the Study of the Complexation of Calix [ 4 ] arene and Calix [ 4 ] reSorCinarene with reSin aCidS by the rp hplC method . binding ConStantS determination

The Host-Guest complexation of octakis-(diphenoxyphosphoryloxy)tetramethylcalix[4]resorcinarene (CR) and 5,17-bis-(N-tolyliminomethyl)-25,27-dipropoxycalix[4]arene (CA) with 6 diterpenoid (resin) acids has been studied by the reversed phase high-performance liquid chromatography (RP HPLC). The chromatographic characteristics (retention time tR and retention factor k’) of resin acids have been determined. The lipophilicity values log P of the acids, binding constants KA (395-682 М-1 for CR and 844-1268 М-1 for CA), as well as Gibbs free energies ∆G (-14.79 – -16.14 kJ/mol for CR and -16.70 – -17.67 kJ/mol for CA) of the complexes with resin acids have been calculated. Molecular modelling of CA complexes has revealed the presence of hydrogen bonds between carboxylic groups of acids and nitrogen atoms of imino groups at the upper rim or oxygen atoms of the hydroxyl groups at the lower rim of the CA macrocycle. Molecular modelling of CR complexes has shown the presence of hydrogen bonds between carboxylic groups of acids and oxygen atoms of diphenoxyphosphoryloxy groups at the upper rim of the CR macrocycle. The effect of log P values on KA values of the CR/CA complexes has been assessed. The linear dependence of the binding constants on the acid lipophilicity indicates a significant role of solvophobic interactions on the complexation. The relationship between supramolecular (KA) and physicochemical (log P, pKa) characteristics of acids has been determined.

Pentacyclic triterpenes -betulin, betulinic, oleanolic and ursolic acids possess the anticancer, anti-inflammatory and antiviral activity.Unfortunately, the biological activity of these compounds is reduced by their poor solubility or bioavailability.To improve these properties the supramolecular Host-Guest complexes of betulinic acid, ursolic acid (K A 140 M -1 ) or oleanolic acid (K A 145 M -1 ) with cyclodextrins were prepared and studied [16][17][18].
Along with cyclodextrins, calixarenes [19] are one of the most important categories of the supramole-cular Hosts for application in pharmacology [20][21][22].Compared to cyclodextrins, calixarenes exhibit a high degree of chemical functionalization, which leads to obtaining compounds with interesting physicochemical and binding properties.There are many conformational isomers of calixarenes, and a large number of cavities of different sizes and shapes, which can be involved in molecular recognition and binding processes.
In this work for the first time the complexation Cr and CA with 6 diterpenoid resin acids -pimaric 1, maleopimaric 2, palustric 3, dehydroabietic 4, abietic 5 and neoabietic 6 (Guest molecules) (Fig. 1) in watercontaining solutions was studied, and stability constants of their supramolecular Host-Guest complexes were determined by the RP HPLC method.To the best of our knowledge, no complexation of any calixarenes with diand triterpenoid acids was described in literature.
We believe that the study of the complexation of CA and Cr with resin acids may be useful for understanding of the process of recognition and binding of diterpenoids in the aqueous medium.The results obtained can be used for developing drug delivery systems for these biologically active acids.

Results and Discussion
The Cr/Ca were registered on the chromatograms by sharp peaks with retention factors k' 8.65 (Cr) and 0.89 (CA).Chromatograms of the resin acids obtained before and after Cr and CA addition in the mobile phase are presented in Fig. 2, 3.
A comparative estimation of the chromatograms presented in Fig. 2, 3 shows that Cr and CA addition to the mobile phase decreases the retention times and changes the elution order of acids.It should be noted CA addition allows separating abietic 5 and neoabietic 6 acids (Fig. 3).Formation of the Host-Guest inclusion complexes weakens the interaction of these ISSN 2308-8303 acids with the stationary phase in the RP HPLC conditions.The linear character of 1/k' vs plots on the calixarene concentration (r = 0.99) indicates formation of the Host-Guest supramolecular complexes with 1:1 stoichiometry.The retention factors k' of acids 1-6 obtained before and after addition of Cr and CA to the mobile phase are presented in Tab. 1.
The binding constants K A and free Gibbs energies ΔG (ΔG = -RT ln K A ) of the Host-Guest calixarene complexes with the acid molecules were calculated by the method described in and are presented in Tab. 2.
As shown in Tab. 2, the binding constants K A of resin acids 1-6 are in the range of 395-682 M -1 for Cr complexes and 844-1268 M -1 for CA complexes.The complexes of resin acids can be stabilized by different supramolecular interactions (hydrogen bonds, van der Waals, solvophobic interaction, etc.).Therefore, the role of hydrophobic interactions for the complexes is confirmed by the binding constants correlation with log P of resin acids (Fig. 4, 5).
Increase of the log P values of acids increases the K A values of their complexes with Cr (Fig. 4), but decreases K A values for the complexes with CA (Fig. 5).
To clarify the nature of supramolecular interactions the molecular modelling of CA and Cr complexes with resin acids were carried out.The Host-Guest complexation with CA is presented in Fig. 6.
As shown in Fig. 6A-6F, all resin acids are included into the macrocyclic cavity of CA.The inclusion of Host-Guest complexes is stabilized by different supramolecular interactions, first of all, hydrogen bonds.In the complexes shown in Fig. 6A, 6C and 6D the hydroxyl group of pimaric, abietic and neoabietic acids, respectively, form hydrogen bonds with a basic nitrogen atom   of the upper rim imino group.The carboxylic groups of dehydroabietic, palustric and maleopimaric acids form hydrogen bonds with oxygen atoms on the CA lower rim (Fig. 6b, 6E, 6F).

Еxperimental Part
The methanol and acetonitrile were obtained from Acros Organics (Thermo Fisher Scientific, New Jersey -USА), and resin acids were obtained from Sig-ma-Aldrich (Sigma-Aldrich Corporation, Sigma-Aldrich Box 14508, St. Louis Missouri, 63178, USA).Cr was synthesized by the method [28] and CA -by the method [29].

rp hplC analysis
The chromatographic experiment was performed on a Hitachi liquid chromatographic system (Hitachi, Ltd., Tokyo, Japan) with an UV detector (λ = 254 nm).The column (250×4.6 mm i.d.) was LiChrosorb RP 18 ISSN 2308-8303 (Merck, Germany).The mobile phases based on Cr and CA (C = 0.01 mM) were prepared by dissolving calixarenes in MeОН/H 2 O/formic acid (75/25/0.01,v/v) (phase A for Cr) and MeCN/H 2 O/formic acid (86/14/ 0.01, v/v) (phase B for CA), respectively.The sample injected was 20 μL.All chromatograms were obtained at 22°C.All measurements were performed in triplicate.The phases A and B were used as blank ones for Cr and CA analysis, respectively.
The binding constants of Cr and CA complexes with acids 1-6 were calculated by the RP HPLC method described in [30] by changing of the retention factor k' values for acids during complexation.

determination of the log P values of resin acids and molecular modelling
The values of log P of acids 1-6 were calculated from the equation: log P = 7.746 .(log k') where coefficient 7.746 was the ratio of the experimental value of log P of abietic acid 6.46 [31] to log k' value of abietic acid 0.834 determined by the RP HPLC method in this work.
Molecular modelling of calixarene complexes with acids 1-6 were carried out using Hyper Chem 8.0 (PM3) [32].The structures were calculated by the semi-empirical method PM3.The RMS (standard deviation of the word root mean square) gradient was equal to 0.01 kcal/A mol.

Conclusions
Using the RP HPLC method it has been shown that the cone-shaped calix [4]arene and calix [4]resorcinarene functionalized at the upper rim with proton accepting imino groups or phosphoryl groups, respectively, form strong Host-Guest inclusion complexes (K A 395-1268 M -1 ) with a series of diterpenoid (resin) acids in water-containing solutions.The complexes are stabilized by the hydrophobic Host-Guest interactions and hydrogen bonds of the Guest COOH groups with basic nitrogen atoms or oxygen atoms of the Host molecule.Calixarenes similar to cyclodextrins can be considered as promising Host molecules in design of drug delivery systems for biologically active resin acids.

Table 1
Retention factors k' of resin acids 1-6 determined before and after CR/CA addition