Acquired Immunodeficiency Syndrome (AIDS) is a malignant infectious disease caused by the Human Immunodeficiency Virus (HIV) [1], [2]. The global prevalence of HIV infection is estimated to be 39.0 million in 2022, indicating the persistent and significant impact on public health worldwide [3]. The current standard of care for AIDS involves antiretroviral therapy (ART), a combination of three or more antiviral medications [4]. This treatment regimen effectively inhibits viral replication, thereby slowing disease progression, extending patient lifespan, and enhancing overall quality of life. However, the therapy was unable to completely eradicate the virus [4]. Moreover, the high mutation rate of HIV-1 and the continuous emergence of new mutant strains have significantly impeded their practical application. Therefore, it is crucial to develop novel anti-HIV drugs with innovative mechanism [5].

RNase H is located within the p66 subunit of reverse transcriptase (RT), facilitating specific hydrolysis of RNA in RNA/DNA heteroduplexes and tRNA precursors to ensure successful synthesis during reverse transcription [6], [7], [8]. Targeting RNase H has been recognized as a promising therapeutic strategy for the treatment of HIV. Over the past, several RNase H inhibitors have been successfully identified, as shown in Fig. 1. The inhibitors of RNase H can be classified into two primary categories, namely active site inhibitors and allosteric inhibitors [9], [10]. The primary mechanism of action for active site inhibitors is the chelation of two metal ions at the RNase H active site [11], [12]. However, these inhibitors often exhibit poor cell permeability and high cytotoxicity [13], [14], [15]. The targeting of allosteric sites provides an opportunity for the development of novel RNase H inhibitors, which is expected to decrease cytotoxicity of active site inhibitors [16], [17], [18], [19], [20], [21].

It has been reported that thiazolopyrimidine derivatives display diverse biological activities including antimicrobial, antitumor, and anti-inflammatory activity [22], [23], [24]. This project was initiated by screening our in house compound library, leading to the identification of the first thiazolo[3, 2-a]pyrimidine-containing RNase H inhibitor A1, which featured moderate inhibitory activity against RNase H with an IC50 of 21.49 μM. Further extensive exploration of the structure–activity relationship (SAR) afforded 31 novel thiazolo[3,2-a]pyrimidine analogs, followed by being evaluated for their inhibitory activity against RNase H, ultimately leading to the generation of compound A28. This analog was found to strongly inhibit the activity of RNase H (IC50 = 4.14 μM), being around 5-fold more potent than that of the hit compound A1. Results from the conducted molecular docking indicated that A28 was snugly projected into the binding allosteric pocket of RNase H. Compared to compound A1, compound A28 could generate three hydrogen bonding forces with Q507, L533 and Q428 of this pocket, respectively. While no hydrogen bonds can be shown in the docking pose of A1 (Fig. 2).