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Study of physico-chemical properties of potential beta-adrenolytics

Study of physico-chemical properties of potential beta-adrenolytics Zámerom práce je stúdium fyzikálno-chemických vlastností vybraných látok, derivátov 2-hydroxy-3-[2-(4-metoxyfenyl)-etylamino]propyl-4-[(alkoxykarbonyl)-amino]-benzoátov a 2-hydroxy-3-[2-(2-metoxyfenyl) etylamino]-propyl-4-[(alkoxykarbonyl)amino]benzoátov s potenciálnym ultra-krátkym beta-adrenolytickým úcinkom. Studované látky sa lísia v polohe substituenta na benzénovom kruhu, v bocnom reazci, ako aj na aromatickom kruhu v polohe 4- s alkyl (metyl- az butyl-) karbamátom. Fyzikálnochemické charakteristiky, ako lipofilita, povrchová aktivita, schopnos adsorpcie, acidobázické vlastnosti, at. sú vemi významné pre objasnenie vzahu medzi struktúrou a biologickou aktivitou lieciva. Tieto parametre slúzia ako základ pre stúdium QSAR. Cieom práce je stanovenie spektrálnych charakteristík v UF oblasti, hodnôt pKa, parametrov lipofility (hodnoty Rf a RM, z chromatografie na tenkej vrstve, retencný cas t´R a kapacitný factor k´ z kvapalinovej chromatografie a experimentálne rozdeovacie koeficienty), povrchového napätie, kritickej micelovej koncentrácie c.m.c., schopnos adsorpcie látok na aktívne uhlie, vyjadrené percentom adsorbovaného mnozstva % ako aj Freundlichovou adsorpcnou izotermou. Získané hodnoty sú korelované s parametrami charakterizujúcimi vekos molekuly, napr. pocet atómov uhlíka v karbamátovej funkcnej skupine. Keywords Kúcové slová: ultra-short-acting beta-blockers, lipophilicity, adsorbability, critical micelle concentration ultra krátko úcinné beta-blokátory, lipofilita, adsorbabilita, kritická micelová koncentrácia INTRODUCTION The studied compounds are derivatives of [(arylcarbonyl)oxy] aminopropanol, with carbamate substitution on benzene ring. The traditional isopropyl or tertiary butyl residues on basic nitrogen atom are replaced with bulky 2- or 4-(methoxyphenyl) ethylamine group. Such structural change enhances the lipophilic character of the molecule. The introduction of a bulky * StankovicM@fpharm.uniba.sk © Acta Facultatis Pharmaceuticae Universitatis Comenianae substituent on the N-atom can lead to compounds with significant 1-adrenoreceptor selectivity (Graham, 2009 & Griffith, 2003). The compounds were prepared at the Department of Chemical drugs of the University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic (Mokrý et al., 2011). These drugs are potential ultra-short-acting beta-adrenergic receptor blocking agents. The short duration of action is realised by incorporation of a metabolically unstable ester group on the benzene ring (Jackman et al. 2002). The list of studied compound is in Table 1. The stability studies of the compounds were examined in acidic and alkaline media, in buffers and due oxidation at room and at elevated temperature chromatographically and Rf values of incipient products and degradation products were detected. Kinetics of acid and base hydrolysis in various solutions at temperature 80°C and 100°C was examined through ultraviolet-area spectrophotometry. Kinetic parameters such as rate constant k, half-life period t1/2 and usable life t90 were determined (Stankovicová et al., 2012). For clarification of relationships between structure and biological activity of potential drugs, it is necessary to state their physicochemical parameters that are related to biological effect such lipophilicity, surface activity, capability of binding on phase boundary, and acidobasic properties. These parameters serve as the base of quantitative structure-activity relationship study, which leads to the projection of new drugs. Spectral characteristics of compounds Ultraviolet spectra of water solutions of compounds were measured in the region from wavelength 200 to 450 nm. The solutions of compounds (c = 1 × 10­5 mol/l) were prepared by diluting of storage solution of compound in methanol c = 0.1% to the volume 100.0 ml by distilled water. The value of absorbance at each maximum was recorded, the values (m2 ·mol­1) and A1%1cm were calculated. Thin-layer chromatography The chromatographic separation of compounds was carried out on silica gel layer Silufol® UV 254 15 × 15 cm. 5 l of examined solution of 0.1% solution of compounds in methanol was applied on the plate. Development distance was 10 cm. A UV light lamp Camag was used for detection at wavelength = 254 nm. Chromatographic system S1: petroleum ether/ diethyl-amine (6.0/3.0 v/v), saturation of chromatographic chamber 20 min, chromatographic system S2: hexane/diethyl-amine (6.0/4.0 v/v) under the same conditions. Liquid chromatography The mobile phase was 90% methanol; pH value adjusted to 6.0 by addition of sodium acetate. The separation was carried out under pressure 7.9 MPa at flow rate 0.6 ml/min and the column temperature was maintained at 25°C. The chromatograms were scanned at wavelength 272 nm. The injection volume was 20 l. The analytes were dissolved in methanol (c = 5 × 10­4 mol/l) and were applied three times. Experimental partition coefficient (P´) The experimental partition coefficient of studied compounds was measured by the shake-flask method in octan-1-ol/phosphate buffer medium at pH = 7.0 using 0.2 ml organic solvent and 10.0 ml of phosphate buffer solution of analysed compound (c = 2.3 × 10­5 mol/l). The concentration of compounds in the aqueous phase after shaking (1 h) and equilibration (1h) was determined spectrophotometrically at wavelength 270 nm. Relative surface activity The relative surface activity of compounds 1 ­ 4 was determined at temperature 22°C by Traube stalagmometric method. Activity of the water solutions of the compounds c = 1 × 10­3 mol/l was measured. The reference solution was distilled water. Critical micelle concentration The critical micelle concentration (c.m.c.) of compounds 5 ­ 8 was determined spectrophotometrically at wavelength 270 nm. The storage solution of compound in methanol c = 0.1% was diluted to volume 25.0 ml by distilled water. The concentration of prepared solutions was from c = 1.1 × 10­5 mol/l to c = 1.95 × 10­4 mol/l. All the measurements were performed at temperature 22°C. The calculation of c.m.c. value was realised from the point of intersection of the two extrapolated linear parts of dependence of absorbance vs concentration. MATERIALS AND METHODS Studied compounds The studied compounds were synthesised as hydrochloride by authors (Mokrý et al., 2011) and they are listed in Table 1. All other chemicals and solvents were of analytical reagent grade. Apparatus The Thermostat Memmert WB 10 (Germany), UV-VIS spectrophotometer Shimadzu UV-1800 (Japan), HPLC system consisting of high pressure pump DeltaChrom SDS 030 Watrex (Slovakia), injector loop with volume 20 l Watrex (Slovakia), column Sepharon SDX C18 with size 250 × 4 mm Watrex (Slovakia), size of grain 7 m, flow UV detector Delta Chrom UVD 200 Watrex (Slovakia), Camag Universal UV Lampe TL-900N (Switzerland), InoLab pH 720 WTW Series pH meter (Germany), WTW pH-Electrode SenTix 61 (Germany). Table 1. List of the studied compounds OH O O N H O CH3 HCl NH O OR Compound 1 2 3 4 5 6 7 8 R CH3 C2H5 C3H7 C4H9 CH3 C2H5 C3H7 C4H9 Position in ring 4-OCH3 4-OCH3 4-OCH3 4-OCH3 2-OCH3 2-OCH3 2-OCH3 2-OCH3 Mr 438.91 452.94 466.96 480.99 438.91 452.94 466.96 480.99 Stankovicová, M. et al. ( ( Dissociation constant (pKa) The pKa values of compounds were determined by the ultraviolet spectrophotometric method. Absorbance values of the solutions of compounds were measured in NaOH (c = 0.1 mol/l), HCl (c = 0.1 mol/l), and six trometamol buffer solutions with pH values 7.2, 7.4, 7.6, 8.0, 8.4 and 9.0 at wavelengths that were responsible for the maximum difference between absorbance values in acidic and alkaline medium. Values of pKa were calculated according to Henderson and Hasselbach equation. The concentration of measured solutions was c = 2.0 × 10­5 mol/l. These were prepared by dilution of storage solutions of compounds in methanol c = 0.1% to volume 25.0 ml by buffer solutions or NaOH and HCl solutions, respectively. Adsorption study The activated carbon was first washed three-times (for 15 min by shaking) with hot distilled water and dried at 110°C for 2 days and then kept in a desiccator containing silica gel. The adsorption of studied compounds 1, 3 and 4 was estimated at pH 7.0 (phosphate buffer). The flasks with 50.0 ml of studied compound solution and with 2 mg of activated carbon were shaken at temperature 20°C for 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 2.0 and 2.5 h. The time duration of another adsorption study was 90 min. The original concentration of studied compounds in buffer solution varied from 4.0 × 10­5 mol/l for time dependence study, and from 1.8 × 10­5 mol/l to 5.1 × 10­5 mol/l. for concentration dependence study. After equilibration, the sample was filtered through thick filter paper. The compound content in the filtrate solution was determined spectrophotometrically in the UV region at wavelength 272 nm. The measured solutions of compounds were prepared by dilution of the storage solution of compound in methanol c = 0.1%. Experimental partition coefficient P´ ( ) ( ( ( (5) Critical micelle concentration straight line y1 = a x1 + b and straight line y2 = c x2 + d ( | | | |) | ( ) m = weight of compound in solution (g), a = volume of aque( ) ous phase (ml), b = volume of organic phase (ml) Mr = relative | | molecular weight, cH2O = concentration of compound in aqueous phase after shaking ) ( ) ( ) ( ) ( pKa value ( ( | | Percent of adsorbed compound pH = pH value of measured solution, A = absorbance in NaOH | | 0.1 mol/l, B = absorbance in buffer solutions, C = absorbance in HCl 0.1 mol/l ( ( (6) (7) (8) cv = concentration of adsorbed compound (mol/l), ceq = concentration of unbounded compound (mol/l) Freundlich adsorption constants k, N (9) CALCULATIONS Retardation factor Rf and RM value (1) ( ) Capacity factor k´ ( ) a = migration distance of the solvent front, ( ) b = migration distance of the analyte ( ) c = equilibrium solute concentration in the bulk (mg/l), m = amount of solute adsorbed (mg/g), k, N = Freundlich constants (2) The linear regression equation (10) was computed by the least squares techniques, where a0 is intercept, a1 is slope. (3) tR = retention time, tM = hold-up time Specific absorbance A1%1cm ( ) ( ) RESULTS AND DISCUSSION The studied compounds are derivatives of 2-hydroxy-3-[2-(4methoxyphenyl)ethylamino]propyl-4-[(alkoxycarbonyl)amino]benzoates and 2-hydroxy-3-[2-(2-methoxyphenyl)ethylamino] propyl-4- [(alkoxycarbonyl)amino]benzoates where the aromatic ring is substituted in para position with alkyl (methyl to butyl) carbamate group. The substances were prepared by ) ( ) ( = molar absorptivity, Mr = relative molecular mass ) ( ) ( ( | ) | ( ) (4) five-step synthesis. The final bases were converted to their hydrochloride salts. The structure of prepared compounds was confirmed by 1H- and 13C-nuclear magnetic resonance spectroscopy as well as Fourier transformation infrared spectrophotometry and mass spectroscopy. The purity of prepared compounds was examined by means of thin layer chromatography (Mokrý et al., 2011). In this paper, the substances were characterized by ultraviolet spectra. The values log were determined as well as the specific absorbance A1%1cm. Ultraviolet absorption spectrum of aqueous methanol solutions of studied compounds between 200 and 450 nm have two absorbance maxima. 2-methoxyderivatives have absorbance maximum max1 around 270 nm and max2 around 212 nm, 4-methoxyderivatives ­ max1 around 272 nm and max2 around 218 nm. The values of molar absorption coefficients and specific absorbance A1%1cm are in Table 2. The absorption bands of the 2-methoxyderivatives are stronger than that of 4-methoxyderivatives. The values of absorption coefficients are used for identification of compounds and values of specific absorbance are used in quantitation of compounds. Chromatographic parameters, the Rf and RM values from TLC for two mobile phases are in Table 2. In both the systems, the stationary phase is more polar than the mobile phase, the Rf values rise with the number of carbon atoms, the RM values decrease, which is in accordance with the lipophilicity of compounds. The Rf values of 2-methoxyderivatives are higher than Rf values of 4-methoxyderivatives in both systems. Lipophilicity of studied compounds was evaluated by HPLC method with reverse-phase system. Chemically modified silica gel with bounded octadecyl groups was used as nonpolar stationary phase. The most favourable mobile phase was methanol 90% with adjusted pH value by sodium acetate at pH = 6.0. The t´R as well as k´ values of compounds rise with the height of relative molar weight Mr. The 2-methoxyderiva- tives in comparison with 4-methoxyderivatives show these higher values. The parameters obtained represent the efficiency of interaction of compound with hydrophobic surface of stationary phase as well as their lipophilicity, which rise with increase of alkyl chain of molecule. The transport of a drug in biological system depends upon its partition coefficient. The experimental partition coefficient P´ is influenced by the ratio of total concentrations of drug in lipid and aqueous phases; however, the drug is present in several forms. The values of experimental partition coefficients log P´of studied compounds are between 1.48 and 2.47 (Table 3). The dependence of log P´ vs number of carbon atoms is linear for 2-methoxyderivatives; these values are in accordance with RM and log k´ values. The theoretical values of log P for basic compounds were obtained by several computation techniques, the values MLOGP were the nearest to the experimental. Results of regression analysis are in Table 4. The pKa value of a compound is, formally, the negative logarithm of its acid dissociation constant Ka. The pKa value is a convenient numerical way to compare the relative acidity or basicity of weakly ionising compounds in aqueous or miscible solvent-aqueous solutions (Newton & Kluza, 1978). The pKa values of studied compounds (Table 3) determined spectrophotometrically are between 8.29 and 9.59, which is in accordance with that of -adrenolytics such as pindolol (8.8), practolol (9.5) and propranolol (9.45) (Newton &Kluza, 1978). The values of relative surface activity [Nm­1] of studied 4-methoxyderivatives determined by Traube stalagmometric method are in Table 3. Because this method was not suitable for determination of concentration dependence of surface activity to evaluate c.m.c. we have chosen ultraviolet spectrophotometry for this. The values of c.m.c. of 2-methoxyderivatives are in Table 3. The dependence of surface activity vs number of carbon atoms is not linear in both groups of compounds. Table 2. UV spectral characteristics and chromatographic parameters of the studied compounds UV spectral characteristics Compound max nm 271 218 271 218 272 218 272 219 270 212 271 212 271 215 271 214 m2 mol ­1 2379 1514 2148 1332 2339 1482 2265 1518 2962 2461 3116 2324 2526 1521 2470 1540 A1%1cm 542 345 474 294 501 317 471 316 675 561 688 513 541 326 513 320 RM S1 0.31 0.14 -0.02 -0.27 0.21 0.02 -0.12 -0.31 S2 0.43 0.23 0.09 -0.05 0.37 0.21 0.10 0.03 Chromatographic parameters t´R min 0.71 0.79 0.91 1.05 0.75 0.83 0.95 1.10 log k´ -0.149 -0.102 -0.041 0.021 -0.125 -0.081 -0.022 0.041 Stankovicová, M. et al. Table 3. The results of the physico-chemical parameters of studied compound Parameters of lipophilicity, dissociation constants, surface tension and critical micelle concentration Compound 1 2 3 4 5 6 7 8 log P´ 1.48 1.92 2.47 2.11 1.77 1.95 2.09 2.26 MLOGP pKa 8.78 8.55 8.95 9.34 9.59 8.61 8.29 8.43 Surface tension [N m­1] 0.0733 0.0741 0.0718 0.0704 Critical micelle conc. [mol/l×104] Results of adsorption study % 60.5 63.5 75.2 k 32.6 31.93 36.34 N 1.150 1.188 1.204 Table 4. Results of the regression analysis Function n r F s a0 a1 a2 RM = f (C) a) 4 0.984 61.004 0.0319 0.460 -0.113 RM = f (C) b) 4 0.996 231.06 0.232 0.570 -0.158 log k´= f (C) a) 4 0.997 322.12 0.0069 -0.186 0.0557 log k´= f (C) b) 4 0.999 831.83 0.0057 -0.210 0.0571 log P' = f (C) a) 4 0.999 960.04 0.0116 1.615 0.161 log P'= f (C) b) 4 0.948 4.400 0.2281 0.385 1.244 -0.200 a) 2-methoxyderivatives; b) 4-methoxyderivatives, n is the number of points, r is the correlation coefficient, s is the standard deviation, F is the F-test. Active charcoal is one of the important adsorbents capable of binding on their surface other substances in relatively large amounts. This property is often used in pharmacy as well as in the study of structures-biological activity relationships, where activated carbon serves as a model substance for the study of hydrophobic interactions (Abe et al., 1990). The activated carbon adsorption property (expressed by the partition coefficient of drugs at infinite dilution) was successfully correlated with drug potencies of local anaesthetics as a parameter for the QSAR (Abe et al., 1990 & Abe et al., 1988). Carbon surface adsorption often follows the Freundlich adsorption isotherms (Abe et al., 1988 & Tanada et al., 1997) in which the logarithm of the adsorbed amount bears a linear relationship with the logarithm of the free drug concentrations. The aim of this work is investigation of the adsorption property of the 4-methoxyderivatives compared to active charcoal according to Freundlich model and according to the amount of the bound substance percents dependending on time. The results are in Table 3. The adsorption of these compounds shows the rise of adsorbed amount depending on time with maximum in time between 80 to 120 min. The time of dura- tion of another adsorption studies was 90 min. The adsorption of compounds rises with number of carbon atoms as well as with relative molecular weight (the size of molecule). The Freundlich model of adsorption was employed to evaluate the course of adsorption in dependence on the concentration of substances. CONCLUSION The obtained results of physico-chemical evaluation of ultrashort-acting beta-adrenolytics may serve as the base for investigation of new drugs in this group of compounds. A study like this was not performed on such compounds. The studied and determined physico-chemical parameters of lipophilicity, surface activity, capability of binding on phase boundary, provide a measure for interfacial hydrophobic ­ hydrophilic interactions that play, together with the acidobasic properties, an important role in various biological processes and may be used as a convenient tool in the QSAR study, investigating drug actions. The work by such experiences also expands the knowledge about derivatives of phenylcarbamic acid. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Facultatis Pharmaceuticae Universitatis Comenianae de Gruyter

Study of physico-chemical properties of potential beta-adrenolytics

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de Gruyter
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1338-6786
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1338-6786
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10.2478/afpuc-2014-0014
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Abstract

Zámerom práce je stúdium fyzikálno-chemických vlastností vybraných látok, derivátov 2-hydroxy-3-[2-(4-metoxyfenyl)-etylamino]propyl-4-[(alkoxykarbonyl)-amino]-benzoátov a 2-hydroxy-3-[2-(2-metoxyfenyl) etylamino]-propyl-4-[(alkoxykarbonyl)amino]benzoátov s potenciálnym ultra-krátkym beta-adrenolytickým úcinkom. Studované látky sa lísia v polohe substituenta na benzénovom kruhu, v bocnom reazci, ako aj na aromatickom kruhu v polohe 4- s alkyl (metyl- az butyl-) karbamátom. Fyzikálnochemické charakteristiky, ako lipofilita, povrchová aktivita, schopnos adsorpcie, acidobázické vlastnosti, at. sú vemi významné pre objasnenie vzahu medzi struktúrou a biologickou aktivitou lieciva. Tieto parametre slúzia ako základ pre stúdium QSAR. Cieom práce je stanovenie spektrálnych charakteristík v UF oblasti, hodnôt pKa, parametrov lipofility (hodnoty Rf a RM, z chromatografie na tenkej vrstve, retencný cas t´R a kapacitný factor k´ z kvapalinovej chromatografie a experimentálne rozdeovacie koeficienty), povrchového napätie, kritickej micelovej koncentrácie c.m.c., schopnos adsorpcie látok na aktívne uhlie, vyjadrené percentom adsorbovaného mnozstva % ako aj Freundlichovou adsorpcnou izotermou. Získané hodnoty sú korelované s parametrami charakterizujúcimi vekos molekuly, napr. pocet atómov uhlíka v karbamátovej funkcnej skupine. Keywords Kúcové slová: ultra-short-acting beta-blockers, lipophilicity, adsorbability, critical micelle concentration ultra krátko úcinné beta-blokátory, lipofilita, adsorbabilita, kritická micelová koncentrácia INTRODUCTION The studied compounds are derivatives of [(arylcarbonyl)oxy] aminopropanol, with carbamate substitution on benzene ring. The traditional isopropyl or tertiary butyl residues on basic nitrogen atom are replaced with bulky 2- or 4-(methoxyphenyl) ethylamine group. Such structural change enhances the lipophilic character of the molecule. The introduction of a bulky * StankovicM@fpharm.uniba.sk © Acta Facultatis Pharmaceuticae Universitatis Comenianae substituent on the N-atom can lead to compounds with significant 1-adrenoreceptor selectivity (Graham, 2009 & Griffith, 2003). The compounds were prepared at the Department of Chemical drugs of the University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic (Mokrý et al., 2011). These drugs are potential ultra-short-acting beta-adrenergic receptor blocking agents. The short duration of action is realised by incorporation of a metabolically unstable ester group on the benzene ring (Jackman et al. 2002). The list of studied compound is in Table 1. The stability studies of the compounds were examined in acidic and alkaline media, in buffers and due oxidation at room and at elevated temperature chromatographically and Rf values of incipient products and degradation products were detected. Kinetics of acid and base hydrolysis in various solutions at temperature 80°C and 100°C was examined through ultraviolet-area spectrophotometry. Kinetic parameters such as rate constant k, half-life period t1/2 and usable life t90 were determined (Stankovicová et al., 2012). For clarification of relationships between structure and biological activity of potential drugs, it is necessary to state their physicochemical parameters that are related to biological effect such lipophilicity, surface activity, capability of binding on phase boundary, and acidobasic properties. These parameters serve as the base of quantitative structure-activity relationship study, which leads to the projection of new drugs. Spectral characteristics of compounds Ultraviolet spectra of water solutions of compounds were measured in the region from wavelength 200 to 450 nm. The solutions of compounds (c = 1 × 10­5 mol/l) were prepared by diluting of storage solution of compound in methanol c = 0.1% to the volume 100.0 ml by distilled water. The value of absorbance at each maximum was recorded, the values (m2 ·mol­1) and A1%1cm were calculated. Thin-layer chromatography The chromatographic separation of compounds was carried out on silica gel layer Silufol® UV 254 15 × 15 cm. 5 l of examined solution of 0.1% solution of compounds in methanol was applied on the plate. Development distance was 10 cm. A UV light lamp Camag was used for detection at wavelength = 254 nm. Chromatographic system S1: petroleum ether/ diethyl-amine (6.0/3.0 v/v), saturation of chromatographic chamber 20 min, chromatographic system S2: hexane/diethyl-amine (6.0/4.0 v/v) under the same conditions. Liquid chromatography The mobile phase was 90% methanol; pH value adjusted to 6.0 by addition of sodium acetate. The separation was carried out under pressure 7.9 MPa at flow rate 0.6 ml/min and the column temperature was maintained at 25°C. The chromatograms were scanned at wavelength 272 nm. The injection volume was 20 l. The analytes were dissolved in methanol (c = 5 × 10­4 mol/l) and were applied three times. Experimental partition coefficient (P´) The experimental partition coefficient of studied compounds was measured by the shake-flask method in octan-1-ol/phosphate buffer medium at pH = 7.0 using 0.2 ml organic solvent and 10.0 ml of phosphate buffer solution of analysed compound (c = 2.3 × 10­5 mol/l). The concentration of compounds in the aqueous phase after shaking (1 h) and equilibration (1h) was determined spectrophotometrically at wavelength 270 nm. Relative surface activity The relative surface activity of compounds 1 ­ 4 was determined at temperature 22°C by Traube stalagmometric method. Activity of the water solutions of the compounds c = 1 × 10­3 mol/l was measured. The reference solution was distilled water. Critical micelle concentration The critical micelle concentration (c.m.c.) of compounds 5 ­ 8 was determined spectrophotometrically at wavelength 270 nm. The storage solution of compound in methanol c = 0.1% was diluted to volume 25.0 ml by distilled water. The concentration of prepared solutions was from c = 1.1 × 10­5 mol/l to c = 1.95 × 10­4 mol/l. All the measurements were performed at temperature 22°C. The calculation of c.m.c. value was realised from the point of intersection of the two extrapolated linear parts of dependence of absorbance vs concentration. MATERIALS AND METHODS Studied compounds The studied compounds were synthesised as hydrochloride by authors (Mokrý et al., 2011) and they are listed in Table 1. All other chemicals and solvents were of analytical reagent grade. Apparatus The Thermostat Memmert WB 10 (Germany), UV-VIS spectrophotometer Shimadzu UV-1800 (Japan), HPLC system consisting of high pressure pump DeltaChrom SDS 030 Watrex (Slovakia), injector loop with volume 20 l Watrex (Slovakia), column Sepharon SDX C18 with size 250 × 4 mm Watrex (Slovakia), size of grain 7 m, flow UV detector Delta Chrom UVD 200 Watrex (Slovakia), Camag Universal UV Lampe TL-900N (Switzerland), InoLab pH 720 WTW Series pH meter (Germany), WTW pH-Electrode SenTix 61 (Germany). Table 1. List of the studied compounds OH O O N H O CH3 HCl NH O OR Compound 1 2 3 4 5 6 7 8 R CH3 C2H5 C3H7 C4H9 CH3 C2H5 C3H7 C4H9 Position in ring 4-OCH3 4-OCH3 4-OCH3 4-OCH3 2-OCH3 2-OCH3 2-OCH3 2-OCH3 Mr 438.91 452.94 466.96 480.99 438.91 452.94 466.96 480.99 Stankovicová, M. et al. ( ( Dissociation constant (pKa) The pKa values of compounds were determined by the ultraviolet spectrophotometric method. Absorbance values of the solutions of compounds were measured in NaOH (c = 0.1 mol/l), HCl (c = 0.1 mol/l), and six trometamol buffer solutions with pH values 7.2, 7.4, 7.6, 8.0, 8.4 and 9.0 at wavelengths that were responsible for the maximum difference between absorbance values in acidic and alkaline medium. Values of pKa were calculated according to Henderson and Hasselbach equation. The concentration of measured solutions was c = 2.0 × 10­5 mol/l. These were prepared by dilution of storage solutions of compounds in methanol c = 0.1% to volume 25.0 ml by buffer solutions or NaOH and HCl solutions, respectively. Adsorption study The activated carbon was first washed three-times (for 15 min by shaking) with hot distilled water and dried at 110°C for 2 days and then kept in a desiccator containing silica gel. The adsorption of studied compounds 1, 3 and 4 was estimated at pH 7.0 (phosphate buffer). The flasks with 50.0 ml of studied compound solution and with 2 mg of activated carbon were shaken at temperature 20°C for 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 2.0 and 2.5 h. The time duration of another adsorption study was 90 min. The original concentration of studied compounds in buffer solution varied from 4.0 × 10­5 mol/l for time dependence study, and from 1.8 × 10­5 mol/l to 5.1 × 10­5 mol/l. for concentration dependence study. After equilibration, the sample was filtered through thick filter paper. The compound content in the filtrate solution was determined spectrophotometrically in the UV region at wavelength 272 nm. The measured solutions of compounds were prepared by dilution of the storage solution of compound in methanol c = 0.1%. Experimental partition coefficient P´ ( ) ( ( ( (5) Critical micelle concentration straight line y1 = a x1 + b and straight line y2 = c x2 + d ( | | | |) | ( ) m = weight of compound in solution (g), a = volume of aque( ) ous phase (ml), b = volume of organic phase (ml) Mr = relative | | molecular weight, cH2O = concentration of compound in aqueous phase after shaking ) ( ) ( ) ( ) ( pKa value ( ( | | Percent of adsorbed compound pH = pH value of measured solution, A = absorbance in NaOH | | 0.1 mol/l, B = absorbance in buffer solutions, C = absorbance in HCl 0.1 mol/l ( ( (6) (7) (8) cv = concentration of adsorbed compound (mol/l), ceq = concentration of unbounded compound (mol/l) Freundlich adsorption constants k, N (9) CALCULATIONS Retardation factor Rf and RM value (1) ( ) Capacity factor k´ ( ) a = migration distance of the solvent front, ( ) b = migration distance of the analyte ( ) c = equilibrium solute concentration in the bulk (mg/l), m = amount of solute adsorbed (mg/g), k, N = Freundlich constants (2) The linear regression equation (10) was computed by the least squares techniques, where a0 is intercept, a1 is slope. (3) tR = retention time, tM = hold-up time Specific absorbance A1%1cm ( ) ( ) RESULTS AND DISCUSSION The studied compounds are derivatives of 2-hydroxy-3-[2-(4methoxyphenyl)ethylamino]propyl-4-[(alkoxycarbonyl)amino]benzoates and 2-hydroxy-3-[2-(2-methoxyphenyl)ethylamino] propyl-4- [(alkoxycarbonyl)amino]benzoates where the aromatic ring is substituted in para position with alkyl (methyl to butyl) carbamate group. The substances were prepared by ) ( ) ( = molar absorptivity, Mr = relative molecular mass ) ( ) ( ( | ) | ( ) (4) five-step synthesis. The final bases were converted to their hydrochloride salts. The structure of prepared compounds was confirmed by 1H- and 13C-nuclear magnetic resonance spectroscopy as well as Fourier transformation infrared spectrophotometry and mass spectroscopy. The purity of prepared compounds was examined by means of thin layer chromatography (Mokrý et al., 2011). In this paper, the substances were characterized by ultraviolet spectra. The values log were determined as well as the specific absorbance A1%1cm. Ultraviolet absorption spectrum of aqueous methanol solutions of studied compounds between 200 and 450 nm have two absorbance maxima. 2-methoxyderivatives have absorbance maximum max1 around 270 nm and max2 around 212 nm, 4-methoxyderivatives ­ max1 around 272 nm and max2 around 218 nm. The values of molar absorption coefficients and specific absorbance A1%1cm are in Table 2. The absorption bands of the 2-methoxyderivatives are stronger than that of 4-methoxyderivatives. The values of absorption coefficients are used for identification of compounds and values of specific absorbance are used in quantitation of compounds. Chromatographic parameters, the Rf and RM values from TLC for two mobile phases are in Table 2. In both the systems, the stationary phase is more polar than the mobile phase, the Rf values rise with the number of carbon atoms, the RM values decrease, which is in accordance with the lipophilicity of compounds. The Rf values of 2-methoxyderivatives are higher than Rf values of 4-methoxyderivatives in both systems. Lipophilicity of studied compounds was evaluated by HPLC method with reverse-phase system. Chemically modified silica gel with bounded octadecyl groups was used as nonpolar stationary phase. The most favourable mobile phase was methanol 90% with adjusted pH value by sodium acetate at pH = 6.0. The t´R as well as k´ values of compounds rise with the height of relative molar weight Mr. The 2-methoxyderiva- tives in comparison with 4-methoxyderivatives show these higher values. The parameters obtained represent the efficiency of interaction of compound with hydrophobic surface of stationary phase as well as their lipophilicity, which rise with increase of alkyl chain of molecule. The transport of a drug in biological system depends upon its partition coefficient. The experimental partition coefficient P´ is influenced by the ratio of total concentrations of drug in lipid and aqueous phases; however, the drug is present in several forms. The values of experimental partition coefficients log P´of studied compounds are between 1.48 and 2.47 (Table 3). The dependence of log P´ vs number of carbon atoms is linear for 2-methoxyderivatives; these values are in accordance with RM and log k´ values. The theoretical values of log P for basic compounds were obtained by several computation techniques, the values MLOGP were the nearest to the experimental. Results of regression analysis are in Table 4. The pKa value of a compound is, formally, the negative logarithm of its acid dissociation constant Ka. The pKa value is a convenient numerical way to compare the relative acidity or basicity of weakly ionising compounds in aqueous or miscible solvent-aqueous solutions (Newton & Kluza, 1978). The pKa values of studied compounds (Table 3) determined spectrophotometrically are between 8.29 and 9.59, which is in accordance with that of -adrenolytics such as pindolol (8.8), practolol (9.5) and propranolol (9.45) (Newton &Kluza, 1978). The values of relative surface activity [Nm­1] of studied 4-methoxyderivatives determined by Traube stalagmometric method are in Table 3. Because this method was not suitable for determination of concentration dependence of surface activity to evaluate c.m.c. we have chosen ultraviolet spectrophotometry for this. The values of c.m.c. of 2-methoxyderivatives are in Table 3. The dependence of surface activity vs number of carbon atoms is not linear in both groups of compounds. Table 2. UV spectral characteristics and chromatographic parameters of the studied compounds UV spectral characteristics Compound max nm 271 218 271 218 272 218 272 219 270 212 271 212 271 215 271 214 m2 mol ­1 2379 1514 2148 1332 2339 1482 2265 1518 2962 2461 3116 2324 2526 1521 2470 1540 A1%1cm 542 345 474 294 501 317 471 316 675 561 688 513 541 326 513 320 RM S1 0.31 0.14 -0.02 -0.27 0.21 0.02 -0.12 -0.31 S2 0.43 0.23 0.09 -0.05 0.37 0.21 0.10 0.03 Chromatographic parameters t´R min 0.71 0.79 0.91 1.05 0.75 0.83 0.95 1.10 log k´ -0.149 -0.102 -0.041 0.021 -0.125 -0.081 -0.022 0.041 Stankovicová, M. et al. Table 3. The results of the physico-chemical parameters of studied compound Parameters of lipophilicity, dissociation constants, surface tension and critical micelle concentration Compound 1 2 3 4 5 6 7 8 log P´ 1.48 1.92 2.47 2.11 1.77 1.95 2.09 2.26 MLOGP pKa 8.78 8.55 8.95 9.34 9.59 8.61 8.29 8.43 Surface tension [N m­1] 0.0733 0.0741 0.0718 0.0704 Critical micelle conc. [mol/l×104] Results of adsorption study % 60.5 63.5 75.2 k 32.6 31.93 36.34 N 1.150 1.188 1.204 Table 4. Results of the regression analysis Function n r F s a0 a1 a2 RM = f (C) a) 4 0.984 61.004 0.0319 0.460 -0.113 RM = f (C) b) 4 0.996 231.06 0.232 0.570 -0.158 log k´= f (C) a) 4 0.997 322.12 0.0069 -0.186 0.0557 log k´= f (C) b) 4 0.999 831.83 0.0057 -0.210 0.0571 log P' = f (C) a) 4 0.999 960.04 0.0116 1.615 0.161 log P'= f (C) b) 4 0.948 4.400 0.2281 0.385 1.244 -0.200 a) 2-methoxyderivatives; b) 4-methoxyderivatives, n is the number of points, r is the correlation coefficient, s is the standard deviation, F is the F-test. Active charcoal is one of the important adsorbents capable of binding on their surface other substances in relatively large amounts. This property is often used in pharmacy as well as in the study of structures-biological activity relationships, where activated carbon serves as a model substance for the study of hydrophobic interactions (Abe et al., 1990). The activated carbon adsorption property (expressed by the partition coefficient of drugs at infinite dilution) was successfully correlated with drug potencies of local anaesthetics as a parameter for the QSAR (Abe et al., 1990 & Abe et al., 1988). Carbon surface adsorption often follows the Freundlich adsorption isotherms (Abe et al., 1988 & Tanada et al., 1997) in which the logarithm of the adsorbed amount bears a linear relationship with the logarithm of the free drug concentrations. The aim of this work is investigation of the adsorption property of the 4-methoxyderivatives compared to active charcoal according to Freundlich model and according to the amount of the bound substance percents dependending on time. The results are in Table 3. The adsorption of these compounds shows the rise of adsorbed amount depending on time with maximum in time between 80 to 120 min. The time of dura- tion of another adsorption studies was 90 min. The adsorption of compounds rises with number of carbon atoms as well as with relative molecular weight (the size of molecule). The Freundlich model of adsorption was employed to evaluate the course of adsorption in dependence on the concentration of substances. CONCLUSION The obtained results of physico-chemical evaluation of ultrashort-acting beta-adrenolytics may serve as the base for investigation of new drugs in this group of compounds. A study like this was not performed on such compounds. The studied and determined physico-chemical parameters of lipophilicity, surface activity, capability of binding on phase boundary, provide a measure for interfacial hydrophobic ­ hydrophilic interactions that play, together with the acidobasic properties, an important role in various biological processes and may be used as a convenient tool in the QSAR study, investigating drug actions. The work by such experiences also expands the knowledge about derivatives of phenylcarbamic acid.

Journal

Acta Facultatis Pharmaceuticae Universitatis Comenianaede Gruyter

Published: Dec 30, 2014

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