STABILITY EVALUATION OF CAFFEINE-8-THIOGLICOLIC ACID AMIDES, WITH DETERMINED ANTIHYPOXIC EFFECTS

This study evaluates a series of caffeine-8-thioglycolic acid amides that were synthesized in the study, for signs of possible degradation. The chemical stability of the test compounds was examined under different conditions of pH and temperature over time. A modified reversed phase-high-performance liquid chromatography method was applied to determine stability and identify possible degradation products. The study identified a new product from oxidative destruction of the test compound through controlled synthesis. UDC Classification: 615.3; DOI: http://dx.doi.org/10.12955/cbup.v5.1107


Introduction
Substituted xanthine derivatives are a class of biologically active substances that are well-defined and widely studied in terms of their pharmacological activity as adenosine receptor antagonists, phosphodiesterase inhibitors, and inducers of histone deacetylase activity (Kalla et al., 2006;Lin et al., 2006;Ito et al., 2002). Due to these and several other known factors, this group have become broadly used in medicine not only as agents affecting the central nervous system, but also as active substances in the field of asthma, bronchitis, and chronic obstructive pulmonary diseases and as diuretics, cardiac stimulants, and renal protective agents (Caramori et al., 2003;Dal Piaz & Giavannoni, 2000;Kiesman et al., 2006). In developing pharmacologically active substances, the stability of an agent is highly relevant because it relates to the duration in which the product maintains its physical, chemical, microbiological, and toxicological properties within previously established limits (Almeida et al., 2013). To evaluate stability, samples of the designed structures are subjected to conditions of temperature, humidity, and light that are known to accelerate the rate of chemical degradation and thus interfere with the microbiological and toxicological quality of the substances (Baby et al., 2004). A preliminary stability study provides information on the behavior of the synthesized molecules for a given period while subjected to different environmental conditions (Almeida et al., 2013). The subject of our study is to evaluate the chemical stability of caffeine-8-thioglycolic acid amides (that were synthesized in the study) under certain conditions of temperature, pH, and presence and absence of air.

Results and Discussion
The test products were synthesized according to a procedure described elsewhere (Mitkov et al., , 2010; Figure 1). Figure 1: The general procedure for synthesis of the test caffeine-8-thioglycolic acid amides 2a-p.

Evaluation of the Stability of the Synthesized Caffeine-8-Thioglycolic Acid Amides
To study the properties of the resulting amide derivatives, the behavior of the structures was followed for degradation processes at different values of pH over time.
For this purpose, compound 2b ( Figure 1) was selected as a model structure, since it was the least spatially hindered amide. The lack of shielding substituents indicated that this amide would be the most susceptible to hydrolysis. Based on the synthesis approach for obtaining the desired caffeine-8-thioglycolic acid amides and the chemical properties of the amide group, a hydrolytic cleavage of the amide bond and formation of the first caffeine-8-thioglycolic acid was expected. The analyzed product, 2b, and the expected caffeine-8-thioglycolic acid, were chromatographically analyzed, individually as well as in a model mixture, to validate the applicability of the RP-Highperformance liquid chromatography (RP-HPLC) method modified by this study.

Validation of the Modified RP-HPLC Analytical Procedure
The method was validated according to guidelines of the International Concil for Harmonisation (ICH) of Technical Requirements for Pharmaceuticals for Human Use (ICH, Q2(R1), Harmonised Tripartite Guideline, 2005). The system suitability (i.e., repeatability of retention times and areas, the number of theoretical plates, and resolution), precision, linearity, accuracy, and selectivity were evaluated during the method validation ( Table 2). The test products were evaluated for accuracy, precision, and selectivity. Precision Six sample solutions (three of Caffeine-8-thioglycolic acid and three of 2b) were prepared and each sample was injected three times. The final results were reported as relative standard deviation (% RSD) of the ratios of the peak area of the tested compound. Linearity A 5-point calibration curve was created, covering the concentration range of 2b, from 0.02 mg ml -1 to 0.1 mg ml -1 . A linear regression of the calibration data was performed. The correlation coefficient of linearity was 0.998 (Figure 2), which indicated a high correlation between the peak areas and the range of concentrations studied. Accuracy A solution of Caffeine-8-thioglycolic acid and a stock solution of compound 2b were prepared. From these, six subsamples (three of the Caffeine-8-thioglycolic acid and three of the stock solution), allowed each solution to be injected onto the column three times. Accuracy is reported as a parameter recovery with relative standard deviations.

Selectivity
The selectivity was determined by the corresponding selectivity factor (α), measured as a ratio of the retention factors (k) of the two peaks in question and visualized as the distance between the apices of the two peaks.

Determination of Stability at Different рН
Chemical Stability By definition, chemical stability is the tendency of a substance to sustain changes or decay caused by internal reactions or effects of air, humidity, heat, light, pressure, or other external factors. The compound presented in this paper had been stored for six months at room temperature with access to air and light. The physical and chemical properties of the compound were determined to be unchanged under these conditions. Hence, the test compound was considered chemically stable.  (Figures 3-4). Stability Determination of Compound 2b at рН 1.0 A 10-mg sample of the model compound was weighed and dissolved in 100 ml of 0.01 mol l -1 HCl (pH 1.0). This solution was stirred at 38 ºC for a total time of 1440 min using liquid nitrogen to assure the absence of air. Aliquots of 20 μl samples were drawn at 30-minute intervals and injected into the RP-HPLC system. Figure 3 presents a representative chromatogram. As presented, no additional peaks were visible for the total 1440 min evaluation period under these conditions. Stability Determination of Compound 2b at рН 12.0 A 10-mg sample of the model compound was weighed and dissolved in 100 ml of 0.01 mol l -1 NaOH (pH 12.0). The obtained solution was stirred at 38 ºC for a total time of 1440 min using liquid nitrogen to assure the absence of air. Aliquots of 20 μl samples were drawn at 30-min intervals and injected into the RP-HPLC system. Figure 4 displays a representative chromatogram. As presented, no additional peaks were visible for the total 1440 min evaluation period under these conditions. These results lead to the conclusion that in both media no hydrolysis occurred within 24 hours. These evaluations were conducted in oxygen-free media. To identify the influence of oxygen on the stability of the target product, evaluations were performed in oxygenated media. The chosen model compound was incubated in both acid and alkali media for 1440 min using the above-discussed conditions, as follows: A 10-mg sample of the model compound was weighed and dissolved in corresponding buffers of pH 1.0 and 12.0, respectively. The obtained solutions were temperature-controlled and stirred in oxygenated media at 38 o C for a total time of 1440 min (24 hours). Aliquot samples of 20 μl of the analyzed solutions were drawn at 30-minute intervals and injected into the apparatus. The corresponding chromatograms were obtained (Fig.5&6). Stability Determination of Compound 2b at рН 1.0 in the Presence of Air Fig. 5 shows the effects of incubating the test compound in acid media, pH 1.0, in the presence of air. An unidentified peak with a retention time close to that of the analyzed structure was observed under the test conditions. The calculated relative retention of the unidentified peak and that of 2b were close to 1. This showed that no separation was achieved under the applied chromatographic conditions. Stability Determination of Compound 2b at рН of 12.0 in the Presence of Air After a 16-hour incubation of the test compound in alkali media, pH 12.0, in the presence of air, the same results to those for the acid media presented. Again, no separation was achieved.
In an attempt to separate and identify the new product, the chromatographic procedure was modified, by changing the mobile phase as shown in Table 3.  In applying the new conditions, a good separation was achieved, as shown in Figure 6. Based on the identified degradation product having a similar chromatographic behavior to that found for 2b, it appears the targeted amides are oxidized through exposure to air into corresponding sulfines. This process is similar to the derivation of corresponding esters from caffeine-8-thioglycolic acid .
To demonstrate this, we synthesized the probable sulfine product, according to the procedure, summarized in Figure 7. The synthesized compound was found to be freely soluble in chloroform, dichloromethane, Dimethylformamide (DMF), and DMSO, but insoluble in non-polar solvents, such as n-hexane. The structure of the synthesized S-oxide was confirmed by FTIR, 1 H-and 13 C-NMR, MS spectra, and elemental analysis. The results were consistent with the assigned structure. The 1 H-NMR spectrum of the compound revealed the signals of the respective protons, which were verified by chemical shifts, multiplicities, and coupling constants. The chemical shifts of the protons and carbon atoms, registered by 1 H-and 13 C-NMR spectra, were compared with simulated values (Banfi et al., 2008, Andrés et.al., 2011, Aires-de-Sousa et al., 2002. A strong correlation between the registered and computed values was observed. The obtained product was analyzed individually and in a model mixture with the initial 2b. Figure 8 shows the result of the analysis. An unidentified peak as a result of the degradation under acid (pH 1.0) and alkali (pH 12.0) media in the presence of oxygen is shown at the same retention time as that for the sulfine product (B; Fig.8).

Conclusion
The stability of an amide derivative of caffeine-8-thiogyolic acid, 2b, was determined under acid (pH 1.0) and alkali (pH 12.0) conditions, at a temperature of 38 o C and in the presence and absence of oxygen. A sulfine degradation product resulted in both pH condition, in the presence of oxygen only.
The structure of the degradation product was determined through controlled synthesis. The findings lead to the conclusion that the only possible degradation process for the test structure is via oxidation at 38 o C in strong acid and alkali media with exposure to air. We propose this to be the most probable metabolic process of the target compound.