CHEM 499 RESEARCH PAPER

Photochemical Reactions in Illuminated Aqueous Solutions of Several Thiazide

Compounds William J. Jackson and John M. Allen

Department of Chemistry, Indiana State University Terre Haute, Indiana 47809

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Photochemical Reactions in Illuminated Aqueous Solutions of Several Thiazide Compounds

William J. Jackson and John M. Allen

Department of Chemistry, Indiana State University

Terre Haute, Indiana 47809

ABSTRACT

Photochemical reactions of drugs can lead to various undesirable effects. The aim of this study was to investigate possible photochemical reactions with two drug species under continuous sunlight range illumination. Spectroscopy methods revealed high absorption in the UV-A region for two thiazide compounds; bendroflumethiazide and hydroflumethiazide. On further investigation with sunlight-range illumination, changes in spectra detail for the illuminated samples were found to undergo photochemical reaction in the form of absorption within the bendroflumethiazide and hydroflumethiazide samples (absorption occurred at 271 nm and 324 nm; and 272 nm and 327 nm respectively. Furthermore, each compound demonstrated very high molar absorptivity coefficients at each maxima indicating high absorptivity at that wavelength (bendroflumethiazide: 271 nm 21,400 M-1cm-1and 324 nm 4,020 M-1cm-1; hydroflumethiazide: 272 nm 17,340 M-1cm-1 and 327 nm 3,460 M-1cm-1). Utilizing high powered liquid chromatography (HPLC) techniques, kinetic information was gathered concerning each thiazide compound.

INTRODUCTION Drug formulations may contain photochemically active ingredients which have proven in some cases to be potentially harmful if exposed to solar or artificial UV-B (290-320 nm) or UV-A (320 – 400 nm) radiation. This depends on the way the energy is dissipated by the molecule. For instance, antibiotic drugs absorb both UV-B and UV-A radiation and transfer the energy to nearby molecules forming reactive oxygen species which cause tissue damage to cells (2). This is one example of a photochemical reaction, but a variety of reactions could occur illustrated in the following scheme:

Drug + hν            Drug* Drug* Drug + Heat Drug* Drug + hν’ Drug* Drug + hν’’ Drug* Drug + M* Drug* Products

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Thiazide compounds (commonly used as diuretics to treat hypertension) show absorption in the UV-B and UV-A region making them of interest to study for photochemical experiments. Due to the potential risk for adverse effects, a study was undertaken designed to evaluate various thiazide compounds in terms of their relative potential to undergo photochemical reactions when illuminated by sunlight-range radiation.

EXPERIMENTAL DETAILS Solutions of bendroflumethiazide and hydroflumethiazide were prepared in 50%/50% CH3CN/H2O. A double-beam UV-2101PC Shimadzu spectrophotometer was used to collect absorption spectra using 1 cm path length matched quartz cells. A 1000 W Xe arc lamp, optical bench, and sample illumination chamber were used as a “solar simulator” for illumination of samples. The lamp output was filtered through a water filter in order to remove IR radiation. Optical filters were utilized to remove wavelengths below 290 nm that are not present in terrestrial sunlight. Purified water used for preparing aqueous solutions was produced by the use of in-house deionized water purified by a Barnstead E-Pure laboratory water purification system with an activated carbon cartridge providing water of > 18 MΩ resistivity. UV-Visible spectroscopy was used to monitor the loss of the drug compounds during photochemical experiments by following changes in absorbance. All methods followed similar methodology outlined in Allen and colleagues’ paper detailing the observation and analysis in the illumination of aqueous solutions of several commercially sunscreen active ingredients (2). The aqueous thiazide solutions were prepared in volumetric flasks by dissolving solid thiazide compounds in 50%/50% CH3CN/H2O to make a 1x10-3 M stock solution for each sample. Various diluted solutions were made thereafter for spectra analysis to determine the molar absorptivity coefficient for each compound (Figs 1 – 4). Very dilute solutions (1x10-6 M) were then prepared for kinetics analysis.

RESULTS Absorption Spectra. The double-beam UV-Visible spectrophotometer used in these experiments provided plots of absorbance versus wavelength throughout the UV and visible regions. A blank containing the solvent (50%/50% CH3CN/H2O) was used to correct absorbance data for any reflection or scattering losses. The Absorption Spectra can be viewed in Appendix – Absorption Spectra. Both of the studied compounds exhibited strong absorbances in the UV region with peaks centered upon the following wavelengths:

Bendroflumethiazide: 271 nm and 324 nm Hydroflumethiazide: 272 nm and 327 nm

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Molar Absorptivity Data. By measuring the absorbance at the wavelengths listed above for solutions at several concentrations for each compound, molar absorptivities were calculated for both of the studied compounds using Beer’s law. The molar absorptivities were determined from the slope of each plot as depicted in the graphs found in Appendix- Molar Absorptivity Data. The calculated values for molar absorptivities for each compound are listed below:

Bendroflumethiazide: 271 nm 21,400 M-1cm-1 and 324 nm 4,020 M-1cm-1 Hydroflumethiazide: 272 nm 17,340 M-1cm-1 and 327 nm 3,460 M-1cm-1

The relatively high values for molar absorptivities indicate that each compound absorbs very strongly at the listed wavelengths. Thus, preparation of very dilute solutions (1x10-6 M) of these compounds was necessary for kinetic studies under first-order conditions were very low absorbance is required. General Photochemical Kinetics Experimental Techniques. A 1x10-6 M solution of each compound was transferred to a Teflon stoppered 1 cm path length quartz cuvette and placed in the sample chamber of the solar simulator. An electric shutter controlled by a timer was used to ensure precise control over the illumination times. Each solution was then illuminated for 5 minutes, the quartz cuvette was transferred to the UV-Visible spectrophotometer, and the absorption spectrum was recorded. This was repeated every 5 minutes until each solution was illuminated for a total of 30 minutes. These spectra can be found in Appendix – Kinetics Graphs.

DISCUSSION

The graphs indicate a steady decrease in the absorbance at the λmax demonstrating loss of the drug molecule. Although the graphs indicate loss of the drug molecules, the data derived from measurements of the absorption spectra as a function of illumination time were inconclusive. The kinetic plots did not exhibit the expected first-order behavior. Unfortunately, the products derived from photochemical reactions also appear to absorb at the same wavelengths as the drug compounds themselves. This resulted in the very complex absorption spectra for the illuminated solutions which were difficult to interpret.

CONCLUSIONS & SUMMARY The photoproducts derived from illumination of the thiazide compounds in solution must be separated from the parent compounds in order to obtain useful results. This will require the use of HPLC. This approach should allow an estimate to be made of the rate at which these compounds are consumed by photochemical reactions in solution. Energy transfer from the thiazide drug molecules to molecular oxygen may yield a variety of reactive oxidant species including singlet molecular oxygen (1O2) and organic peroxyl radicals (RO2·) which have been shown to cause oxidative damage to biological systems (1). Damage from reactive oxygen species has been observed for amino acids,

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nucleic acids, proteins, and lipids. Further, the formation of 1O2 is frequently the first step in adverse biological photosensitivity reactions with many drugs.

APPENDIX Absorption Spectra

Figure 1

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Figure 2

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Molar Absorptivity Data

y = 2.1400E+4x – 1.52E-2

slope = 2.1400E+4

Figure 3

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y = 4.020E+3x - 6.2E-3

slope = 4.020E+3

Figure 4

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y = 1.7340E+4x + 6.E-3

slope = 1.7340E+4

Figure 5

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y = 3.460E+3x + 4.E-3

slope = 3.460E+3

Figure 6

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Kinetics Graphs

Figure 7

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Figure 8

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References 1R.S. Ray, S. Mehrotra, U. Shankar, G. Suresh Babu, P. C. Joshi, and R. K. Hans. 2001. Evaluation of UV-Induced Superoxide Radical Generation Potential of Some Common Antibiotics. Drug and Chemical Toxicology, 24(2), 191-200. 2Allen, J.; Gossett, C.; and Allen S. 1996. Photochemical Formation of Singlet Molecular Oxygen in Illuminated Aqueous Solutions of Several Commercially Available Sunscreen Active Ingredients. Chem. Res. Toxicol., 9(3), 605-609.