, 44 West Culture Road, Jinan 250012, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2985-8890","authenticated-orcid":false,"given":"Erik","family":"De Clercq","sequence":"additional","affiliation":[{"name":"Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, K.U. 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