Research Article - (2016) Volume 7, Issue 8

Spectroscopic Studies and Applications of the Reactions of Some Anti-Diabetic and Anti- Hypertensive Drugs with Rose Bengal

Zayed MA* and Farrag YS
Chemistry Department, Faculty of Science, Cairo University, Giza, Egypt
*Corresponding Author: Zayed MA, Chemistry Department, Faculty of Science, Cairo University, Giza, Egypt, Tel: 002-01005776675 Email:

Abstract

Simple, rapid and sensitive spectrophotometric methods were developed and validated for the microdetermination of terazosin HCl, doxazosin mesylate and pioglitazone HCl drugs in pure and pharmaceutical dosage forms. These methods are based on ion-pair formation reaction between these drugs and a chromogenic reagent Rose Bengal (RBeng). These reactions were studied under various conditions and the optimum parameters were selected. The spectrophotometric microdeterminations have been done at λmax=570 nm for terazosin HCl and pioglitazone HCl and at λmax=575 nm for doxazosin mesylate. Under proper conditions the suggested procedures were successfully applied for microdetermination of these drugs in pure and in pharmaceutical dosage forms. The values of SD, RSD, recovery %, LOD, LOQ and Sandell sensitivity refer to the high accuracy and precession of the applied procedures. The results obtained were compared with the data obtained by the official methods, referring to confidence and agreement with rose bengal procedure results. The solid drugs-reagent ion-pairs were prepared, separated and their structures were investigated using elemental analysis, FT-IR, 1H-NMR and thermal analyses and the results confirm the structures proposed by the stoichoimetric ratio in the solution work. The biological activities of the drugs and their solid ion-pairs against some types of (G-) and (G+) bacteria and fungai were studied and compared with each other. It is found that terazosin-RBeng and pioglitazone-RBeng reaction products have antibacterial effect higher than the parent drugs, but doxazosin-RBeng reaction product has almost the same antibacterial effect of the parent drug.

Keywords: Terazosin HCl; Doxazosin mesylate; Pioglitazone HCl; Spectrophotometric microdetermination; Rose Bengal Reagent; Spectroscopic study

Introduction

Terazosin (TRZ) HCl dihydrate has an IUPAC name (1-(4-amino-6, 7- dimethoxyquinazoline-2-yl)-4-[[(2RS)-tetrahydrofuran-2-yl] carbonyl] piperazine hydrochloride dihydrate [1]) and structure given in Figure 1. It has a general formula C19H25N5O4.HCl.2H2O, and mole mass=459.9 g.mol-1.

pharmaceutica-TRZ-HCl-dehydrate

Figure 1: Structure of TRZ HCl dehydrate.

Doxazosin (DOX) mesylate has an IUPAC name (1-(4-amino-6, 7- dimethoxy-2- quinazolinyl)-4-[(2, 3-dihydro-1, 4-dioxin-2-yl) carbonyl] piperazine methanesulphonate) [2] and structure given by Figure 2. It has a general formula C23H25N5O5.CH3SO3H, and mol mass=547.6 g mol-1.

pharmaceutica-Structure-DOX-Mesylate

Figure 2: Structure of DOX Mesylate.

TRZ HCl dihydrate and DOX mesylate both are highly selective potent alpha-1 adrenoreceptor antagonists used in the treatment of hypertension [3] and benign prostatic hyperplasia [4,5]. The methods available for the determination of TRZ in pure, pharmaceutical and biological samples included titrimetric method [1], high performance liquid chromatography (HPLC) [3], spectrophotometric methods [6], potentiometric sensors [7] and stripping voltammetry [8].

A number of studies were described for the determination of DOX in pure, pharmaceutical and biological samples. These methods include reversed phase (RP)-HPLC [9], liquid chromatography (LC)-mass spectrometry (MS) [10], spectrophotometric methods [11] and voltammetry [12].

Pioglitazone (PIOG) HCl has an IUPAC name (5-[[4-[2-(5-Ethyl-2 pyridyl) ethoxy] phenyl] methyl]-2, 4-thiazolidinedione hydrochloride) and structure given in Figure 3; it has a general formula C19H20N2O3S.HCl, and mol mass=392.9 g mol-1 [13].

pharmaceutica-Structure-PIOG-HCl

Figure 3: Structure of PIOG HCl.

PIOG is indicated as an adjunct to diet and exercise to improve glycemic control in adults with type 2 diabetes mellitus in multiple clinical settings [14]. It can also be used as a stopper in horseracing [15].

PIOG was determined in pure, pharmaceutical and biological samples using various methods alone or in a combination with other drug substances. These methods include HPLC [16], LC/MS/MS [17], spectrophotometric methods [18], Potentiometric Sensors [19], and voltammetry [20].

Rose Bengal (RBeng) dye has an IUPAC name (4, 5, 6, 7- tetrachloro-2', 4', 5', 7'-tetra iodo fluorescein disodium salt) and possible structure given in Figure 4. It is an anionic water soluble xanthene dye [21].

pharmaceutica-RBeng-disodium-salt

Figure 4: Structure of RBeng disodium salt.

Various spectrophotometric methods were reported; where RBeng was used as a reagent for the determination of different drugs [22,23].

This present work is concerned with the spectrophotometric study of the reaction between TRZ, DOX and PIOG drugs with RBeng reagent, and how to use these reactions as new, simple and sensitive methods for the microdetermination of these drugs. It also contains spectroscopic studies on the solid reaction products for their structures investigation and a study of their biological activities.

Experimental

Materials and reagents

All chemicals used were of analytical reagent grade (AR), and of highest purity available. They included PIOG HCl, an authentic sample was kindly supplied by Elrazy Pharmaceutical Co, Ismailia (Egypt), TRZ HCl dihydrate and DOX mesylate, and authentic samples were kindly supplied by national organization for drug and control research (NODCAR), Cairo (Egypt). Itrin tablets (Kahira Pharm. & Chem. IND. CO, Egypt, under license from ABBOTT Laboratories) labeled to contain 2 mg TRZ per tablet, Dosin (EIPICO, Egypt) labeled to contain 4 mg DOX per tablet and Glustin (Takeda Pharmaceutical Company Limited, Japan) labeled to contain 30 mg PIOG per tablet, were collected from local market in Cairo, Egypt. RBeng disodium salt reagent was supplied from BDH Chemicals Ltd, Poole, England.

Absolute ethanol (99.8%, Sigma Aldrich, Germany), phosphoric acid (88%, BDH, England), acetic acid (El Salam for chemical industries, Egypt), boric acid (ADWIC), sodium hydroxide (Merck, Germany) and distilled water, obtained from all glass equipment, were used.

Instruments

The spectrophotometric measurements in solutions were carried out using Spectrophotometer, Thermo fisher scientific, model Evolution 60 v2 recording spectrophotometer, USA, UV-Vis ranged from 190 to 1100 nm, with matched quartz cell of 1 cm optical length. Elemental microanalysis of the separated solid ion-pairs, for C, H and N were performed in the Microanalytical Centre, Cairo University using Elementar CHNS analyzer, model Vario EL III. Infrared Spectra were recorded on FTIR 4100, Jasco spectrophotometer in wavenumber region 4000-400 cm-1. The spectra were recorded as KBr pellets. The 1H-NMR spectra were recorded with a varian-300 MHz in DMSO-d6 as solvent, where the chemical shifts were determined relative to the solvent peaks. The thermal analyses (TGA, DTG and DTA) were carried out in dynamic nitrogen atmosphere (20 mL min-1) with a heating rate of 10°C min-1 using Shimadzu system of DTG-60H thermal analyzers.

Solutions

Stock solution of (1×10-3 M) of the three drugs were prepared by dissolving the accurately weighed amount of the pure drugs (0.0393 g, 0.0459 g, 0.0548 g for PIOG, TRZ and DOX, respectively) in the appropriate volume of absolute ethanol for PIOG, distilled water for TRZ and (ethanol: distilled water) mixture (1:3) for DOX and the volume was completed to 100 mL volumetric flask. Diluted solutions were prepared by accurate dilution from the stock solutions to get the desired concentrations. Solution of 1×10-3 M RBeng disodium salt was prepared by dissolving the accurately weighed amount in the appropriate volume of distilled water and the volume completed to 250 mL volumetric flask.

The universal buffer (0.04 M acid mixture of acetic, boric and phosphoric acids) solutions of different pH values (2.00 to 11.05) were prepared as recommended by Britton and Robinson [24].

Sample Solutions

Ten tablets of Itrin (2 mg/tablet) and Glustin (30 mg/tablet) were powdered well separately. Equivalent amount of powder to two tablets of Itrin and one tablet of Glustin were weighed and dissolved in sufficient amount of distilled water and absolute ethanol, respectively, with gentle warming. The resulting solutions were filtered. The solutions were transferred to 100 mL volumetric flask after cooling and the volume completed to the mark with the appropriate solvent.

For dosing (4 mg/tablet), 20 tablets were powdered well. Equivalent amount of powder to 2 tablets were weighed and dissolved in about 60 mL ethanol with gentle warming for 30 min. The resulting solutions were filtered. Ethanol was evaporated to volume equal to 5 mL. The solution was transferred to 100 mL volumetric flask after cooling and the volume completed to the mark with distilled water and filtered if necessary.

Procedures

Selection of suitable wavelength procedure: The spectra of 1×10-4 M of the three drugs and 0.25×10-4 M of RBeng were measured separately at 200-400 nm, and 450-650 nm, respectively in order to determine the λmax of each of them. On the other hand 1 mL of 10-3 M solution of RBeng was added to 1 mL of 10-3 M solution of the standard drugs solution in 10 mL measuring flask. The mixture obtained was scanned in the wavelength range 450-650 nm using the reagent as a blank in order to determine the λmax of the formed products.

Selection of suitable pH, time and temperature procedures: To select the optimum pH value; the spectra of mixture of 1×10-4 M of the standard drugs and 1×10-4 M of RBeng were measured at 450-650 nm using the reagent as a blank at different pH values using buffer solutions. A suitable amount of ethanol was added to the solutions to dissolve any formed precipitates. To select the optimum time and temperature; these mixtures were measured also at different time intervals and different temperature values using the reagent as a blank.

Effect of ethanol volume: Effect of ethanol volume was studied on DOX reaction product with RBeng; where different volumes of ethanol (0.5-5 mL) were added to mixture of 1×10-4 M of the standard drug and 1×10-4 M of RBeng. The spectrophotometric measurements were recorded using the reagent as a blank to select the optimum ethanol volume.

The stoichiometric ratio of reaction: The stoichiometry of these reactions was also studied applying Job’s continuous variation method (CVM) [25] and molar ratio method (MRM) [26].

Calibration curve: RBeng solution (1×10-3 M) was added to variable concentrations of the standard drugs (4.599-41.39 μg mL-1, 10.95-54.76 μg mL-1 and 11.79-31.43 μg mL-1 for TRZ, DOX and PIOG, respectively) under proper selected conditions and the volume was completed to 10 mL H2O. The absorbance was plotted against drugs concentrations at selected λmax=570 nm for TRZ and PIOG and 575 nm for DOX.

Within- and In-between-day measurements: The effect of long time on the spectra of the standard drugs was carried out on five replicate experiments, at different concentrations of the standard drugs under the proper selected conditions.

Application of suggested procedures: The suggested procedures were applied for microdetermination of TRZ, DOX and PIOG in pharmaceutical Itrin, Dosin and Glustin Tablets, respectively using RBeng reagent in comparison with the official methods [6,27,28] respectively. To variable concentrations of the drugs in their pharmaceutical forms (3.334-10.00, 14.55-33.96 and 13.84-24.91 μg mL-1 for TRZ, DOX and PIOG, respectively) RBeng was added under previously mentioned proper selected conditions. The spectra of the obtained mixtures were measured applying the suggested procedures.

Preparation of the solid drugs-reagent ion-pairs: The solid ion-pairs of TRZ, DOX and PIOG drugs with RBeng were prepared, by addition of a warm solution of appropriate weight of RBeng of 0.5088 g (0.5 mmol), dissolved in least amount of water; to warm solutions of 0.2300 g (0.5 mmol) TRZ, 0.2738 g (0.5 mmol) DOX, dissolved in least amount of water and 0.1965 g (0.5 mmol) PIOG, dissolved in least amount of ethanol. The resulted solid ion-pairs were appeared as precipitates. These precipitates leaved with gentle warming for 10 min, filtered, dried and recrystallized from ethanol. The melting points of these ion-pairs were measured.

Results and Discussion

Spectrophotometric studies on the reaction of RBeng with the selected standard drugs

Figure 5 shows the spectra of: RBeng 0.25×10-4 M and TRZ (10-4 M) - RBeng (10-4 M), PIOG (10-4 M) - RBeng (10-4 M), DOX (10-4 M) - RBeng (10-4 M) reaction products using reagent as a blank.

pharmaceutica-Vis-absorption-spectra

Figure 5: Vis. absorption spectra of: (1) RBeng 0.25×10-4 M, (2) TRZ (10-4 M) - RBeng (10-4 M) reaction product, (3) PIOG (10-4 M) - RBeng (10-4 M) reaction product, (4) DOX (10-4 M) - RBeng (10-4 M) reaction product.

From Figure 5, λmax=570 nm is selected as a suitable wavelength for microdetermination of TRZ and PIOG with RBeng reagent and λmax=575 nm is selected as a suitable wavelength for microdetermination of DOX with RBeng reagent, which are away from that of RBeng reagent (λmax=545 nm).

Effect of pH (2.00-11.05) on the spectrum of TRZ-RBeng and PIOG-RBeng products was studied spectrophotometrically at λmax=570 nm. The results show that the maximum absorbance attained at pH 5 for both of them with the highest molar absorptivity, ε=1.442 x 104 and 1.488 x 104 L mol-1 cm-1 for TRZ and PIOG, respectively.

Effect of temperature was studied in the temperature range 25-70°C on absorption spectrum of TRZ-RBeng and PIOG-RBeng reaction products at λmax=570 nm, pH 5 and in the range 26-45°C on absorption spectrum of DOX-RBeng reaction product at λmax=575 nm. These results show that the optimum temperatures are found to be; room temperature (25 ± 2 °C), 35 ± 1°C and 30-35 °C, for TRZ, PIOG and DOX, respectively with molar absorptivity ε =1.198×104, 1.640×104 and 2.070×104 L mol-1 cm-1, respectively.

Studying the effect of time (0-60 min.) on the formation of the three products at the selected conditions shows that the three reaction products are stable over one hour and the time has no significant effect on their stabilities.

For DOX-RBeng reaction product an extra ethanol had to be added to prevent any formation of precipitates, but this addition affects the absorbance, so ethanol volume effect was studied and the results show that the absorbance remains constant at 1.5 and 2 mL of ethanol, so ethanol volume of 2 mL is chosen as the optimum volume for the microdetermination.

Stoichiometric ratios of drugs-RBeng reaction products

The Stoichiometric ratio of RBeng reaction with the selected drugs, which depends on ion-pair formation, was studied by the CVM [25] and MRM [26]. The results obtained from the MRM of the reaction of RBeng with variable [TRZ] refer to stoichiometric ratio between RBeng reagent and TRZ drug, (R:D), equal to (1:1). For DOX, the results obtained from CVM and MRM of the reaction of DOX with variable [RBeng] refer to stoichiometric ratio (1:1) and the results obtained from the MRM of the reaction of RBeng with variable [DOX] refer to stoichiometric ratio, equal to (1:2).

For PIOG, the results obtained from CVM refer to stoichiometric ratio, equal to (1:1) and the results obtained from the MRM of the reaction of RBeng with variable [PIOG] refer to stoichiometric ratios, (1:1) and (1:2).

Depending upon these results; the proposed reaction for ion-pair formation between TRZ, DOX and PIOG drugs and RBeng reagent may be given by the schemes 1, 2 and 3, respectively.

pharmaceutica-TRZ-RBeng-ion-pair-formation

Scheme (1): Proposed reaction for TRZ-RBeng ion-pair formation

pharmaceutica-DOX-RBeng-ion-pair-formation

Scheme (2): Proposed reaction for DOX-RBeng ion-pair formation.

pharmaceutica-PIOG-RBeng-ion-pair-formation

Scheme (3): Proposed reaction for PIOG-RBeng ion-pair formation.

Calibration curve

The calibration curves of TRZ, DOX and PIOG drugs and RBeng reagent were constructed by plotting the absorbance against variable concentrations of the drugs under the selected proper conditions. These calibration curves were constructed at λmax=570 nm for TRZ and PIOG and at 575 nm for DOX. The results show that the three calibration curves are rectilinear in the concentration range of 4.599-41.39 μg mL-1, 10.95-54.76 μg mL-1 and 11.79-31.43 μg mL-1, for TRZ, DOX and PIOG, respectively (Table 1).

Parameters Drug
TRZ DOX PIOG
Reagent RBeng
Temperature (°C) Room Temperature (25 ± 2) 30–35 35 ± 1 ºC
λmax (nm) 570 575 570
pH 5 - 5
Linearity (μg mL-1) 4.599–41.39 10.95–54.76 11.79–31.43
LOD (μg mL-1) 0.7731 1.303 1.051
LOQ (μg mL-1) 2.343 3.947 3.184
R2 0.9998 0.9994 0.9982
Regression equation Y=0.0398x + 0.0044 Y=0.0334x + 0.175 Y=0.0391x + 0.0861
Molar absorptivity (L moL-1 cm-1) 1.831 x 104 1.826 x 104 1.534 x 104
SD 0.0503–0.1501 0.0650–0.4604 0.1030–0.2798
RSD % 0.1414-1.767 0.2651–1.099 0.4344–1.082
Sandell sensitivity (μg cm-2) 0.0054 0.0055 0.0065
Recovery % 98.57–101.14 97.87–101.6 98.52–101.4

Table 1: Analytical parameters for spectrophotometric determination of standard TRZ, DOX and PIOG drugs by proposed RBeng method.

From Table 1, the high values of the molar absorptivities (1.831×104, 1.826×104 and 1.534×104 L mol-1 cm-1) indicate the sensitivity of the proposed methods. The correlation coefficient values are found to be 0.9998, 0.9994 and 0.9982; which supports the linearity of the curves. Also the mean recovery values obtained are in the ranges of 98.57-101.14%, 97.87-101.6% and 98.52-101.4%; which indicate the high accuracy of the applied procedures in determination of standard TRZ, DPX and PIOG drugs, respectively. The low values of standard deviation (SD) (0.0503-0.1501, 0.0650-0.4604 and 0.1030-0.2798) and relative standard deviation (RSD %) (0.1414-1.767%, 0.2651-1.099% and 0.4344-1.082%), for n=5, indicate the high precision of the applied procedures under the selected proper conditions. The values of the limit of detection (LOD) are found to be 0.7731, 1.303 and 1.051 μg mL-1 for TRZ, DOX and PIOG, respectively, and limit of quantification (LoQ) are found to be 2.343, 3.947 and 3.184 μg mL-1 for TRZ, DOX and PIOG, respectively. The low values of Sandell sensitivity (S.S=0.0054, 0.0055 and 0.0065 μg cm-2) refer to the high sensitivity of the proposed methods. From these parameters it is concluded that, the proposed spectrophotometric methods can be applied successfully for the determination of TRZ, DOX and PIOG drugs, respectively, in the concentration range mentioned above with a high accuracy, precision and sensitivity.

Within- and In-between-day measurements

The results obtained from within- and in-between-day measurements are shown in Tables 2 and 3, respectively.

Drug [wt.] taken [wt.] found (μg mL-1) ± SD Recovery (%) SDa RSD(%)a
(μg mL-1)
TRZ 29.18 28.97 ± 0.2058 98.67–100.4 0.2058 0.7107
34.05 33.44 ± 0.3384 97.55–99.59 0.3384 1.014
DOX 32.85 33.09 ± 1.085 100.4–101.3 1.085 3.279
38.33 37.88 ± 0.9191 98.21–99.46 0.9191 2.425
41.99 42.92 ± 0.8022 101.5–103.1 0.8022 1.869
47.31 46.56 ± 0.9286 97.50–99.33 0.9286 1.998
52.56 52.37 ± 1.470 99.13–100.5 1.47 2.806
PIOG 25.54 25.69 ± 0.3228 98.99–102.7 0.3228 1.254
27.5 28.05 ± 0.1877 101.2–102.7 0.1877 0.6694
31.48 31.37 ± 0.3707 98.19–100.6 0.3707 1.18

Table 2: Within-day spectrophotometric microdetermination of standard TRZ, DOX and PIOG drugs by the proposed RBeng method. aMean values for six determinations for TRZ, four determinations for DOX and five determinations for PIOG, within 5 h.

Drug [wt.] taken [wt.] found (μg mL-1) ± SD Recovery (%) SDa RSD(%)a
(μg mL-1)
TRZ 29.18 30.46 ± 0.3421 103.0–106.9 0.3421 1.123
34.05 35.12 ± 0.3524 101.9–104.7 0.3524 1.002
DOX 32.85 32.59 ± 1.293 96.68–101.6 1.293 3.279
PIOG 25.54 25.36 ± 0.3285 98.13–100.4 0.3285 1.297
27.5 28.25 ± 0.2658 101.6–105.0 0.2658 0.9368
31.48 31.41 ± 0.6141 98.19–100.6 0.6141 1.95

Table 3: In-between-day spectrophotometric microdetermination of standard TRZ, DOX and PIOG drugs by the proposed RBeng method. aMean values of four determinations for five days for TRZ and DOX and of four determinations for six days for PIOG.

From Tables 2 and 3, the within- and in-between-day recovery percentage, SD and RSD % values for the three standard drugs indicate that, the proposed methods are reproducible and RBeng can be successfully applied for determination of standard TRZ, DOX and PIOG drugs via the proposed ion-pair formation reaction.

Application of the applied procedures in comparison with the official methods

The RBeng reagent was successfully applied for the microdetermination of TRZ in Itrin, 2 mg/tablet, DOX in Dosin, 4 mg/ tablet and PIOG in Glustin, 30 mg/tablet under proper conditions at λmax=570 nm for TRZ and PIOG and λmax=575 nm for DOX. The results obtained are compared with the official methods [6,27,28], for TRZ, DOX and PIOG, respectively and are presented in Table 4.

Sample Proposed method Official methods F-test t-test
[Drug] μg mL-1 [Drug] μg mL-1
Taken Found Recovery SDa Mean ± SD Taken Found Recovery (%)b SDb Mean ± SD
(%)a
TRZ in ItrinTablet(2mg/Tablet) 3.334 3.332 99.93 0.0208 100.52 ± 0.0521 4 3.986 99.65 0.0367 99.71 ± 0.0652 1.566 (6.256)* 22.45 (2.26)*
5.002 4.791 98.04 0.0417 8 7.978 99.73 0.0824
6.669 6.783 101.7 0.0136 12 11.99 99.95 0.0696
8.336 8.551 102.6 0.1348 14 13.93 99.5 0.0714
10 10.04 100.3 0.0498        
DOX inDosin Tablet(4mg/Tablet) 14.55 14.66 100.7 0.1349 99.56 ± 0.3463 4 3.994 99.84   99.46 ± 0.3900 1.268 (6.944)* 0.3788 (2.45)*
19.41 19.59 100.9 0.2691 6 5.938 98.96
24.26 23.78 98.03 0.3912 8 7.974 99.68
29.11 28.84 99.07 0.3712 10 9.934 99.34
33.96 33.63 99.03 0.5652      
PIOG inGlustinTablet(30 mgTablet) 13.84 13.53 97.8 0.0968 100.0 ± 0.1772 20 19.92 99.58   99.79 ± 0.1976 1.243 (6.256)* 1.837 (2.26)*
16.6 16.65 100.3 0.2005 30 29.95 99.83
19.37 19.6 101.2 0.1825 40 39.99 99.97
22.14 22.29 100.7 0.2222      
24.91 24.92 100.1 0.1841      

Table 4: Spectrophotometric microdetermination of TRZ, DOX and PIOG drugs in pharmaceutical formulations by proposed RBeng method. aAverage of five determinations. bAverage of six determinations for TRZ, of three determinations for DOX, *The values between brackets are the tabulated F- and t-values at P=0.05 and confidence limit 95% [29].

From Table 4, the values of the recovery and the low values of SD of the proposed methods indicate the high accuracy and precision of the proposed methods for the microdetermination of TRZ, DOX and PIOG drugs in the pharmaceutical dosage forms. The accuracy and the precision of the proposed methods are compared with those obtained from the official methods by student's t-test and F-test, respectively, at confidence limit 95% and P=0.05 [29]. The obtained values of F-test and t-test indicate that there is no significant difference between the accuracy and the precision of the proposed and the official methods and hence the reliability of the proposed methods for the routine analysis of TRZ, DOX and PIOG drugs in pure and in their pharmaceutical formulations.

Structure identification of drugs-RBeng products by different physicochemical methods of analyses

The structures of drugs-RBeng solid ion-pairs have been identified by different physicochemical tools which are elemental analyses (C, H, and N); IR, 1H-NMR and thermal analyses (TGA, DTGA and DTA). The products spectra are compared with those of the drugs and RBeng aiming chiefly to shed light on the mechanism of the reaction between the three drugs and RBeng in solution.

Elemental Analyses (EA) of the solid ion-pairs

The elemental analyses results, analytical and physical data of RBeng-drugs ion-pairs are given in Table 5. From these data; the general formulae of the formed solid ion-pairs are determined and their mole masses are calculated.

Ion-pair R:D m.p Elemental analysis
(ºC) Found (calcd %)
C H N
TRZ–RBeng 1:1 248 33.08 3.14 5.94
(C39H27Cl4I4N5O9) -34.36 -2.2 -5.14
Mol Mass=1359.06 - - -
DOX–RBeng 1:2 232 41.4 3.12 6.8
(C66H52Cl4I4N10O15) -42.28 -2.793 -7.473
Mol Mass=1874.62      
PIOG–RBeng 1:2 180 41.8 2.15 3.09
(C58H42Cl4I4N4O11S2) -42.32 -2.509 -3.325
Mol Mass=1685.46 - - -

Table 5: Analytical and physical data of RBeng-drug ion-pairs.

FT-IR analysis

The FT-IR of RBeng refers to the bands of ν (C=O) stretching of the carboxylate at 1550.49 and 1492.63 cm-1, these bands are shifted to lower values of wavenumbers, (1540.85-1511.92 cm-1 and 1490.7-1447.31 cm-1), for the three drugs ion-pairs [30].

The FT-IR of TRZ refers to the bands of ν (C=O) stretching of the amide at 1633.41 cm-1, and ν (C-O) stretching of the hydro-furan ring at 1111.79 and 1078.98 cm-1 [30]. These bands are shifted to lower values of wavenumber in the corresponding TRZ-RBeng ion-pair, ν (C=O) stretching of the amide at 1627.63 cm-1 and ν (C-O) stretching of the hydro-furan ring at 955.55 cm-1. The FT-IR of DOX refers to the bands of, ν (C=O) stretching of the amide at 1635.34 cm-1, ν (C-O) of the six membered ring at 1214.93 and 1171.54 cm-1 and ν (sulfoxide) stretching at 1042.34 cm-1 [30]. These bands are shifted to lower values of wavenumber in the corresponding DOX-RBeng ion-pair, ν (C= O) stretching of the amide at 1631.48 cm-1, ν (C-O) stretching of the six membered ring at 1108.87 and 1034.62 cm-1 and ν (sulfoxide) stretching is disappeared. The FT-IR of PIOG refers to the bands of ν (C-H) stretching of the aliphatic alkane at 2742.28 and 2615.97 cm-1, ν (C= O) stretching of the amide at 1742.37 and 1690.3 cm-1. These bands are shifted to lower values of wavenumber in the corresponding PIOG-RBeng ion-pair, ν (C-H) stretching of the aliphatic alkane are disappeared, ν (C=O) stretching of the amide at 1760.69 and 1701.87 cm-1. The data of the IR spectra of RBeng, the three drugs and their reaction products are listed in Table 6.

.
Compound C-Hstreching ofaliphaticalkane C=Ostreching ofthe amide C=Ostreching ofcarboxylate C-Nstreching C-O ofsixmembered ring C-Ostrechingofhydrofuranring Sulfoxidestreching C-OStreching ofcarboxylate
RBeng - - 1550.49and1492.63 - - - - 950.734
TRZ 2963.09 and2682.5 1633.41 - 1283.39and1244.83 - 1111.79and1078.98 - -
TRZ–RBeng Prod. 2929.34and2364.3 1627.63 1540.85and1448.28 1234.22 - 955.55 - 1107.9
DOX 2943.8 1635.34 - 1266.04,1236.15 1214.93and1171.54 - 1042.34 -
DOX-RBeng Prod. 2928.38 1631.48 1541.81and1490.7 1261.22–1233.25 1108.87and1034.62 - disappeared 985.447
PIOG 2742.28and2615.97 1742.37and1690.3 - - - - - -
PIOG–RBeng Prod. disappeared 1760 69and1701.87 1511.92and1447.31         1041.37

Table 6: FT-IR characteristic peaks of the drugs (TRZ, DOX and PIOG), RBeng reagent and their ion-pairs.

The shift of the bands frequencies of some groups of the reagent and the drugs into lower and higher wavenumbers may be attributed to the electrostatic attraction between the cationic drugs and the anionic form of RBeng reagent.

These data confirm the proposed structures of drugs-RBeng ionpair in schemes (1-3).

1H-NMR analysis

The 1H-NMR spectra of RBeng; show two peaks with chemical shifts 7.410 and 7.912 ppm. These peaks appear in the 1H-NMR spectra of TRZ-RBeng ion-pair at chemical shifts 7.373 and 7.913 ppm; DOX-RBeng ion-pair at chemical shifts 7.395 and 7.918 ppm and PIOG-RBeng ion-pair with a chemical shift range 7.394-7.713 ppm. This refers to the presence of RBeng in these ion-pairs.

The 1H-NMR spectra of TRZ; show peak with chemical shift 12.451 ppm; which corresponds to the proton of NH2 group and peak with chemical shift 8.620 ppm; which corresponds to the proton of NH+ group. These peaks are shifted to 6.961 ppm (H of NH2 group) or disappeared (H of NH+ group) in the 1H-NMR spectra of TRZ-RBeng ion-pair. The 1H-NMR spectra of DOX show peak with chemical shift 11.725 ppm; which corresponds to the proton of NH2 group, two peaks with chemical shifts 8.702 and 8.812 ppm; which corresponds to the proton of NH+ group and peak with chemical shift 2.328 ppm; which corresponds to H of the CH3 of the mesylate group. These peaks are shifted to 6.704 ppm (H of NH2 group) or disappeared (H of NH+ group and H of CH3 of the mesylate group) in the 1H-NMR spectra of DOX-RBeng ion-pair. The 1H-NMR spectra of PIOG; show peaks in the chemical shift range 7.929-8.698 ppm; which corresponds to the protons of pyridine ring. These peaks are shifted to 8.420 ppm in the 1H-NMR spectrum of PIOG-RBeng ion-pair.

These changes in chemical shifts may be due to the electrostatic attraction between the cationic drugs and the bulky anionic reagent (small charge/unit volume); which is different from the electrostatic attraction between the cationic drugs and the small anionic part, chloride in case of TRZ and PIOG and mesylate in case of DOX, (has bigger charge/unit volume). These data confirm the proposed structure of drugs-RBeng ion-pair in schemes (1-3).

Thermal analyses

The TGA curve of RBeng shows that it decomposes in three steps. The first step may be related to the loss of 3Cl radicals. The second step may be related to the loss of Na2CO3, 2I2 and 0.5Cl2. The third step may be related to the loss of C18H2O leaving CO as a remaining part.

The TGA of TRZ refers to the decomposition of this drug in three steps. The first step may be related to the loss of two water molecules. The second step may be attributed to the loss of C7H13NO2 radical. The third step may be attributed to the loss of C10H11N3O2.HCl molecule. It appears as strong exothermic peak in DTA; which may refers to chemical rearrangement and/or chemical recombination of the fragments to give the final formula. The TGA of TRZ-RBeng shows three steps of the thermal decomposition. The first step may be due to the loss of Cl2. The second step may be attributed to the loss of C14H18N4O2 (from TRZ molecule). The third step is accompanied by two peaks at the DTA, endothermic peak and followed by exothermic one, so this step may occur at two stages. The first stage may be the loss of Cl2 and 2I2 and the second one may be the breaking of the ion pair which leads to the loss of C20H3O5 and formation of C5H9NO2 as a final residue.

The TGA of DOX shows that it decomposes in three main steps. The first step may be related to the loss of C11H11NO3 radical. The second step may be attributed to the loss of C2H7N.CH3O3S. The third step may be attributed to the loss of C10H10N2O2 molecule. It appears as strong exothermic peak in DTA; which may refers to chemical rearrangement and/or chemical recombination of the fragments to give the final Chemical formula. The TGA of DOX-RBeng refers to thermal decomposition in four steps. The first step may be due to the loss of Cl2. The second step may be attributed to the loss of C14H18N4O2 (from one of DOX molecules). The third step may be attributed to the loss of C14H18N4O2 (from the other DOX molecule) and the loss of Cl2 (from RBeng molecule). The fourth step is accompanied by two peaks at the DTA, endothermic peak and followed by exothermic one, so this step may occur at two stages. The first stage may be the loss of 2I2 and the second one may be the breaking of the ion pair which leads to the loss of C20H3O5 and 2C9H10NO3.

The TGA of PIOG shows that it decomposes in two main steps. The first step may be related to the loss of CH3 and C12H12NO3S.HCl radicals. The second step may be attributed to the loss of pyridine molecule C5H5N leaving CH2 as a final residual. The TGA of PIOGRBeng ion-pair refers to thermal decomposition in two steps. The first step may be due to the breaking of the ion-pair and the loss of 2PIOG molecules. The second step is accompanied by two peaks at the DTA, small endothermic peak followed by strong exothermic one, so this step may occurs at two stages. The first stage may be the loss of 2I2 and 2Cl2 and the second one may be the loss of C19H12O2 and CO2 leaving H2O as a final part. Thermal Analyses results of the drugs (TRZ, DOX and PIOG), RBeng reagent and their ion-pairs are given in Table 7.

Compound TG rangeºC(DTGmaxºC) DTAºC Calcd (Estim)% Assignment ResidueCalcd. (Estim)%
Mass loss Total mass loss
RBeng 35-214 (65.27) 114.1endothermic 10.46 (11.14) 97.27 (98.61) Loss of 3Cl' CO2.75 (1.39)
215-671 (403.9) 522.4exothermic 63.82 (63.55) Loss of Na2CO3, 2I2 and Cl
671-954 (911.9) 821.1endothermic 22.99 (23.92) Loss of C18H2O
TRZ 72-139 (114.1) 117.9endothermic 7.827 (6.765) 90.99 (89.63) Loss of 2H2O C2H7N9.78 (10.37)
204-358 (291.6) 345.9exothermic 30.66 (29.94) loss of C7H13NO2
358-640 (564.1) 558.7exothermic 52.51 (54.65) Loss of C10H11N3O2.HCl
TRZ–RBeng ion-pair 47-150 (59.91) 63.64exothermic 5.22 (4.32) 91.66 (92.41) Loss of Cl2 C5H9NO28.46 (8.13)
150-277 (244.4) 239.9exothermic 20.16 (21.17) Loss of C14H18N4O2 (from TRZ molecule)
277-632 (585.8) 506.7endothermic 67.03 (66.17) Loss of Cl2, 2I2
588.2exothermic Loss of C20H12O5
DOX 218-350 (319.3) 347.4endothermic 37.47 (35.33) 96.90 (94.75) Loss of C11H11NO3 NH33.10 (5.54)
350-482 (414.6) 432.8exothermic 25.57 (24.09) Loss of C2H7N.CH3O3S
482-659 (598.9) 603.6exothermic 34.68 (35.33) Loss of C10H10N2O2
DOX-RBeng ion-pair 63-174 (75.34) 82.61exothermic 3.79 (3.19) 100 (99.39) Loss of Cl2 -
174-260 (249.8) 241.7exothermic 14.62 (14.77) Loss of C14H18N4O2 (from one of DOX molecules)
260-350 (304.3) 267.8exothermic 18.40 (17.38) Loss of C14H18N4O2 (from the other DOX molecule) and Cl2 (form RBeng molecule)
350-651 (572.39) 547.6endothermic 63.53 (64.05) Loss of 2I2
635.8exothermic Loss of 2C9H10NO3 and C20H3O5
PIOG 68-334 (291.9) 299.0endothermic 76.61 (75.53) 96.72 (95.24) Loss of CH3 and C12H12NO3S.HCl CH23.56 (4.76)
410-600 (537.0) 533.0exothermic 20.11 (19.71) Loss of C5H5N
PIOG–RBeng ion-pair 40-309 (284.0) 178.4exothermic 42.36 (43.36) 99.67 (98.40) Loss of 2PIOG molecules H2O1.07 (1.31)
309-584 (483.9) 341.6endothermic 57.28 (55.04) 2I2 and 2Cl2
497.7 Loss of CO2 and C19H12O2

Table 7: Thermal Analyses results of the drugs (TRZ, DOX and PIOG), RBeng reagent and their ion-pairs.

Biological activity

RBeng has a bacteriostatic action where it prevents the growth of some bacteria. The drugs under study are anti-hypertensive (TRZ and DOX) or anti-diabetic (PIOG) drugs, but they may also have some biological activity and adding RBeng to their structure may affects this activity. The biological activity of TRZ HCl, DOX mesylate and PIOG HCl drugs and their solid ion-pairs with RBeng were determined using a modified Kirby-Bauer disc diffusion method towards two types of bacteria (Escherichia coli (G-) and Staphylococcus aureus (G+)) and two types of fungus (Aspergillus flavus and Candida albicans ). Ampicillin was used as a reference compound for antibacterial activities and Amphotericin B was used as a reference compound for antifungal activities. Both the antibacterial and antifungal activities were evaluated by measuring the inhibition zone (mm/mg sample) (Table 8).

  Antimicrobial effect(% relative to Ampicillin) Antifungal effect(% relative to Amphotericin B)
Sample Escherichiacoli(G-) Staphylococcusaureus(G+) Aspergillusflavus Candidaalbicans
TRZ 45.45 55.56 0.00 47.37
TRZ–RBeng 72.73 94.44 0.00 0.00
PIOG 0.00 0.00 0.00 0.00
PIOG–RBeng 63.64 83.33 0.00 0.00
DOX 54.55 72.22 0.00 63.16
DOX-RBeng 54.55 66.67 0.00 0.00

Table 8: Comparison between the biological activity of standard drugs and their products with RBeng.

From Table 8; it is found that the antibacterial activities of TRZ and PIOG increased after adding RBeng to their structures. It is found that TRZ-RBeng and PIOG-RBeng ion-pairs are biologically more active than the parent drugs. The antibacterial activity toward E. coli and S. aureus , in comparison with the standard found in the local market (Ampicillin), are 72.73% and 94.44% (for TRZ-RBeng ion-pair), 63.64% and 83.33% (for PIOG-RBeng ion-pair), respectively. DOXRBeng ion-pair has almost the same bacterial activity of the parent drug. Increasing the biological activity of the products refers to the biological activity of RBeng itself found in their entities. From studying the antifungal activity of the drugs, it is found that, only TRZ and DOX, have antifungal activity toward C. albicans , in comparison with the standard found in the local market (Amphotericin B) (47.37% and 63.16% respectively). It is can be concluded that the drugs under study and their solid ion-pairs with RBeng are more antibacterial agents than antifungal agents.

Conclusion

This manuscript involved fast (not time consuming), cheap and reliable spectrophotometric procedures for the determination of TRZ HCl, DOX mesylate and PIOG HCl drugs depending on color reactions between them and a chromogenic reagent (RBeng). The solid reaction products were prepared, separated and characterized by elemental, spectroscopic (FT-IR, 1H NMR) and thermal techniques. Antibacterial activity of the drugs and their reaction products with RBeng were tested by diffusion agar method and compared with each other. It is found that TRZ-RBeng and PIOG-RBeng reaction products have antimicrobial effect higher than the parent drugs, but DOXRBeng reaction product has almost the same antimicrobial effect of the parent drug. Increasing the antimicrobial effect of the products refers to the antimicrobial effect of RBeng itself in their entities. It is also found that the drugs and their solid ion-pairs with RBeng are more antibactrial agents than antifungal agents.

References

Citation: Zayed MA, Farrag YS (2016) Spectroscopic Studies and Applications of the Reactions of Some Anti-Diabetic and Anti-Hypertensive Drugs with Rose Bengal. Pharm Anal Acta 7:503.

Copyright: © 2016 Zayed MA et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.