Supramolecular Organization of Neutral and Ionic Forms of

Article pubs.acs.org/crystal

Supramolecular Organization of Neutral and Ionic Forms of Pharmaceutically Relevant Glycyrrhizic AcidAmphiphile SelfAssembly and Inclusion of Small Drug Molecules Ewa Tykarska,*,† Stanisław Sobiak,† and Maria Gdaniec*,§ †

Department of Chemical Technology of Drugs, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland Faculty of Chemistry, Adam Mickiewicz University, Grunwaldzka 6, 60-780 Poznan, Poland

§

S Supporting Information *

ABSTRACT: The first structural characterization of glycyrrhizic acid, its monoammonium salt (AGA), and the complex of the salt with p-aminobenzoic acid in the solid state is reported. X-ray crystallography reveals that neutral and ionic forms of GA have similar supramolecular organization, with aglycon groups protruding from the 2D hydrogenbonded sugar platform. Interpenetration of these assemblies leads to the generation of intersecting channels where solvent and guest molecules are enclosed. It is worth noting that small solvent molecules or cations can functionally replace three water molecules which are an integral part of the sugar platform in GA. The crystal structures indicate the drug and cation binding sites and explain the similar properties of glycyrrhizic acid and its salts. The presented structures provide information about the supramolecular organization of amphiphilic GA molecule in the solid state, giving a guide into a possible aggregation mode in gels and solutions.

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INTRODUCTION The potency and therapeutic effects of drugs can be affected by modifying the drug delivery system.1−10 Formation of supramolecular complexes of active pharmaceutical ingredients is among the best known methods of improving drug stability, water solubility, bioavailability, and toxicity. Cyclodextrins are the best known and widely used drug-complexing agents that have been shown to influence both the physicochemical parameters and the pharmacokinetics of drugs.7−10 There is, however, a constant search for new supramolecular complexing agents,11−13 and 18β-glycyrrhizic acid (GA) and its salts emerge as promising compounds in this field of pharmaceutical applications.14−23 GA (Scheme 1) is a natural saponin and a principal, biologically active component extracted from the licorice roots in the form of glycyrrhizin, a mixture of its potassium, calcium,

and magnesium salts. The molecule consists of a lipophilic triterpenoid aglycon conjugated with a hydrophilic disaccharide glycon containing β(1→2) linked D-glucuronic acids. GA, being mainly recognized for its remarkable medicinal properties,24−31 has been recently the subject of renewed interest, related to its ability to form water-soluble complexes with hydrophobic molecules.15−23 Moreover, the synergistic impact of GA on the therapeutic activity of drugs has been reported. The mechanism of supramolecular complex formation by GA is a matter of controversy,14 and it should be different from that of cyclodextrins, as no stringent restrictions are imposed on the size of guest molecules. On the other hand, the GA molecule, with its open-chain amphiphilic structure, has micelle-forming ability in water and water−alcoholic solutions, and thereby, the ability to enhance the solubility of poorly soluble drugs in aqueous media might be related to enclosure of drug molecules within the micelles. In view of the significant interest in the biological activities and physicochemical properties of GA, its salts, and complexes, the very limited structural information on this natural compound is indeed surprising. The basic motivation for this study was to look at the aggregation mode of GA in the crystalline state through determination of its crystal structure, in order to get more insight into the possible supramolecular organization of GA in solutions and gels. As the monoammonium salt of GA (hereafter AGA) shows similar properties to GA, the crystal structure of this salt was also determined to compare the aggregation modes of the neutral and anionic forms of GA. In

Scheme 1

Received: February 3, 2012 Published: February 28, 2012 © 2012 American Chemical Society

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dx.doi.org/10.1021/cg300160c | Cryst. Growth Des. 2012, 12, 2133−2137

Crystal Growth & Design

Article

view of reports that GA, or AGA, can be used as a complexforming agent for some active pharmaceutical ingredients, an attempt was made to cocrystallize GA and its salt with small drug molecules, water-soluble and insoluble. The only successful preparation was with AGA and a slightly watersoluble p-aminobenzoic acid (PABA), a vitamin which has a carboxylic group complementary with the carboxylic groups located on the terpene and disaccharide parts of the host.

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EXPERIMENTAL SECTION

All reagents (analytical grade) were obtained from commercial suppliers and used without further purification. In water, in the concentration range required for crystallization experiments, most triterpene saponins including GA and its monoammonium salt form colloidal suspensions or gels, and thus, their crystallization is not a trivial task. The concentration of GA and AGA for crystallization experiments was chosen in the 5−50 mM range. To determine the best crystallization conditions, diverse organic solvents, including alcohols, carboxylic acids, ethers, and ketones, were added to aqueous suspensions of glycyrrhizic acid or its monoammonium salt at 90 °C until a colloidal suspension did not appear on cooling to room temperature. This experimentally determined minimum solvent concentration was the starting point for subsequent crystallization attempts using the hanging drop vapor diffusion technique. The crystalline material was obtained from solutions where the concentration of GA or AGA was at least 10 mM. The crystallization conditions were further optimized on a larger scale by either slow evaporation or vapor diffusion methods. Single crystals of 1 were grown from a 50 mM solution of GA in a 1:1 (v/v) H2O/ propionic acid mixture by a slow evaporation method at room temperature. Crystals of 2 and 3 (20 and 25 mM AGA, respectively) were obtained at room temperature by the vapor diffusion method. AGA (2) or a 1:4 molar mixture of AGA and PABA (3) was dissolved in a 70% methanol solution on heating and equilibrated with 90−95% methanol. The diffraction intensity data for crystals 1−3 were collected at 130 K with an Oxford Diffraction SuperNova diffractometer using microsource and mirror-monochromitized Cu Kα radiation (λ = 1.5418 Å) and processed with the Oxford Diffraction CrysAlis Pro software.32 All structures were solved by direct methods and refined by full matrix least-squares on F2 (SHELXS-97 and SHELXL-97).33 The structures 1−3 exhibit an extensive disorder of solvent molecules whereas the GA and PABA molecules are quite ordered. GA exhibits minor disorder in 1 where the carboxylic group of the B sugar occupies two sets of sites that are related by rotation around the C−C bond. In 1 the only ordered solvent molecule is the propionic acid molecule connected via the cyclic hydrogen-bond motif to the triterpenoid carboxylic group. The remaining solvent molecules are disordered over two or three positions with partial occupancies. In 2 and 3, sites I and II of the sugar platform (Figure 1) are occupied by an ordered ammonium ion and methanol molecule, respectively, and site III is disordered, being occupied by methanol or a water molecule. In both crystal structures, an extensive disorder of the methanol and water solvent was modeled. The H atoms from C−H groups were included in calculated positions with C−H = 0.93−0.98 Å and refined as riding on their corresponding carbon atoms, with Uiso (H) = 1.2U eq (C) or 1.5Ueq(methyl C). The H atoms from O−H and N−H groups, whenever possible, were located in difference electron-density maps, taking into account possible hydrogen-bond interactions. The summary of the structure determination for 1−3 is given in Table 1. Hydrogen bonds for 1−3 are given in Tables S1−S3 (Supporting Information).

Figure 1. Structure of GA: (a) the X-ray structure showing mutual orientation of the inner A and the outer B sugar units; three balls represent water molecules placed at sites I, II, and III. Hydrogen bonds are indicated by dashed lines. (b) A space-filling illustration of the concave, L-shaped structure.

of GA (1), AGA (2), and AGA-PABA (3) were unstable in air, and the crystals collapsed immediately when removed from the mother liquor. X-ray crystallography clearly showed that, despite variation in the ionization state of the host and inclusion of a relatively large organic molecule, the crystal packing in the three examined crystalline forms 1−3 is nearly identical. As shown in Figure 1, the connection of a rigid, concave-shaped triterpenoid unit to a disaccharide group generates the L-shaped GA molecule with the β(1→2) linked D-glucuronic acid units as the base of the letter L. The two sugar rings are differently oriented relative to the triterpenoid, with the inner (A) sugar ring supplementing its concave shape and the outer (B) sugar ring strongly twisted relative to the inner sugar unit. Our results also confirm the βconfiguration of the two glycosidic bonds in the GA molecule.34 A closer inspection of the molecular structure reveals that a plane passing through the carbon atoms attached to the carboxylic groups divides the GA molecule in such a way that these groups are all located on one side of the plane whereas sugar hydroxyl groups are found on the opposite side. This seems to be an important feature of the GA molecular structure, as it has been shown35 that the carboxylic trio in potassium β-glycyrrhizinate catalyzes a pH dependent hydrolysis of nonionic ester surfactants. Moreover, three of the four ether oxygen atoms and the carbonyl O atom of GA are grouped on the same side of the plane as carboxylic groups and exposed as acceptors of hydrogen bonds (Figure 1). GA is a triacid, and as shown by the structures 2−3 of its monoammonium salts, its deprotonation starts with the carboxylic group of the inner glucuronic unit. As can be seen in Figures 2 and S3 (Supporting Information), the L-shaped GA molecules, or GA monoanions, form in the crystal a characteristic pattern of alternating hydrophilic and hydrophobic areas, that result from the crystal packing of hydrogen-bonded GA assemblies. These assemblies can be seen as bilayers consisting of the hydrophilic interior formed by a two-dimensional sugar platform and aglycon fragments protruding from its surface on both sides. The triterpenoid groups are not densely arranged on this surface, leaving enough space for interpenetration of adjacent bilayers. However, their mutual penetration is limited and the carboxylic group of the aglycon does not reach the sugar platform of the

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RESULTS AND DISCUSSION All crystallizations were carried out from water solutions with an addition of methanol (AGA) or propionic acid (GA) to overcome gelation problems. The highly solvated crystal forms 2134



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Table 1. Crystal Data and Refinement Details for 1−3 empirical formula CCDC no. formula weight crystal system temp, K space group unit cell dimens., Å

volume, Å3 Z calc density, g/cm3 absorption coefficient, mm−1 reflections collected ind reflections data/restraints/ parameters goodness-of-fit on F2 final R indices [I > 2σ(I)] R indices (all data) largest peak and hole, e A−3

1

2

3

C42H62O16·4.26CH3CH2COOH·3.94H2O 859196 1203.71 orthorhombic 130 P212121 a = 10.6054(2) b = 11.3456(2) c = 52.5014(5) 6317.2(2) 4 1.266 0.857

C42H61O16·NH4·6.25CH3OH·2.04H2O 859197 1077.09 orthorhombic 130 P212121 a = 10.3718(2) b = 11.2995(2) c = 50.3536(6) 5901.3(2) 4 1.212 0.811

C42H61O16·NH4·C7H7NO2·3.53CH3OH·1.82H2O 859198 1122.95 orthorhombic 130 P212121 a = 10.4464(2) b = 11.2468(2) c = 51.0762(5) 6000.9 (2) 4 1.243 0.815

27074 10604 10604/255/1003

29554 10159 10159/0/689

26670 10263 10263/0/716

1.048 R1 = 0.0716,

1.046 R1 = 0.0654,

1.103 R1 = 0.0701

wR2 = 0.1984 R1 = 0.0721, wR2 = 0.1990 0.74, −0.42

wR2 = 0.1855 R1 = 0.0656, wR2 = 0.1860 0.68, −0.27

wR2 = 0.1905 R1 = 0.0712 wR2 = 0.1913 0.88, −0.32

from the neutral (1) or ionic (2−3) diglucuronic units. A noticeable feature of these platforms are hydrophilic hollows found between hydrogen-bonded zigzag chains (Figure 3) of

Figure 3. Space-filling illustration of the sugar platform showing hydrophilic hollows between hydrogen-bonded zigzag chains and the three sites occupied by water molecules, ammonium ions, or solvent OH groups.

diglucuronic units where water molecules, solvent hydroxy groups, and ammonium ions are accommodated. The hydrogen-bonded water molecule from this area, that is placed in the anti position relative to the carboxylic group of the sugar A (Figure 1a, site I), is replaced by the ammonium ion in 2 and 3. This ion now binds to the carboxylate group, and only a minor reconstruction of the hydrogen-bond system in this region is required to accommodate H2O/NH4+ and COOH/COO− replacements (Figure 4). Within the hydrophilic hollow of the sugar platform, there are three distinct sites denoted as I− III, with position I occupied by the ammonium cation (AGA) or a water molecule (GA) and positions II and III occupied by either a water molecule or a hydroxy group of an alcoholic solvent. The interaction patterns of the chemical units accommodated at these three sites suggest a possibility of

Figure 2. Schematic presentation of the supramolecular organization of the host molecules. Molecules shown in gray belong to neighboring bilayers.

neighboring bilayer but stops at the level of the triterpenoid ring B. This arrangement leads to a system of intersecting channels filled with guest molecules that run along the crystal a and b axes. These channels account for ca. 42% of the crystal volume. The size of the channels can be slightly modified, mainly by small shifts in the direction perpendicular to the bilayers. To explain the unexpected similarities in the crystal packing of GA and its monoamonium salt, a closer look was taken at the structure of the hydrogen-bonded sugar platforms constructed 2135



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Figure 4. Comparison of the hydrogen-bond systems in GA and AGA. The arrows indicate directions of hydrogen bonds. Dashed lines represent hydrogen bonds.

their replacement by an additional ammonium cation in the diammonium salt or by metal ions. The crystal structure of the only complex obtained by cocrystallization of AGA with a pharmaceutical compound, the AGA-PABA complex, shows that the PABA molecule replaces some of the solvent molecules located in the hydrophobic area of the intersecting channels (Figure S2). In 2 there are four methanol molecules bridging two adjacent bilayers by forming hydrogen bonds between the triterpenoid carboxylic group in one bilayer and the sugar platform of the other one. The PABA molecule replaces three of these methanol molecules by binding via the R22(8) motif to the aglycon carboxylic group and through the amino group to the methanol attached to the sugar platform. There are no close contacts between the PABA molecules in the channels. This structure might serve as an illustration of the GA complexation mechanism of compounds poorly soluble in water.

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CONCLUSION In summary, the three reported crystal structures provide for the first time detailed information related to the molecular structure and the assembly mode of the pharmaceutically relevant natural product, glycyrrhizic acid. This experimentally determined structure should be useful in modeling interactions of GA with biological macromolecules.36 We also believe that the observed supramolecular structure of GA, featuring alternating hydrophobic and hydrophilic areas, can form a solid basis for modeling GA micelles and their complexes with drug molecules.

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ASSOCIATED CONTENT

S Supporting Information *

Hydrogen bonds tables, atom labeling system, and crystallographic information files (CIF). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +48 61 8546639. Telephone: +48 61 8546632. E-mail: [email protected]. Telephone: +48 61 8291273. Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Yoon, H.-J.; Jang, W.-D. J. Mater. Chem. 2010, 20, 211−222. (2) Ajazuddin, S. S. Fitoterapia 2010, 81, 680−689. 2136



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(36) Mollica, L.; De Marchis, F.; Spitaleri, A.; Dallacosta, C.; Pennacchini, D.; Zamai, M.; Agresti, A.; Trisciuoglio, L.; Musco, G.; Bianchi, M. E. Chem. Biol. 2007, 14, 431−441.

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Supramolecular Organization of Neutral and Ionic Forms of

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