Radiolabeling and In-Silico Study of 131 I-(4-fluorobenzoyl-3-methylthiourea) as Radiopharmaceuticals for Breast Cancer Theranostics

The chemicals produced from thiourea are actively being studied as anticancer possibilities. In complexes with radionuclides like Iodine-131, the 1-(4-Fluorobenzoyl)- 3-methyl thiourea is a promising ligand for theragnostic applications. This study aimed to label 1-(4-fluorobenzoyl-3-methylthiourea) with iodine-131 and observe its interaction with breast cancer receptors. The radiolabeling of 131 I-(4-fluorobenzoyl-3-methylthiourea) uses the radioiodination method with Chloramine-T, and an in-silico investigation of breast cancer receptors was conducted. According to the results of molecular docking using AutoDockTools, this radiopharmaceutical molecule has the best activity on the HER2 receptor (PDB ID: 3PP0) compared to the native ligand and control positive, with a binding affinity of -6.13 kcal/mol and a Ki value of 32.05 mM. According to the molecular dynamics data using Desmond, the radiopharmaceutical molecule 131 I-(4-Fluorobenzoyl-3-methylthiourea) displays good stability starting from the 50ns range. The indirect radioiodination method has successfully labeled 1-(4-Fluorobenzoyl-3-methylthiourea) with iodine-131.


INTRODUCTION
Iodine-131 ( 131 I) is a gamma (γ) and beta (β) ray emitting radionuclide. 131I produces γ energy of 364 keV and β of 0.67 MeV to emit γ and β rays and has a halflife of 8 days.The 131 I is an ideal radionuclide for cancer diagnosis and nuclear medicine therapy which is often used by labeling compounds that have pharmacological activity against cancer cells 1 .Iodine-131 has advantages such as its therapeutic applications, selective delivery, and availability, but it also has disadvantages including radiation emission, low specificity, and low molar activity that need to be carefully considered in the development and use of radiopharmaceuticals for diagnosis and therapy [2][3][4] .
Radiopharmaceuticals are drugs containing radioisotopes that are safe to administer to humans for diagnosis or therapy.The use of radiopharmaceuticals for imaging organ function and disease states is a unique capability in nuclear medicine 5 .Radionuclides or radioisotopes are atoms that have excess nuclear energy, making them unstable.This excessive energy can be utilized in one of three ways: emitted from the nucleus as gamma (γ) radiation; transferred to one of its electrons to release it as a conversion electron; or used to create and emit new particles (alpha (α) particles or beta (β) particles) from the nucleus.Radiopharmaceuticals used for diagnostic purposes are those with gamma emitters, while radioactive atoms emit particles or are used for internal radiotherapy 6 .
Tiourea is one compound that is widely used in new drug discovery research.Tiourea compounds have pharmacological activity as an anticancer 7 .Some of the advantages of thiourea derivatives in cancer treatment include their potent anticancer properties, high selectivity, and low toxicity.The mechanism of interaction that occurs with cancer cells involves the Pratama et al. | 28   ability of thiourea derivatives to favor the formation of hydrogen bonding NH moieties, increasing their solubility in an aqueous medium and allowing better cellular uptake [8][9][10] .Thiourea is an organic compound containing carbon, hydrogen, nitrogen, and sulfur atoms.The structure of thiourea is similar to urea, except that the S atom in thiourea is replaced by an O atom in urea 11 .Derivates of thiourea compounds have good cytotoxic effects on breast cancer cells 12 .One of the derivatives of thiourea compounds is 1-(4-fluorobenzoyl-3methylthiourea), where It had an IC-50 value of 251 g/mL 13 .This compound is synthesized from Nmethyl thiourea and 4-fluorobenzoyl chloride and has been shown to have anticancer activity 14 .The radiolabeling of 131 I-(4-fluorobenzoyl-(3methylthiourea) is a promising approach for the development of a new radiopharmaceutical candidate for cancer diagnosis and therapy.The use of molecular docking and molecular dynamic simulation is essential to study the interaction of this candidate with its target protein, as it provides insights into the binding mode, affinity, and stability of the compound, which are critical factors in the development of effective radiopharmaceuticals.

Instruments and Materials
The tools used in this research include several software.For molecular docking, AutodockTools 1.5.6 was utilized, MarvinSketch 21.17.0 for ligand preparation, Molegro and Pymol for protein preparation, Discovery Studio 20.1 for visualization, Desmond software (academic license, D.E.Shaw Research, New York) for molecular dynamic simulation, and web-based programs including Protein Data Bank and PubChem for download the protein and ligand, pkCSM for toxicity prediction.Meanwhile, the hardware used in this research was a portable computer with specifications AMD A4-9125 Radeon R3 2.30 GHz, 8 GB Ram 64-bit Operating System of Windows 10 and Intel i7 9700k GTX 1070 Ti 8GB DDR5 Ram 32GB LPX DDR4 2666MHz OS Linux Ubuntu.The instruments used for work in the PRTRRB ORTN lab included microtubes, erlenmeyer, beaker, measuring cup, stirring rod, micropipette (Eppendorf), 0.2 and 0.5 mL microcentrifuge tubes, a set of paper chromatography and paper electrophoresis equipment, analytical balance (Metler Toledo), oven (Memmert), digital shaking dry bath (Thermo Scientific), laminar airflow cabinet for radioactivity (Comecer), refrigerator -20°C (Samsung), dose calibrator (Biodex), RadioTLC Scanner (Bioscan), H-NMR spectrophotometer (JEOL).

Preparation and Analysis of Receptors
The three receptors were obtained from the Protein Data Bank (https://www.rcsb.org/pdb).The downloaded receptors were analyzed using a web-based server (https://www.ebi.ac.uk/pdbsum).The results of the analysis were seen based on the fit with the Ramachandran plot parameters.The receptors were prepared by removing water residues and adding hydrogen atoms and separating the receptors with their native ligands 15 .

Ligand Structure Preparation
The ligand was drawn and prepared using MarvinSketch software (https://chemaxon.com/marvin) by protonation at blood pH (7.4) and confirmed.Then the ligands were saved in the '.mol2' file format 16 .

Docking Validation
Validation of the docking method is done by redocking each of the native ligands to the receptor (PDB ID 3ERT), HER2 (PDB ID 3PP0), and NUDT5 (PDB ID 5NQR).The parameter for this validation result is based on the Root Mean Square Deviation (RMSD) value with a good value of <2 Å 17 .

Molecular Docking
Molecular docking uses AutodockTools 1.5.6 software to see the interaction between ligands and proteins.The prepared ligand must first be converted into PDBQT format in order to do docking.Then adjust the grid box the same as during docking validation.Ligand analysis was performed with the default docking system in this application and using LGA for 100 runs.The parameter used in docking is binding energy 17 .

Drug Scan and Toxicity Profile Prediction
Drug scan analysis was performed according to Lipinski's parameters of medicine (Lipinski's Rule of Five).In these parameters, it is considered that a good drug must have a lipophility <5, molecular weight <500 g/mol, hydrogen acceptor <10, hydrogen donor <5, and molar refractory between 40-130 14 .Pharmacokinetic and toxicity profiling of compounds carried out by the open-sourced pkCSM website (https://structure.bioc.cam.ac.uk./pkcsm) 17 .

Molecular Dynamic Simulation
Molecular dynamics simulations were performed by the Desmond application to get the stability of the compounds.This simulation was carried out on TIP3P (Transferable Intermolecular Potential with 3 Points) type water and 0.15 M NaCl to mimic a physiological ionic concentration.The TIP3P is favored for its simplicity and efficiency, making it suitable for simulations that require a large number of water molecules and where computational resources may be limited.It provides a good approximation of water's physical properties that are relevant for a wide range of biological and chemical systems 18 .System energy was minimized to obtain the lowest energy configuration and followed by equilibration for 100 ps before a production run of frames at temperature 300 K and standard pressure (1.01325 bar) in 200ns using the orthorhombic box (10 Å x 10 Å x 10 Å) and NPT ensembles 19 .NPT stands for the isothermal-isobaric ensemble, where: N stands for the number of particles in the system, which is kept constant; P stands for the pressure of the system, which is kept constant; and T stands for the temperature of the system, which is also kept constant.Neutralization of the protein-ligand complex was performed by adding Na + and Cl -ions.Noose-Hoover and Martyna-Tobias_klein algorithms were used 20 .

Synthesis of 1-(4-fluorobenzoyl-3-methylthiourea)
N-methylthiourea (0.032 mol) was mixed with 20 mL of tetrahydrofuran solvent in a 250 mL flatbottom flask.Triethylamine was added to the catalyst.Next, 0.016 mol of 4-fluorobenzoyl chloride in 15 mL of tetrahydrofuran was dripped into the mixture in the flat bottom flask using a separatory funnel while stirring using a magnetic stirrer.The mixture was refluxed for 8 hours and analyzed every hour by Thin Layer Chromatography (TLC) using an eluent of methanol: chloroform 9:1.Reflux was stopped when a single spot was obtained on the TLC plate and then evaporated with a rotary evaporator until the solvent disappeared due to evaporation.The results obtained were added to saturated sodium bicarbonate solution while stirring until there was no foam.The synthesis product was washed with 2 x 100 mL of distilled water, and then filtered with a Buchner funnel 7 .
This study uses indirect labeling to label histamine (concentration 2.2 mg/mL) with Na 131 I (radioactivity 100-300 μCi).Chloramine T (5 mg/mL) is added to the solution, and the reaction is stopped by adding sodium metabisulphite (300 mg/mL).4fluorobenzoyl-3-methylthiourea is activated by dissolving it in dioxane (20 μg/μL), and then added to an iodination tube containing 131 I-histamine.The conjugation is tested by electrophoresis.Purification of 131 I-(4-fluorobenzoyl-3-methylthiourea) iodohistamine is done using toluene extraction, and the radiochemical purity is tested by electrophoresis 21 .

Selection of Chromatographic System for Determination of Radiochemical Purity of 131 I-(4fluorobenzoyl-3-methylthiourea)
The purity of 131 I-(4-fluorobenzoyl-3methylthiourea) radiochemical was determined by ascending paper chromatography and electrophoresis method.Paper chromatography used six stationary phase variations namely TLC SG, Whatman 1 and Whatman 3 MM with various mobile phases; chloroform: ethanol (90:10), (70:30) and (1:1); methanol: water (70:30) and (80:20).This method was chosen to separate 131 I-(4fluorobenzoyl-3-methylthiourea) from impurities 131 I2 and free iodine ( 131 I -).For comparison, the assay was performed by paper electrophoresis.Cellulose acetate paper was used as the stationary phase and Whatman 1 used phosphate buffer solution (0.05 M, pH 7.4) as the electrolyte.Electrophoresis was performed for 60 minutes at 300 V.The paper for paper chromatography and TLC was dried for 5 minutes hanging with a lamp.Then, these papers were enumerated using a RadioTLC Scanner.The data were processed using Excel to calculate the percentage of radiochemical purity of 131 I-(4-fluorobenzoyl-3-methylthiourea) in solution.The 131 I-(4-fluorobenzoyl-3-methylthiourea) labeled compound meets the radiochemical purity requirement if the percentage is ≥ 95% 21 .

Effect of pH
The initial optimization stage was carried out by varying the pH from 7-10 with a fixed conjugation time

Receptor Quality Analysis and Preparation
Receptors analysis was performed by viewing the Ramachandran plot profiles of the proteins available on the website www.ebi.ac.uk/pdbsum by entering the PDB IDs of the breast receptors used, namely estrogen (3ERT), HER2 (3PP0), and NUDT5 (5NQR).Ramachandran plots aim to assess the stereochemical quality of the protein structure by showing the phi-psi dihedral angle distribution for all residues in the structure (except at the chain ends) and visualizing it in three dimensions.Therefore, each amino acid residue is specified as a region in this Ramachandran plot. 12.
In this Ramachandran plot, the most favored regions value parameter is used, which indicates the most favored core regions with an ideal value of >90% and disallowed regions with a recommended value of <15% 14 .In Figure 1, it can be seen that the proteins 3ERT, 3PP0, and 5NQR have the most favored regions of 91.2%, 93.6%, and 91.1% respectively and all three have the same disallowed regions value of 0.0%.These results indicate that the three receptors are of good quality and stable because they fulfill the requirements of the Ramachandran plot.The red, brown, and yellow regions represent the favored, allowed, and "generously allowed" regions.
The three proteins used in this study were pretreated by removing solvents and other residues such as water that can interfere with ligand-protein interactions and ease the work of hardware and adding hydrogen bonds to the protein to make it polar 22 .The structures of the three proteins can be seen in Figure 2.

Docking Validation
The validation method is conducted via determination of Root Mean Square Deviation (RMSD) value.The parameter shows the magnitude of the change in the interaction between the protein with the crystallographic natural ligand compared with the redocking result.The docking method can be trusted or declared valid if the RMSD value ≤ 2 Å 22 .In Table 1, it can be seen that the RMSD values of the three proteins are below 2 Å so that the method can be declared valid and can perform testing on the test compound.
In Figure 3, it can be seen the overlay comparison between the natural ligand crystallography with redocking results with a very small difference distance because the RMSD value of the redocking results of the three proteins is below 2 Å

Ligand Structure Preparation
The ligand was drawn and pretreated using MarvinSketch software with protonation done by equalizing the pH to the blood pH (7.4).The best conformation of the protonated ligand was used.The stability of the ligand and protein interaction will be greatly influenced by the conformation of the ligand itself 23 .Visualization of ligand and radioligand can be seen in Figure 4.

Molecular Docking
Molecular docking simulation was performed by docking the test ligand, 131 I-(4-fluorobenzoyl-3methylthiourea) complex, to the three receptors, 3ERT, 3PP0, and 5NQR, with the original ligand, 1-(4-fluorobenzoyl-3-methylthiourea) and breast cancer drug, doxorubicin.The grid box used in this docking test simulation is the grid box used during the method validation of each receptor.The value of Gibbs binding affinity (ΔG) and inhibition constant (Ki) contained in the file with '.dlg' format indicate show the stability of the bond between ligand and receptor.This simulation uses the Lamarckian genetic algorithm (LGA) with 100 times run conformation.The advantages of using the Lamarckian Genetic Algorithm in AutoDock compared to other methods, such as pure genetic algorithms or simulated annealing, include efficiency, balance between exploration and exploitation, avoidance of local minima, better for complex landscapes, and adaptability.
Control PositiveTo visualize the interaction between ligand and amino acid residue, it can be seen using Discovery Studio software from the .pdbfile of the Pratama et al. | 33  best docking result that has been simulated before.
Interactions observed include hydrogen bonds and hydrophobic bonds.3D and 2D visualization of the best receptor can be seen in Figure 5 and the amino acid residues can be seen in Table 3.
In receptor 3PP0, the test ligand forms one hydrogen bond (TRY A:137) and 7 hydrophobic bonds, some matching the natural ligand (ASN A:135 and TYR A:91).The comparator ligand has 3 hydrogen bonds (TYR A:217, GLY A:70, GLN A:39) and 11 hydrophobic bonds, with 3 matching the natural ligand (TYR A:137, ASN A:135, TYR A:91).Doxorubicin, used for comparison, forms 4 hydrogen bonds (TYR A:217, GLN A:39, ASP A:95, ASN A:72) and 18 hydrophobic bonds, with 3 matching the natural ligand (TYR A:91, ASN A:135, TYR A:137).Hydrogen and hydrophobic bonds are crucial for the biological activity of drugs, affecting their properties and stability 24 .

Drug Scan, Pharmacokinetic and Toxicity Profile Prediction
Drug scans are conducted based on similarity to existing drugs using Lipinski's rules of five parameters.These scan aim to characterize the physicochemical properties including water solubility, intestinal permeability, and oral bioavailability.Table 4 shows that the radiopharmaceutical compound passed the test of all Lipinski's rules of five parameters compared to its control positive(doxorubicin), which only matched the LogP parameter.
Overall, based on Table 4, the 4F and 131 I-4F appear to fit within the typical profile of drug-like molecules more closely than Doxorubicin, at least regarding the criteria presented here.Doxorubicin, however, is an established chemotherapy medication, indicating that despite its divergence from the typical "drug-like" range in molar refractivity and LogP, it is still a therapeutically effective compound.This illustrates that while guidelines like the "Rule of Five" are helpful, there are successful drugs that do not meet all the criteria, emphasizing the importance of considering the full context of each compound's pharmacodynamics and pharmacokinetics.Molecular weight in physicochemical properties can affect the absorption and distribution of the compound, the compound will be easier to diffuse through the cell membrane and distributed throughout the cell to bind to the receptor if the molecular weight of the compound is lower.The LogP value affects the ability of the compound to dissolve in solvents such as oils, fats, and also other nonpolar solvents.Meanwhile, hydrogen donor and acceptor values affect the biological activity of the compound.Molar refractivity relates to the total polarisability of the compound 23 .
In drug development, predicting pharmacokinetic and toxicity profiles is key to assessing a drug candidate's effectiveness and side effects.Key parameters include CaCO2, intestinal absorption, VDss, BBB permeability, CYP3A4 and OCT2 interactions, AMES toxicity, hepatotoxicity, and LD50.Radioligand shows promising results: a high CaCO2 value (0.975), strong intestinal absorption (over 80%), a low volume of distribution (below -0.15), and adequate BBB penetration, as seen in Table 5.It's neither a substrate nor an inhibitor of CYP3A4 and OCT2.Toxicity-wise, it's non-mutagenic, non-carcinogenic, and not hepatotoxic 25 .

Molecular Dynamic Simulation
Molecular dynamics simulation in this study uses Desmond software to see the stability of the tested radioligand by looking at the RMSD (Root Mean Square Deviation) and RMSF (Root Mean Square Fluctuation) graphs.Molecular dynamics itself is an application to see information from the interactions that occur between ligands and proteins in a flexible state involving atomic and molecular interactions in a certain time span so this stage can be called an advanced stage of molecular docking.In this stage, we can also see the stability of the protein enzyme, protein structure, protein folding, conformational changes, and ion transport 26 .
Compounds that are continued at the molecular dynamics stage are compounds that are tested by molecular docking with proteins or receptors with better binding affinity values than their natural ligands, namely radiopharmaceutical compounds on 3PP0 receptors.Graphs of RMSD and RMSF values can be seen in Figures 6 and 7.  From the RMSD graph above, it can be seen the stability of each ligand analyzed against protein 3PP0.For radioligand as a test ligand, it can be seen that it starts to stabilize from 50ns onwards.There is a slight fluctuation in a certain time range but can return to its stable area.The RMSD values can be seen in Table 6.The RMSF values can be seen in Table 7.In addition to seeing the stability, RMSF can also see the interaction of the radioligand with the residue.The interaction of the radioligand with amino acid residues can be seen in Figure 7, including interaction with SER_40 (RMSD 2.232), GLY_70 (RMSD 2.908), and ALA_171 (RMSD 2.822 Protein RMSF can see the flexibility of the ligand and local residues that can serve for evaluation and see the atomic fluctuations of the ligand.Figure 8 shows the RMSF of the tested radioligand 131 I-(4-Fluorobenzoyl-3-methylthiourea).Meanwhile, Figure 9 shows the amino acid residues interacting with the radioligand with a darker orange color indicating more than one specific contact between the residue and the radioligand during 200ns.Protein structure fluctuations in molecular dynamics are influenced by factors such as protein architecture and fold, temperature and environmental conditions, atomic fluctuations and protein flexibility, solvent interactions, and residue-level fluctuations 27,28 .In general, protein structure fluctuations in molecular dynamics are influenced by both internal and external factors.Internally, the amino acid composition, protein fold and secondary structures, intramolecular interactions, and conformational states dictate flexibility and stability.
Externally, environmental conditions like temperature, pH, ionic strength, solvent properties, and pressure play significant roles.Additionally, ligand binding, post-translational modifications, and external mechanical forces or biomolecular interactions can considerably affect protein dynamics.These factors collectively determine how a protein behaves and responds to its environment.
The parameters used in addition to RMSD and RMSF in molecular dynamics also include the radius of gyration (rGyr), NS34, Molecular Surface Area (MolSA), Solvent Accessible Surface Area (SASA), Polar Surface Area (PSA) which can be seen in Figure 9 It can be seen that the RMSD and rGyr of the tested radioligands are stable with no excessive fluctuations during 200ns, there is only an increase at the beginning for RMSD.In the IntraHB plot, there is an interaction characterized by fluctuating graphs at the beginning and middle of the time.For the MolSA plot, it is stable throughout the time, while in the SASA plot, there are fluctuations at 30ns and 140ns.Meanwhile, the PSA plot is stable with slight fluctuations including at times 45ns and 100ns.

Synthesis of 4-fluorobenzoyl-3-methylthiourea
This method synthesizes amides, reacting amine nucleophiles with benzoyl chloride in a basic atmosphere.Specifically, 4-Fluorobenzoyl chloride is used, conditioned with triethylamine to achieve pH 8, and heated at 100°C for about 7 hours.The reaction employs 0.016 mol of 4-Fluorobenzoyl chloride and 0.032 mol of N-Methylthiourea.HCl, a by-product, reacts with excess N-Methylthiourea to produce a soluble thiourea salt.Post-reaction, the product is recrystallized to purify and remove impurities.The resulting crystal is yellowish-white, odorless, and soluble in DMSO, with a weight of 2.2736 grams and a recovery rate of 78.81% from the theoretical yield of 2.885 grams.

Radioiodination 131 I-(4-fluorobenzoyl-3methylthiourea)
Radioiodination is a method for labeling substances using γ-emitting iodine radioisotopes, producing high-specific compounds with low concentrations.It involves oxidizing Sodium Iodide to I + , which forms hydrated iodonium ions or hypoiodic acids through electrophilic substitution processes 29,30 .The compound 1-(4-Fluorobenzoyl-3-methylthiourea) was not labeled directly by iodine-131 due to the same chromatogram profile.Instead, it was labeled indirectly using conjugation with histamine.This indirect labeling was achieved through radioiodination, where histamine was labeled with Iodine-131 to 131 I-histamine.The compounds were tested for radiochemical purity.This is different from the in-silico results where the 1-(4-Fluorobenzoyl-3-methylthiourea) compound can be characterized directly by iodine-131.This is because in the in-silico study, the conditions used are optimal conditions where iodine-131 can bind to the 1-(4-Fluorobenzoyl-3-methylthiourea) compound.In practice, the optimal conditions that have been adjusted in silico cannot be achieved due to the limitations of nonoptimal conditions as in the in-silico setting.Ligand conditions that still have impurities can also affect the direct labeling results.

Selection of Chromatographic System for Determination of Radiochemical Purity of 131 I-(4fluorobenzoyl-3-methylthiourea)
The initial step in radiolabeling 131I-(4-Fluorobenzoyl-3-methylthiourea) involves selecting suitable mobile and stationary phases to separate the compound from its impurities, mainly free Iodine-131 and reduced Iodine ( 131 I2).The effectiveness of this separation is assessed using paper chromatography.Trials with various eluents revealed that a chloroformethanol mixture can distinguish 131 I2 from the labeled 131 I-(4-Fluorobenzoyl-3-methylthiourea), but it's ineffective in separating free Iodine-131.These findings are summarized in Table 8.
The electrophoresis method was used to determine the radiochemical purity of 131 I-(4-Fluorobenzoyl-3-methylthiourea) based on its chemical properties and the compounds to be separated.The results showed that free Iodine-131 with a negative charge was at the anode (right peak), while 131 I-(4-Fluorobenzoyl-3-methylthiourea) was at the cathode (left peak), and 131 I2 as a neutral charge was at the center peak, as shown in Figure 13    Conjugation time is an important parameter in labeling optimization to see how long it takes for 131 I-Histamine and 1-(4-Fluorobenzoyl-3-methylthiourea) to conjugate optimally.The variation of conjugation time was carried out at 2, 22, and 24 hours and the radiochemical purity obtained was 97.82%, 99.56%, and 99.54%, respectively.According to Figure 15, the most optimal conjugation time was at the 22nd hour with the highest radiochemical purity.To ensure stability and integrity, radiopharmaceuticals must be formulated and maintained at an appropriate pH.Ideally, the pH of parenteral radiopharmaceuticals should be the same as that of blood, i.e. pH = 7.4; however, due to the high buffering capacity of the blood, the pH can vary between 2 and 9 31 .

Effect of pH
The pH variation was carried out to see the radiochemical purity obtained from various pH.As can be seen from Figure 16, pH variations of 7, 8, and 10 were used with the respective radiochemical purity of 95.92%, 96.53%, and 87.03%.The optimal pH range used in the labeling of 1-(4-Fluorobenzoyl-3methylthiourea) compounds by iodine-131 is pH 7-8 with the most optimal pH being at pH 8.The pH of the labeled compound should ideally be around the pH of blood (7.4) intended for intravenous use 32 .To determine the optimal yield, the amount of ligand was varied to 25, 50, and 100 μg.The resulting radiochemical purity was 99.10%, 98.77%, and 96.47%, respectively.Based on the data in Figure 17, the optimum amount of 1-(4-Fluorobenzoyl-3methylthiourea) for labeling is 25 μg.The fewer ligands used, the higher the purity of the labeling results achieved.This can happen because the amount of ligand used in the reaction will affect the mole ratio between the ligand and the radioisotope.In addition, the use of fewer ligands can also help reduce the possibility of unwanted radioactive contamination in the labeled product.

Figure 9 .
Figure 9. (a) Representation of Time in Contact of 3PP0 Amino Acid Residue with Radioligand (b) Properties of Radioligands

Figure 16 .
Figure 16.Variation of Radiochemical Purity against pH Condition

Figure 17 .
Figure 17.Variation of Radiochemical Purity against the Number of Ligands.

Table 1 .
Docking method validation results

Table 2 .
Binding Affinity and Inhibition Constant Values of Docking Results Against Receptors 3ERT, 3PP0, and 5NQR

Table 3 .
Ligand-Residue Amino Acid Bonds on the 3PP0 Receptor

Table 4 .
Drug Scan Analysis

Table 5 .
Predicted Pharmacokinetic and Toxicity Profile

Table 6 .
Mean, Minimum and Maximum RMSD Values of Complex 3PP0

Table 7 .
Average, Minimum and Maximum RMSF Values of Complex 3PP0