Design, Synthesis, In Silico Studies, and HIV‐1 RT Inhibitory Activity of 1,3‐Oxazolidine Pyrimidine Nucleoside Analogues

ABSTRACT

A series of 1,3‐oxazolidine nucleoside analogues (1‐6) was designed and synthesized for their anti‐HIV activity. The purpose of this study was to determine whether the antiviral activity of the pyrimidine ring is affected by oxazolidine ring substitution. The synthesized compounds were tested for their HIV‐1 RT inhibitory activity by a colorimetric HIV reverse transcriptase (HIVRT) test. The fluoro (S4) and bromo (S5) derivatives of these compounds showed the highest HIV‐1 reverse transcriptase IC₅₀ values, 19.27 and 22.80 µM, respectively. Modest activity was observed for the remaining compounds. The molecular docking experiments with the crystal structure of HIV‐1 reverse transcriptase (PDB ID: 1RT1) revealed that S4 and S5 have good binding affinities of −7.6 and −7.3 kcal/mol, respectively, with multiple hydrogen bonds and π‐interactions with key residues in the active site. The properties of all the synthesized compounds were found to be in accordance with Lipinski Rule of Five and showed a favorable ADME profile, indicating good drug‐likeness, acceptable solubility, and potential oral bioavailability. Halogen substitution enhances electronic dispersion and hydrophobic interactions, but the oxazolidine scaffold is superior to thiazolidine scaffolds. Halogenated oxazolidine compounds are generally promising HIV‐1 reverse transcriptase inhibitors.

Previously synthesized 1,3‐oxazolidine pyrimidine nucleoside analogues (S1–S6) were evaluated for HIV‐1 reverse transcriptase inhibition using molecular docking, ADMET prediction, and an in vitro colorimetric HIV‐RT assay. Compounds S4 and S5 exhibited the strongest binding affinities and moderate enzyme inhibition (IC₅₀ = 19.27 and 22.80 µM), identifying them as preliminary hit compounds for further structural optimization and biological evaluation.

1. Introduction

The nucleoside analogues are one of the most successful antiviral drug classes because they are able to mimic the structure of the naturally occurring nucleosides and to disrupt the production of viral nucleic acids [1]. Structural modification of the nucleobase or carbohydrate moiety has yielded a wide range of clinically useful antiviral and anticancer agents in the last few decades that are more potent, selective, and have better pharmacokinetic properties [2]. In particular, replacement of the native ribofuranose ring with heterocyclic bioisosteres has proved an effective strategy for increasing metabolic stability, changing conformational flexibility and optimizing the interactions with biological targets [3, 4]. Among them, 1,3‐oxazolidine nucleoside analogues with both nitrogen and oxygen in a five‐membered pseudo‐sugar ring have received significant interest due to their favorable stereo electronic properties, increased chemical stability and promising antiviral activity. These heterocyclic backbones also serve as flexible scaffolds for the rational substitution of heteroatoms with enhanced biological activity and optimization of the scaffold [5, 6].

Human immunodeficiency virus (HIV) continues to be one of the major public health issues since the discovery of HIV‐1 and HIV‐2 as the causes of acquired immune deficiency syndrome (AIDS) [7]. The World Health Organization (WHO) estimates that about 40.8 million people were living with HIV globally by the end of 2024, with Sub‐Saharan Africa, Asia, and South America having the highest prevalence. HIV strikes CD4⁺ T lymphocytes, gradually worsening the immune system and making the host vulnerable to opportunistic infections and cancers [8]. HIV strikes CD4⁺ T lymphocytes, gradually worsening the immune system and making the host vulnerable to opportunistic infections and cancers. Despite major advances in treatment, HIV infection has become a manageable chronic disease, but new variants of the virus that are resistant to current medications are constantly emerging, while toxicities associated with the drugs are a concern [9].

Reverse transcriptase (RT) is one of the most fully validated targets for HIV therapy among the viral proteins essential for HIV replication. This multifunctional enzyme also serves to convert viral single‐stranded RNA to double‐stranded DNA, a key step in viral insertion into the host genome [10]. Inhibition of HIV‐1 RT, therefore plays a major role in the discovery of new anti‐HIV‐1 drugs and is a major point of focus in research. Highly active antiretroviral therapy (HAART), which includes nucleoside reverse transcriptase inhibitors (NRTIs), non‐nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors, and more recently, integrase inhibitors, has significantly decreased HIV‐related morbidity and mortality [11, 12]. However, long‐term treatment often leads to viral resistance, side effects, and decreased long‐term treatment adherence, which demonstrates that structurally novel RT inhibitors with better pharmacological properties remain a therapeutic necessity [13].

Nucleoside reverse transcriptase inhibitors (NRTIs) work by mimicking the function of a natural nucleoside; then, after they are phosphorylated by cellular enzymes, they inhibit the activity of reverse transcriptase and stop chain elongation in the synthesis of viral DNA. The structure optimization of nucleoside analogues is an ongoing field of medicinal chemistry, and in particular, modifications to the pseudo‐sugar have led to significant enhancements in enzymatic recognition, conformational preference, metabolic stability, and antiviral activity. The incorporation of heteroatoms into the carbohydrate framework is also an attractive approach to the creation of new bioisosteric nucleosides with improved physicochemical and biological properties in this context [14, 15, 16].

We reported previously the design and biological evaluation of 1,3‐thiazolidine‐based pyrimidine nucleoside analogues, which showed that the modification of the sugar by heterocycles can provide compounds with promising anti‐HIV activity. Based on those results, the present study examines the effects of bioisosteric replacement of the sulfur atom in the 1,3‐thiazolidine ring with oxygen, resulting in a series of novel pyrimidine nucleoside analogues 1,3‐oxazolidine. The heteroatom substitution was designed to change the electronic environment, hydrogen‐bonding capability, conformational behavior and molecular recognition within the HIV‐1 reverse transcriptase binding pocket. In contrast with the earlier study that examined sulfur‐containing thiazolidine analogues, the effect of the incorporation of oxygen was investigated systematically in this work for antiviral activity and binding affinity to HIV‐1 reverse transcriptase.

Based on this, six novel 1,3‐oxazolidine pyrimidine nucleoside analogues (S1–S6) were designed, synthesized, and structurally characterized [17]. The anti‐HIV potential of these molecules was thoroughly examined using molecular docking, ADMET prediction, and biological evaluation against HIV‐1 reverse transcriptase. The present work highlights new perspectives on the structure–activity relationships of oxazolidine‐based nucleoside analogues and illustrates the potential of pseudo‐sugar engineering of heterocycles as a viable approach to develop next‐generation anti‐HIV drugs.

2. Materials and Methods

2.1. Instrument and General Materials

2.1.1. Chemicals and Reagents

The reagents and solvents used in this study were all of analytical reagent (AR) grade and purchased from commercial suppliers. These were used without further purification unless otherwise stated. Tetrahydrofuran (THF) was freshly distilled over sodium/benzophenone in order to ensure anhydrous conditions. Water and air‐sensitive reactions were performed under a nitrogen atmosphere.

2.1.2. Reaction Conditions and Spectroscopic Analysis

All reactions were performed in typical Schlenk techniques under an anhydrous atmosphere of dry nitrogen to ensure anhydrous conditions throughout the synthesis. The uncorrected melting points were measured using Thomas Hoover's melting point apparatus. The reactions were monitored via thin‐layer chromatography (TLC) employing aluminum sheets and silica gel 60 F254 (Merck). The synthesized compounds were purified by column chromatography on a silica gel (230–400 mesh) column. Thin films, either on KBr plates (oils) or KBr discs (solids), were used to obtain infrared spectra (t_max) with a Perkin‐Elmer FT‐IR spectrophotometer. NMR data were collected on ¹H (500 MHz) and ¹³C (125 MHz) NMR spectrometers (Bruker) in DMSO‐d6 with TMS as an internal standard. Karlsruhe Agilent 1100 ES‐MS Gemany was used for recording mass spectra. The elemental analysen systeme GmbH VarioEL V3.00 element analyzer was used for the microanalyses.

2.2. Synthesis Methodology

The synthesis of target compounds followed the reaction routes outlined in the scheme.

2.2.1. Synthesis of 1,3‐Oxazolidine Nucleoside Analogues (S1–S6)

L‐serine methyl ester hydrochloride (L) was first protected throughout the synthesis by reacting with di‐tert‐butyl dicarbonate (Boc₂O) at room temperature when 4‐dimethylaminopyridine (DMAP) and triethylamine were present. N‐tert‐Butoxycarbonyl‐4 methoxycarbonyl‐1,3‐oxazolidin‐2‐one (M) was produced in the process. Washing with 5% hydrochloric acid and water, followed by chloroform extraction, was used to remove excess DMAP [18]. The compound M with both the lactam and ester functions was stereo selectively reduced with lithium triethylborohydride (Super Hydride) in anhydrous THF at 0 °C, and thus N‐tert‐butoxycarbonyl 4‐hydroxymethyl‐1,3‐oxazolidin‐2‐ol(N) [19, 20] was synthesized. The main hydroxyl group of compound N was tritylated with trityl chloride in pyridine to give N‐tert‐butoxycarbonyl‐4 trityloxymethyl‐1,3‐oxazolidin‐2‐ol (O). Subsequently, the C2‐hydroxyl of compound O was acylated under the same conditions with acetic anhydride to afford N‐tert‐butoxycarbonyl‐2‐acyloxy‐4‐trityloxymethyl‐1,3‐oxazolidine (P) (Figure 1) [21].

FIGURE 1.

Synthesis of 1‐(2R,4S‐4‐hydroxymethyl‐1,3‐oxazolidine‐2‐yl)‐pyrimidine nucleoside analogues S1‐6.

The Vorbrüggen coupling technique was used to glycosylate the pyridones Q1‐Q6 with substance P. The pyrimidine bases were refluxed with hexamethyldisilazane (HMDS) in the presence of a catalytic amount of ammonium sulfate, and excess HMDS was removed under reduced pressure. To produce the protected nucleoside analogues (R1‐R6), the silylated bases (Q1‐Q6) were treated with compound P in dry acetonitrile using a Lewis acid catalyst, such as SnCl₄ or TMSOTf [22, 23]. Dichloromethane was then added at room temperature to the mixture, and trifluoroacetic acid (TFA) was added to remove N‐tert‐butoxycarbonyl (Boc) and trityl (Tr) protecting groups [24]. The obtained crude product was refined, and 1,3‐oxazolidine derivatives of the required nucleosides were obtained in the form of a white powder after the product was washed with diethyl ether after evaporation under reduced pressure. The reaction conditions of the complete synthesis step are published in our previous work [25].

2.3. In Silico Studies

2.3.1. Molecular Docking Process

The binding ability of the 1,3‐oxazolidine nucleoside analogues (S1‐S6) with HIV‐1 Reverse Transcriptase (PDB ID: 1RT1) was investigated using molecular docking studies using AutoDock Vina v1.2.0, via command prompt interface, were carried out, to compare their docking scores with the reference drug and the cocrystal ligand [26].

2.3.2. Ligand Preparation

The 3D structures of the ligands were obtained using the ChemDraw software and saved as SDF files and then converted to PDB format with Openbabel (https://sourceforge.net/projects/openbabel/) using the same software. The ligands were then prepared using AutoDock Tools (ADT) with Gasteiger charges, non‐polar hydrogens, and assignment of the torsion degrees of freedom. Docking required the structures to be prepared in PDBQT format, which were saved in this format [27].

2.3.3. Protein Preparation

HIV‐1 reverse transcriptase structure in complex with small molecule inhibitors (PDB ID: 1RT1) was downloaded from the RCSB Protein Data Bank (https://www.rcsb.org/). The structure was selected for its good resolution (2.55 Å) and R‐value (0.197) [26].

The stereochemical quality of the protein structure was analyzed with the PROCHECK server (https://www.ebi.ac.uk/thornton‐srv/software/PROCHECK/) by using Ramachandran plot analysis. In the docking studies, the protein was prepared by removing water molecules and any heteroatoms, adding polar hydrogen atoms, and adding Kollman charges for the protein using AutoDock Tools v1.5.6, and the structure was saved as a PDBQT file [28].

2.3.4. Blind Docking Protocol

Blind docking permitted the full evaluation of all protein surface ligand binding sites without any preconceived idea of the active site. Therefore, a grid box was designed to cover the protein structure that enables the ligand to explore the receptor surface. The geometric center was used to determine the center of the grid, and the grid size was set to 80 ų × 80 ų × 80 ų to ensure a wide coverage of the protein, while providing the flexibility to explore the protein's conformation and the flexibility of the ligand. This approach is particularly well suited for the detection of canonical and possible allosteric binding sites and to reduce site‐selection bias (Table 1) [29].

TABLE 1.

Docking grid parameters for molecular docking simulations (Vina configuration).

Receptor file	Centre X	Centre Y	Centre Z
1RT1.pdbqt	−1.875054	−34.514098	24.359913

2.3.5. Docking Execution

AutoDock Vina v1.2.0 was used for docking through the command prompt, using the docked configuration file. The output consisted of the binding affinity values (in kcal/mol) and several binding poses for each ligand. For each compound, one of the lowest binding energies (the best pose) was chosen for further analysis [30].

2.3.6. Interaction Analysis

The docked complexes were visualized and analyzed using discovery studio visualizer. The different types of key interactions, like hydrogen bonding, π–π stacking, π–alkyl, π—sigma, and hydrophobic interactions, were explored, and the amino acid residues involved in binding were identified. This docking procedure gave information on the binding orientation, binding affinity, and possible inhibitory interactions of the synthesized nucleoside analogues with HIV‐1 reverse transcriptase, which could be helpful for further biological evaluation [31].

2.3.7. Docking Validation

Docking validation involved re‐docking the co‐crystal ligand into the 1RT1 binding site using the same parameters. The root‐mean‐square deviation (RMSD) between the re‐docked and experimental ligand conformation was found to be lower, indicating the accuracy of the protocol in reproducing the natural binding mode. Protein‐ligand interaction profiles were analyzed with discovery studio visualizer 2021, and the best‐scoring protein‐ligand poses were shown in PyMOL [32].

2.3.8. ADME Prediction

The synthesized compounds (S1‐S6) were predicted using ADMETLab 3.0 (https://admetlab3.scbdd.com/). The important pharmacokinetic properties, such as the lipidophilicity (LogP), aqueous solubility (LogS), human intestinal absorption (HIA), volume of distribution (VD), CYP1A2 inhibition, clearance (CL), and half‐life (T₁/₂), were calculated. Additionally, properties that are described by Lipinski's Rule of Five (RO5), such as molecular weight, hydrogen bond donors (HBD), hydrogen bond acceptors (HBA), and LogP, were analyzed to assess the drug‐likeness and potential for oral bioavailability [28].

2.4. Anti‐HIV Activity

Anti‐HIV activity of the synthesized compounds was evaluated by an HIV‐1 reverse transcriptase (RT) colorimetric assay kit and compared with the standard drug Nevirapine. The assay was conducted following the manufacturer's guidelines (Sigma‐Aldrich). HIV‐1 reverse transcriptase catalyses the synthesis of DNA using a hybrid poly(A)/oligo(dT) template‐primer. The amount of DNA synthesized was determined by a sandwich ELISA‐based colorimetric detection method that reflects RT enzymatic activity [33]. The concentration of test compounds ranged from 3.13 to 100 µg/mL, while the reference drug Nevirapine was tested between 12.5 and 400 ng/mL. The percentage inhibition of the RT activity was determined at such concentrations, and the IC₅₀ values were calculated using the GraphPad Prism software by nonlinear regression. The reference standard used was an approved non‐nucleoside reverse transcriptase inhibitor (NNRTI), Nevirapine, in the appropriate assay conditions.

2.5. Statistical Analysis

HIV‐1 reverse transcriptase inhibition assays were conducted three times (n = 3). The results are presented as mean ± standard deviation (SD), and the IC₅₀ values were determined from the dose–response curve by nonlinear regression.

3. Result

3.1. Synthesis Results

The target 1,3‐oxazolidine nucleoside analogues (S1‐S6) were synthesized using a sequence of well‐defined protection, reduction, and glycosylation processes, beginning with L‐serine methyl ester as the chiral precursor. The oxazolidinone ring formed the main heterocyclic framework, and the subsequent lithium triethylborohydride reduction successfully transformed the carbonyl functionalities into the appropriate alcohol, demonstrating the strong stereoselective hydride transfer capabilities. Stepwise tritylation and acetylation allowed for selective hydroxyl group protection, resulting in site‐specific functionalization prior to glycosylation. The Vorbrüggen condensation of the acyloxymethyl oxazolidine intermediate with silylated pyrimidine bases in the presence of Lewis acids (SnCl₄ or TMSOTf) produced the desired nucleoside analogues, confirming the feasibility of the oxazolidine sugar scaffold in nucleoside synthesis. The final acid‐catalyzed deprotection produced pure nucleoside analogues in a satisfactory overall yield. Mechanistically, each step was highly selective, keeping the configuration at the C4‐position and confirming the synthetic value of oxazolidine intermediates as stable sugar mimics for creating modified nucleosides. The synthesized compounds were characterized using FTIR, ¹H NMR, and CHN elemental analysis to confirm their structural integrity and purity. The detailed spectral and analytical data for compounds S1–S6 have been reported in our previously published article [25].

3.2. In Silico Studies

3.2.1. Protein Validation

The structure of HIV‐1 Reverse Transcriptase (PDB ID: 1RT1) was analyzed on the stereochemical properties by Ramachandran plot analysis. Out of 828 non‐Glycine, non‐Proline residues, 723 residues (87.3%) were in the most favored regions (A, B, L), indicating optimal backbone conformations. The number of additional residues in the allowed regions (a, b, l, p) is 96 (11.6%), and 8 (1.0%) in the generously allowed regions. Notably, there was a single residue (0.1%) outside of the allowed region, with relatively small deviation from the ideal angles of torsion.

The total number of residues in the protein was 954, of which 53 were glycine and 67 were proline; six end residues were not included in the main analysis. The results are indicative of a well‐refined and structurally sound protein model appropriate for downstream molecular docking and computational studies (Figure 2, Table S1 ).

FIGURE 2.

Ramachandran plot of HIV‐1 Reverse Transcriptase (PDB ID: 1RT1) showing the distribution of φ (phi) and ψ (psi) dihedral angles of amino acid residues.

3.2.2. Docking Analysis

The molecular docking studies were carried out using the crystal structure of HIV‐1 reverse transcriptase (PDB ID: 1RT1) to find out the binding mode of the synthesized compounds (S1–S6) in the NNRTI binding pocket of the HIV‐1 reverse transcriptase. The docking scores and interaction profiles were compared with those of the reference ligand (Nevirapine) and the co‐crystallized ligand (Table 2, Figure 3).

TABLE 2.

Molecular docking scores and interaction profiles of synthesized compounds (S1‐S6), reference, and co‐crystal with HIV‐1 reverse transcriptase (PDB ID: 1RT1).

Compound code	Docking score	Number of hydrogen bonds
S1	−6.8	5
S2	−7.1	2
S3	−6.9	2
S4	−7.6	5
S5	−7.3	2
S6	−6.7	1
Reference	−7.2	2
Co‐crystal Ligand	−7.0	2

FIGURE 3.

2D interaction of compound S4, S5, reference, and co‐crystal with (1RT1) protein.

The best docking scores of S4 with −7.6 kcal/mol, S5 with −7.3 kcal/mol, and S2 with −7.1 kcal/mol among the synthesized compounds were obtained. The docking scores of the reference compound and co‐crystallized ligand were −7.2 and −7.0 kcal/mol, respectively, while the docking scores of S1, S3, and S6 were comparatively low, ranging from −6.8 to −6.9 kcal/mol.

The most biologically active compound (S4, IC₅₀ = 19.27 µM) formed five hydrogen bonds in the active site, as revealed by detailed interaction analysis. In particular, the carbonyl oxygen of the pyrimidine ring engaged in a pair of conventional hydrogen bonds with Gln91 (2.65 Å) and Arg143 (2.32 Å), and the oxazolidine oxygen formed another hydrogen bond with Gly141 (3.04 Å). The complex was further stabilized through additional carbon‐hydrogen bonds with Pro52 (3.58 Å), Val90 (3.51 Å), and π‐anion and π‐alkyl interactions with Glu89 and Pro140, respectively. S5, in contrast, made two typical hydrogen bonds with the oxazolidine O and Arg143 (3.03 Å), and with the pyrimidine carbonyl O and Gly141 (2.97 Å), as well as a π‐anion interaction with Glu89 (3.39 Å). The co‐crystallized ligand also hydrogen‐bonded to Trp535 (2.69 Å) and Leu533 (2.23 Å); the reference ligand hydrogen‐bonded to Leu92 and Gly93, and formed hydrophobic interactions with several other residues.

The compounds S2 and S1 exhibited good docking properties, but did not produce measurable HIV‐RT inhibition in vitro. Specifically, the docking score of S2 was −7.1 kcal/mol, which is relatively high, while that of S1 was −6.8 kcal/mol, and it was not biologically active because it formed five hydrogen bonds. The above observations show that the binding affinity from docking alone is not always correlated with the experimental inhibitory activity. The absence of activity can be caused by different orientations of the ligand in the binding pocket, insufficient binding to important hydrophobic regions, poor binding kinetics, or physicochemical properties not considered by docking calculations. Hence, molecular docking can be used as a qualitative tool that can be used to predict possible binding modes, but it's not a direct measure of the biological potency.

The overall results of the docking are partially in agreement with the experimental HIV‐RT inhibition assay. S4 and S5 showed good binding interactions and good enzyme inhibition, which made them good lead compounds for further optimization. The 2D interactions of all the docked ligands are in supplementary file Figures S1‐S8.

3.2.3. Docking Validation

The superimposition of the re‐docked pose and the experimentally resolved ligand conformation gave a root mean square deviation (RMSD) of 1.62 Å. RMSD values < 2.0 Å indicate that the docked and native binding modes have a high similarity, which confirms the accuracy and reliability of the docking approach. The validated protocol was deemed to be appropriate for the next step of docking the synthesized compounds (Figure 4).

FIGURE 4.

Docking validation of the target protein showed an RMSD of 1.62 Å between the re‐docked and co‐crystallized ligand.

3.2.4. ADMET Studies

The synthesized compounds (S1‐S6) were predicted to have ADMET properties to determine their drug‐likeness and pharmacokinetic behavior (Table 3). All the compounds follow Lipinski's Rule of Five, and their molecular weights range from 213 to 290 Da; the number of hydrogen bond acceptors is seven, the number of hydrogen bond donors is three, and LogP is between −1.074 and −0.735, which shows good drug‐like properties. The predicted LogS values (−0.442 to −1.810) indicated moderate aqueous solubility, and the relatively low CaCO2 permeability values indicated poor passive membrane diffusion, which is expected to account for the polar nature of nucleoside analogues. The human intestinal absorption predicted is different for the series and is relatively higher for S3 and S5.

TABLE 3.

Lipinski's rule of 5 and ADME properties of synthesized compounds.

	Lipinski's rule of 5				Absorption			Distribution	Metabolism	Elimination
Comp Code	MW	HBA	HBD	LogP	CaCO2 Per	LogS	HIA	VD	CYP1A2 inhibitor	CL	T1/2
S1	213	7	3	−1.074	−5.638	−0.442	0.056	−0.227	0.000118	4.77	1.721
S2	227	7	3	−0.823	−5.394	−0.751	0.005	−0.226	1.16E‐05	7.82	1.20
S3	245	7	3	−0.735	−5.612	−1.198	0.229	−0.207	1.10E‐06	8.31	1.22
S4	231	7	3	−0.894	−5.411	−0.541	0.057	−0.258	1.10E‐05	7.81	1.41
S5	290	7	3	−0.777	−5.433	−1.810	0.204	−0.179	0.000119	5.71	1.74
S6	247	7	3	−0.828	−5.460	−1.301	0.059	−0.159	8.09E‐05	7.21	1.22

Compounds exhibited low values for predicted VoD (VOD) from −0.258 to −0.159, indicating a limited tissue distribution. The CYP1A2 inhibition was not predicted for any analogue, suggesting low risk of inhibition of CYP‐mediated drug–drug interactions. The clearance ranged between 4.77 and 8.31, with an elimination half‐life of 1.20–1.74 h, indicating rapid clearance. The physicochemical and pharmacokinetic properties of the synthesized compounds were satisfactory and consistent with drug‐like characteristics, warranting additional biological evaluation.

3.3. In Vitro Anti‐HIV Activity Evaluation

Anti‐HIV activity of the synthesized 1,3‐oxazolidine nucleoside analogues (S1‐S6) was evaluated by a colorimetric HIV Reverse Transcriptase (HIVRT) test kit (Sigma‐Aldrich) following the manufacturer's standard protocol. Nevirapine, a standard non‐nucleoside reverse transcriptase inhibitor (NNRTI), had an IC₅₀ value of 112.2 nM, which indicated strong inhibition. Each compound was analyzed at six different concentrations (3.13, 6.12, 12.5, 25, 50, and 100 µg/mL). Among all the chemicals tested, S4 (R = F) and S5 (R = Br) exhibited the highest HIV‐1 RT inhibitory effect, with the activity being dose‐dependent. The compound with the highest inhibitory activity was compound S4 with 88.16% inhibition at 100 µg/mL, while the compound with the lowest inhibitory activity was compound S5 with 71.25% inhibition at 100 µg/mL. The calculated IC₅₀ values for S4 and S5 were found to be 19.27 and 22.80 µM, respectively, which were significantly lower than that of nevirapine, the reference drug (100 µM). Substances S1, S2, S3, and S6, on the other hand, exhibited only weak or insignificant inhibition, and the maximum inhibition values were below 12%, even at the highest tested concentration (100 µg/mL). The analogues of these compounds were considered inactive or poor inhibitors in the conditions studied because the values of IC₅₀ could not be determined because of low inhibitory response (Table 4). The compounds S4 and S5 showed a dose‐response inhibition of HIV‐1 reverse transcriptase activity. S4 and S5 were the two synthesized compounds with moderate HIV‐1 reverse transcriptase inhibitory activity, with IC₅₀ values of 19.27 and 22.80 µM, respectively. Both compounds were significantly less potent than the reference compound, Nevirapine, with an IC₅₀ of 112.2 nM. The dose‐response curve has been provided in Figures S9‐S11.

TABLE 4.

Anti‐HIV activity of synthesized compounds (% inhibition and IC₅₀ values).

Compounds	R	%Inhibition/Concentration (µg/mL)						IC50 (µM)
		3.13	6.12	12.5	25	50	100
S1	H	1.50	4.30	5.87	6.52	8.52	11.25	NA
S2	CH3	1.00	1.02	1.65	1.89	3.16	4.03	NA
S3	SH	0.51	0.55	0.65	0.71	0.85	1.16	NA
S4	F	16.32	26.24	43.65	58.65	77.95	88.16	19.27
S5	Br	10.65	21.68	32.57	44.68	59.65	71.25	22.80
S6	Cl	0.72	0.88	0.89	0.98	1.11	1.31	NA
Nevirapine								112.2(nM)

4. Discussion

The synthesized 1,3‐oxazolidine derivatives (S1–S6) were docked with HIV‐1 reverse transcriptase (RT), and their in vitro inhibitory activity against HIV‐1 RT was evaluated. All synthesized compounds were structurally characterized using spectroscopic techniques. The docking studies done on the crystal structure of HIV‐1 RT (PDB: 1RT1) showed moderate docking scores ranging from −6.7 to −7.6 kcal/mol, which were almost similar to the reference compound Nevirapine (−7.2 kcal/mol) and the co‐crystallized ligand (−7.0 kcal/mol). The synthesized compounds were analyzed for their binding affinity with the target protein, and compound S4 was predicted to have the highest docking score of −7.6 kcal/mol, followed by S5 with a score of −7.3 kcal/mol, while slightly lower docking scores were predicted for S1, S2, S3, and S6.

The in vitro HIV‐1 RT inhibition assay showed that only S4 and S5 showed measurable enzyme inhibitory activity with IC₅₀ values of 19.27 and 22.80 µM, respectively. Both compounds were, however, significantly less active than the reference inhibitor, Nevirapine (IC₅₀ = 112.2 nM). However, compounds S1, S2, S3, and S6 exhibited little inhibition over the range of concentrations tested despite having moderate docking scores. These results show that the docking methods don't directly relate to the experimental inhibitory activity. For instance, S2 has a favorable docking score; however, it is inactive in the enzyme assay, whereas multiple hydrogen bonds were formed with S1, but it has very little inhibition. These discrepancies imply that the biological activity is not only determined by binding affinity, but also by other factors, such as ligand orientations, dynamics of interactions, and physicochemical properties.

Based on structure–activity relationship (SAR) analysis, halogen substitution affected the HIV‐1 RT inhibition of the present series. The substituted compounds S4 and S5, with fluoro and bromo, respectively, showed greater inhibitory activity than the analogues that lack them or have an electron‐donating group. The same behavior was seen in our previous work on 1,3‐thiazolidine pyrimidine derivatives, which showed that the halogenated compounds were also comparatively more active inhibitors of HIV‐RT [17]. This observation indicates that halogen substitution might be more beneficial for interactions in the HIV‐1 RT binding pocket, but further analogues and mechanistic studies are needed to define a structure–activity relationship.

The comparison of the present oxazolidine derivatives with the previously reported thiazolidine analogues indicates that the replacement of sulfur by oxygen in the heterocyclic ring may have an effect on binding interactions and biological activity. However, the differences between the two series are relatively small, and additional research is needed to determine whether one of the scaffolds is better than the other.

Overall, the computational and enzymatic data suggest that S4 and S5 are worth further consideration as potential early hit compounds for HIV‐1 reverse transcriptase inhibitors, but not as antiviral candidates. These compounds showed activity that could be measured as a degree of enzyme inhibition, but activity was still far less than for Nevirapine. Thus, there is a need for further optimization of the structure, cytotoxicity assays, cell‐based antiviral assays, and detailed pharmacological studies in order to validate their therapeutic potential and to explore the possibility of developing these compounds into more potent HIV‐1 RT inhibitors.

5. Conclusion

The anti‐HIV activities of 1,3‐oxazolidine nucleoside analogues were significant, with moderate anti‐HIV activities being observed for the fluoro (S4) and bromo (S5) derivatives, respectively, when compared to the reference drug. These results were validated by molecular docking, displaying that the interactions in the active site of HIV‐1 reverse transcriptase were enhanced, underscoring the beneficial impact of the halogen substitution. The oxazolidine scaffold was found to be a more potent inhibitor compared to thiazolidines, highlighting its attractive antiviral lead. Optimization by molecular dynamics studies and structure‐directed modifications is required to improve potency and selectivity.

6. Limitation and Future Prospective

The number of oxazolidine nucleoside analogues synthesized in the current study is limited, and their structures are not diverse enough to provide for a detailed study of structure–activity relationships. Biological evaluation mainly included an HIV‐1 reverse transcriptase enzyme inhibition assay, which produced a moderate inhibitory effect against the reference drug, but no validation was carried out in cell‐based antiviral or cytotoxicity models. The drug‐likeness and ADME properties were studied solely by in silico prediction, without any experimental pharmacokinetic studies. Moreover, the information from molecular docking was scarce and did not allow for mechanistic interpretation. Future work should be focused on expanding the chemical library through structural modification, followed by molecular dynamics simulation to confirm stability and interaction. In addition, extensive in vitro cell‐based antiviral assays, cytotoxicity testing, and experimental ADME studies should be performed to assess the pharmacological relevance and to optimize leads.

Authors Contributions

Shimoga Nagaraj Sriharsha: conceptualization, investigation, supervision, and project administration. Zaid Mahmood Mohamed: data curation and software. Ammar A. Razzak Mahmood: formal analysis and software. Sibghatullah Muhammad Ali Sangi and Sadik Shaik: validation and visualization. Sreeharsha Nagaraja and Sheeba Kumari: data curation and review. Mahalakshmi Suresha Biradar, Vidhya Thirunavukkarasu: writing original draft and supervision. Pavan Kumar Sreenivasalu, Monirul Islam, and Muhammad Shahzad Chohan: writing, review, and software.

Funding

This work was supported by the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia [Grant No. KFU264639].

Conflicts of Interest

The authors declare no conflicts of interest.

Animal, Human, or Tissue Experiments

The studies do not contain experiments using animals, humans, or tissue.

Supporting information

Supporting File 1: cbdv71638‐sup‐0001‐SuppMat.docx

Data Availability Statement

Data are provided in the supplementary file.