ABSTRACT

Objectives:

Triazolopyrimidinones are compounds used in medicinal chemistry. In this study, three novel triazolopyrimidinone derivatives were synthesized as drug candidates: (5-(chloromethyl)-2-(4-methoxyphenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(3H)-one) (S1-TP), 2-(4-methoxyphenyl)-5-(piperidinomethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(3H)-one) (S2-TP), and 2-(4-methoxyphenyl)-5-(morpholinomethyl)-[1,2,4]triazolo[1,5-a] pyrimidin-7(3H)-one) (S3-TP). Their electrochemical properties were investigated for the first time using voltammetric techniques on carbon graphite electrodes. Moreover, stability tests for each drug candidate were performed on different days. After revealing the electrochemical properties of the drug candidates, their effect on double-stranded (ds) DNA was examined by measuring the oxidation currents of the guanine of dsDNA before and after the interaction.

Materials and Methods:

An electrochemical setup that included a pencil graphite electrode as the working electrode, an Ag/AgCl reference electrode, and a platinum wire as the auxiliary electrode was used in this study. Experiments for optimum pH, scan rate, and concentration of drug candidates were conducted. The interaction between Ss-TP and dsDNA was evaluated using differential pulse voltammetry. The stability of each drug candidate was tested on various days.

Results:

A comprehensive characterization of the S1-TP, S2-TP, and S3-TP compounds was performed for the first time. This study showed that the electrochemical oxidation of S1-TP and S2-TP was irreversible and diffusion-controlled. In addition, the transfer of electrons in S3-TP was controlled by adsorption. The interaction between Ss-TP and dsDNA resulted in notable changes in the peak potentialof dsDNA. The dsDNA peak potential shifted negatively after interaction with S1-TP, S2-TP, and S3-TP. Under optimum conditions, the detection limits for S1-TP, S2-TP, and S3-TP were 1.5 µg/mL, 1.0 µg/mL, and 2.0 µg/mL, respectively.

Conclusion:

From our experimental data, we concluded that these molecules can be used as drug molecules because of their remarkable effects on DNA.

INTRODUCTION

Triazolopyrimidinones, a class of fuzed pyrimidinone-triazole heterocyclic ring systems, are considered to be privileged scaffolds in medicinal chemistry. A wide range of bioactivities of triazolopyrimidinone-bearing compounds has been reported as FABPs, ferrochelatase, and per-arnt-sim kinase inhibitors.^1,2 In addition, triazolopyrimidinones and triazolopyrimidines with purine bioisosteric analogs have been reported to have anticancer activity through various mechanisms.³ Fandzloch et al.⁴ reported that triazolopyrimidine ruthenium (II) complexes show anticancer activity on various cancer cell lines, and these complexes bind to the minor groove of DNA or intercalate it. In another study, triazolopyrimidine copper (II) complexes and their DNA intercalating capacity were analyzed using absorption and fluorescence spectra.⁵ The results suggested that the complexes were intercalated into DNA strands and damaged through metallonuclease activity. Harrison et al.⁶ discovered a selective and highly soluble triazolopyrimidine derivative molecule as an NLRP3 inflammasome inhibitor using an in silico pharmacophore model, which could be used as an inhibitor for the treatment of inflammatory diseases.

Drugs and drug candidates can interact with DNA in several ways, and the interaction between them can be determined using various instrumental methods such as circular dichroism (CD), nuclear magnetic resonance (NMR), fourier transform infrared spectroscopy (FTIR), viscosity measurements, infrared spectroscopy (IR), mass spectrometry (MS), molecular docking, and electrochemical methods.^{7,8,9,10,11,12,13,14,15} Morawska et al.¹¹ developed a voltammetric method to observe the electrochemical behavior of tenofovir and its interaction mechanism with ds/ssDNA and compared their method with spectrophotometric analyses, where the electrochemical method showed better analytical performance compared with spectrophotometry in terms of limit of detection (LOD) and linear range. In another study, the interaction mechanism of mitoxantrones and DNA molecules was studied using FTIR, ultraviolet-visible spectroscopy (VIS), and CD. According to spectroscopic results, mitoxantrone possibly binds to DNA from guanine (N7), thymine (O2), and cytosine (O2) locations.¹⁶ Electrochemical methods are preferred because of their rapidness, high selectivity, low instrumentation cost, simple operation, and portability. They could reveal the chemical properties and potential toxic effects of drug candidates and determine the metabolic processes.¹⁷

The main interaction modes between the drug molecules and DNA can be simply classified as covalent and non-covalent binding. Among them, covalent binding with DNA is irreversible, inhibits the functions of DNA, and leads to cell death. In contrast, non-covalent binding is reversible, and they are subclassified as electrostatic, groove, and intercalative bindings. Electrostatic binding results from the interactions of positively charged ligands with the negatively charged DNA phosphate backbone structure. Groove binding is also sub-categorized as minor and major groove binding in which small ligands bind to the minor or major groove of DNA by van der Waals or hydrogen bonds. Small ligands can bind to DNA via unique binding sites, and this mode is called intercalation. In this mode, intercalators containing planar heterocycle groups could slide and stack between base pairs of DNA and stabilize the duplex without breaking the base pair or forming covalent bonds.

Our study is the first to analyze the electrochemical properties of novel triazolopyrimidine derivatives as purine analog chemical structures. First, we investigated the electrochemical properties of novel drug candidates, and then their interaction with dsDNA was analyzed using voltametric methods as differential pulse voltammetry  (DPV) and cyclic voltammetry (CV). Experimental parameters, e.g., pH, concentration of drug candidates, and scan rate, were examined to reveal the analytical properties of these novel drug candidates. Stability tests were performed under optimal storage conditions and within different days to observe the shelf life of the drug candidates.

MATERIALS AND METHODS

RESULTS AND DISCUSSION

CONCLUSION

In conclusion, in this article, we for the first time studied the full characterization of S₁-TP, S₂-TP, and S₃-TP compounds. Importantly, S₁-TP is reported for the first time in the literature, whereas S₂-TP and S₃-TP compounds were reported in the literature only for their screening activity against FABPs (Patent no; WO 2010/056630 A1). We have introduced the synthetic pathway and full spectral characterization data of these compounds to the literature. Our study focused on the electrochemical behaviors of Ss-TP and their interactions with dsDNA using DPV and CV. We showed that the interaction of Ss-TP-dsDNA resulted in significant changes in the dsDNA peak potential. The dsDNA peak potential shifted negatively after interaction with S₁-TP, S₂-TP, and S₃-TP. The shift of the dsDNA peak potential reveals the interaction of Ss-TP with DNA supporting the binding in between. Moreover, the shift in the peak potential of dsDNA toward more positive values indicates that the DNA-drug interaction mechanism is intercalation, whereas the shift toward more negative values indicates that the DNA-drug interaction mechanism is an electrostatic mode. Our study also showed that the electrochemical oxidation processes of S₁-TP and S₂-TP were irreversible and controlled by diffusion. In addition, the electron transfer process was an adsorption-controlled process for S₃-TP. We believe our study can provide critical information for understanding the DNA-drug interaction, which could be very advantageous for analyzing new drug compounds and their potential effects on target biomolecules.

Materials

Salmon sperm DNA used in this experiment (Sigma Aldrich, purity ≥ 98%) was dissolved in deionized water to prepare stock solutions. 3-Amino-5-(methylthio)-4H-1,2,4-triazole (Merck, purity ≥ 97%) and chloroform (Alfa Aesar-Acros Organics, purity ≥ 99%) were used as received without further purification. The buffers were prepared using analytical grade chemicals from various companies such as Carlo Erba, Alfa Thermo Fisher Scientific, and Isolab. In the experiments, we used 0.5 M acetate (ACB, pH: 3.8, 4.8, 5.6) and 0.05 M phosphate (PBS, pH: 7.4) buffers containing 0.02 M NaCl and 0.05 M Tris-EDTA (TE, pH: 8.0) buffer.

Instrumentation

Analytical thin layer chromatography (TLC) was performed using Merck silica gel F-254 plates. Melting points were employed with Stuart SMP 30 (Staffordshire, ST15 OSA). NMR spectra were reported with a Varian AS 400 Mercury plus NMR (Varian Inc.) spectrometer at 400 MHz for ¹H and 100 MHz for ¹³C using DMSO-d6 as the solvent. The coupling constants (J) are presented in Hertz (Hz) without an internal standard. Splitting patterns were designated as follows: s (singlet); d (doublet); t (triplet); p (pentet); and m (multiplet). HR-MS was performed on Agilent 6200 Series TOF and 6500 Series Q-TOF LC/MS System with ESI (+) ionization. Microwave irradiation (MW) synthesis of the compounds was conducted on a Milestone MicroSYNTH (Milestone S.r.l.) microwave apparatus. A PalmSens4 handheld analyzer with PSTrace 5.8 software was used in electrochemical studies. Pencil graphite electrodes were employed as the working electrodes. To complete the three-electrode system, a platinum wire and an Ag/AgCl electrode were employed as auxiliary and reference electrodes, respectively.

Experimental

General synthesis of the drug candidates

The synthesis steps are shown in Figure 1.

Synthesis of the substituted triazole

5-(4-Metoxyphenyl)-3-amino-1,2,4-triazole was synthesized according to reference.¹⁸ In the first step, aminoguanidine bicarbonate and 4-metoxybenzoyl chloride reacted. Later, the amide derivative was cyclized to yield 5-(4-metoxyphenyl)3-aminotriazole.

Synthesis of S₁-TP

5-(4-Metoxyphenyl)-3-amino-1,2,4-triazole (10 mmol) and ethyl 4-chloroacetoacetate (20 mmol) were mixed in 18 mL acetic acid (MW, 20 min, 180 °C). The formed solid was filtered, rinsed with acetic acid, and dried. These steps yielded S₁-TP (5-(chloromethyl)-2-(4-methoxyphenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(3H)-one, which was used in the next steps without purification.

Synthesis of S₂-TP and S₃-TP

In the last step of the synthesis, the nucleophilic substitution of the obtained S₁-TP with piperidine and morpholine yielded S₂-TP and S₃-TP. S₁-TP (1 mmol) and amine derivatives, namely, piperidine/morpholine (2 mmol), were stirred in 16 mL dimethylformamide (DMF) in the presence of 1.5 mmol caesium carbonate (Cs₂CO₃) using MW irradiation (150 W, 15-30 min, 95 °C). The excess Cs₂CO₃ was filtered, and the filtrate was concentrated under reduced pressure. This mixture was further purified by column chromatography over silica gel 60 (70-230 mesh American Standard Test Sieve Series, Merck) with chloroform: methanol (10:2), and the compounds were recrystallized from methanol or acetone. The crude yield was 40-45%.

S₂-TP: 2-(4-Methoxyphenyl)-5-(piperidinomethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(3H)-one

S₃-TP: 2-(4-Methoxyphenyl)-5-(morpholinomethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(3H)-one

Electrochemical investigation of drug candidates

+ 1.4 V potential for 30 s was applied to activate and clean the PGEs. 1000 µg/mL was prepared with TE buffer and diluted with ACB. 1000 µg/mL) were prepared in DMF and diluted with appropriate buffers. These solutions were then added to the electrochemical measuring cell. Activated PGEs were dipped in these solutions, and DPV measurements were performed.

Interaction

Solutions containing 50 µg/mL of dsDNA and 10 µg/mL of drug candidates were mixed in ACB (pH: 3.8 for S₁-TP/DNA, S₂-TP/DNA; and pH: 5.6 for S₃-TP/DNA). The solutions were then placed in a thermal shaker at 600 rpm and 45 °C for 30 min. Then, 100 µL of this interaction solution was added to the tubes. The PGEs were immersed in the interaction solutions for 30 min. Then, DPV measurements were performed.

Measurement

DPV and CV measurements were performed from + 0.4 and + 1.4 V at a scan rate of 100 mV/s with 0.5 s interval time. The experimental steps are illustrated in Figure 2.

Synthesis of the compounds

Three novel drug candidates were synthesized follows:

5-(Chloromethyl)-2-(4-methoxyphenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(3H)-one (S1-TP)

Yellow solid; yield, 52 %; m.p., 113 °C; ¹H-NMR (DMSO-d_6, 400 MHz) δ 8.02 (d, J= 8.7 Hz, 2H), 7.07 (d, J= 8.8 Hz, 2H), 6.16 (s, 1H), 4.66 (s, 2H), 3.81 (s, 3H) ppm; ¹³C-NMR (DMSO-d_6, 100 MHz) δ 161.6, 156.1, 151.8, 128.7, 114.8, 100.4, 55.8ppm; C₁₃H₁₁ClN₄O₂ HRMS m/z: 290.0552 (Calcd for 290.05705)

2-(4-Methoxyphenyl)-5-(piperidinomethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(3H)-one (S2-TP)

Yellow solid; yield, 30 %; m.p., 128 °C; ¹H-NMR (DMSO-d₆, 400 MHz) δ 8.01 (d, J= 8.7 Hz, 2H), 6.99 (d, J= 8.8 Hz, 2H), 5.63 (s, 1H), 3.79 (s, 3H), 3.30 (s, 2H), 3.23 (s, 2H), 2.34-2.42 (m, 2H), 1.50 (p, J= 5.5 Hz, 4H), 1.34-1.43 (m, 2H) ppm; ¹³C-NMR (DMSO-d₆, 100 MHz) δ 162.39, 160.28, 159.72, 158.74, 128.12, 125.96, 114.21, 94.35, 65.34, 55.59, 54.70, 26.19, 24.47 ppm; C₁₈H₂₁N₅O₂ HRMS m/z: 339.16979 (Calcd for 339.16952).

2-(4-Methoxyphenyl)-5-(morpholinomethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(3H)-one (S3-TP)

Yellow solid; yield, 35 %; m.p., 103 °C; ¹H-NMR (DMSO-d_6, 400 MHz) δ 8.02 (d, J= 8.9 Hz, 2H), 6.99 (d, J= 8.9 Hz, 2H), 5.66 (s, 1H), 3.79 (s, 3H), 3.53-3.65 (m, 4H), 3.28 (s, 2H), 2.37-2.47 (m, 4H) ppm; ¹³C-NMR (DMSO-d_6, 100 MHz) δ 161.27, 160.51, 160.34, 159.55, 158.65, 128.16, 125.82, 114.23, 94.76, 66.76, 64.72, 55.59, 53.91 ppm; C₁₇H₁₉N₅O₃ HRMS m/z: 341.14814 (Calcd for 341.14879).

The designed compounds were synthesized in three steps (Figure 1). The structures of the final compounds were determined by spectral analyses the spectroscopic data confirmed the proposed structures. In the ¹H NMR spectra, the hydrogen atom of the heterocyclic ring was observed as a singlet signal between δ 5.63-6.16 ppm. The methylene protons, attached to the heterocyclic ring at 5 positions, were observed as singlets with two proton integrals between δ 4.66-3.28 ppm. The proton signals of the benzene ring and cyclic amines were identified in the expected chemical shifts with expected divisions. The observed carbon signals in the ¹³C NMR of the compounds were in accordance with those of the target compounds. The amide carbon signal was observed between δ 161-162 ppm in ¹³C NMR spectrums.

The purity of the compounds was determined using HRMS spectra. The HRMS data were in accordance with the molecular formula and a found value within 0.003 m/z unit of the calculated value of a parent-derived ion. S₂-TP and S₃-TP were introduced earlier in the literature,¹⁹ while the full spectral characterization of these compounds was introduced in this article.

Electrochemical properties of the drug candidates

In this section, the electrochemical behaviors of the drug candidates were analyzed using DPV. Because pH is important for the metabolism of drug molecules, the effect of pH on the oxidation signals of drug candidates was examined, and the obtained results are shown in Figure 3.

For the pH study in DPV, drug candidates were prepared using buffers with pH ranging from 3.8 to 7.4. As shown in Figure 3A, S₁-TP and S₂-TP produced stable responses, and the highest currents were obtained at pH 3.8. S₃-TP showed the highest electrochemical signal at pH 5.6. Thus, pH 5.6 was chosen as the dilution buffer for S₃-TP (Figure 3A).

At pH 3.8, the oxidation peak potentials of S₁-TP were detected at 1.03 V and 1.15 V. Because it is more stable and higher, the signal obtained at 1.03 V was chosen as the main oxidation signal for further studies. At pH 3.8, S₂-TP produced two oxidation signals at 0.79 V and 1.11 V. At pH 5.6, the oxidation peak potentials of S₃-TP were observed at 0.76 V and 1.04 V (Figure 3B). All oxidation signals shifted to lower potentials with pH. These shifts in the peak potentials of drugs demonstrate that protons participate in the oxidation process of drugs.²⁰

As shown in Figure 3B, all the drug candidates have oxidation capacity. The triazolopyrimidinone structure could form triazolopyrimidine by H atom shifting and tautomerization. Several factors could contribute to the stability between the two tautomers, e.g., substitution, aromaticity, hydrogen bonding, and solvation. The redox mechanism of these novel agents on graphite electrode surfaces could involve the oxidation of the phenol groups of the compounds. Heteroatoms in the substituted amines might also contribute to the oxidation potency of the compounds, e.g., they could change the oxidation potency. Considering the phenolic tautomerization of the heteroaromatic ring and the amine substitution, the title compound could possess oxidative properties.

In the second part of our study, DPV measurements were performed at different concentrations of drug candidates at 100 mV/s to determine the analytical concentration ranges for the drug candidates (Figure 4).

The formulas for calculating the LOD and limit of quantification (LOQ) depend on the specific method used, but generally, the LOD is calculated as 3 times the standard deviation of the response (ss) divided by the slope of the calibration curve (m), while the LOQ is calculated as 10 times the standard deviation of the response divided by the slope of the calibration curve’.

LOD = 3 s/m

LOQ = 10 s/m

LOD and LOQ were determined from Figure 4. For S₁-TP and S₂-TP, LOD and LOQ were calculated from the concentrations 5, 10, 15, and 20 µg/mL. For S₃-TP, LOD and LOQ were calculated from the concentrations 2.5, 5, 10, and 20 µg/mL. LOD and LOQ for S₁-TP are 1.5 µg/mL and 5.0 µg/mL, respectively (Figure 4A). The LOD and LOQ for S₂-TP are 1.0 µg/mL and 3.4 µg/mL, respectively (Figure 4B). The LOD and LOQ for S₃-TP are 2.0 µg/mL and 6.8 µg/mL, respectively (Figure 4C). The correlation coefficients were 0.9990, 0.9988, and 0.9930 for S₁-TP, S₂-TP, and S₃-TP, respectively.

The effects of scan rate (V) on peak current (I_p) were studied using a CV between 25 and 150 mV/s, as shown in Figure 5.

As shown in Figure 5A, the anodic peak current (Ip_a) has a linear relationship with the scan rate (ν):

S₁-TP: Ip_a (µA) = 0.374v + 17.666 (R²= 0.9990) (Equation 1)

S₂-TP: Ip_a (µA) = 0.1316v + 6.2628 (R²= 0.9880) (Equation 2)

S₃-TP: (µA) = 0.2254v + 2.2687 (R²= 0.9980) (Equation 3)

As shown in Figure 5B, the peak current (Ip_a) also has a linear relationship with the root of the scan rate (ν^1/2):

S₁-TP: Ip_a (µA) = 203.97v^(1/2) − 7.7443 (R²=0.9928) (Equation 4)

S₂-TP: Ip_a (µA) = 72.771v^(1/2) − 2.9923 (R²=0.9977) (Equation 5)

S₃-TP: Ip_a (µA) = 122.95v^(1/2) − 13.05 (R²=0.9920) (Equation 6)

In Figure 5C, log(Ip_a) and log(ν) linear relationship is presented within the scan rate range between 25 and 150 mV/s:

S₁-TP: log Ip_a = 0.5716 log v + 2.3213 (R²=0.9937) (Equation 7)

S₂-TP: log Ip_a = 0.5996 log v + 1.899 (R²= 0.9987) (Equation 8)

S₃-TP: log Ip_a = 0.8568 log v + 2.2551 (R²= 0.9978) (Equation 9)

According to the literature, these slope values are close to the theoretical value, e.g., 0.5, indicating diffusion-controlled processes, whereas for the theoretical value 1, the process is adsorption-controlled.²¹ The slopes of Equations 7 and 8 were determined to be 0.5716 and 0.5996, respectively, which indicates that electrochemical oxidation of S₁-TP and S₂-TP are diffusion-controlled processes. According to Equation 9, the slope is 0.8568, which proves that the electrode process was adsorption controlled for S₃-TP.

Interaction

The intrinsic electroactivity of adenine and guanine bases is generally used as an indicator of drug-DNA interactions. In the next step of our study, we studied the interaction of drug candidates with dsDNA. 100 µg/mL were prepared with TE buffer (pH: 8.0).

The stock drug solutions were prepared with DMF and diluted with ACB (pH: 3.8 for S₁-TP and S₂-TP, and pH: 5.6 for S₃-TP). The final concentrations of dsDNA and Ss-TP were 50 µg/mL and 10 µg/mL, respectively. All solutions were placed in a thermal shaker, where stirring was applied at 600 rpm and 45 °C for 30 min. Then, 100 µL of the solution was transferred into tubes, and the electrodes were immersed in these tubes for 30 min. Oxidation signals were measured with DPV in the range between 0 and 1.4 V at a scan rate of 50 mV/s in the absence and presence of drug candidate molecules. Analytical signals associated with the guanine bases of DNA were obtained at ~1.0 V vs. Ag/AgCl.

In Figure 6A, two distinct oxidation signals associated with dsDNA in ACB (pH: 3.8) were obtained at + 0.92 V and + 1.17 V. After interaction with S₁-TP, the peak potential of dsDNA shifted to + 0.89 V and + 1.16 V, respectively. On the other hand, after interaction with S₂-TP, one of the peak potentials of dsDNA shifted negatively from + 0.92 V to + 0.89 V. Oxidation signals of dsDNA in ACB (pH: 5.6) were obtained at + 0.92 V and + 1.18 V. In the presence of S₃-TP, the peak potentials of dsDNA slightly shifted toward smaller values, e.g., + 0.90 V and + 1.16 V (Figure 6C). dsDNA oxidation potentials shifted negatively after interacting with S₁-TP, S₂-TP, and S₃-TP. Here, positive or negative shifts in peak potential can reveal the interaction mechanism between the drug candidate and dsDNA. Positive peak potential shifts are associated with intercalative binding, whereas negative peak potential shifts are associated with electrostatic binding.²² Bilge et al.²³ reported the interaction mechanism between ibrutinib (IBR) and dsDNA using electrochemical and molecular docking techniques. Voltametric studies have indicated that the peak potential of IBR shifts toward less positive potentials because of electrostatic interaction.²³ In our study, the shift in the peak potential toward negative could be attributed to the irreversible electrode process.²⁴ The reason for this shift could be explained by the electrostatic binding between Ss-TP and dsDNA.

In Figure 6A, the peak currents of dsDNA were found as 3.95 µA and 4.79 µA with the relative standard deviation (RSD) 5.45%, 3.76% which decreased to 1.30 µA and 3.11 µA after the interaction with RSD 3.12%, 4.87% (n= 5). In Figure 6B, the peak currents of dsDNA were found as 4.94 µA and 4.86 µA with RSD 1.68% and 4.54%, respectively, which increased, e.g., 6.92 µA with RSD 3.95% and 7.62% and 8.08 µA with RSD 1.49% and 5.23%, due to the binding of S₂-TP to dsDNA, changing the dsDNA structure. Figure 6C demonstrates that S₃-TP caused a significant change in the oxidation currents of dsDNA. Here, the peak currents of dsDNA are as 5.42 µA with RSD 2.97% and 5.58 µA with RSD 7. 66%, while after interaction, we determined three peaks at 3.07 µA with RSD 5.22%, 1.66 µA with RSD 9.13%, and 5.03 µA with RSD 4.68%.

Stability

Stability is one of the most important factors related to the efficacy of drug candidates. To evaluate the stability of the drug candidates, we performed DPV (Figure 7). Stock solutions of drug candidates were freshly prepared and stored in the dark at room temperature (25 °C). Stock solutions of drug candidates measured within 0, 5, 7, 14, and 30 days. Here, S₁-TP and S₃-TP exhibited good stability for 30 days of storage without significant percentage changes in current values, e.g., we observed a slight reduction in the percentage of current for S₁-TP between days 0 and 30. At the end of day 30, the percentage of the current values for S₁-TP was determined to be 91%. A noticeable change in the percentage current of S₂-TP was observed on day 30, i.e., 48%. In contrast, minute changes in the current values of S₂-TP were observed between days 7 and 30, e.g., the current value of S₃-TP decreased to 86% at day 30. These results proved that the stock solutions of drugs were stable for 30 days, except S₂-TP, which could be very advantageous for long-term use by retaining their pharmaceutical properties.