Derivatives of uracil, which is a pyrimidine nucleobase that occurs as a component of ribonucleic acid (RNA), have been a cornerstone of medicinal chemistry for decades. The  derivatives are fantastic scaffolds for optimizing a biological activity. They are capable of various tautomeric changes, extensive coordination chemistry, and targeted functionalization across almost any part of the heterocyclic ring  [1]. Thus, the uracil scaffold turns into an ideal foundation for novel drugs, which can be engineered with a thorough understanding of the correlation between the molecular structure and the biological activity at the molecular level.

Uracil and uracil derivatives stand out among numerous pyrimidine nucleic acids due to their core role in biochemical processes and a wide range of pharmacological activities. They now exhibit antimicrobial, acaricidal, analgesic, pronounced anti-inflammatory and anticancer properties  [2–7].

Both natural pyrimidines and their synthetic analogues are widely used for therapeutic purposes (fig. 1). The era of anticancer and antiviral chemotherapy was established when halogenated uracil derivatives were introduced in clinical practice. The derivatives included idoxuridine (1), the first topical antiviral drug for herpetic keratitis) and cytarabine (2), which remains a core medication in acute leukemia treatments and works by inhibiting DNA polymerase.

Invention of pyrimidine antimetabolites, primarily fluorinated derivatives, was essential for the history of antitumor therapy. Fluorouracil (3) and its prodrug floxuridine (4) are the most heavily researched and clinically standardized drugs that belong to this class. They are toxic to cells because they block thymidylate synthase and are subsequently incorporated into nucleic acids (fig. 1). Success of these drugs in oncology results in continuous search of novel, improved and selective analogues that can overcome resistance to drugs and possess an improved safety profile  [8].

It should also be noted that the pyrimidine scaffold can act both as a pharmacophore, and as a vehicle that transports a biologically active fragment to specific targets. Specifically, incorporation of uracil as a recognition site resulted in the development of potent inhibitors for human carbonic anhydrase (hCA). It shows that precise design ensures high affinity and selectivity to certain isoforms of the enzyme  [9].

When exploring novel biologically active compounds, the effect of physicochemical properties and subsequent pharmacological effects of uracil derivatives on their potential for tautomeric and conformational transformations requires particular focus. Uracil exists in multiple tautomeric states: three lactam-lactim variations (U1–3) with different exocyclic hydroxyl group positions and a dienolic variation (U4). Furthermore, rotation of the hydroxyl group around the single C–O bond  [10] creates various rotamers (U5–6) (fig. 2).

 Diketo (U1) and keto-enol (U2, U3) tautomers have been successfully identified using dispersed fluorescence spectroscopy in supersonic jets  [11]. Experts have long argued over the precise configuration of the latter.

Previous calorimetric experiments and theoretical assessments have shown that diketo tautomer U1 is the predominant structure in the gas phase and low-polarity environments. Compared to tautomer U1, dienolic tautomer U4 is less stable by 20 ± 10 kcal/mol, whereas keto-enolic tautomer U3 is less stable by 19 ± 6 kcal/mol  [12].

Earlier quantum chemical modeling, utilizing density functional theory (B97-D3/aug-cc-pVDZ) within the Polarizable Continuum Model (PCM) across various media with different polarity values (1 < ε < 109) demonstrated that neither solvation dynamics nor the attachment of potent donor-acceptor groups (such as nitro and amino substituents) at position С(5) fundamentally shift the tautomeric preferences of the unsubstituted uracil ring  [10]. However, certain interactions within the molecule can substantially alter tautomeric stability.

Therefore, in the 5-nitro derivative, a strong NO⋅⋅⋅HO intramolecular hydrogen bond stabilizes the dienol tautomer, reducing its relative energy from 17.9 kcal/mol (in the absence of such a bond, see Figure 2 to 5.4 kcal/mol relative to the most stable diketo-tautomer.

Earlier, as part of a systematic study of organosilicon compounds, we proposed an approach for synthesizing N-sulfoalkylated uracil derivatives via the reaction of 1,3-propanesultone with N-silylated uracil analogs (fig. 4) [13]. This approach has provided an efficient route to zwitterionic compounds 5 and 6. These compounds contain a sulfopropyl group, which can adjust the physical, chemical, and pharmacokinetic traits of the molecule such as its solubility and capacity to for intermolecular interactions (fig. 3).

This paper continues our prior investigation into discovering original central nervous system (CNS) drugs with proven effectiveness. Our focus on CNS conditions (ischemic stroke, Alzheimer disease, Parkinson disease, and anxiety-depressive disorder) is driven by urgent medical needs. It is also supported by our experimental data showing that synthesized sulfoalkylated heterocycles can exhibit neuroprotective and nootropic effects  [14–20].

As pharmacological responses to the administered substance are determined by the complex chemical arrangement of a molecule, including its gross formula, bond sequences, spatial arrangement and 3D folding of the molecule, establishing the correlation between the spatial structure and biological activity of the compound is the cornerstone of medical chemistry.

Thus, the goal of this research was to conduct an in-depth structural analysis (including tautomerism and conformational profiles) of two groups in the condensed phase: previously synthesized N-sulfopropylated uracil derivatives (7–13) and novel 5- and 6-amino uracil derivatives (14–16) that show potential for medical applications. Furthermore, we performed an in silico evaluation of the potential biological activity of compounds 7–16 and their tautomeric forms. The obtained data will be helpful in choosing and subsequent examination of the potential leaders.

MATERIALS AND METHODS

fig. 1 and fig. 5 provide the data used by OpenBabel to model the three-dimensional structures of the tautomers [21]. Subsequently, the CREST 3.0.2 program [22] and the GFN2-xtb method were used to perform a conformational search, identifying the most optimal arrangement of atoms with the total energy. Further, for the obtained conformer structures, geometry was optimized and thermodynamic parameters were calculated at 298 K using the ωB97X-3c composite method, which provides a sufficiently high level of results with moderate computational costs. It is also integrated into the Orca 6.1.0 program [23].

This method calculates total energy using the ωB97X meta-GGA functional, which incorporates range-separation  [24]. The vDZP (double-zeta) basis set modeled valence electrons, core electrons were treated using a pseudopotential, and dispersion interactions were captured via the D4 correction [25]. The CPCM (conductor-like polarizable continuum) model was used to describe solvent effects.

The biological activity profile of the tested compounds was evaluated using the 2024 version of the PASS (Prediction of Activity Spectra for Substances) program [26]. The predicted biological activity profiles of the analyzed compounds were evaluated using PharmaExpert software, version 2024 [27]. PASS 2024 predicts 9,274 activities with the average forecast accuracy of the AUC, computed using cross-validation, with a single peak value of 0.929. The spectrum of anticipated biological activity includes 575 pharmacological effects, 5,422 mechanisms of action, 160 activities relate to anti-target actions, and 226 terms detailing how these compounds interact with metabolic enzymes. The prediction was generated using a probability threshold Pa greater than 0.3.

RESEARCH RESULTS

36 possible tautomeric structures were selected for compounds 7–16 (fig. 5), detailed below, to evaluate their stability and define the Quantitative Structure–Activity Relationship (QSAR). The results of the comprehensive study are presented below.

Quantum chemical research

tab. 1 summarizes the calculated values of total energy, thermodynamic properties for investigated compounds 7–16, their tautomers, and tautomeric forms of uracil U1–6 (fig. 1, fig. 5). As shown in tab. 1, tautomer a with two keto groups demonstrates the highest stability across all examined cases, including that of uracil. It is followed by less stable tautomer b, featuring a hydroxyl at position 4 and a keto group at position 6. The current results align with prior research results regarding the tautomeric forms of uracil  [10].

Study of biological activity in silico

In silico (QSAR) data show that the studied molecules can produce several pharmacological effects. tab. 2 provides some of the most representative data regarding the predictions for diketo tautomer a.

1) pharmacological effects and related mechanisms of action (with indented sub-points showing the link between the effect and the mechanism) with a threshold Pa>0.3 (effects column);

2) molecular mechanisms of action where Pa > 0.5 (mechanism column);

3) prediction of terms related to the interaction of the compounds with metabolic enzymes using a probability threshold of Pa>0.5 (column metabolism); prediction of terms related to the effect of the compound anti-target with a threshold of Pa>0.3 (anti-target column).

Let us evaluate the predicted effects in decreasing order of likelihood, prioritizing those for which mechanisms of action are established. This combination makes experimental proof of the predicted effect much more probable.

The antiviral activity (Antiviral) is most likely to be predicted, including against HIV Antiviral (HIV). Additionally, the predicted mechanism of action (DNA directed DNA polymerase inhibitor) is highly likely to be experimentally proven, though slightly less certain than the overall effect itself.

The predicted antiviral and DNA-directed DNA polymerase inhibitory effects are most evident in the tautomers of compound 8. All of the calculated tautomers show a high probability of both antiviral activity (Pa>0.75) and DNA-directed DNA polymerase inhibition (Pa>0.45). The combination is predicted with the least probability for tautomers within a series 14.

In most cases, tautomers a and c are more likely to be present than tautomer b. The probability of DNA-directed DNA polymerase inhibition for series 10 does not meet the designated prediction threshold of Pa>0.3. Despite the high probability of antiviral activity in compounds 12b, c,  the probability of inhibition of DNA-directed DNA polymerase is below the minimum prediction threshold of Pa>0.3. A number of tautomers of compound 15 are characterized by a high probability of antiviral activity (Pa>0.6). The effect is more pronounced for tautomers a (Pa=0.733), and tautomers a and c (Pa=0.550 and Pa=0.617, respectively) in case of DNA-directed DNA polymerase. It should be noted that this particular pharmacological activity was deemed unlikely for tautomer 9b (Pa > 0.3). In series 16, tautomer d exhibits the lowest likelihood of both antiviral activity (Pa=0.547) and DNA-directed DNA polymerase inhibition (Pa=0.398). Antibacterial activity (Antibacterial) may also become a promising area of biological research. This is especially true for the management of viral ophthalmic conditions (Antibacterial, ophthalmic), as well as bacteria that cause tuberculosis (Antituberculous). Cell wall synthesis inhibitor and Hyaluronate lyase inhibitor are predicted to be mechanisms of action associated with these effects. Both effects and mechanisms are predicted to occur with similar likelihood across all data series, with an average probability of effects in the Pa range from 0.4 to 0.6, and mechanisms in the Pa range from 0.3 to 0.4.

Also, many compounds are predicted to exhibit anti-ischemic properties (Antiischemic), including as part of myocardial ischemia therapies (Myocardial ischemia treatment). Heparin-like activity (Heparin and LMW heparins) is predicted to be the mechanism of action. Tautomers of compounds 7, 9 and 14 most frequently exhibit predicted anti-ischemic effects on the myocardium in the Pa range from 0.3 to 0.4. The overall anti-ischemic effect for the majority of the evaluated compounds is predicted to occur within a Pa range of 0.3 to 0.5. Most of the tautomers are also predicted to exhibit a heparin-like activity, with a Pa score ranging from 0.3 to 0.5, the only exceptions being the tautomers of compounds 15, 16.

Some other predicted effects deserve particular attention as well:
1. Antitumor activity against different types of tumors and activity associated with an enhanced antitumor effect (Antineoplastic enhancer) (9b, c, 10a, c, 11c).
2. Treatment of atherosclerosis (Atherosclerosis treatment) together with inhibition of sodium/bile acid cotransporter inhibitor (Sodium/bile acid cotransporter inhibitor) — 7b, 8с.
3. Antiulcer effect (Antiulcerative) together with an antacid and cytoprotective action (Cytoprotectant) for 7a, c.

Tautomer 15b is highly promising in predicting the average probability of treatment for neurodegenerative diseases, especially Alzheimer’s disease with predicted mechanisms of action such as inhibition of beta-amyloid aggregation and predicted antimyelogenic effect.

It seems important that no inhibition of the main metabolic enzymes is predicted for compounds 7–16. For some compounds, CYP 2B6 (7a, 8a, 11a, 14a) and CYP 2C19 (7a, 8a, 10a, 11a, 13a, 14a, 15a, d) cytochromes are predicted to be induced. Based on the calculations, tautomers 7b, c can serve as substrates for GST A1–1, while tautomers 9b, c may act as substrates for GST A, with Pa>0,5.

For several tautomeric forms of the investigational compounds (specifically 7a, 9a-c, and 15d), computational models predict the inhibition of phosphofructokinase-1 (Phosphofructokinase-1 inhibitor) with a probability between 0.3 and 0.4 Pa, which suggests an impact on anti-targets. This type of interaction can induce a toxic effect on the blood (Hematotoxic) and hemolytic anemia (Anemia, hemolytic). These conditions, combined with phosphofructokinase-1 activity, are predicted to occur within a range of Pa 0.3 to Pa 0.6 (for compounds 9a, b, 13d, 16a, d). The tautomeric variations b and c of compound 9 differ in how they affect beta DNA polymerase activity. As a result, side effects like peripheral neuropathy and pancreatitis can develop.

Once these interactions are experimentally confirmed, the associated compounds or tautomers can be ruled out from further investigation.

DISCUSSION OF RESULTS

A comprehensive study, consisting of quantum chemical analysis and in silico biological profiling of various derivatives of N-sulfopropylated uracils (7–16) and their tautomers, identified key structural trends. These characteristics are vital for understanding the structural dynamics of these compounds and designing novel targeted therapeutics, particularly for central nervous system (CNS) diseases.

Tautomeric preferences in the condensed phase.

Quantum chemical calculations reveal pronounced dominance of diketo tautomer а for all tested compounds, as well as for unsubstituted uracil. When the relative Gibbs energy is set to 0 for tautomer а, and changes within the broad range of 36 to 122 kJ/mol for forms b and c, depending on the nature of a substituent (tab. 1).

Tautomers b were closest in energy to the diketo form (e. g., with ΔΔG setting to 38.0 kJ/mol for 7b and 36.0 kJ/mol for 14b), whereas the dienolic forms d and e were substantially less stable, with ΔΔG reaching 125 kJ/mol. These findings align with the established ideas that lactam forms of pyrimidines dominate in polar media. Furthermore, they demonstrate that attaching a sulfopropyl group at either the N1 position (for compounds 7–13) or the N3 position (for compounds 14–16) does not alter the tautomeric preferences of the pyrimidine basic structure. Furthermore, the experimental ΔΔG values for uracil (60.3 kJ/mol for U1 vs U2 and 46.2 kJ/mol for U1 vs U3) agree well with existing literature  [10], with a difference of ~11–19 kcal/mol) ≈ 46–79 kJ/mol). Under normal physiological conditions (an aqueous environment at pH 7.4), compounds 7–16 will almost entirely exist as diketo form a. Consequently, this specific structure should serve as the primary model for interpreting both biological test results and molecular simulations.

The effect of substituents on the stability of tautomers.

Although the overall stability order of tautomers (a > b > c) applies to all compounds, the relative energy values differ significantly (tab. 1). Compound 14 (ΔΔG = 36.0 kJ/mol) demonstrates the most significant stabilization of keto-enol form b compared to the diketo form. As a 6-amino derivative, its position-6 amino group can establish an intramolecular hydrogen bond with the C4 carbonyl group within the enol form. In contrast, the difference is more significant for the 5-amino (15) and 5,6-diamine (16) derivatives, measuring 42.8 and 91.5 kJ/mol respectively, which can be attributed to electronic and steric effects. For the halogen derivatives (10 — Br, 11 — Cl), form b exhibits lower stability compared to unsubstituted uracil (ΔΔG is ~45 kJ/mol vs. 38 kJ/mol for 7b). It demonstrates that electron-withdrawing substituents exert a destabilizing effect on the keto-enol tautomer. Compound 9 (5-nitro derivative) is given special attention as the difference between 9a and 9b is 38.5 kJ/mol, which is comparable with unsubstituted uracil.

Previous studies highlight that an intramolecular NO···HO hydrogen bond significantly stabilizes the dienolic tautomer of 5-nitrouracil decreasing the difference in energy values up to ~22 kJ/mol  [10]. Our calculations show that the dienolic form (C2-OH, C4-OH) of 9c yields a much higher ΔΔG value of 67.0 kJ/mol. The observed discrepancy may be due to the fact that  [10] evaluated the complete dienolic form with possible additional stabilization, whereas the investigated compounds contain only one enol fragment. Nevertheless, the data obtained show that even strong acceptor substituents do not change the global tautomeric equilibrium in a condensed medium.

Prediction of biological activity in silico: key effects and the role of tautomerism.

Based on the predicted activities in tab. 2, most tautomers consistently show a strong antiviral effect, with compound 12a achieving Pa value of up to 0.890. The combination of antiviral activity and DNA-directed DNA polymerase inhibition is predicted with a high probability for tautomers 8a, 10a, 11a, 12a, 13a, 14a (Pa > 0.55), and therefore requires special attention. This directly indicates a possible mechanism of action similar to known antimetabolites (cytarabine, idoxuridine), and allows to consider compounds of series 14 (5-methyluracil, thymine) and 13 (bromine derivative) as a promising basis for the development of new antiviral drugs, including those with an activity against HIV (Pa > 0.8 for 8a and 13a). It should be noted that this activity is significantly lower for amino derivatives 14–16 (Pa < 0.6). The reduction is likely caused by an altered electron density in the heterocycle and a decreased ability to form hydrogen bonds with the active polymerase center.

A comparison of different tautomeric forms shows that in the studied series, the most likely antiviral activity is in diketo form a and in some cases in form c (for example, Pa = 0.766 for 8c), whereas form b gives lower Pa values in all cases. This may indicate the importance of preserving two carbonyl groups for binding to the target. For compounds 14–16 (amino derivatives), tautomer a is also preferred, but its absolute Pa values are lower. Thus, when planning further research, one should focus on the diketo form, which is highly likely to actually exist in the aquatic environment.

In addition to antiviral activity, an antibacterial effect (specifically ophthalmic effect with Pa of up to 0.648), an anti-ischemic effect (with Pa of up to 0.564) and heparin-like activity are predicted. It is noteworthy that treatment of neurodegenerative diseases (Alzheimer’s disease) with a mechanism of inhibition of beta-amyloid aggregation (Pa = 0.442) and an anti-amyloidogenic effect (Pa = 0.349) was predicted for tautomer 15b. As our aim was to search for neuroprotectors, this compound requires a primary experimental study in models of Alzheimer’s disease. However, note that the relative energy values of keto-enol tautomer 15b are 42.8 kJ/mol higher as compared to the stable diketo form of 15a. Under normal physiological conditions, the equilibrium concentration of compound 15b is quite low (~ 10–7), which can limit its pharmacological efficacy. Perhaps the latter can be realized in the process of a reversible tautomeric transition, or the diketo form will exhibit activity, and the prediction for tautomer 15b reflects structural similarity with the active conformer. This issue requires additional investigation with the help of molecular docking methods.

Analysis of side effects and toxicity based on the prediction of interactions with anti-targets and metabolic enzymes.

Most compounds are not expected to inhibit the primary cytochrome P450 (CYP) enzymes. However, specific tautomers 7a, 8a, 10a, 11a, 13a, 14a, 15a, d are predicted to induce CYP2B6 and CYP2C19 activity (with Pa 0.51–0.59). This can lead to drug interactions when used concomitantly with substrates of these enzymes. In addition, a number of tautomers (9a, b, 13d, 16a, d) are predicted to have a hemotoxic effect (with Pa values of up to 0.43) and hemolytic anemia due to probable inhibition of phosphofructokinase-1. Tautomers 9b, c require particular focus, as they are additionally predicted to cause peripheral neuropathy (Pa = 0,431) and inhibit DNA polymerase β (Pa = 0.414). Compounds with the nitro group in position 5 (9) should be subjected to a thorough toxicological assessment. Likewise, tautomers 9b, c were predicted to carry a risk of pancreatitis (Pa ~0,48), aligning with the established toxicity of nitroaromatic compounds.

CONCLUSIONS

 Evaluation of our experimental data alongside worldwide advancements in medical chemistry of nitrogen-containing heterocycles have shown that uracil analogs, specifically those containing amino and sulfoalkyl groups, hold substantial, yet underexplored, potential for the development of novel therapeutic agents. The conducted study found a correlation between the tautomeric forms of N-sulfopropylated uracils and their expected biological activities. Dominance of diketo form a in condensed environments makes interpreting SAR data easier and allows researchers to use just one low-energy conformation for molecular modeling. PASS forecasts indicate a number of promising activities, specifically antiviral and anti-amyloidogenic ones, but require experimental confirmation.