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. 2026 Sep 28. Online ahead of print. doi: 10.1039/d6ra03750j

Quinazoline-tethered thiazolo[3,2-a]pyrimidines and their cyclic chalcone derivatives as HIV-1 reverse transcriptase inhibitors: green synthesis, molecular docking, and in silico studies

Sobhi M Gomha a,✉, Sami A Al-Hussain b, Yaser A M Sulaiman c, Sayed M Riyadh d, Basant Farag e, Mohammad Alhilal f, Suzan Alhilal g, Magdi E A Zaki b,✉
PMCID: PMC13618335  PMID: 42807970

Abstract

Nitrogen-fused heterocycles have attracted considerable research interest in pharmaceutical, medicinal, organometallic, and materials chemistry. In this work, two new series of quinazoline-linked thiazolo[3,2-a]pyrimidines and their cyclic chalcone derivatives were synthesized using choline hydroxide as an environmentally friendly catalyst. The synthesized compounds were evaluated for their inhibitory activity against HIV-1 reverse transcriptase (RT). Compound 11e showed the highest inhibitory activity, with an IC50 value of 0.599 µg mL−1 (1.305 µM). Molecular docking was performed to examine the possible binding modes and interactions within the active site of the enzyme. However, only a weak correlation was observed between the docking scores and experimental IC50 values (Pearson's r ≈ −0.18), indicating that the docking results provide qualitative binding information rather than a direct prediction of the inhibitory activity. In silico ADMET analysis also indicated generally favorable oral drug-like properties. These findings suggest that the synthesized compounds, particularly 11e, may serve as preliminary leads for the further development of HIV-1 RT inhibitors.

Citrus lemon juice-mediated green synthesis afforded quinazolinyl biphenylsulfone imines with dual AChE/BuChE inhibitory activity. Compound 3m emerged as the lead, supported by docking and ADMET analyses.graphic file with name d6ra03750j-ga.webp

1. Introduction

Ribonuclease H (RNase H) is located within the subunit of reverse transcriptase (RT) and is considered a crucial enzyme in HIV replication. The significant challenges in their HIV inhibition applications are the high mutation rate and continuous formation of novel mutant strains. The current standard approach for managing HIV infection is antiretroviral therapy (ART).1 Therefore, the development of novel anti-HIV drugs with innovative mechanisms of action is a crucial target. Thiazolo[3,2-a] pyrimidines are popular motifs that serve as isosteric analogues of purines and were recently reported as allosteric inhibitors of RNase H with low cytotoxicity compared with active-site inhibitors.2,3 Also, these scaffolds have been implicated in a variety of therapeutic areas, including as 5-hydroxytryptamine (5-HT2A) receptor antagonists,4 inhibitors of cell division cycle 25B (CDC25B) phosphatase,5 xanthine oxidase inhibitors,6 tyrosinase inhibitors (a copper-containing enzyme that oxidizes tyrosine into melanin pigments in the skin, hair, and eyes),7 and inhibitors of the Bcl-2 family of proteins.8 The wide range of activities of thiazolo[3,2-a]pyrimidines as pharmacophores has revealed their potential as anticancer, anti-inflammatory, antimicrobial, antioxidant, anti-Alzheimer, antidiabetic, antitubercular, anticonvulsant, anti-Parkinsonian, and antimalarial agents.9,10

The conformationally restricted cyclic chalcones, with three-dimensional structures and different degrees of conjugation, are considered bioactive molecules capable of forming covalent and H-bond interactions. Recently, they have played a crucial role as follicle-stimulating hormone (FSH) agonists, peroxisome proliferator-activated receptor (PPAR) binders, cystic fibrosis transmembrane conductance regulator (CFTR) inhibitors,11 and anticancer agents.12 In materials science, the physicochemical properties of cyclic chalcones have been exploited in various applications, including high-contrast mechano-chromic luminescence,13 optical compounds,14 forensic reagents,15 photo-switches,16 semiconductors,17 anion sensors,18 and thermostable polymeric materials.19

Volatilization of hazardous chemicals has a detrimental effect on human health and the environment. Ionic liquids play a major role in addressing this global environmental problem and promoting the development of green chemistry.19 These ionic liquids have privileged properties, including high solubility, low volatility, biodegradability, and recyclability.20,21 An environmentally benign example of ionic liquids is choline hydroxide (ChOH), which possesses several advantageous properties, including a high basic character, ready availability, low cost, high polarity, and solubility in water and ethanol.22 Recently, ChOH was used as an alternative to conventional basic catalysts under mild and efficient conditions for the synthesis of fused heterocycles23,24 and thiazolidinone-based cyclic chalcones.25 Our on-going commitment to developing this technique with a sustainable profile directed our attention towards the synthesis of thiazolo[3,2-a]pyrimidines and their cyclic chalcone derivatives and the study of their biological activities against HIV-1 RT. The absorption, distribution, metabolism, excretion, and toxicity (ADMET) prediction of these compounds provided further motivation to investigate their in silico drug-likeness properties. This report continues our program aimed at the synthesis of bioactive candidates.26–36

2. Results and discussion

2.1. Chemistry

Our research aimed to prepare quinazoline as a framework skeleton from a benzoxazine-based molecule using an environmentally benign non-toxic catalyst. The ring opening of benzoxazine by a nitrogen nucleophile in the presence of a basic catalyst is a common procedure in the synthesis of quinazoline.37–39

The detailed synthetic route for the target compounds is summarized in Schemes 1–3. Scheme 1 outlines the preparation of quinazoline-tethered thiazolo[3,2-a]pyrimidine derivatives 5, 6, and 7a–f from benzoxazinone 1 and the corresponding heterocyclic amines under choline hydroxide-mediated conditions, including the substituent scope and isolated yields. Scheme 2 presents the proposed mechanism involving amine deprotonation, benzoxazine ring opening, cyclization, and dehydration. Scheme 3 describes the synthesis of cyclic chalcone derivatives 11a–f through the condensation of compound 5 with different aromatic aldehydes 10a–f, with reaction conditions and isolated yields included.

Scheme 1. Detailed synthetic route of the quinazoline-tethered thiazolo[3,2-a]pyrimidine derivatives 5, 6, and 7a–f.

Scheme 1

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Scheme 2. Proposed mechanism of the benzoxazine ring opening and quinazoline ring formation.

Scheme 2

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Scheme 3. Detailed synthesis of the cyclic chalcone derivatives 11a–f.

Scheme 3

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The inherent synthetic strategy adopted in our study was initiated by fusing 2-methyl-4H-benzo[3,1]oxazin-4-one (1) with 7-amino-5H-thiazolo[3,2-a]pyrimidine-3,5(2H)-dione (2),40 6-amino-2-thioxo-2,3-dihydropyrimidin-4(1H)-one (3) or 7-amino-3-aryl-5H-thiazolo[3,2-a]pyrimidin-5-ones (4a–f)41–43 in the presence of choline hydroxide (ChOH) under thermal conditions to furnish 7-(2-methyl-4-oxoquinazolin-3-yl)-5H-thiazolo[3,2-a]pyrimidine-3,5(2H)-dione (5), 2-methyl-3-(6-oxo-2-thioxo-1,2,3,6-tetrahydropyrimidin-4-yl)quinazolin-4(3H)-one (6) or 7-(2-methyl-4-oxoquinazolin-3-yl)-3-aryl-5H-thiazolo[3,2-a]pyrimidin-5-one (7a–f), respectively, as depicted in Scheme 1.

The molecular structures of compounds 5, 6, and 7a–f were confirmed based on their spectral data and elemental analyses. The IR spectra of the isolated products lacked the lactone carbonyl absorption characteristic of benzoxazine and revealed the lactam amide absorption of quinazoline at υ = 1682–1692 cm−1.37 The 1H NMR spectrum of compound 5 showed a singlet signal at δ = 3.87 ppm, integrating for 2 protons and assignable to the (CH2–CO) groups of the thiazolidinone ring. The pyrimidine-H at position 5 in compound 6 resonated at δ = 6.81 ppm, while the (NH–CO–NH–CS) residue resonated at δ = 10.82 and 11.54 ppm. Additionally, compounds 7a–f revealed a singlet signal at δ = 6.91–6.96 ppm, attributed to the (CH) of the thiazole ring.35

Alternative routes to compounds 5 and 7a involved treating compound 6 with chloroacetic acid (8) or phenacyl bromide (9), respectively, in an ethanolic solution of choline hydroxide under the same reaction conditions (Scheme 1).

The proposed mechanism for the ring opening of benzoxazine and ring closure to form quinazoline39 is depicted in Scheme 2. Choline hydroxide promotes the initial step of the reaction via deprotonation of the heterocyclic amine, thereby increasing its nucleophilicity. The resulting anion attacks the lactone group of benzoxazine and affords intermediate (A), which abstracts a proton from the oxonium ion of choline hydroxide and is converted into intermediate (B). Ring opening of the latter intermediate gives intermediate (C). Deprotonation and ring closure of intermediate (D) yield the respective intermediate (E), which undergoes dehydration to yield the isolated products 5, 6, and 7a–f (Scheme 2).

To demonstrate the generality of the above-mentioned environmentally benign procedure, we extended its scope to the preparation of cyclic chalcones. Thus, heating compound 5 with different aromatic aldehydes 10a–f in an ethanolic solution of choline hydroxide yielded the respective 2-arylidine-7-(2-methyl-4-oxoquinazolin-3-yl)-5H-thiazolo[3,2-a]pyrimidine-3,5(2H)-dione derivatives (11a–f), as shown in Scheme 3.

The consecutive steps of this condensation reaction are initiated by deprotonation of the active methylene group (CH2–CO) in the thiazolidinone ring with the aid of choline hydroxide, affording intermediate F. The resulting carbanion then attacks the aldehydic carbonyl group to form the corresponding tetrahedral addition intermediate G. Subsequent dehydration of this intermediate furnishes the isolated cyclic chalcones 11a–f (Scheme 3).25

Two different choline hydroxide-mediated conditions were used depending on the nature of the transformation. Fusion conditions were applied for the synthesis of compounds 5, 6, and 7a–f, since this step involves benzoxazine ring opening followed by quinazoline ring formation, which requires stronger thermal activation. This method afforded the target products in good isolated yields. In contrast, compounds 11a–f were obtained through condensation of the active methylene group of compound 5 with aromatic aldehydes under milder ethanolic reflux conditions. These conditions were more suitable for the chalcone-forming step because they gave good yields, avoided high-temperature fusion, and allowed easier work-up. Therefore, while fusion was necessary for the ring-opening/cyclization step, ethanolic reflux was the preferable condition for preparing the cyclic chalcone derivatives 11a–f.

Although the same catalyst, choline hydroxide, was used in both steps, the synthesis was carried out stepwise to allow the isolation and full structural characterization of intermediate 5 before aldehyde condensation. A one-pot protocol for the direct preparation of compounds 11a–f from benzoxazinone 1, amine 2, and aldehydes 10a–f would be synthetically attractive and may reduce the need for work-up and minimize waste generation. This approach will be investigated in future optimization studies.

The use of choline hydroxide provides several green-chemistry advantages, because it is a readily available, non-volatile, and strongly basic ionic liquid that can promote the reactions without using conventional inorganic bases. In the present protocol, the synthesis of compounds 5–7a–f was performed under fusion conditions, reducing the need for a reaction solvent, while compounds 11a–f were prepared in ethanol. The reactions also afforded good isolated yields, supporting the practical efficiency of the method. However, the greenness of the process should be considered cautiously, since the fusion step requires a relatively high temperature (230 °C) for 6 h, which increases energy demand, and some products required recrystallization from EtOH/DMF. Therefore, although choline hydroxide improves the sustainability profile compared with many conventional basic catalysts, further optimization is still needed to reduce energy consumption, minimize DMF use, evaluate catalyst recyclability, and assess waste generation and atom economy more quantitatively.

2.2. In silico docking study

To gain insights into molecular interactions and docking scores, the newly synthesized ligand molecules were subjected to molecular docking studies against HIV-1 reverse transcriptase.44 The docking results for the newly synthesized compounds are presented alongside those of the reference drug (zidovudine), as shown in Tables 1, 2, and S1.

Table 1. Docking scores (kcal mol−1), interactions of the docked complexes, and RMSD (Å) values of the compounds 7a–f, 11a–f, and zidovudine within the binding site of the target enzyme.

Cpd Docking score (kcal mol−1) Docked complex interactions Donor atom Acceptor atom RMSD (Å)
7a −6.375 H-bond S — 1.073
1 (THR 80)
7b −6.898 H-bond C — 1.115
1 (ASP 30)
Arene–H
1 (ILE 47)
1 (GLY 48)
7c −6.428 Arene–H — — 1.310
1 (ALA 28)
7d −6.422 Arene–H — — 1.144
1 (ALA 28)
7e −6.776 H-bond — O 1.264
1 (ASP 29) —
1 (ASP 30)
Arene–H
1 (ILE 47)
1 (PRO 81)
7f −6.762 H-bond — O 1.270
1 (ASP 29)
Arene–H
1 (ILE 47)
11a −6.732 H-bond S — 0.581
1 (ASP 25) C —
1 (ASP 30)
Arene–H
1 (ALA 28)
1 (ASP 29)
11b −6.916 H-bond S — 1.079
1 (THR 80)
11c −6.709 Arene–H — — 1.611
1 (ALA 28)
11d −6.571 H-bond — O 1.158
1 (ASP 29) —
1 (ASP 30)
Arene–H
1 (ILE 47)
1 (GLY 48)
11e −6.815 Arene–H — — 1.110
1 (ASP 29)
1 (GLY 49)
11f −6.838 Arene–H — — 1.474
1 (ASP 29)
Co-crystallized ligand −5.144 H-bond — O 1.386
1 (ASP 29) O —
1 (ASP 30)
Arene–H
1 (ALA 28)
Zidovudine −5.796 H-bond N — 1.712
1 (ASP 30) — O
1 (ASP 30)
Arene–H
1 (ILE 47)
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Table 2. 2D and 3D ligand interactions of the ligands with 7a–f, 11a–f, and zidovudine at the 6DV4 binding site of the target enzyme.

Cpd 2D ligand interactions 3D ligand interactions
7b graphic file with name d6ra03750j-u1.jpg graphic file with name d6ra03750j-u2.jpg
7e graphic file with name d6ra03750j-u3.jpg graphic file with name d6ra03750j-u4.jpg
7f graphic file with name d6ra03750j-u5.jpg graphic file with name d6ra03750j-u6.jpg
11b graphic file with name d6ra03750j-u7.jpg graphic file with name d6ra03750j-u8.jpg
11e graphic file with name d6ra03750j-u9.jpg graphic file with name d6ra03750j-u10.jpg
11f graphic file with name d6ra03750j-u11.jpg graphic file with name d6ra03750j-u12.jpg
Co-crystallized ligand graphic file with name d6ra03750j-u13.jpg graphic file with name d6ra03750j-u14.jpg
Zidovudine graphic file with name d6ra03750j-u15.jpg graphic file with name d6ra03750j-u16.jpg
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As shown in Table 1, the tested compounds exhibited stronger binding affinities toward the target enzyme than the reference ligand, with acceptable RMSD values of less than 2 Å. The docking scores of the investigated ligands ranged from −6.375 to −6.916 kcal mol−1, compared with that of zidovudine (−5.796 kcal mol−1). The highest binding interactions were observed for compounds 7b, 7e, 7f, 11b, 11e, and 11f. Compounds 7a and 11b exhibited an H-bond interaction between the S atom of the thiazole moiety and the residue THR 80. Compound 7b showed a hydrogen-bond interaction between the C5 of the thiazole moiety and the side-chain residue ASP30. On the contrary, when comparing the docking poses of 7e and 11d with that of the co-crystallized ligand, it is evident that both molecules occupy similar positions and establish two H-bond acceptor interactions between the O atom and the vital backbone residues of ASP29 and ASP30. The predicted binding modes of compound 7f at the active site of the target enzyme suggested a hydrogen-bond acceptor interaction between the coumarin moiety and ASP29. Similarly, 11a ligand was connected to the protein through two hydrogen bonds involving the protein residues ASP25 and ASP30.

The results of the docking analyses showed high docking scores and strong relevant binding modes for the tested compounds with the target enzyme, in agreement with their in vitro enzyme inhibitory activity. The tested compounds were able to maintain strong binding with the target receptors and a similar binding mode for the HIV-1 RT enzyme, albeit at slightly different orientations.

The docking results provide qualitative insights into the possible binding modes and ligand–enzyme interactions; however, docking scores alone should not be considered direct predictors of experimental HIV-1 RT inhibitory activity.

2.2.1. Assessment of physicochemical characteristics

The physicochemical features are critical in determining whether a molecule becomes a therapeutic candidate and how well it performs during the significant phases of drug development.45 A compound is considered to exhibit drug-likeness molecule (DLM) properties when it satisfies the requirements outlined in Lipinski's “Rule of Five” (Ro5). The establishment of physicochemical filters as protocols to identify compounds with good drug-likeness, like Lipinski's “Rule of 5”, is a technique that has been frequently used.46 To assess the physicochemical parameters and DLM properties of the investigated compounds, the SwissADME tool was used,47 and the results are summarized in Table 3. A molecule is considered to meet the Ro5 requirements if its molecular weight is less than 500 and its log P0/w (octanol–water partition coefficient) is less than 5, and the hydrogen-bond donors and acceptors are less than 5 and 10, respectively.46 According to the Lipinski rule, a therapeutic candidate or oral bioactive drug should not have more than one violation.48 All the synthesized compounds fell within the specified ranges according to the predicted ADME data obtained from the computations.49 The tested compounds are predicted to obey Lipinski's Rule of Five and to exhibit intestinal absorption properties in humans.

Table 3. Physicochemical characteristics of the compounds 7a–f and 11a–f.
Cpd M wt, g mol−1 log P0/w No. of H-bond donors No. of H-bond acceptors TPSA, Å2 log S F (sp3) Rotatable bonds
7a 386.0 3.050 0 6 69.26 −4.528 0.048 2
7b 400.1 3.619 0 6 69.26 −5.117 0.091 2
7c 420.0 3.892 0 6 69.26 −5.375 0.048 2
7d 463.9 4.097 0 6 69.26 −5.490 0.048 2
7e 431.0 3.066 0 9 112.4 −5.082 0.048 3
7f 454.0 2.829 0 8 99.47 −5.150 0.042 2
11a 414.0 2.403 0 7 86.33 −4.518 0.045 2
11b 444.0 2.533 0 8 95.56 −4.791 0.087 3
11c 448.0 3.267 0 7 86.33 −5.022 0.045 2
11d 432.0 2.558 0 7 86.33 −4.758 0.045 2
11e 459.0 2.379 0 10 129.4 −4.972 0.045 3
11f 464.0 3.683 0 7 86.33 −5.792 0.038 2
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As shown in Table 3, the optimal range for each property is as follows: M. Wt. between 150 and 500 g mol−1, lipophilicity (log P0/w) under 5, polarity (TPSA) between 20 and 130 Å2, solubility (log S) higher than −6, and flexibility of no more than 6 rotatable bonds.50

Topological polar surface area (TPSA) is widely used as a physicochemical descriptor to estimate polarity and, in turn, to assess properties that influence membrane permeation and transport. In the radar visualization, the shaded pink zone marks the desirable window associated with oral drug-likeness, integrating constraints on molecular size (SIZE), lipophilicity (LIPO), polarity/TPSA, aqueous solubility (INSOLU), saturation (INSATU), and conformational flexibility (FLEX), as depicted in Fig. 1 and S2.

Fig. 1. Bioavailability radar of the compounds 7b, 7e, 7f, 11b, 11e and 11f.

Fig. 1

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The oral bioavailability graph (Fig. 1) provides an overview of these properties, including potential size, lipophilicity, total polar surface area, degree of saturation, and molecular flexibility.

2.2.2. In silico pharmacokinetic profile ADMET

The ADMET properties of compounds 7a–f and 11a–f were computationally predicted, and the results are summarized in Table 4. The predicted human intestinal absorption values ranged from 89.87 to 100%, while the predicted fraction unbound ranged from 0.283 to 0.37. Identifying CYP isoforms potentially involved in drug metabolism is important because polymorphically expressed isoforms, including CYP2C9 and CYP2C19, may contribute to interindividual pharmacokinetic variability. The CYP superfamily plays a major role in metabolic biotransformation and drug elimination, and inhibition of these enzymes may contribute to pharmacokinetic drug–drug interactions.51–56 The computational models classified compounds 7a–d as potential CYP3A4 substrates and predicted them to be inhibitors of CYP1A2, CYP2C19, and CYP2C9. Compounds 7e and 11f were predicted to be CYP3A4 substrates and inhibitors of CYP1A2 and CYP2C9, whereas compounds 7f and 11a–d were predicted to be CYP3A4 substrates and CYP1A2 inhibitors. Compound 11e was predicted to be a CYP3A4 substrate. Predicted total-clearance values ranged from −0.47 to 0.115 log mL min−1 kg−1. The toxicity model predicted no skin sensitization for the tested compounds. All these values are computational estimates and therefore require experimental confirmation.

Table 4. ADMET characteristics of the synthesized compounds 7a–f and 11a–f and zidovudine.
Cpd Absorption Distribution Metabolism Excretion Toxicity
HIA (% absorbed) Fraction unbound
Total clearance (log mL min−1 kg−1) Skin sensitization
7a 100 0.348 CYP3A4 substrate, CYP1A2, CYP2C19, and CYP2C9 inhibitors 0.115 No
7b 100 0.37 0.057
7c 99.65 0.363 −0.017
7d 99.58 0.362 −0.039
7e 100 0.325 CYP3A4 substrate, CYP1A2, and CYP2C9 inhibitors 0.071
7f 100 0.344 CYP3A4 substrate and CYP1A2 inhibitor 0.013
11a 100 0.291 −0.323
11b 94.51 0.305 −0.291
11c 100 0.31 −0.455
11d 100 0.325 −0.47
11e 89.87 0.283 CYP3A4 substrate −0.421
11f 100 0.326 CYP3A4 substrate, CYP1A2, and CYP2C9 inhibitors −0.364
Zidovudine 69.94 0.065 — 0.052
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The in silico ADMET analysis predicted high intestinal absorption, moderate fraction-unbound values, several potential CYP450 interactions, and no skin sensitization. These computational results suggest potentially favorable pharmacokinetic profiles; however, they do not confirm oral bioavailability and require experimental validation from medicinal researchers.

2.3. HIV-1 RT inhibition activity

RT is a key target enzyme for the life cycle of retroviruses like HIV, where it plays a role in viral replication and propagation within host cells. Thus, RT inhibitors as antiviral drugs could block viral replication, offering improved tolerability and resistance profiles. The ability of compounds 7b, 7e, 7f, 11b, 11e, and 11f to inhibit RT was evaluated using zidovudine as the reference drug, and the results are summarized in Table 5. These compounds were selected as representative derivatives to cover different substituent effects, including electron-donating, electron-withdrawing, and extended aromatic/coumarin motifs, as well as favorable docking interactions. Therefore, the SAR discussion is limited to these experimentally tested compounds, and testing the full synthesized library will be required for a more comprehensive SAR evaluation.

Table 5. HIV-1 RT inhibitory activities of the compounds 7b, 7e, 7f, 11b, 11e, 11f and zidovudine expressed as IC50 values in µg mL−1 and µM.

graphic file with name d6ra03750j-u17.jpg
No. Ar IC50 (µg mL−1) IC50 (µM)
7b 4-CH3C6H4 1.5336 ± 0.066 3.834 ± 0.165
7e 4-NO2C6H4 5.5463 ± 0.238 12.868 ± 0.552
7f graphic file with name d6ra03750j-u18.jpg 2.0074 ± 0.086 4.422 ± 0.189
11b 4-CH3OC6H4 6.2751 ± 0.269 14.133 ± 0.606
11e 4-NO2C6H4 0.599 ± 0.026 1.305 ± 0.057
11f graphic file with name d6ra03750j-u19.jpg 0.9005 ± 0.034 1.941 ± 0.073
Zidovudine — 0.2024 ± 0.009 0.758 ± 0.034
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Although the tested compounds showed favorable docking scores within the HIV-1 RT binding site, the docking results did not fully correlate with the in vitro inhibitory activity. For the biologically tested compounds 7b, 7e, 7f, 11b, 11e, and 11f, the correlation between docking score and IC50 values was weak (Pearson's r ≈ −0.18). Notably, compound 11b showed the best docking score (−6.916 kcal mol−1) but relatively weak HIV-1 RT inhibition (IC50 = 14.133 µM), whereas 11e displayed the strongest RT inhibition (IC50 = 1.305 µM) despite not having the best docking score. Compound 11e was the most active derivative in the synthesized series, with an IC50 value of 1.305 µM, although it was less potent than zidovudine (IC50 = 0.758 µM).

Therefore, docking was used mainly to support the proposed binding modes and key ligand–enzyme interactions, while the experimental RT inhibition data remain the primary basis for ranking the compounds according to their activity.

Zidovudine was used as the reference compound in this assay to provide a standard comparison with an established nucleoside reverse transcriptase inhibitor. Since zidovudine requires intracellular phosphorylation to its active triphosphate form in cellular systems, its use here should be interpreted only within the context of the present enzyme-based assay. Therefore, the comparison with zidovudine is intended as an assay reference and does not imply cellular antiviral efficacy. Further studies using additional direct RT inhibitor controls, together with cellular antiviral and cytotoxicity assays, will be required to confirm the pharmacological relevance of these compounds.

2.3.1. Structure–activity relationship (SAR)

Because only compounds 7b, 7e, 7f, 11b, 11e, and 11f were experimentally evaluated, the SAR discussion is limited to this tested subset (Table 5). Within the 7-series, the 4-methylphenyl derivative 7b showed the strongest HIV-1 RT inhibition, followed by the coumarin-substituted derivative 7f, whereas the 4-nitrophenyl derivative 7e was less active. These results suggest that, among the tested 7-series compounds, the methyl substituent and extended coumarin system were more favorable than the strongly electron-withdrawing nitro group. Within the tested 11-series, the 4-nitrophenyl derivative 11e was the most active compound, followed by the naphthyl derivative 11f, whereas the 4-methoxyphenyl derivative 11b was less active. Thus, nitro substitution and extended aromaticity appeared to be more favorable than methoxy substitution in this limited subset. These observations represent preliminary SAR trends only; experimental evaluation of the remaining synthesized compounds, including 7c and 7d, is required before broader conclusions can be drawn.

These results identify the tested compounds, particularly 11e and 11f, as preliminary leads for further optimization as HIV-1 RT inhibitors. However, cell-based HIV replication assays, cytotoxicity evaluation in relevant host cells, CC50 determination, selectivity index calculation, and resistance-profiling studies are required to confirm their antiviral relevance and therapeutic potential.

3. Experimental

3.1. Chemistry

3.1.1. Materials and instruments

The melting points of the newly synthesized compounds were determined using an electrothermal Gallenkamp apparatus (IA 9000). FTIR spectra were recorded on a Pye-Unicam SP300 spectrophotometer using KBr discs. 1H and 13C NMR spectra were acquired on a JEOL 500 MHz spectrometer (500 MHz for 1H and 125 MHz for 13C). Mass spectra were obtained using a Shimadzu GCMS-Q1000-EX at an ionization voltage of 70 eV. Elemental (CHNS) analyses were performed using an Elementar Vario ELIII analyzer (Germany).

3.1.1.1. General procedure for the reactions of 2-methyl-4H-benzo[3,1]oxazin-4-one with heterocyclic amines

7-Amino-5H-thiazolo[3,2-a]pyrimidine-3,5(2H)-dione (2), 6-amino-2-thioxo-2,3-dihydro pyrimidin-4(1H)-one (3) or 7-amino-3-aryl-5H-thiazolo[3,2-a]pyrimidin-5-ones (4a–f) (1 mmol each) was treated with 2-methyl-4H-benzo[3,1]oxazin-4-one (1) (0.16 g, 1 mmol) and choline hydroxide (5 mL). The reaction mixture was heated in an oil bath at 230 °C for 6 hours (monitored by TLC). After cooling, ethanol (5 mL) was added and the resulting precipitate was filtered and recrystallized from an EtOH/DMF mixture to give product 5, 6 or 7a–f, respectively.

3.1.1.1.1. 7-(2-Methyl-4-oxoquinazolin-3-yl)-5H-thiazolo[3,2-a]pyrimidine-3,5(2H)-dione (5)

Yellow powder (0.28 g, yield = 85%), mp 217–219 °C; IR (KBr): υ = 1680, 1668 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 1591 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), cm−1; 1H-NMR (DMSO-d6): δ = 2.47 (s, 3H, CH3), 3.87 (s, 2H, CH2), 6.82 (s, 1H, pyrimidine-H), 7.05–7.76 (m, 4H, Ar–H) ppm; 13C-NMR (DMSO-d6): δ = 17.1 (CH3), 34.2 (C-2 thiazole), 85.4 (C-5 pyrimidine), 116.9, 121.0, 121.6, 131.8, 134.3, 143.1, 146.2, 151.9, 156.3 (Ar–Cs), 163.7, 169.5, 174.7 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O) ppm; MS (70 eV): m/z = 326 (M+, 34), 184 (100); anal. calcd for C15H10N4O3S (326.05): C, 55.21; H, 3.09; N, 17.17%. Found: C, 55.36; H, 3.21; N, 17.29%.

3.1.1.1.2. 2-Methyl-3-(6-oxo-2-thioxo-1,2,3,6-tetrahydropyrimidin-4-yl)quinazolin-4(3H)-one (6)

Yellow powder (0.24 g, yield = 85%), mp 233–235 °C; IR (KBr): υ = 2260, 2130 (NH), 1682, 1660 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 1593 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), 1530 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 S) cm−1; 1H-NMR (DMSO-d6): δ = 2.47 (s, 3H, CH3), 6.81 (s, 1H, pyrimidine-H), 7.45–7.96 (m, 4H, Ar–H), 10.82 (s, 1H, NH), 11.54 (s, 1H, NH) ppm; 13C-NMR (DMSO-d6): δ = 17.3 (CH3), 85.4 (C-5 pyrimidine), 119.9, 125.6, 127.8 (2C), 133.3, 146.1, 156.2, 158.9 (Ar–Cs), 164.7, 167.5 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 174.3 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 S) ppm; MS (70 eV): m/z = 286 (M+, 21), 64 (100); anal. calcd for C13H10N4O2S (286.05): C, 54.54; H, 3.52; N, 19.57%. Found: C, 54.38; H, 3.41; N, 19.40%.

3.1.1.1.3. 7-(2-Methyl-4-oxoquinazolin-3-yl)-3-phenyl-5H-thiazolo[3,2-a]pyrimidin-5-one (7a)

Yellow powder (0.32 g, yield = 83%), mp 206–208 °C; IR (KBr): υ = 1690, 1665 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 1593 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), cm−1; 1H-NMR (DMSO-d6): δ = 2.47 (s, 3H, CH3), 6.36 (s, 1H, pyrimidine-H), 6.95 (s, 1H, thiazole-H), 7.02–8.01 (m, 9H, Ar–H) ppm; 13C-NMR (DMSO-d6): δ = 16.9 (CH3), 85.5 (C-5 pyrimidine), 112.6 (C-2 thiazole), 116.3, 116.8, 120.4, 120.9, 122.5, 122.9, 123.4, 128.9, 131.0, 131.5, 132.6, 134.4, 141.4, 152.3 (Ar–Cs), 163.8, 168.9 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O) ppm; MS (70 eV): m/z = 386 (M+, 18), 90 (100); anal. calcd for C21H14N4O2S (386.08): C, 65.27; H, 3.65; N, 14.50%. Found: C, 65.35; H, 3.42; N, 14.39%.

3.1.1.1.4. 7-(2-Methyl-4-oxoquinazolin-3-yl)-3-(p-tolyl)-5H-thiazolo[3,2-a]pyrimidin-5-one (7b)

Yellow powder (0.33 g, yield = 83%), mp 213–215 °C; IR (KBr): υ = 1690, 1666 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 1597 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), cm−1; 1H-NMR (DMSO-d6): δ = 2.35 (s, 3H, Ar-CH3), 2.47 (s, 3H, CH3), 6.37 (s, 1H, pyrimidine-H), 6.91 (s, 1H, thiazole-H), 7.14–7.91 (m, 8H, Ar–H) ppm; 13C-NMR (DMSO-d6): δ = 17.6 (CH3), 22.4 (Ar-CH3), 85.4 (C-5 pyrimidine), 110.1 (C-2 thiazole), 120.7, 126.1, 126.7, 127.4 (2C), 128.5 (2C), 129.1 (2C), 131.8, 146.2, 148.6, 151.9, 156.3 (Ar–Cs), 163.8, 169.4 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O) ppm; MS (70 eV): m/z = 400 (M+, 47), 90 (100); anal. calcd. for C22H16N4O2S (400.10): C, 65.99; H, 4.03; N, 13.99%. Found: C, 66.08; H, 3.92; N, 14.09%.

3.1.1.1.5. 7-(2-Methyl-4-oxoquinazolin-3-yl)-3-(4-chlorophenyl)-5H-thiazolo[3,2-a]pyrimidin-5-one (7c)

Yellow powder (0.34 g, yield = 82%), mp 241–243 °C; IR (KBr): υ = 1691, 1666 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 1594 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), cm−1; 1H-NMR (DMSO-d6): δ = 2.47 (s, 3H, CH3), 6.33 (s, 1H, pyrimidine-H), 6.95 (s, 1H, thiazole-H), 7.36–8.02 (m, 8H, Ar–H) ppm; 13C-NMR (DMSO-d6): δ = 17.3 (CH3), 85.1 (C-5 pyrimidine), 113.1 (C-2 thiazole), 120.8, 122.3, 126.1, 126.7, 127.4, 128.5, 129.1, 133.8, 134.8, 142.1, 146.2, 148.6, 151.9, 156.3 (Ar–Cs), 164.1, 168.5 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O) ppm; MS (70 eV): m/z = 420 (M+, 23), 198 (100); anal. calcd for C21H13ClN4O2S (420.04): C, 59.93; H, 3.11; N, 13.31%. Found: C, 60.05; H, 3.28; N, 13.49%.

3.1.1.1.6. 7-(2-Methyl-4-oxoquinazolin-3-yl)-3-(4-bromophenyl)-5H-thiazolo[3,2-a]pyrimidin-5-one (7d)

Yellow powder (0.38 g, yield = 82%), mp 230–232 °C; IR (KBr): υ = 1691, 1668 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 1594 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), cm−1; 1H-NMR (DMSO-d6): δ = 2.47 (s, 3H, CH3), 6.33 (s, 1H, pyrimidine-H), 6.69 (s, 1H, thiazole-H), 7.26–7.94 (m, 8H, Ar–H) ppm; 13C-NMR (DMSO-d6): δ = 17.3 (CH3), 85.2 (C-5 pyrimidine), 110.9 (C-2 thiazole), 120.7, 122.3, 126.1, 126.8, 127.4, 128.5, 131.1, 133.8, 134.7, 142.1, 146.3, 148.6, 151.9, 156.4 (Ar–Cs), 164.1, 168.4 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O) ppm; MS (70 eV): m/z = 463 (M+, 32), 154 (100); anal. calcd for C21H13BrN4O2S (463.99): C, 54.21; H, 2.82; N, 12.04%. Found: C, 54.08; H, 2.68; N, 11.97%.

3.1.1.1.7. 7-(2-Methyl-4-oxoquinazolin-3-yl)-3-(4-nitrophenyl)-5H-thiazolo[3,2-a]pyrimidin-5-one (7e)

Yellow powder (0.34 g, yield = 80%), mp 264–266 °C; IR (KBr): υ = 1690, 1665 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 1592 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), cm−1; 1H-NMR (DMSO-d6): δ = 2.47 (s, 3H, CH3), 6.32 (s, 1H, pyrimidine-H), 6.71 (s, 1H, thiazole-H), 7.19–8.31 (m, 8H, Ar–H) ppm; 13C-NMR (DMSO-d6): δ = 17.8 (CH3), 85.4 (C-5 pyrimidine), 111.1 (C-2 thiazole), 120.8, 123.1, 126.1, 126.8, 127.4, 127.9, 133.5, 140.8, 142.6, 146.2, 148.6, 149.7, 151.9, 156.3 (Ar–Cs), 163.7, 169.5 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O) ppm; MS (70 eV): m/z = 431 (M+, 47), 138 (100); anal. calcd for C21H13N5O4S (431.07): C, 58.46; H, 3.04; N, 16.23%. Found: C, 58.34; H, 3.12; N, 16.38%.

3.1.1.1.8. 7-(2-Methyl-4-oxoquinazolin-3-yl)-3-(2-oxo-2H-chromen-3-yl)-5H-thiazolo[3,2-a]pyrimidin-5-one (7f)

Yellow powder (0.36 g, yield = 80%), mp 250–252 °C; IR (KBr): υ = 1692, 1665 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 1594 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), cm−1; 1H-NMR (DMSO-d6): δ = 2.47 (s, 3H, CH3), 6.34 (s, 1H, pyrimidine-H), 6.92 (s, 1H, thiazole-H), 7.08–7.93 (m, 9H, Ar–H and coumarin-H) ppm; 13C-NMR (DMSO-d6): δ = 17.4 (CH3), 85.4 (C-5 pyrimidine), 116.1 (C-2 thiazole), 118.4, 120.8, 125.6, 126.2, 126.8, 127.4, 128.5, 130.4, 133.8, 134.3, 140.2, 142.1, 146.2, 148.6, 151.9, 153.6, 156.3, 158.4 (Ar–Cs), 163.7, 167.4, 169.5 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O) ppm; MS (70 eV): m/z = 454 (M+, 30), 123 (100); anal. calcd for C24H14N4O4S (454.07): C, 63.43; H, 3.11; N, 12.33%. Found: C, 63.38; H, 3.22; N, 12.44%.

3.2.1. Alternative synthesis of compounds 5 or 7a

2-Methyl-3-(6-oxo-2-thioxo-1,2,3,6-tetrahydropyrimidin-4-yl)quinazolin-4(3H)-one (6) (0.286 g, 1 mmol) was mixed with chloroacetic acid (8) (0.094 g, 1 mmol) or phenacyl bromide (9) (0.2 g, 1 mmol) in absolute ethanol (30 mL) containing choline hydroxide (0.24 g, 2 mmol). The reaction mixture was heated under refluxing for 6 hours and the resulting precipitate was filtered and crystallized from an EtOH/DMF mixture to afford an authentic sample of compound 5 or 7a, respectively.

3.2.1.1. Reactions of compound 3 with aldehydes (10a–f)

An equimolar amount of 7-(2-methyl-4-oxoquinazolin-3-yl)-5H-thiazolo[3,2-a]pyrimidine-3,5(2H)-dione (5) (0.33 g, 1 mmol) and the appropriate aldehyde (10a–f) (1 mmol each) was dissolved in 30 mL of absolute ethanol containing choline hydroxide (0.24 g, 2 mmol). The reaction mixture was heated under reflux for 6 hours (monitored by TLC). After completion, water was added to the mixture, and the precipitated solid was collected by filtration, washed with water, and recrystallized from EtOH to give the pure products 11a–f.

3.2.1.1.1. 2-Benzylidine-7-(2-methyl-4-oxoquinazolin-3-yl)-5H-thiazolo[3,2-a]pyrimidine-3,5(2H)-dione (11a)

Yellow powder (0.35 g, yield = 85%), mp 247–249 °C; IR (KBr): υ = 1690, 1665 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 1593 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), cm−1; 1H-NMR (DMSO-d6): δ = 2.47 (s, 3H, CH3), 6.88 (s, 1H, pyrimidine-H), 7.11–7.92 (m, 9H, Ar–H), 8.36 (s, 1H, CH Created by potrace 1.16, written by Peter Selinger 2001-2019 C) ppm; 13C-NMR (DMSO-d6): δ = 17.0 (CH3), 85.4 (C-5 pyrimidine), 117.6, 121.4, 122.3, 123.9, 125.7, 128.8, 130.1, 131.8, 132.0, 134.3, 142.3, 145.1, 150.2, 151.9, 159.0 (Ar–Cs), 163.7, 168.5, 174.6 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O) ppm; MS (70 eV): m/z = 414 (M+, 35), 115 (100); anal. calcd for C22H14N4O3S (414.08): C, 63.76; H, 3.41; N, 13.52%. Found: C, 63.56; H, 3.27; N, 13.38%.

3.2.1.1.2. 2-(4-Methoxybenzylidine)-7-(2-methyl-4-oxoquinazolin-3-yl)-5H-thiazolo[3,2-a]pyrimidine-3,5(2H)-dione (11b)

Yellow powder (0.38 g, yield = 85%), mp 236–238 °C; IR (KBr): υ = 1691, 1666 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 1593 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), cm−1; 1H-NMR (DMSO-d6): δ = 2.47 (s, 3H, CH3), 3.82 (s, 3H, OCH3), 6.75 (s, 1H, pyrimidine-H), 7.02–7.91 (m, 8H, Ar–H), 8.45 (s, 1H, CH Created by potrace 1.16, written by Peter Selinger 2001-2019 C) ppm; 13C-NMR (DMSO-d6): δ = 18.4 (CH3), 54.2 (OCH3), 84.6 (C-5 pyrimidine), 116.8, 120.6, 126.8 (2C), 127.3 (2C), 128.3 (2C), 133.5, 135.4, 143.2, 146.2, 151.8, 156.3, 158.3 (Ar–Cs), 163.7, 168.5, 173.8 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O) ppm; MS (70 eV): m/z = 444 (M+, 24), 62 (100); anal. calcd for C23H16N4O4S (444.09): C, 62.15; H, 3.63; N, 12.61%. Found: C, 62.26; H, 3.52; N, 12.49%.

3.2.1.1.3. 2-(4-Chlorobenzylidine)-7-(2-methyl-4-oxoquinazolin-3-yl)-5H-thiazolo[3,2-a]pyrimidine-3,5(2H)-dione (11c)

Yellow powder (0.37 g, yield = 83%), mp 263–265 °C; IR (KBr): υ = 1690, 1665 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 1593 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), cm−1; 1H-NMR (DMSO-d6): δ = 2.47 (s, 3H, CH3), 6.81 (s, 1H, pyrimidine-H), 7.20–7.88 (m, 8H, Ar–H), 8.43 (s, 1H, CH Created by potrace 1.16, written by Peter Selinger 2001-2019 C) ppm; 13C-NMR (DMSO-d6): δ = 18.6 (CH3), 84.4 (C-5 pyrimidine), 116.8, 120.6, 126.7 (2C), 127.2 (2C), 128.6 (2C), 133.5, 135.4, 143.4, 146.6, 151.9, 156.4, 158.2 (Ar–Cs), 163.7, 168.5, 174.1 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O) ppm; MS (70 eV): m/z = 448 (M+, 26), 138 (100); anal. calcd for C22H13ClN4O3S (448.04): C, 58.87; H, 2.92; N, 12.48%. Found: C, 58.69; H, 2.81; N, 12.35%.

3.2.1.1.4. 2-(4-Fluorobenzylidine)-7-(2-methyl-4-oxoquinazolin-3-yl)-5H-thiazolo[3,2-a]pyrimidine-3,5(2H)-dione (11d)

Yellow powder (0.36 g, yield = 83%), mp 244–246 °C; IR (KBr): υ = 1692, 1665 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 1598 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), cm−1; 1H-NMR (DMSO-d6): δ = 2.47 (s, 3H, CH3), 6.82 (s, 1H, pyrimidine-H), 7.01–7.96 (m, 8H, Ar–H), 8.41 (s, 1H, CH Created by potrace 1.16, written by Peter Selinger 2001-2019 C) ppm; 13C-NMR (DMSO-d6): δ = 18.4 (CH3), 84.4 (C-5 pyrimidine), 116.8, 117.5, 120.6, 126.7 (2C), 127.5, 128.6, 130.4, 131.5, 133.5, 143.4, 146.6, 151.9, 156.4, 160.2 (Ar–Cs), 163.7, 168.5, 174.3 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O) ppm; MS (70 eV): m/z = 433 (M+, 23), 76 (100); anal. calcd for C22H13FN4O3S (433.07): C, 61.11; H, 3.03; N, 12.96%. Found: C, 61.19; H, 2.97; N, 13.05%.

3.2.1.1.5. 2-(4-Nitrobenzylidine)-7-(2-methyl-4-oxoquinazolin-3-yl)-5H-thiazolo[3,2-a]pyrimidine-3,5(2H)-dione (11e)

Yellow powder (0.37 g, yield = 80%), mp 273–275 °C; IR (KBr): υ = 1690, 1668 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 1594 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), cm−1; 1H-NMR (DMSO-d6): δ = 2.47 (s, 3H, CH3), 6.87 (s, 1H, pyrimidine-H), 7.29–8.13 (m, 8H, Ar–H), 8.36 (s, 1H, CH Created by potrace 1.16, written by Peter Selinger 2001-2019 C) ppm; 13C-NMR (DMSO-d6): δ = 18.4 (CH3), 83.9 (C-5 pyrimidine), 116.4, 120.4, 123.7, 126.1, 126.8, 127.3, 128.4, 129.1, 133.5, 136.4, 141.1, 146.9, 151.9, 156.3, 158.3 (Ar–Cs), 163.7, 168.5, 173.7 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O) ppm; MS (70 eV): m/z = 459 (M+, 27), 122 (100); anal. calcd for C22H13N5O5S (459.06): C, 57.51; H, 2.85; N, 15.24%. Found: C, 57.72; H, 3.03; N, 15.37%.

3.2.1.1.6. 2-(Naphthalen-1-ylmethylene)-7-(2-methyl-4-oxoquinazolin-3-yl)-5H-thiazolo[3,2-a]pyrimidine-3,5(2H)-dione (11f)

Yellow powder (0.37 g, yield = 80%), mp 259–261 °C; IR (KBr): υ = 1690, 1666 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 1598 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), cm−1; 1H-NMR (DMSO-d6): δ = 2.47 (s, 3H, CH3), 6.85 (s, 1H, pyrimidine-H), 7.03–7.73 (m, 11H, Ar–H), 8.39 (s, 1H, CH Created by potrace 1.16, written by Peter Selinger 2001-2019 C) ppm; 13C-NMR (DMSO-d6): δ = 18.1 (CH3), 84.5 (C-5 pyrimidine), 116.9, 122.6, 124.5 (2C), 125.3 (2C), 126.8 (2C), 127.3 (2C), 128.4 (2C), 131.2, 133.5, 135.4, 141.5, 143.8, 146.2, 151.9, 156.3, 158.2 (Ar–Cs), 163.5, 168.5, 173.4 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O) ppm; MS (70 eV): m/z = 464 (M+, 25), 77 (100); anal. calcd for C26H16N4O3S (464.09): C, 67.23; H, 3.47; N, 12.06%. Found: C, 67.11; H, 3.30; N, 11.95%.

3.2. Docking study

3.2.1. Ligand preparation

The synthesized structures were first constructed using ChemDraw Professional 16.0 and subsequently subjected to energy minimization using the MMFF94× force field. Geometry optimization was continued until the gradient convergence criterion of 0.1 kcal mol−1·Å−1 was reached.

3.2.2. Protein preparation

The enzyme's X-ray crystal structure (PDB ID: 6DV4, resolution: 1.14 Å) was downloaded in PDB format from the Protein Data Bank.57 The enzyme was prepared for docking study through the following steps:

(1) Water molecules were removed, and hydrogen atoms were added to the structure using the usual geometry. Broken bonds were then reconnected, and potential structural issues were corrected.58

(2) The Alpha Site Finder was used to identify the largest binding site. Dummy atoms were generated based on the enzyme structure, and the protein binding pocket was identified.

(3) The identified binding pocket was saved in PDB format and used to dock the most effective drugs and estimate the ligand–enzyme interactions at the active site.

(4) Binding poses were generated with the Triangle Matcher placement approach and ranked using the London dG scoring function, focusing on contacts between the ligand and key active-site residues.

(5) The resulting complexes were then examined using 2D interaction maps and 3D molecular views to identify and confirm ligand–amino acid interactions. Docking parameters, scores, and interaction profiles were reported following the previously described literature workflow.59,60

Protein and ligand preparation, together with redocking of the co-crystallized ligand, were performed to validate the docking protocol. The ligand was redocked into its native binding site, and the predicted binding pose was compared with its crystallographic orientation. The co-crystallized ligand (CCL) showed an RMSD of 1.386 Å, which was below the acceptable threshold of 2.0 Å, confirming the reliability of the docking protocol.

3.2.3. In silico physicochemical and ADMET

To predict the physiological and ADMET parameters, the SMILE representations of the synthesized compounds were uploaded to the pkCSM web server. Using pkCSM, the pharmacokinetic profiles of the selected synthesized compounds were predicted.61 The pkCSM web server, which is publicly available (https://structure.bioc.cam.ac.uk/pkcsm), provides an extensive information platform for quickly assessing pharmacokinetic62,63 and toxicity properties.64

3.3. HIV-1 RT inhibition activity

The antiviral activities of the tested compounds against RT were evaluated using an RT assay kit.65

Negative control: 0.5% DMSO in reaction buffer (without inhibitor, maximal RT activity).

Background control: all components except RT (to measure the non-specific signal).

Positive control: the reference drug at several concentrations (for validation).

3.3.1. Assay procedure

3.3.1.1. RT reaction

(i) Set up the reaction mixture (for each well):

• 10 µL template–primer complex

• 10 µL nucleotide mix (with biotin-dUTP)

• 10 µL diluted compound or control

• 10 µL HIV-1 RT enzyme (diluted in 1× reaction buffer)

“Final reaction volume = 40 µL per well.”

(ii) Add the components to the plate as follows (duplicates/triplicates recommended):

• Columns 1–2: background control (RT replaced with buffer)

• Columns 3–4: negative control (0.5% DMSO, no inhibitor)

• Columns 5–20: tested compounds and the reference drug at various concentrations.

(iii) Incubate the plate at 37 °C for 60–90 min (with the plate covered to prevent evaporation).

(iv) Stop the reaction by adding 10 µL of stop solution (if provided), or proceed directly to the detection step.

3.3.1.2. Detection (can be done immediately after RT step)

(a) Transfer 50 µL of each reaction mixture to the corresponding well of the pre-coated 96-well plate.

(b) Incubate for 60 min at 37 °C on a shaker (gentle shaking).

(c) Wash three times with 300 µL per well of 1× wash buffer (soak for 30 s during each wash and tap dry).

(d) Add 100 µL per well of anti-biotin HRP conjugate. Incubate for 60 min at 37 °C.

(e) Wash five times as described in step (c).

(f) Add 100 µL per well of TMB substrate. Incubate for 10–20 min at room temperature in the dark (color develops).

(g) Stop reaction with 100 µL per well of stop solution. Color changes from blue to yellow.

(h) Measure absorbance at 450 nm (reference 620–650 nm) within 30 min.

3.3.1.3. Calculations and data processing

(1) Average the duplicate/triplicate readings.

(2) Subtract the background (wells without RT):

Corrected absorbance = (A) sample – (A) background.

(3) Normalize activity:

(%) Activity = [corrected (A)sample/corrected(A) negative control] × 100

(%) Inhibition = 100 − (%) activity

3.2.4. HIV-1 RT inhibition assay full protocol

The anti-HIV-1 RT activity was evaluated using a commercial HIV-1 reverse transcriptase assay kit. The assay was performed in a 96-well format using a template–primer complex, a nucleotide mixture containing biotin-dUTP, HIV-1 RT enzyme, and different concentrations of each tested compound. The compounds were dissolved in DMSO and diluted with reaction buffer, keeping the final DMSO concentration at 0.5%. Zidovudine was used as the positive control, while wells containing 0.5% DMSO without an inhibitor served as the negative control. Background wells contained all assay components except HIV-1 RT.

The reaction mixture contained 10 µL of the template–primer complex, 10 µL of the nucleotide mixture, 10 µL of the tested compound or control, and 10 µL of the diluted HIV-1 RT enzyme. After incubation at 37 °C for 60–90 min, the reaction mixture was transferred to the pre-coated detection plate, followed by incubation with an anti-biotin HRP conjugate and TMB substrate. Absorbance was measured at 450 nm with reference wavelength correction at 620–650 nm.

Each compound was tested in triplicate at concentrations of 0.01, 0.1, 1, 10, and 50 µg mL−1. Background absorbance was subtracted, and percentage inhibition was calculated relative to the negative control. IC50 values were obtained from dose–response curves by nonlinear regression analysis and are reported as µg mL−1 and µM.

4. Conclusion

Two novel series of quinazoline-tethered thiazolo[3,2-a]pyrimidines, namely 3-aryl-5H-thiazolo[3,2-a]pyrimidin-5-ones (7a–f) and 2-arylidine-5H-thiazolo[3,2-a]pyrimidine-3,5(2H)-diones (11a–f), were efficiently synthesized using a choline hydroxide-mediated protocol. Selected representative compounds were evaluated for HIV-1 RT inhibition, showing IC50 values ranging from 1.305–14.133 µM, compared with zidovudine (0.758 µM). Among the synthesized derivatives, compound 11e was the most active member of the series, with an IC50 value of 0.599 µg mL−1 (1.305 µM). Molecular docking provided qualitative insights into possible ligand–target interactions, with docking scores ranging from −6.375 to −6.916 kcal mol−1 compared with zidovudine (−5.796 kcal mol−1). However, the docking scores did not fully correlate with the experimental HIV-1 RT inhibition data and should therefore be interpreted as supportive binding-mode information rather than direct predictors of activity. In silico ADMET predictions suggested generally favorable properties, including high intestinal absorption (89.87–100%), a moderate fraction unbound (0.283–0.37), total clearance values ranging from −0.47 to 0.115 log mL min−1 kg−1, and no predicted skin sensitization. Overall, these findings identify the prepared scaffolds, particularly 11e, as promising preliminary leads for further optimization as HIV-1 RT inhibitors. Nevertheless, additional cell-based antiviral assays, cytotoxicity evaluation, CC50 determination, selectivity index calculation, and resistance-profile studies are required to validate their antiviral relevance and therapeutic potential.

Conflicts of interest

There are no conflicts to declare.

Supplementary Material

RA-OLF-D6RA03750J-s001

Acknowledgments

This work was supported and funded by the Deanship of Scientific Research at Imam Mohammad Ibn Saud Islamic University (IMSIU) (grant number IMSIU-DDRSP2601).

Data availability

The data supporting the findings of this study are available within the article and its supplementary information (SI). Additional data related to this work are available from the corresponding author upon reasonable request. Supplementary information: additional data supporting the molecular docking and in silico physicochemical analyses presented in the article. See DOI: https://doi.org/10.1039/d6ra03750j.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

RA-OLF-D6RA03750J-s001
RA-OLF-D6RA03750J-s001.pdf (5.8MB, pdf)

Data Availability Statement

The data supporting the findings of this study are available within the article and its supplementary information (SI). Additional data related to this work are available from the corresponding author upon reasonable request. Supplementary information: additional data supporting the molecular docking and in silico physicochemical analyses presented in the article. See DOI: https://doi.org/10.1039/d6ra03750j.

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