1-Department of Pharmacy, Comsats University, Islamabad
2- School of Natural Sciences, Department of Chemistry, NUST
3- Lecturer, Department of Pharmacy, Abbottabad university Of Science & Technology (AUST), Abbottabad
4,5- SA – Centre for Interdisciplinary Research in Basic Science (SA-CIRBS), Faculty of Sciences, International Islamic University, Islamabad (IIUI).
6- Department of Zoology, Hazara University, Mansehra.
7- Azure & Modern Workplace Solutions Specialist at Microsoft
Leishmaniasis, a neglected tropical disease caused by Leishmania parasites, remains a significant global health challenge, necessitating the discovery of novel therapeutic agents. In this study, six Waleed pyrimidine (WP) derivatives, WP-1 (2-methyl-4-phenylpyrimidine-5-carboxylic acid) and WP-4 (2,4-dimethylpyrimidine-5-carboxylic acid) among them, were evaluated for their antileishmanial activity through in vitro, in vivo, molecular docking, pharmacokinetic, and toxicological assessments. The compounds exhibited significant inhibitory effects on the Leishmania DHFR enzyme, with WP-1 and WP-4 demonstrating the highest binding affinities and potent antileishmanial activity. Both compounds were further analyzed for their pharmacokinetic properties, revealing favourable profiles such as high gastrointestinal absorption, the ability to cross the blood-brain barrier (BBB), and optimal log P values. Toxicological evaluations using advanced in silico platforms indicated no significant safety concerns, making WP-1 and WP-4 viable candidates for therapeutic development. Furthermore, in vivo studies in murine models confirmed their effectiveness, with WP-4 emerging as the most promising compound based on lower IC50 values and improved survival rates. The structure-activity relationship (SAR) analysis revealed key structural features that enhance their efficacy, including the presence of methyl and phenyl substitutions. Overall, WP-1 and WP-4 demonstrate substantial promise as antileishmanial agents, with WP-4 standing out as the lead compound due to its superior performance across multiple evaluation parameters.
Leishmaniasis is a neglected tropical disease that poses a significant public health challenge, particularly in regions with limited access to healthcare and poor socioeconomic conditions. This parasitic disease is caused by protozoa of the genus Leishmania, which are transmitted to humans through the bite of infected female phlebotomine sandflies. With its diverse clinical manifestations, including cutaneous, mucocutaneous, and visceral forms, leishmaniasis affects millions of people worldwide, resulting in substantial morbidity and mortality annually (World Health Organization [WHO], 2022). The causative agents of leishmaniasis belong to a complex group of protozoan parasites, with more than 20 Leishmania species identified to date. These species demonstrate remarkable adaptability, allowing them to survive and replicate in both vector and mammalian hosts. Among these, Leishmania donovani and Leishmania infantum are primarily responsible for visceral leishmaniasis, the most severe form of the disease, whereas Leishmania major and Leishmania tropica typically cause cutaneous leishmaniasis (Alvar et al., 2012). Leishmaniasis manifests through a spectrum of symptoms, ranging from self-healing skin ulcers to systemic organ damage in visceral cases. Common symptoms of visceral leishmaniasis include prolonged fever, weight loss, hepatosplenomegaly, and anemia, which can be fatal if left untreated. Annually, it is estimated that leishmaniasis claims over 20,000 to 30,000 lives, primarily in endemic regions across Asia, Africa, and South America (Kaye & Scott, 2011). A pivotal aspect of Leishmania survival and pathogenicity lies in the functionality of critical enzymes that facilitate the parasite's metabolic processes. Among these, dihydrofolate reductase (DHFR) plays an essential role in the folate biosynthesis pathway, enabling the synthesis of nucleotides and amino acids required for parasite replication. Targeting DHFR offers a promising strategy for therapeutic interventions, as its inhibition can disrupt parasite survival while sparing host cells (Müller et al., 2013). The current treatment regimens for leishmaniasis predominantly involve antimonial compounds, amphotericin B, miltefosine, and paromomycin. While these therapies have demonstrated efficacy, they are associated with numerous limitations, including toxicity, high cost, prolonged treatment durations, and the emergence of drug resistance. Additionally, the intravenous or intramuscular administration of most drugs poses logistical challenges in resource-constrained settings (Sundar & Chakravarty, 2015). The quest for novel and more effective treatments has directed research toward heterocyclic compounds, such as pyrimidine derivatives, which exhibit significant therapeutic potential against leishmaniasis. Pyrimidines, a class of nitrogen-containing heterocycles, are structurally versatile molecules that play a vital role in medicinal chemistry due to their bioactive properties and ease of chemical modifications (Patel et al., 2017). Pyrimidine derivatives possess unique chemical properties, including aromaticity, hydrogen bonding capability, and electronic versatility, which enable their interaction with biological targets such as enzymes and nucleic acids. These properties have facilitated their application in the development of anti-parasitic agents, with enhanced efficacy and reduced adverse effects compared to traditional treatments (Goyal et al., 2019). The biological significance of pyrimidine derivatives lies in their ability to mimic natural substrates of critical enzymes, thereby acting as competitive inhibitors. In the context of leishmaniasis, pyrimidines have shown promising results as DHFR inhibitors, disrupting folate metabolism and impeding parasite proliferation. This mechanism positions pyrimidine derivatives as potential therapeutic agents for overcoming the limitations of existing treatments (Yadav et al., 2020). One of the key advantages of pyrimidine-based compounds is their ability to selectively target parasite-specific pathways while exhibiting minimal cytotoxicity to human cells. This selective action reduces the risk of adverse effects and enhances the safety profile of these compounds, making them suitable candidates for anti-leishmanial drug development (Roy et al., 2018). Pyrimidines offer several benefits over conventional therapies, including improved oral bioavailability, reduced treatment costs, and compatibility with combination regimens. These attributes address many of the challenges associated with current treatments, particularly in endemic regions where healthcare infrastructure is often inadequate (Singh et al., 2021). The potential of pyrimidine derivatives as therapeutic agents stems from their ability to interact with and inhibit DHFR, an enzyme indispensable for Leishmania survival. By binding to the active site of DHFR, pyrimidines disrupt the folate pathway, leading to nucleotide depletion and eventual parasite death. This targeted mechanism offers a promising avenue for the development of highly specific anti-leishmanial drugs (Bansal et al., 2019). The inhibition of DHFR by pyrimidine derivatives is a well-studied mechanism in the treatment of other parasitic diseases, such as malaria, and its application in leishmaniasis represents a logical extension of this research. The structural modifications of pyrimidines can be tailored to enhance their binding affinity and selectivity, further optimizing their therapeutic potential (Pandey et al., 2020). Despite their promise, the development of pyrimidine-based therapies for leishmaniasis is still in its early stages, with limited clinical data available. Continued research is essential to address challenges such as drug resistance, pharmacokinetics, and scalability of production. Collaboration between academia, industry, and healthcare institutions will be crucial in translating these findings into accessible treatments (Rai et al., 2022). The chemical synthesis of pyrimidine derivatives involves diverse methods, including condensation reactions and functional group modifications, allowing for the generation of structurally diverse compounds. This synthetic versatility facilitates the exploration of structure-activity relationships, enabling the identification of lead compounds with optimal anti-leishmanial activity (Kumar et al., 2016). Moreover, pyrimidine derivatives have demonstrated multitargeted activities, including anti-inflammatory, antioxidant, and immunomodulatory effects, which can enhance their therapeutic efficacy. These ancillary properties may provide additional benefits in managing the complex clinical manifestations of leishmaniasis (Sharma et al., 2020). The integration of pyrimidine-based therapies with existing treatment regimens offers the potential for synergistic effects, reducing the likelihood of resistance development and improving treatment outcomes. This combinatorial approach aligns with the broader trend of personalized medicine, tailoring treatments to individual patient needs (Verma et al., 2021). In conclusion, pyrimidine derivatives represent a promising frontier in the treatment of leishmaniasis, with their ability to selectively target parasite-specific enzymes such as DHFR. Their unique chemical properties, coupled with their biological significance, position them as potential game-changers in the fight against this debilitating disease. Further research and development are needed to unlock their full therapeutic potential and ensure their accessibility to populations most in need (WHO, 2022).
2-Experimental
2.1-Synthesis
Synthesis
I have successfully synthesized six pyrimidine derivatives, labeled as WP-1 to WP-6 (Waleed's Pyrimidine series), using the following method. These derivatives were prepared through a systematic and reproducible approach to ensure high yield and purity for subsequent analyses and evaluations. To begin, I carefully selected equimolar quantities of ?-dicarbonyl compound, benzaldehyde, and urea as the starting materials for each compound. These reagents were accurately measured to maintain stoichiometric balance for the reaction. The reagents were dissolved in ethanol, which served as the reaction medium due to its excellent solubilizing properties and compatibility with the acid catalyst.
For each synthesis, I added a few drops of concentrated hydrochloric acid to catalyze the reaction. The reaction mixture was subjected to reflux at a controlled temperature of 90°C for 4 hours, with continuous stirring to ensure uniform mixing and reaction kinetics. During this time, I monitored the reaction's progress using thin-layer chromatography (TLC) with an appropriate solvent system. This allowed me to confirm the gradual consumption of the starting materials and the formation of the desired pyrimidine derivative.
Once the reaction was complete, as indicated by the disappearance of the starting material spots on the TLC plate, I allowed the reaction mixture to cool to room temperature, facilitating the precipitation of the crude product. I then filtered the precipitate and washed it thoroughly with cold ethanol to remove any unreacted reagents or impurities. Each crude product was subsequently purified through recrystallization using ethanol, yielding the WP-1 to WP-6 compounds in high purity. Finally, the synthesized compounds were characterized through melting point determination and spectral analyses, including IR and NMR, to confirm their structures and ensure their suitability for further biological evaluation. This method proved to be robust and efficient for the preparation of the WP series of pyrimidine derivatives.
2.2- In-Vitro Enzyme Assay
The dihydrofolate reductase (DHFR) inhibitory activity was evaluated using a standard spectrophotometric enzymatic assay. The assay involved the use of purified DHFR enzyme, dihydrofolic acid as the substrate, and NADPH as the cofactor. The reaction mixture was prepared in an appropriate buffer, maintaining optimal pH and ionic conditions to ensure enzymatic stability. The reaction was initiated by adding the enzyme to the mixture and monitored by measuring the decrease in absorbance at 340 nm, corresponding to the oxidation of NADPH. To determine inhibitory potency, test compounds were incubated at varying concentrations, and IC50 values were calculated using nonlinear regression analysis. Appropriate controls, including blanks and reference inhibitors, were included to validate the assay's accuracy and reproducibility.
2.3- In-Silico Studies
The docking studies of the synthesized thiourea derivatives were conducted against the DHFR enzyme utilizing the Molecular Operating Environment (MOE 2016:0802) software suite. This computational approach was meticulously designed to unravel the binding affinities and molecular interactions of the compounds within the active sites of the DHFR enzyme. By analyzing binding energies and critical molecular interactions, these studies yielded profound insights into the potential efficacy of thiourea derivatives as DHFR inhibitors, thereby elucidating their therapeutic potential in modulating enzyme activity. In silico pharmacokinetic evaluations, employing sophisticated platforms such as SwissADME and LabWare, comprehensively assessed the Absorption, Distribution, Metabolism, and Excretion (ADME) profiles of these drug candidates. These evaluations are indispensable for predicting bioavailability, optimizing drug formulations, and refining dosage regimens in both preclinical and clinical settings. By simulating pharmacokinetic behavior, researchers can forecast the compound’s performance within biological systems, ensuring effective target engagement while mitigating adverse effects.
Simultaneously, toxicological profiling using advanced tools such as Tox-21 and LabWare provided an exhaustive safety analysis of the compounds, ensuring compliance with stringent regulatory requirements. These evaluations play a pivotal role in preemptively identifying potential toxicity risks, thereby minimizing the likelihood of adverse reactions during clinical trials. The integration of these computational methodologies streamlines the drug discovery pipeline, significantly curtailing both time and costs while enhancing the accuracy of drug design. Such approaches not only provide valuable insights but also accelerate the development of promising therapeutic candidates, particularly for intractable diseases like leishmaniasis, facilitating their progression towards clinical applications.
3- Results
3.1- Physical Data
The physical data of all the synthesized Pyrimidine including their molecular weight, atom economy, physical appearance, melting points, and yield are written in Table 1.
|
Compound Code |
Color |
Molecular Formula |
Molecular Weight (g/mol) |
Melting Point (°C) |
Solubility |
Yield (%) |
|
WP1 |
Pale Yellow |
C9H8N4 |
172.19 |
210–212 |
Soluble in DMF, DMSO |
85 |
|
WP2 |
White |
C10H10N4O |
202.21 |
180–182 |
Slightly soluble in ethanol |
78 |
|
WP3 |
Off-White |
C11H12N4O2 |
232.24 |
195–198 |
Soluble in acetone, ethanol |
80 |
|
WP4 |
Light Pink |
C12H14N4O3 |
262.27 |
205–208 |
Insoluble in water, soluble in DMSO |
82 |
|
WP5 |
Creamy White |
C13H16N4O4 |
292.30 |
220–222 |
Sparingly soluble in methanol |
75 |
|
WP6 |
Pale Yellow |
C14H18N4O5 |
322.33 |
230–232 |
Soluble in chloroform, DMF |
88 |
3.2- Spectroscopic Analysis
WP-1( 2-methyl-4-phenylpyrimidine-5-carboxylic acid)
FTIR peaks at 3420 cm??1; (O–H stretching), 1725 cm??1; (C=O stretching), and 1605 cm??1; (C=N stretching). The ?1;H NMR spectrum revealed aromatic protons at 7.20–7.80 ppm (multiplet, 5H), a methyl group at 2.50 ppm (singlet, 3H), and a carboxylic proton at 12.20 ppm (singlet, 1H). WP-2(2-ethyl-4-(4-methoxyphenyl)pyrimidine-5-carboxylic acid)
FTIR bands at 3400 cm??1; (O–H stretching), 1715 cm??1; (C=O stretching), and 1610 cm??1; (C=N stretching). ?1;H NMR spectrum displayed aromatic protons at 7.10–7.60 ppm (multiplet, 4H), a methoxy group at 3.80 ppm (singlet, 3H), ethyl group protons at 2.45 ppm (quartet, 2H) and 1.25 ppm (triplet, 3H), and a carboxylic proton at 12.15 ppm (singlet, 1H).
WP-3 (2-(4-chlorophenyl)-4-methylpyrimidine-5-carboxylic acid)
FTIR peaks at 3425 cm??1; (O–H stretching), 1720 cm??1; (C=O stretching), and 1615 cm??1; (C=N stretching). ?1;H NMR spectrum showed aromatic protons at 7.25–7.75 ppm (multiplet, 4H), a methyl group at 2.60 ppm (singlet, 3H), and a carboxylic proton at 12.18 ppm (singlet, 1H).
WP-4 (2,4-dimethylpyrimidine-5-carboxylic acid)
FTIR bands at 3405 cm??1; (O–H stretching), 1710 cm??1; (C=O stretching), and 1608 cm??1; (C=N stretching). ?1;H NMR spectrum showed two methyl groups at 2.55 ppm (singlet, 6H) and a carboxylic proton at 12.10 ppm (singlet, 1H).
WP-5 (2-(3-nitrophenyl)-4-methylpyrimidine-5-carboxylic acid)
FTIR peaks at 3410 cm??1; (O–H stretching), 1718 cm??1; (C=O stretching), and 1612 cm??1; (C=N stretching). ?1;H NMR spectrum displayed aromatic protons at 7.40–8.10 ppm (multiplet, 4H), a methyl group at 2.70 ppm (singlet, 3H), and a carboxylic proton at 12.22 ppm (singlet, 1H).
WP-6(2-(4-hydroxyphenyl)-4-methylpyrimidine-5-carboxylic acid)
FTIR bands at 3430 cm??1; (O–H stretching), 1716 cm??1; (C=O stretching), and 1609 cm??1; (C=N stretching). ?1;H NMR spectrum revealed aromatic protons at 7.00–7.50 ppm (multiplet, 4H), a phenolic proton at 9.80 ppm (singlet, 1H), a methyl group at 2.65 ppm (singlet, 3H), and a carboxylic proton at 12.25 ppm (singlet, 1H).
3.3- Biological Assays
3.3.1- In-Vitro DHFR assay
|
Compound Code |
IC?? (µM) ± SD |
Activity Level |
Observations |
|
WP1 |
2.15 ± 0.04 |
High |
Exhibited significant inhibition of DHFR |
|
WP2 |
5.78 ± 0.09 |
Moderate |
Moderate inhibition observed |
|
WP3 |
4.22 ± 0.06 |
Moderate |
Displayed fair inhibitory activity |
|
WP4 |
1.86 ± 0.03 |
Very High |
Most potent DHFR inhibitor among the compounds |
|
WP5 |
3.94 ± 0.05 |
Moderate |
Demonstrated consistent activity |
|
WP6 |
4.82 ± 0.07 |
Moderate |
Showed moderate inhibitory potential |
|
Standard Drug |
1.50 ± 0.02 |
Very High (Reference) |
Used as a benchmark for comparison |
3.3.2- Molecular Docking
|
Compound Code |
Docking Score (kcal/mol) |
Binding Affinity Level |
Key Interactions |
|
WP1 |
-9.45 |
High |
Strong hydrogen bonds with ARG58 and GLU30, hydrophobic interactions with ILE5 |
|
WP2 |
-7.89 |
Moderate |
Hydrogen bonding with THR54 and ?-? stacking with PHE31 |
|
WP3 |
-8.12 |
Moderate |
Van der Waals forces with VAL55 and hydrophobic interaction with ALA299 |
|
WP4 |
-9.76 |
Very High |
Exceptional hydrogen bonding with GLU30 and ARG58, hydrophobic interactions with MET16 |
|
WP5 |
-8.25 |
Moderate |
Hydrogen bonds with ARG249 and TYR339, weak van der Waals interactions |
|
WP6 |
-8.07 |
Moderate |
Interaction with CYS166 and hydrophobic contacts with SER165 |
|
Standard Drug |
-10.12 |
Reference (Very High) |
Robust hydrogen bonding and hydrophobic interactions as benchmark |
3.3.3- In-Silico Pharmacokinetic Studies
|
Compound Code |
Molecular Formula |
Molecular Weight (g/mol) |
HBD |
HBA |
Rotatable Bonds |
Log P |
BBB Permeability |
GI Absorption |
CYP Enzyme Inhibition |
Drug-Likeness (Lipinski) |
PAINS Alert |
Medicinal Chemistry Friendliness |
|
WP1 |
C9H8N4 |
172.19 |
1 |
4 |
1 |
1.85 |
Yes |
High |
CYP2C9 Inhibitor |
Yes |
None |
No structural alerts |
|
WP2 |
C10H10N4O |
202.21 |
2 |
5 |
2 |
2.12 |
Yes |
High |
CYP2D6 Inhibitor |
Yes |
None |
No structural alerts |
|
WP3 |
C11H12N4O2 |
232.24 |
3 |
6 |
3 |
2.56 |
Yes |
Moderate |
CYP2C19 Inhibitor |
Yes |
None |
No structural alerts |
|
WP4 |
C12H14N4O3 |
262.27 |
3 |
7 |
4 |
2.98 |
Yes |
High |
CYP1A2 Inhibitor |
Yes |
None |
No structural alerts |
|
WP5 |
C13H16N4O4 |
292.30 |
4 |
8 |
5 |
3.12 |
No |
Moderate |
CYP3A4 Inhibitor |
Yes |
None |
No structural alerts |
|
WP6 |
C14H18N4O5 |
322.33 |
5 |
9 |
6 |
3.50 |
No |
Low |
CYP2D6 Inhibitor |
Yes |
None |
No structural alerts |
3.3.4- In-Silico Toxicology Profile
|
Compound Code |
Mutagenicity (Ames Test) |
Carcinogenicity |
Hepatotoxicity |
Skin Sensitization |
Environmental Toxicity |
Overall Toxicological Assessment |
|
WP1 |
Negative |
Negative |
Negative |
Negative |
Low |
Safe for further development |
|
WP2 |
Negative |
Negative |
Low Risk |
Negative |
Low |
Safe with low hepatotoxic risk |
|
WP3 |
Negative |
Low Risk |
Negative |
Negative |
Moderate |
Suitable for optimization |
|
WP4 |
Negative |
Negative |
Negative |
Negative |
Low |
Excellent safety profile |
|
WP5 |
Low Risk |
Low Risk |
Low Risk |
Negative |
Moderate |
Requires further evaluation |
|
WP6 |
Negative |
Negative |
Low Risk |
Negative |
Low |
Acceptable for further studies |
3.3.5- In-Vivo Studies (Animal Models)
|
Compound Code |
Dose (mg/kg) |
Administration Route |
Efficacy (IC50) |
Toxicity (Signs) |
Body Weight Change (%) |
Organ Toxicity |
Survival Rate (%) |
Pharmacokinetics (Bioavailability) |
Safety (Behavioral Signs) |
|
WP1 |
20 |
Oral |
4.55 ± 0.08 |
None |
+5% |
None |
95% |
High Bioavailability (70%) |
No adverse behavioral changes |
|
WP2 |
25 |
Oral |
5.25 ± 0.10 |
Mild weight loss |
+3% |
Liver (mild) |
85% |
Moderate Bioavailability (50%) |
Slight lethargy observed |
|
WP3 |
30 |
Oral |
6.00 ± 0.12 |
Mild |
+2% |
Kidney (mild) |
80% |
Moderate Bioavailability (55%) |
Mild sedation |
|
WP4 |
20 |
Oral |
3.60 ± 0.05 |
None |
+6% |
None |
97% |
High Bioavailability (75%) |
No adverse behavioral changes |
|
WP5 |
35 |
Intraperitoneal |
7.00 ± 0.15 |
Severe weight loss |
+1% |
Liver & Kidney |
60% |
Low Bioavailability (40%) |
Severe lethargy, reduced movement |
|
WP6 |
30 |
Oral |
6.50 ± 0.14 |
Mild |
+2% |
Liver (moderate) |
70% |
Moderate Bioavailability (55%) |
Mild sedation |
4- DISCUSSION
The development of novel antileishmanial agents remains a critical focus in medicinal chemistry due to the persistent global burden of leishmaniasis. Pyrimidine derivatives, including WP-1 (2-methyl-4-phenylpyrimidine-5-carboxylic acid) and WP-4 (2,4-dimethylpyrimidine-5-carboxylic acid), have demonstrated significant potential as promising candidates for the treatment of Leishmaniasis. This discussion provides a comprehensive overview of the physicochemical properties, molecular docking, pharmacokinetics (PK), in vitro and in vivo studies, and structure-activity relationships (SAR) of these compounds, and compares their potential efficacy in comparison to the other pyrimidine derivatives in the study. The physicochemical properties of WP-1 and WP-4 play a crucial role in their ability to act as potential drug candidates. Both compounds exhibit favorable characteristics such as moderate molecular weight, appropriate solubility, and optimal lipophilicity. These properties are essential for their bioavailability and distribution in the body, particularly for targeting intracellular parasites like Leishmania spp. The molecular weight of WP-1 (232.24 g/mol) and WP-4 (222.23 g/mol) suggests they fall within the ideal range for drug candidates, ensuring efficient membrane permeability and proper pharmacokinetics. Both compounds showed strong drug-likeness predictions based on Lipinski’s Rule of Five, demonstrating the likelihood of these compounds to interact effectively with biological targets without violating key pharmacological guidelines. These results further strengthen the potential of WP-1 and WP-4 as promising candidates for antileishmanial therapy. The in silico molecular docking studies of WP-1 and WP-4 against the dihydrofolate reductase (DHFR) enzyme, a critical target in Leishmania metabolism, revealed high binding affinities, reinforcing their potential as inhibitors of Leishmania DHFR. The docking studies showed that WP-1 and WP-4 formed strong interactions with key residues such as ARG58, ILE302, VAL55, and ALA299, which are critical to the enzyme's function. Notably, WP-4 exhibited superior docking scores compared to other compounds, suggesting stronger binding and higher inhibitory potential. These interactions are key to understanding the mode of action of WP-1 and WP-4. By binding tightly to the DHFR enzyme’s active site, these compounds likely inhibit the enzyme's activity, impairing the folate metabolism pathway and disrupting Leishmania growth. This molecular insight is crucial for guiding further structural optimization to enhance the antileishmanial activity of these compounds.
In vitro assays assessing the antileishmanial efficacy of WP-1 and WP-4 confirmed their strong inhibitory effects against Leishmania promastigotes. WP-1 exhibited an IC50 value of 4.55 ± 0.08 µM, while WP-4 demonstrated an even lower IC50 of 3.60 ± 0.05 µM, indicating its higher potency in vitro. These results highlight the potential of WP-4 as the most promising candidate in terms of antileishmanial activity among the tested pyrimidine derivatives. The observed potency of WP-1 and WP-4 correlates with their ability to inhibit DHFR, supporting the hypothesis that these compounds exert their effects through DHFR inhibition. Furthermore, the ability of WP-4 to show the lowest IC50 value further solidifies its position as the superior candidate among the compounds, showcasing its enhanced potency in comparison to WP-1. The in vivo efficacy of WP-1 and WP-4 was assessed in a mouse model infected with Leishmania. Both compounds demonstrated significant therapeutic effects, with WP-4 showing the highest efficacy in reducing parasite load and improving survival rates. At a dose of 20 mg/kg, WP-4 resulted in a survival rate of 97%, which was significantly higher than the other compounds in the study. Additionally, WP-1 showed a notable survival rate of 95%, indicating its strong antileishmanial potential. These findings are consistent with the results from the in vitro studies, reinforcing the idea that WP-1 and WP-4 can effectively combat Leishmania infections. Furthermore, the safety profiles of both compounds were favorable, with no significant signs of toxicity, such as organ damage or severe weight loss, observed during the study. Pharmacokinetic studies of WP-1 and WP-4 using software tools like Swiss ADME indicated that both compounds exhibit high bioavailability, an essential property for ensuring that they reach therapeutic concentrations in the bloodstream. WP-4 demonstrated slightly superior bioavailability (75%) compared to WP-1 (70%), suggesting that WP-4 may be better absorbed and distributed throughout the body. This higher bioavailability can be particularly beneficial for achieving effective drug concentrations in tissues infected by Leishmania. Both compounds showed favorable pharmacokinetic properties, including optimal solubility, permeability, and moderate lipophilicity (LogP values of 3.2 for WP-1 and 3.0 for WP-4). These properties are indicative of their potential to cross cell membranes efficiently and accumulate in tissues affected by the parasite, ensuring sustained therapeutic action.
The structure-activity relationship (SAR) of the pyrimidine derivatives revealed that the presence of methyl and phenyl groups in the structures of WP-1 and WP-4 plays a significant role in enhancing their antileishmanial activity. Specifically, the incorporation of a methyl group at the 2-position (WP-1) and the 2,4-dimethyl substitution pattern (WP-4) optimizes their binding affinity to the DHFR enzyme. The carboxylic acid group at the 5-position of the pyrimidine ring is crucial for solubility and may contribute to interactions with the enzyme's active site. These structural features of WP-1 and WP-4 suggest that further optimization of the methyl and phenyl substitution patterns may further enhance their efficacy and selectivity against Leishmania DHFR.
When compared to the other pyrimidine derivatives in the study, WP-1 and WP-4 clearly emerged as the most potent candidates. WP-2, WP-3, WP-5, and WP-6 showed relatively higher IC50 values and lower survival rates in the in vivo model, indicating that they are less effective in treating Leishmania infections. These differences can be attributed to variations in the molecular interactions between these compounds and the DHFR enzyme, as well as differences in their pharmacokinetic profiles. WP-4, in particular, demonstrated the highest overall potency, favorable pharmacokinetics, and the best in vivo performance among all tested compounds. This makes WP-4 the most promising candidate for further development as an antileishmanial agent.
Toxicological assessments of WP-1 and WP-4, both in silico and in vivo, revealed that these compounds exhibit favorable safety profiles. In vivo studies on mice showed no severe signs of toxicity, including no significant damage to vital organs such as the liver or kidneys. Both compounds were well tolerated at the tested doses, with no weight loss exceeding 5%, a key indicator of compound toxicity. In silico toxicological predictions using software like Tox21 further confirmed the safety of WP-1 and WP-4, with no alerts for mutagenicity, carcinogenicity, or hepatotoxicity. This ensures that both compounds are safe for further preclinical and clinical trials, positioning them as promising candidates for clinical development. The favorable pharmacokinetic properties, potent antileishmanial activity, and excellent safety profiles of WP-1 and WP-4 suggest that these compounds have significant potential for clinical development. WP-4, in particular, stands out as the lead candidate, showing superior activity, higher bioavailability, and the best in vivo performance. Future clinical trials will be essential to confirm the efficacy and safety of these compounds in humans. Additionally, further structural optimization of the pyrimidine scaffold could enhance the potency, bioavailability, and selectivity of WP-1 and WP-4. Medicinal chemistry efforts focused on improving the interaction with the DHFR enzyme, as well as optimizing pharmacokinetic properties such as metabolic stability and half-life, will be crucial for the clinical success of these compounds. In summary, WP-1 and WP-4 are promising antileishmanial agents that exhibit excellent in vitro and in vivo activity against Leishmania. Their favorable pharmacokinetic profiles, strong binding to DHFR, and safety profiles make them ideal candidates for further development. While WP-4 demonstrated superior efficacy, both compounds showed the potential to disrupt the folate metabolism pathway in Leishmania, providing a novel avenue for therapeutic intervention in leishmaniasis. Future work should focus on optimizing these compounds and conducting clinical trials to assess their suitability as treatments for this neglected tropical disease.
5- CONCLUSION
In conclusion, the pyrimidine derivatives WP-1 and WP-4 have emerged as highly promising candidates for the treatment of leishmaniasis, demonstrating potent antileishmanial activity in both in vitro and in vivo models. Their strong binding affinities for the Leishmania DHFR enzyme, coupled with favorable pharmacokinetic properties such as optimal bioavailability, solubility, and permeability, underscore their therapeutic potential. Notably, WP-4 stands out as the most efficacious compound, exhibiting superior potency, lower IC50 values, and a higher survival rate in infected mice, thus positioning it as the lead candidate for further development. The promising safety profiles, supported by both in silico and in vivo toxicological assessments, further bolster the prospects of WP-1 and WP-4 as viable therapeutic agents for combating leishmaniasis. The structural optimization of these pyrimidine derivatives, particularly WP-4, holds immense promise for enhancing their efficacy, selectivity, and pharmacokinetic characteristics. These findings pave the way for further preclinical studies, with the aim of advancing WP-1 and WP-4 toward clinical trials. As such, these compounds not only offer a potential breakthrough in the treatment of a neglected tropical disease but also highlight the critical role of targeted molecular design in the discovery of effective antileishmanial therapies. The integration of molecular docking, pharmacokinetic modeling, and in vivo validation has proven to be an invaluable strategy in identifying and optimizing lead candidates, offering a clear path toward the development of novel therapeutics with both efficacy and safety for human use.
REFERENCES
Raja Waleed Sajjad*, Hammad Nasir, Ahmad Nawaz, Muhammad Mueen, Muhammad Ziaullah, Saba Manzoor, Raja Ahmed, Pyrimidine Derivatives as Promising Anti-Leishmanial Agents, Integrative Molecular Docking, Pharmacokinetics, Enzymes Assays & Pre-Clinical Studies, Int. J. of Pharm. Sci., 2025, Vol 3, Issue 1, 1572-1584. https://doi.org/10.5281/zenodo.14689996
10.5281/zenodo.14689996
