NMR and molecular modelling studies on the interaction of fluconazole with β-cyclodextrin
© Upadhyay et al 2009
Received: 11 May 2009
Accepted: 10 August 2009
Published: 10 August 2009
Fluconazole (FLZ) is a synthetic, bistriazole antifungal agent, effective in treating superficial and systemic infections caused by Candida species. Major challenges in formulating this drug for clinical applications include solubility enhancement and improving stability in biological systems. Cyclodextrins (CDs) are chiral, truncated cone shaped macrocyles, and can easily encapsulate fluconazole inside their hydrophobic cavity. NMR spectroscopy has been recognized as an important tool for the interaction study of cyclodextrin and pharmaceutical compounds in solution state.
Inclusion complex of fluconazole with β-cyclodextrins (β-CD) were investigated by applying NMR and molecular modelling methods. The 1:1 stoichiometry of FLZ:β-CD complex was determined by continuous variation (Job's plot) method and the overall association constant was determined by using Scott's method. The association constant was determined to be 68.7 M-1 which is consistent with efficient FLZ:β-CD complexation. The shielding of cavity protons of β-CD and deshielding of aromatic protons of FLZ in various1H-NMR experiments show complexation between β-CD and FLZ. Based on spectral data obtained from 2D ROESY, a reasonable geometry for the complex could be proposed implicating the insertion of the m-difluorophenyl ring of FLZ into the wide end of the torus cavity of β-CD. Molecular modelling studies were conducted to further interpret the NMR data. Indeed the best docked complex in terms of binding free energy supports the model proposed from NMR experiments and the m-difluorophenyl ring of FLZ is observed to enter into the torus cavity of β-CD from the wider end.
Various NMR spectroscopic studies of FLZ in the presence of β-CD in D2O at room temperature confirmed the formation of a 1:1 (FLZ:β-CD) inclusion complex in which m-difluorophenyl ring acts as guest. The induced shift changes as well as splitting of most of the signals of FLZ in the presence of β-CD suggest some chiral differentiation of guest by β-CD.
The principle objective of the work embodied in this article was to study the complexation of fluconazole with β-cyclodextrin in aqueous solution. Complexation of pharmaceutical compounds with cyclodextrins (CDs), to form a host-guest complex in solution as well as solid states, results in altered physicochemical properties of the guest, like solubility, stability, dispersibility, volatility, masking of undesirable properties and so on, which are desirable for their use as pharmaceuticals . Moreover, these host-guest complexes are considered as new entities and are required to be characterized for their approval as drugs. CDs are chiral saccharides that exhibit chiral recognition i.e. they form diastereomeric complexes, usually of different stability, with enantiomeric species . The separation of enantiomers of a racemic drug is of great importance to the pharmaceutical industry because in a racemic drug, only one enantiomer is usually desired. CDs are all-purpose molecular containers for organic, inorganic, organometallic, and metal organic compounds that may be neutral, cationic, anionic or even radical.
The CDs belongs to the family of cyclic oligosaccharides, and have been studied extensively as a host in supramolecular chemistry. The three major types of CDs are crystalline, homogeneous, nonhygroscopic, consisting of six (α-), seven (β-), and eight (γ-) D-glucose units, respectively, attached by α-(1→4) glycosidic linkages (figure 1) [4, 5]. Each of the chiral glucose units is in the rigid 4C1-chair conformation, giving the macrocycle shape of a hollow truncated cone with all the secondary hydroxyl groups located on the wider rim, while all the primary hydroxyl groups on the narrower rim (figure 1). The primary and secondary hydroxyls on the outside of the CDs make it water-soluble. The non-bonding electron pairs of the glycosidic oxygen bridges are directed toward the inside of the cavity, producing a high electron density and lending it a Lewis base character. These features suggest that the CD cavity is relatively hydrophobic compared to the exterior faces which are hydrophilic.
Structure determination is of particular importance for supramolecular host-guest complexes, which are the basis of most cyclodextrin applications in medicine, catalysis, separation and sensor technology and also food chemistry. NMR spectroscopy has been recognized as an important tool for the structural elucidation of organic compounds, particularly in solution state in view of its application in drug discovery. This technique also gives information on the topology of the interaction between the guest and β-CD; furnishing information not only on the structure of inclusion complexes but also deriving the stoichiometry and association or binding constant of guest:β-CD complexes . 2D COSY and ROESY experiments are important in cyclodextrin related studies, as they complement each other, COSY provides information on coupling of protons while 2D ROESY gives same information through space, i.e., the two nuclei are at 3–4 Å from each other (intermolecular distance) [5, 7].
Here we present a study highlighting the interaction between antifungal drug fluconazole and β-cyclodextrin, using NMR as a spectroscopic tool. In order to better understand the structure of the inclusion complex between the two chemical species, we have complemented these studies with molecular modelling simulations.
Fluconazole (FLZ) was obtained from Dr. Reddy's Ltd., India while β-cyclodextrin was a generous gift from DKSH India Pvt. Ltd. and these were used as received. All other reagents were of analytical reagent grade.
Samples were prepared in 99.96% D2O for NMR analysis. All the 1H NMR spectra were recorded with a Bruker Avance 400 MHz spectrometer operating at 300 K and were acquired with a spectral width of 5995.204 Hz, 128 scans and 65536 data-points. Both 2D COSY and ROESY experiments were acquired on a Bruker DRX 500 MHz using 5 mm BBI 1H-BB probe or a Varian Inova 500 MHz, equipped with a triple resonance, Z pulsed field gradient probe. 2D COSY spectra displaying 1H-1H cross correlation for free FLZ, free β-CD and FLZ:β-CD mixture were acquired using 2048 data-points with 128 increments and 18 scans for each increment. 2D ROESY spectrum of FLZ:β-CD was acquired using 2048 data-points, 256 increments, 8 scans for each increment and 500 ms mixing time. 1H NMR spectra of five samples containing mixtures of β-CD and FLZ with FLZ/β-CD molar ratios ranging from 0.2 to 1.2 was recorded. As there was no separate peak for free as well as complexed form of FLZ, we presume that it undergoes rapid exchange between free and bound state on the NMR time scale. The resonance at 4.7 ppm due to residual solvents (H2O and HDO) was used as internal reference. Chemical shift (δ) reported in ppm and chemical shift changes (Δδ) was calculated by using the formula: Δδ = δ(complex) - δ(free).
Molecular modelling studies
where the f terms fractionally count atomic contacts of specific types and the C's are coefficients that weight the term contributions to affinity estimate. The individual terms are: hb, interactions between hydrogen bond donor-acceptor pairs; ion, ionic interactions; mlig, metal ligation; hh, hydrophobic interactions; hp, interactions between hydrophobic and polar atoms; aa, an interaction between any two atoms. The docked complex was finally energy-minimized keeping both the ligand and the receptor flexible.
Results and discussion
Determination of the FLZ:β-CD inclusion complex stoichiometry by continuous variation method (Job's Plot)
Determination of association constant (Ka)
where [FLZ] is the molar concentration of the guest, Δδobs the observed chemical shift change for a given [FLZ] concentration, Δδmax the chemical shift change between a pure sample of complex and the free component at saturation.
1H NMR (500 MHz) chemical shift (δ) data of pure β-CD and β-CD:FLZ mixtures
β-CD/FLZ = 0.2
β-CD/FLZ = 0.4
β-CD/FLZ = 0.6
β-CD/FLZ = 0.8
β-CD/FLZ = 1.2
Unambiguous resonance assignments of FLZ protons were made on the basis of 1H NMR data, 2D COSY and 2D ROESY spectra. Some of the peaks that were completely not distinct in the spectrum of the pure FLZ separated well in the presence of β-CD aiding the assignment. FLZ has one difluoro substituted phenyl ring and two triazole rings and its 1H NMR chemical shift appeared separately. Here, we are giving the 1H NMR assignment of pure FLZ. The two singlets at δ = 8.23 and 7.74 were assigned as H-8, 10 and H-9, 11 resonance, consistent with the two protons of the two equivalent triazole rings of FLZ. Two highly coupled resonance patterns (probably multiplet) appeared near δ = 6.9 (H-1, 3) and 6.7 (H-2), integrating with the ratio of 2:1 protons of three consistent aromatic protons of the m-difluorophenyl ring of FLZ. An AB pattern appeared at δ = 4.9 (H-4, 5, 6, 7), totally integrating for the four protons consistent for the two sets of β-methylene protons of FLZ.
In presence of β-CD, all the aromatic protons deshielded, indicating the proximity of these protons' to an electronegative atom like oxygen. The shielding of β-CD cavity protons and concomitant deshielding of the most of the aromatic protons of FLZ suggest that this ring is inserted in the β-CD cavity [11–13]. More detailed indications concerning the geometry of the inclusion complex can be derived from the evidence of spatial proximities between protons of β-CD and FLZ. Partial contour plot of 2D (1H-1H) ROESY spectra of inclusion complex of β-CD and FLZ is shown in figure 7. Analysis of ROESY data revealed that both the cavity protons of β-CD (H-5' and H-3') share weak NOE's with triazole ring protons (H-8, 10 and H-9, 11). H-5' protons of β-CD show NOE's with H-1 (weak), H-2 (weak) and H-3 (very weak). On the other hand H-3' protons of β-CD show NOE's with H-1 (strong), H-3 (strong) and H-2 (weak-to-strong). Collectively, these data suggest that the m-difluorophenyl moiety penetrates deep into the β-CD cavity from the wider rim side. The inclusion of m-diflurophenyl ring from narrower rim side is not possible based on the weak NOE's with H-5'. The penetration of triazole ring was ruled out on the basis of weak NOE's cross peaks between triazole ring protons and β-CD protons.
In summary, our NMR experiments confirm that FLZ forms 1:1 complex with β-CD in aqueous medium. Employing experimental and theoretical methods, the present work unambiguously determined the geometrical inclusion parameters of FLZ with β-CD. The ROESY experiments showed that the complex is formed with the m-difluorophenyl ring of FLZ inside the β-CD torus cavity. These results were also supported by molecular modelling which also highlight a hydrogen bond formation between the host and the guest providing stability to the complex.
List of abbreviations
- 1H NMR:
proton Nucleic Magnetic Resonance spectroscopy
Rotating frame Overhauser Effect SpectroscopY
Nuclear Overhauser Effect
Molecular Operating Environment.
SKU and GK are recipients of Research Associateship from the centre of excellence, Department of Biotechnology, Government of India, awarded Prof. A. Surolia. SKU is grateful to Prof. A. Surolia and Dr. Monica Sundd for teaching and training me in NMR.
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