INCLUSION OF ALKYLPARABENS IN CYCLODEXTRINS
INCLUSION OF ALKYLPARABENS IN CYCLODEXTRINS
M R Caira1, E J C De Vries1, M Bogdan2, D Bogdan2, S I Farcas2
1Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
2National Institute for R&D of Isotopic and Molecular Technologies, P.O. Box 700, Cluj-Napoca, Romania
ABSTRACT. The alkyl esters of 4-hydroxybenzoic acid, with formula HO-C6H4-COOR and R = Me, Et, n-Pr, n-Bu, are well known as antibacterial preservatives. Owing to their poor aqueous solubilities, their inclusion in cyclodextrins has been investigated by numerous workers as a means of enhancing their dissolution properties. This paper reports salient results of a systematic study of the inclusion of the alkylparabens in native and methylated cyclodextrins, with emphasis on the thermal and structural properties of the solid inclusion complexes. Techniques used to study the interaction between the various hosts and guests included X-ray diffraction, thermal analysis, FTIR spectroscopy and high-resolution NMR spectroscopy.
1. Introduction
Inclusion of drugs in cyclodextrins (CDs) is an attractive, widely used strategy for improving their performance [1]. Increased drug solubility, as well as physical and chemical stability are some of the advantages offered by this approach. The special features of cyclodextrins (cyclic oligosaccharides composed of -1,4-linked glucopyranose units) which enable them to encapsulate and solubilise hydrophobic guest molecules are their toroidal shape, hydrophobic interior cavity and hydrophilic exterior (Figure 1).
Fig. 1 Structures of the native cyclodextrins: from left to right -, -, and -cyclodextrin.
The guest molecules relevant to the present study are the alkylparabens, alkyl esters of 4-hydroxybenzoic acid (Figure 2), commonly employed as antibacterial preservatives in cosmetics, food products and pharmaceutical formulations [2]. These compounds, which are used singly or in combination, have broad spectrum antimicrobial activity in the pH range 4-8 and are most effective against yeasts and moulds. For the series shown in Figure 2, the aqueous solubilities at 25C are very low, ranging from 0.016-0.001M as the chain length of the alkyl moiety increases [3]. Consequently, methods for improving their solubility have been sought and one strategy for achieving this is cyclodextrin inclusion. Recent reports documenting increased aqueous solubility of alkylparabens effected by cyclodextrins include those on the use of heptakis(2,6-di-O-methyl-3-O-acetyl)--cyclodextrin [4] and 2-hydroxypropyl--cyclodextrins with different degrees of substitution [5].
Fig. 2 Structures of the guest molecules, methyl-, ethyl-, n-propyl- and n-butylparaben.
Our interest in the area of cyclodextrin-drug complexation prompted us to investigate the physical stabilities and structures of solid inclusion compounds formed between alkylparabens and a range of cyclodextrins [6]. Apart from the pharmaceutical interest, the availability of this homologous series of guests allowed investigation of the mode of guest inclusion as a function of alkyl-chain length. Here we report some pertinent results gleaned primarily from thermal analysis and X-ray diffraction. Additional information on the interaction between the four alkylparabens shown in Figure 2 and -cyclodextrin in solution was obtained by NMR experiments. Owing to the large mass of available data we have accumulated on this topic and limited space, only selected aspects of the work are alluded to here. Full accounts which describe the detailed structures and thermal decompositions of the complexes with native and derivatised cyclodextrins are in preparation and will be published in extenso elsewhere as indicated under individual sections of this paper.
2. General Experimental Procedures
Proton NMR experiments were performed at 298K in D2O solution and at 300 MHz on a Varian-Gemini 300 spectrometer to study the interaction between -cyclodextrin and each of the four guests. The continuous variation method was used to determine complex stoichiometry in solution. This involved plotting measured changes in chemical shifts for both host and guest protons, obs[X], against the mole fraction ratio r. Computation of the association constants followed. The procedures used were described in detail recently [7].
Solid inclusion complexes with - and -cyclodextrins as hosts were generally prepared by kneading and co-precipitation methods, using the individual pure alkylparabens as guests in 1:1 molar ratio with the host. With the methylated cyclodextrins DIMEB [heptakis(2,6-di-O-methyl)--cyclodextrin] and TRIMEB [heptakis(2,3,6-tri-O-methyl)--cyclodextrin], the technique of incubating a solution of the drug and cyclodextrin at 50-60C was employed, leading to crystallization of inclusion complexes.
Putative complexes were examined by hot stage microscopy, thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). Recorded powder X-ray diffraction (PXRD) patterns were compared with reference patterns [8] to establish complex formation and possible space groups. Single crystal X-ray diffraction was applied to complexes that yielded crystals of adequate quality. Intensity data were collected on a Nonius Kappa CCD diffractometer with the crystals generally cooled in a stream of nitrogen to improve diffraction quality. Structures were solved by a variety of methods including Patterson search and isomorphous replacement techniques. For structure refinement, full-matrix least-squares methods were employed.
3. Interaction between the parabens and -cyclodextrin
In solution, inclusion complex formation between the parabens and -cyclodextrin was inferred from the NMR experiments which showed upfield shifts of the host H3, H5 and H6 protons, the first two of which are located inside the cavity and the last at the narrow primary rim of the macrocycle. No significant shifts were observed for the host H1, H2 and H4 protons. Distinct peaks were not observed for a bound and a free form.This observation implied that complexation is a dynamic process, the included drug undergoing fast exchange (relative to the NMR time scale) between the free and bound states and only the shifts of the spectral lines were observed. Therefore the exchange rate between the free and bound states must exceed the reciprocal of the largest observed shift difference (in Hz) for any proton of the guest molecule [9]. The stoichiometries of the complexes were determined using the continuous variation methodby following the changes in the chemical shifts of the host protons which showed the greatest variations viz. H3, H5 and H6. The Job plot of the -CD proton shifts is more accurate than that of the paraben
proton shifts, as the -CD signals are strengthened by the seven chemically equivalent protons (one from each glucose monomer). The Job plots for the -CD H3, H5 and H6 protons are unremarkable, having an almost symmetric appearance, indicating that only one type of complex had formed [9]. For each -CD-paraben complex, the Job plot showed a maximum at r = 0.5, indicating the existence of a complex with 1:1 stoichiometry within the range of concentrations investigated. The association constants for the 1:1 complexes were evaluated by a non-linear least-squares regression analysis of the observed chemical shift changes of the drug and -CD NMR lines, as a function of -CD concentration. For consistency, K was evaluated from the observed differences in chemical shifts for the H3, H5, and H6 protons of -CD in each case. The overall association constants (K) obtained were 1631, 938, 460 and 2022 M-1 for complex formation with methyl-, ethyl-, propyl- and butylparaben respectively. The (H5) (range 0.24-0.28 ppm) was generally found to exceed (H3) (range 0.13-0.26 ppm) and it was concluded that the guests penetrate the cavity fairly deeply and that the host primary side is also involved in inclusion. The general decrease in K with increasing guest alkyl chain length (up to propyl-) is consistent with this part of the guest finding itself outside the cavity in the hydrophilic water environment. However the butylparaben complex is inconsistent with this trend and the calculated association constant suggests that this guest has the tightest fit within the CD cavity.
Regarding complex structure in solution, some conclusions were drawn from the complexation-induced shifts. The signals of the included paraben were shifted by complexation to a variable extent and a similar trend for each of the guest proton resonances was observed, suggesting a comparable mode of insertion of each paraben guest in the -CD cavity. An increase in the concentration of the CD for each -CD-paraben mixture caused down-field shifts of the alkyl chain and ester moiety signals and up-field shifts of the aromatic ring protons. The latter was interpreted as a consequence of the inclusion of the phenyl ring in the host while the former suggested that the alkyl protons lay outside the host cavity. The guest position is consistent with the up-field shifts of the -CD cavity protons, as the -CD protons experience anisotropic shielding attributed to the inclusion of the guest aromatic ring. Regarding guest orientations, further consideration of the available data suggested that the guest hydroxyl groups are located at the host primary rim for the higher homologues propyl- and butylparaben, while the reverse orientation for methylparaben was inferred. Near equality of the chemical shifts for the aromatic protons of ethylparaben prevented definite conclusions regarding its orientation in the host cavity.
Complex formation in the solid state via both kneading and co-precipitation methods was confirmed by a variety of methods [10], PXRD proving to be the most definitive. DSC traces of 1:1 physical mixtures of each paraben with -CD showed the corresponding fusion endotherm of the guest. The latter disappeared in
the traces for kneaded and co-precipitated products, where only peaks for complex dehydration and final decomposition (around 300C) were evident. UV spectrophotometry indicated 1:1 host-guest stoichiometry in each case and TGA enabled water contents to be assessed, leading to a range of 7.0-7.3 water molecules per complex unit. FTIR spectra were also useful, showing significant shifts of the guest C=O absorption band to higher frequencies in the complexes relative to the pure guests. Comparison of experimental PXRD traces with reference patterns [8, 10] immediately furnished proof of inclusion complex formation as well as indicating probable crystal packing arrangements. All four inclusion complexes yielded a PXRD pattern consistent with channel-packing of dimeric -CD inclusion complexes.
Single crystals of each of the four crystalline inclusion complexes were grown and attaempts were made to determine their detailed structures by X-ray diffraction. Table 1 lists the unit cell parameters for the complexes crystallized at ambient temperature (20C). All four crystallize in the monoclinic space group C2 with Z = 4, implying a unit cell content of two dimeric -CD inclusion complexes. Crystals of the complexes with ethylparaben and butylparaben diffracted weakly and only a set of imprecise unit cell data could be recorded.
Table 1.
Crystal data for -CD complexes with the four guest alkylparabens
Complex formula / CDM7.2H2O / CDE7.0H2O / CDP7.0H2O / CDB7.3H2Oa/Å / 18.8632(4) / 18.88(1) / 19.137(2) / 19.05(1)
b/Å / 24.4542(45) / 24.44(1) / 24.523(1) / 24.48(1)
c/Å / 15.5942(5) / 15.73(1) / 15.793(1) / 15.73(1)
/ / 110.668(1) / 109.8(1) / 109.52(2) / 110.9(1)
The near equality of unit cell data for the complexes is consistent with the inference of isostructurality from PXRD analysis. Detailed single crystal X-ray analysis was only possible for CDM7.2H2O and CDP7.0H2O. However, in the latter case, severe disorder of the guest prevented its modelling despite the well-ordered nature of the host molecule. This phenomenon has been observed previously with channel-packed inclusion complexes crystallizing in the space group C2. The only reliable structure available is therefore that of CDM7.2H2O [11], whose dimeric 1:1 host-guest unit is shown in Figure 3(a). The guest M was found to be disordered over two sites with equal statistical occupancy within the host cavity. In this structure, the two halves of the dimeric complex are related by a horizontal two-fold rotation axis and the guest hydroxyl groups are located at the host primary rim. Here they engage in hydrogen bonding with water molecules and hydroxyl groups of neighbouring counterparts. This orientation is the reverse of that established in solution, where at low solute concentration the secondary side of
the host may be exposed to the aqueous environment, allowing hydration of the guest hydroxyl group. The dimeric complex units pack in the ‘channel-mode’, characterized by linear channels produced when close-packed layers of dimers stack in exact alignment.
(a) (b)
Fig. 3 Structures of the dimeric -CD complex units in (a) CDM7.2H2O (monoclinic) and (b) CDM14.0H2O (triclinic). All disordered components of the guest molecules are shown.
Earlier, we had alluded to the possibility that cyclodextrin inclusion complexes should in principle display polymorphism [8]. This was realized by our recent discovery that when the complex CDM is crystallized at reduced temperature (7C), a modification crystallizing in the triclinic space group P1 results. The structure of the complex unit in this crystal (Figure 3(b)) is also a dimer containing disordered guests in the same orientation as found in the monoclinic form. As indicated in a recent communication [11], the isolation of two crystalline forms of a cyclodextrin inclusion complex by systematic variation of crystallization conditions is a novel finding of general significance. The monoclinic and triclinic crystals were furthermore shown to have significantly different temperatures of dehydration and complex decomposition. A full account of the interaction between the parabens and -cyclodextrin in solution and in the solid state is in preparation [12].
4. Interaction between the parabens and -cyclodextrin
Attempted complexation between the parabens and -cyclodextrin by the kneading method led to crystalline powders whose PXRD patterns matched those of physical mixtures of the components. However, inclusion complexes with H:G stoichiometry 1:2 were obtained by the co-precipitation method [6]. These complexes displayed similar thermal behaviour to the inclusion complexes with -CD in that the DSC traces were devoid of the guest melting endotherms. Definitive identification of these species was obtained by noting that their common
PXRD traces were practically superimposable on the reference pattern for the unique tetragonal family of -CD inclusion complexes with a ~ 23.8, c ~ 23.2Å crystallizing in space group P4212 [8].
5. Interaction between the parabens and dimethylated -CD (DIMEB)
Inclusion complexes of H:G stoichiometry 1:1 were obtained in crystalline form and were characterized by HSM, TGA, DSC, PXRD and single crystal X-ray diffraction methods. Here we focus on preliminary results of the X-ray study. Table 2 lists crystal data for these complexes which crystallize in the orthorhombic space group P212121 with four molecules per unit cell in each case. The complexes clearly fall into two distinct isostructural series.
Table 2.
Crystal data for DIMEB (DMB) complexes with the four guest alkylparabens
Complex formula / DMBM3.7H2O / DMBE4.0H2O / DMBP3.9H2O / DMBB3.7H2Oa/Å / 10.6014(1) / 10.6560(1) / 15.1399(2) / 15.3735(2)
b/Å / 15.4760(2) / 15.3073(2) / 18.8943(3) / 18.8114(2)
c/Å / 48.2438(6) / 49.0417(6) / 28.4009(5) / 28.3989(4)
The presence of two crystal packing modes implied by the above data is consistent with the detailed modes of guest inclusion (shown schematically in Figure 4) determined from single crystals. In the first isostructural pair of complexes (guests M, E), the guest hydroxyl group is located at the host secondary rim while the aromatic portion occupies the central part of the host cavity. The guest orientation
is reversed in the case of P and B, the greater portion of their respective phenolic groups extending from the host primary sides.
Fig. 4 Schematic diagram showing the modes of guest inclusion in the host DIMEB.
In contrast to the -CD complexes of the parabens which are dimeric, the DIMEB complexes are monomeric and thus both ends of the DIMEB host molecule can provide a favourable aqueous environment to the guest hydroxyl group, enabling the two guest orientations to occur. However, the specific orientations adopted may only be explained after a detailed study of the host-guest interactions in the four
complexes has been undertaken. Such a study is underway and a full account is in preparation [13].
6. Interaction between the parabens and permethylated -CD (TRIMEB)
Crystalline inclusion complexes between TRIMEB (TMB) and the parabens were isolated and characterized using the same methods as for the DIMEB complexes. Table 3 lists crystal data for the TMB complexes, all of which crystallize in space group P212121 with four molecules per unit cell. In this series the complex with methylparaben has unique unit cell data while complexes with E, P and B are isostructural, corresponding to a ‘screw-channel’ crystal packing mode [8].
Table 3.
Crystal data for TRIMEB (TMB) complexes with the four guest alkylparabens
Complex formula / TMBM2.6H2O / TMBE5.0H2O / TMBP5.2H2O / TMBB5.6H2Oa/Å / 10.718(1) / 14.886(2) / 14.863(1) / 14.866(2)
b/Å / 26.353(1) / 22.024(3) / 21.862(2) / 21.967(2)
c/Å / 30.018(2) / 27.602(1) / 27.627(3) / 27.635(4)
Comparison of the host geometries in the four structures showed that TMB in the complex with methylparaben has a distinctly different conformation from the common one found in the other three complexes. As shown schematically in Figure 5, the complex with ethylparaben is unique in having the guest phenolic group situated at the primary side of the host molecule. In the other three complexes, the alkyl chains are located at, or protrude through, the host primary rim. Again these preferred modes of guest inclusion require detailed investigation and full details will be published elsewhere [13].
Fig. 5 Schematic diagram showing the modes of guest inclusion in the host TRIMEB.
7. Concluding remarks
The most significant result that emerged from a study of the inclusion of the parabens in -CD was the finding that in the solid state two distinct crystals of the complex with methylparaben can form, depending on the temperature conditions
during crystallization. This is the first well-documented example of this phenomenon for cyclodextrin inclusion complexes [11]. The fact that these complexes have different physical properties indicates that crystallization temperature should be considered more critically in future as an experimental variable as it may enable a richer variety of cyclodextrin inclusion complexes to be generated. A systematic study of the effect of temperature on the crystallization of analogous complexes is in progress [14].