THE STUDY OF COMPLEXATION OF 5,17-BIS-(N-TOLYLIMINO-METHYL)-25,27-DIPROPOXYCALIX[4]ARENE WITH BENZOIC ACIDS BY RP HPLC AND MOLECULAR MODELING METHODS

The Host-Guest complexation of 5,17-bis-(N-tolyliminomethyl)-25,27-dipropoxycalix[4]arene with benzoic acids has been studied by reversed-phase high-performance liquid chromatography (RP HPLC) method (the mobile phase – MeCN/H 2 O, 86/14 v/v, the column support – LiChrosorb RP 18, UV detector, l = 254 nm). The study of the chromatographic behaviour of 5,17-bis-(N-tolyliminomethyl)-25,27-dipropoxycalix[4]arene and benzoic acids, as well as determination of their main chromatographic characteristics – the retention times t R and capacity factors k’ have been performed. On the basis of the data obtained the lipophilicity log P, as well as the binding constants and Gibbs free energies of the complexes of 5,17-bis-(N-tolyliminomethyl)-25,27-dipropoxycalix[4]arene with benzoic acids have been calculated. The binding constants and Gibbs free energies of the complexes of 5,17-bis-(N-tolyliminomethyl)-25,27-dipropoxycalix[4]arene with benzoic acids are in the range of 335-910 М -1 or -14.38 – -16.85 kJ/mol, respectively. The influence of the benzoic acids lipophilicity log P and pKa values on the binding constants K A of the complexes has been examined. It has been found that decrease of the log P and pKa values increases the binding constants K A of the complexes. Molecular modeling of the complexes revealed the presence of hydrogen bonds between carboxylic groups of the acids and nitrogen atoms of imino-groups at the upper rim or oxygen atoms of the hydroxyl groups at the lower rim of the calixarene macrocycle. A linear dependence of the binding constants from the acid lipophilicity log P indicates a significant role of solvatophobic interactions during the complexation process.

Benzoic acids are widely used as drugs, and their salts as carriers of specific anions. In medical practice benzoic acid is used as an antibacterial and antifungal medicine for skin diseases and mycosis. Benzoic acid is a constituent of Whitfield's ointment, which is used for treating fungal skin diseases such as tinea, ringworm, and athlete's foot [18]. Its esters (from methyl to amyl) are used in the perfume industry. Hydrochloride of β-diethylaminoethyl ester of p-aminobenzoic acid is known as Novocain drug with a moderate anesthetic activity and a wide spectrum of the therapeutic action. p-Аmіnоbеnzоic acid (PABA) is the growth factor of microorganisms and its derivatives are used as effective antibacterial agents. PABA has been referred to as Vitamin B 10 . Some bacteria in the human intestinal tract such as E. coli generate PABA from chorismate [19].
Information on the supramolecular Host-Guest interaction of CA with benzoic acids will be useful in design of calixarene based sensors or drug delivery systems for the biorelevant acids.

Еxperimental Part
The RP HPLC study was performed on a Hitachi chromatograph (Hitachi, Ltd., Tokyo, Japan) consisting of a high-pressure pump connected to a Rheodyne sample 7120 injector with a 20 μL loop (Rheodyne, Berkeley) and an ultraviolet-visible detector. The column (250×4.6 mm i.d.) was packed with Li-Chrosorb RP-18 (Merck, Germany). Acetonitrile was obtained from Acros Organics. Сarboxylic acids were purchased from Sigma-Aldrich. CA was synthesized by the method [20]. The acetonitrile-water (86:14, v/v) mixture was used as a blank mobile phase. The calixarene based mobile phases were prepared by dissolving CA in the acetonitrile-water (86:14, v/v) mixture to obtain the CA concentration of 0.05-0.6 mМ. The analytes for injections were dissolved in the same acetonitrile-water (86:14, v/v) mixture (C = 0.01 mM). The amount of the sample injected was 20 μL. All chromatograms were recorded at 22°C. The UV detector was operated at 254 nm. The mobile phase contain-Chart ing the CA additive was equilibrated for 3 h before the analysis. Under these conditions the column was saturated with the CA additive. The dead time t 0 was measured with NaNO 2 .
Lipophilicity log P of acids 1-19 (Таble) was calculated from equation log P = 3.438 (log k'). The coefficient 3.438 is the ratio of log P value of benzoic acid 1 (1.87) [21] to its log k' (0.544) determined by RP HPLC in this work (Table). Free Gibbs energy DG is determined by the equation DG = -RT ln K A .
Molecular modelling of the CA complexes with acids 1-19 were carried out using Hyper Chem, 8.0 program [http://www.hyper.com/Download/AllDownloads/ tabid/470/Default.aspx]. The structures were opti-mized by the semi-empirical PM3 method. The RMS gradient was equal to 0.01 kcal/A mol.

Results and Discussion
Cаlіxаrеne CA and benzoic acids 1-19 in the given analysis conditions were registered on the chromatograms as sharp peaks. Cаlіxаrеne CA retention time t R and сapacity factor k' were 5.67 min and 0.89, respectively. A linear isotherm character (Fig. 1) reflects reversible adsorption of calixarene CA on the LiChrosorb-RP18 support.
The chromatographic characteristics of acids 1-19retention times t R , retention volumes V R , capacity factors k', their log P, as well as binding constants К А and free Gibbs energies DG of their complexes with CA are presented in Таble.
Binding constants of the supramolecular Host-Guest complexes of calixarene with benzoic acids 1-19 were determined by RP HPLC method in accordance with [20]. The method consists of the choice of analysis conditions of the Host and Guest, determination of the retention time t R аnd the capacity factor k' of the Host, determination of the retention time t R аnd the capacity factor k' of the Guest before and after Host addition to the mobile phase. Addition of cаlіxаrеne to the mobile phase decreases the capacity factor k' values of benzoic acids. The linear character plots of k'ns the cаlіxаrеne concentration (R 2 = 0.95-0.99) testifies formation of Host-Guest supramolecular complexes with 1:1 stoichiometry [20].
The binding constant K A of the calixarene complex with the Guest molecule (the ratio of 1:1) can be calculated by equation (1): whеre k 0 ' і k' -are capacity factors of the Guest molecule determined in the absence and the presence of CA in the mobile phase.  As shown in Table 1, the binding constants K A of the complexes are in the range of 335-910 М -1 . The lowest K A values were observed for hydroxybenzoic acids (335-403 М -1 ) and toluic acids (401-448 М -1 ). The highest K A values were observed for phthalic (534-749 М -1 ) and nitrobenzoic (608-910 М -1 ) acids. It should be noted that the binding constants of CA with benzoic acid 1 is higher compared to its complex with β-cyclodextrin modified by three MeO-groups [22] (K A = 650 М -1 and 550 М -1 , respectively).
Calixarene Host-Guest complexes are stabilized by various non-valence interactions -hydrogen bonds, Van der Waals, π-π, C-H...π, solvatophobic interactions [8]. It has been found that there are the linear dependence of the binding constants K A from the lipophilicity log P of o-chlorobenzoic, o-nitrobenzoic, o-hydroxybenzoic, o-aminobenzoic, o-toluic acids (Fig. 2), as well as m-nitrobenzoic, m-chlorobenzoic, m-aminobenzoic, m-toluic acids (Fig. 3). It is interesting to note that increase of log P of the acids decreases K A values of the complexes.
The binding constants K A are strongly depended on pKa values of benzoic acids. These dependences for the Host-Guest complexes of meta and para substituted benzoic acids are demonstrated by Fig. 4 and Fig. 5, respectively. Unfortunately, for p-substituted acids such correlation has not been observed.
As it is shown in Fig.4, 5, the increase of рКа values of the acids decreases К А of their complexes with СА. Unfortunately, for o-substituted acids such correlation was not observed.
To clarify the nature of the supramolecular Host-Guest interactions the molecular modeling of CA complex with benzoic acid and o-, m-, p-phthalic acids was carried out. The inclusion complex of benzoic acid 1 shows two approximately energy equal structures A (∆E=-29.38 kcal/ mol) and B (∆E=-31.15 kcal/mol) with one hydrogen bond between the Host and Guest molecules (Fig. 6).
In the structure A the hydrogen bond of the OH group of benzoic acid 1 with basic nitrogen of the imino group of CA is observed. In the structure B the OH group of the acid forms a hydrogen bond with the oxygen atom at the lower rim of the macrocycle.
Energy minimized structures of CA complexes with o-phthalic (C), m phthalic -(D) and p-phthalic (E) acids are presented in Fig. 7.
Calixarene complexes with o-phthalic C and m-phthalic D acids are stabilized by two hydrogen bonds. Thus, one carboxyl group forms a hydrogen bond with the imino nitrogen atom, and another with the oxygen atom of the hydroxyl groups at the lower rim of the macrocycle. Complex E with p-phthalic acid having the most distant carboxyl groups is stabilized by two hydrogen bonds with the distal imino nitrogen atoms.   It should be noted that the Host-Guest complexation does not change the flattened-cone conformation of calixarene confirming complementarity of benzoic acids to the molecular architecture of the calixarene macrocycle.

Conclusions
5,17-bis-(N-tolyliminomethyl)-25,27-dipropoxycalix [4]arene containing two imino groups on the up-per rim of the macrocycle forms the Host-Guest inclusion complexes with benzoic acids, which are characterized by binding constants 335-910 М -1 in acetonitrile-water solution. The data obtained provide the basis for creating a highly sensitive sensor for recognition and binding of biologically active benzoic acids in the biological media. Calixarene is a promising compound in design of the sensor devices or drug delivery systems for biorelevant acids.