Determination of lipophilicity of some new 1 , 2 , 4-triazole derivatives by RP-HPLC and RP-TLC and calculated methods

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Introduction
Molecular transport through lipid membranes (intestinal absorption, blood-brain barrier penetration) of potential drugs is directly related to their chemical and physical properties.The most important properties are lipophilicity, number of hydrogen bonds, surface properties 1 .
Lipophlicity is well-known as a prime physic-chemical descriptor of drug (potential drug) with relevance to their biological properties.It plays the main role in the control of pharmacokinetic and pharmacodynamic properties of biological active compounds.Lipophilicity is usually expressed as a partition coefficient (P) or its decimal logarithm (logP) between a non-aqueous and aqueous phase 2 .The determination of partition coefficient using the shake flask equilibration method is associated with 102 many problems (poor reproducibility, time consuming experiments, high purity of analytes is required).The reversed-phase HPLC or TLC are very popular alternative methods in logP determined due to its high-throughput, small amount of solutes needed and a wide applicable range.In case of HPLC method the lipophilicity index is derived from the retention factor and in case of TLC method from   values.The extrapolated and   values at pure water as mobile phase (  ,   0 ) were used as the lipophilicity parameters.Both HPLC and TLC methods are used to determine the lipophilicity and to predict the relationship between molecular structure and its biological activity [3][4][5][6][7] .A linear relationships between the retention parameters and the concentration (ϕ) of organic modifier in the aqueous mobile phase has to be established for chromatographic measurement of lipophilicity and it can be represented as the dependence (1) 8 : where  is solute retention factor at a specific mobile phase composition, ϕ is the volume fraction of organic solvent in the water-organic solvent mixture,  is the slope of the regression curve, the intercept   (lipophilicity index) is the retention parameter of solutes for pure water as the eluent.
The relationship between the retention factor (  ) and the mobile phase composition in this case looks as follows 9 : where similarly the   is the retention factor of substance.Subsequent chromatographic lipophilicity parameter is ϕ 0 for both methods respectively [10][11][12] : where, ϕ 0 represents the ratio of the slope and intercept of Eq. (1) and Eq. ( 2).The ϕ 0 parameter is the concentration of the organic modifier, which causes the  is equal to zero, i.e. the amount of solute in the mobile phase and stationary phase are equal ( = 1) 10 .
In order to determine the lipophilicity properties some novel 1,2,4-triazole derivatives, the chromatographic analysis in reversed phase system was conducted.Previous studies have shown that the examined compounds show antibacterial activity 13,14 .The inhibitory activities against Grampositive bacteria on the basis of minimal inhibitory concentration (MIC, µg/mL) values showing the following compounds: 6,7,8,9,10 and 16,18 13,14 .
The aim of this work was the determination of lipophilicity of the 1,2,4-triazole derivatives by RP-HPLC and RP-TLC methods (  or   0 , , ϕ 0 and the scores corresponding to the first principal components of  and   or   and   0 values) and using different calculation methods (, , , , , , , 2, 3and logPChS) [15][16][17] . 1.2 software calculates log P values as the sum of group contribution and correlation factors ( parameter) 15,18 .These have been obtained by fitting calculated  with experimental  for a training set more than twelve thousand, mostly drug-like molecules.
In this way hydrophobicity values for 35 small simple "basic" fragments have been obtained, as well as values for 185 larger fragments, characterizing intramolecular hydrogen bonding contribution to  and charge interactions.Molinspiration methodology for  calculation is very robust and is able to process practically all organic and most organometallic molecules.
calculation method is implemented as increment system adding contributions of every atom based on its atom type 16 .All together the  predicting engine distinguishes 368 atom types which are composed of various properties of the atom itself (atomic no and ring membership) as its direct neighbours (bond type, aromaticity state and encoded atomic no).More than 5000 compounds with experimentally determined  values were used as training set to optimize the 369 contribution values associated with the atom types.The program  2.1 from Virtual Computational Chemistry Laboratory provides interactive on-line prediction of  (, , , , , 2, 3) 17 .The method is based on atom -type E -state indices and associative neural network modeling and was developed by Tetko et al.This method combines electronic and topological characteristics to predict the lipophilicity of the molecules analyzed.

Results and discussion
1,2,4-triazole derivatives shown in Table 1 were analyzed by RP-TLC and RP-HPLC methods using methanol-water and acetonitrile-water as mobile phases (mobile phase composition are shown in Table 2).The linear relationships between the  (or   ) values and volume fraction of methanol (or acetonitrile) were obtained in given analytical range (Table 2).The parameters of these relationships are presented in Table 3a (for methanol-water system) and Table 3b (for acetonitrile-water system).The goodness of fit the linear equation for experimental data was defined by Jaffe 19 .The excellent goodness of fit the linear equations (1-2) for experimental data for all compounds in methanol-water system was obtained (see Table 3a).For acetonitrile-water system, the excellent goodness of fit the Eq. ( 2) for experimental data in 17 out of 18 cases.In case of RP-HPLC method the correlation coefficient of the equation ( 1) > 0,98 in 12 out of 15 cases.
The values of   and   0 are the retention parameters of a substance in pure water (obtained by extrapolation of and   to pure water using equations 1 and 2).In case of HPLC method the compounds with number 7, 16 and 17 have rejected (asymmetric and wide peaks were obtained).The retention time values were ambiguous and the determination of   parameter was subject to large error of 50%.Similarities in the structure are visible for the first three pairs of compounds.The substituent R 1 for the compounds 1, 2, 3 is a phenyl, while the rest of compounds of analogous structure (13, 14, 15) is pyridine-2-yl.The presence of nitrogen in the heterocyclic structure of the analyzed compounds (proton-acceptor properties) to increase their affinity to the polar mobile phase (mixtures of methanol and water or acetonitrile and water).The lipophilicity determined experimentally (  ,   0 ) in each case are higher for phenyl substituent (Table 3).The average of differences between the   0 for these pairs of compounds (1, 2, 3 and 13, 14, 15) equal:
The next group are compounds 4 -7 and 16 -18.The average of differences between the   0 values obtained for methanol and acetonitrile for these compounds is ∆ = 0.9746.The lowest   0 values were obtained for compounds 4, 6 and 16 (these substances have the lowest value of molecular weight) and the highest   0 values were obtained for compounds 7 and 18.The compound 7 has the longest carbon chain of substituent R 2 and the 18 has p-chlorophenyl substituent and the highest molecular weight.
In case the methanol-water system (in HPLC method) the highest   value was obtained for 4, but in case of acetonitrile-water eluent the most lipophilic compound is 6.The compounds 8, 9, 10 and 12 have additional heterocyclic ring in their structure.There are most lipophilic substances among all analyzed compounds.The values of   and  0 from methanol-water system are higher than 3.2 and for acetonitrile are higher than 2.0.The most lipophilic compound of this group is 9 (Table 3).
The significant influence of used organic modifiers (methanol and acetonitrile) on the chromatographic lipophilicity parameters (  ,   0 ) were observed.The values of   ,   0 for all compounds are higher for methanol-water chromatographic system than the acetonitrile-water system.As it is well known acetonitrile elution strength is smaller than methanol.Methanol and acetonitrile belong to two different groups of solvents based on the Snyder's selectivity triangle 20 .The methanol is in the second group and acetonitrile in the sixth.According to Karger at al 21 the protondonor solubility parameter for methanol is high (acetonitrile does not have proton-donor properties) and the proton-acceptor solubility parameter is also much higher for methanol than for acetonitrile.Based on obtained results stronger affinity of 1,2,4-triazole derivatives to acetonitrile than to methanol were observed.
The slope () from Eq. ( 1) and Eq. ( 2) is negative in all cases (Table 3), and it is suggested that the hydrophobic surface of the molecule interacts the non-polar adsorbent 22 .Generally the absolute value of the slope is lower for acetonitrile than for methanol as organic modifier of eluents.The lines of Eq. (1) and Eq. ( 2) are more steep for methanol-water mobile phase than for acetonitrile-water mobile phase.
As it is shown in Table 2 the concentration range for methanol or acetonitrile is not the same for all compounds.Therefore, a range of concentrations was selected (for methanol 45%-70% and for acetonitrile 25%-50%, in both cases a concentration increased by 5%) and missing values of  (and   ) were calculated by extrapolation on the basis of a linear equation.Selection of these intervals were performed in order to extrapolate it was always in the direction of lower concentrations of organic modifier.
As shown in Table 4 poor correlations between chromatographic lipophilicity parameters and computed logP values were obtained.The best correlations between the chromatographic lipophilicity parameter (  or  0 ) and the calculated logP values were obtained for logP calculated by 2.1 program.The high value of the correlation coefficient for relationships between the 1/ and ϕ 0 were observed for methanol and acetonitrile for HPLC method (r ≥ 0,97) (see Table 4).Weaker correlations between 1/  and ϕ 0 were obtained for TLC methods (r ≥0,80) than HPLC one.These results may indicate that the lipophilic interactions between the non-ionizable 1,2,4-triazole and 1,3,4-thiadiazole derivatives and stationary phases occur in this case 23 .Moreover in case of TLC method a high value of correlation coefficient (r = 0,95) for relationships:   .2/  was obtained.This may indicate the presence of residual specific interactions between analyzed substances and adsorbent.The eigenvalues and cumulative variance for  and   values were collected in Table 5.The value of cumulative proportion for the first two principal components were compared.They explain 99,84% of variance in the case of acetonitrile as modifier, 99,47% of variance in the case of methanol (HPLC).In the case of TLC method these values are 98,70% (for acetonitrile) and 97,77% (for methanol).This is probably related to the precision of the measurements.
. values M R and logk .Eigenvalues of the covariance matrix and cumulative proportion for

Conclusions
The lipophilicity of 1,2,4-triazole derivatives were obtained both chromatographically and by calculating methods.The linear correlation between retention parameter (,   ) and the concentration of organic modifier were received using RP-HPLC and RP-TLC methods.The values of   and   0 are the retention parameters obtained for pure water by extrapolation.Based on the relationship between the structure of analyzed compounds and their retention parameters better results were obtained for TLC method than HPLC.The   and   0 values obtained are less for acetonitrile modifier than methanol in both chromatographic methods.A stronger affinity the 1,2,4-triazole and derivatives for acetonitrile than for methanol were observed.
The best correlations between the chromatographic indices of lipophilicity and calculated by  2.1 program were obtained.The 1/ (or 1/  ) and ϕ 0 parameters are most suitable for the assessment of lipophilicityof the 1,2,4-triazole derivatives.

Materials
1,2,4-triazole derivatives (Table 1) were synthesized in the laboratory at the Department of Organic Chemistry in Medical University of Lublin.Two binary solvent systems: methanol-water and acetonitrile-water were used.Solvents (methanol and acetonitrile) were LiChrosolv (Merck, Darmstadt, Germany) for liquid chromatography grade and bidistilled water was used as the diluter.
Chromatograms were detected at 254nm.All experiments were repeated triplicate and the final results were its arithmetic mean.Dead time was measured for uracil (Calbiochem -Merck, Darmstadt, Germany).

Thin Layer Chromatography
Thin-layer chromatography was performed on 10x10cm TLC plates coated with RP-18254 using methanol-water and acetonitrile-water mixtures as mobile phase (Table II).0,5% ofthe solutions wereappliedto the plates and they were developed to a distance of 9cm at room temperature in a horizontal chambers DS II (Chromdes, Lublin, Poland).After drying the chromatograms were visualized at a wavelength of254nm.Each experiment was performed three time.

PCA calculations
The necessary calculations were performed using Microsoft Excel 2002.The PCA analysis were facilitated using the Pooptools TM program 24 .

Table 2 .
The solvent mixtures used as mobile phases (n -number of points)

Table 3a .
Parameters of the equations (1,2) for methanol-water system

Table 4 .
The correlation matrix concerning results obtained experimentally and theoretical lipophilicity parameters.