NMR and molecular modelling studies on the interaction of fluconazole with beta-cyclodextrin

Abstract

Background: 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.

Results: Inclusion complex of fluconazole with beta-cyclodextrins (beta-CD) were investigated by applying NMR and molecular modelling methods. The 1:1 stoichiometry of FLZ:beta-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:beta-CD complexation. The shielding of cavity protons of beta-CD and deshielding of aromatic protons of FLZ in various 1H-NMR experiments show complexation between beta-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 beta-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 beta-CD from the wider end.

Conclusion: Various NMR spectroscopic studies of FLZ in the presence of beta-CD in D2O at room temperature confirmed the formation of a 1:1 (FLZ:beta-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 beta-CD suggest some chiral differentiation of guest by beta-CD.

Figure 1

(a) Chemical structure of the host β-cyclodextrin (β-CD); (b) truncated cone shape of β-CD; and (c) guest fluconazole (FLZ).

Figure 2

Job's plot (continuous variation method) of FLZ:β-CD inclusion complex showing 1:1 stoichiometry. Δδ = δ_(complex) - δ_(free).

Figure 3

A typical Scott's plot for FLZ:β-CD inclusion complex showing overall association constant (K_a) = 68.7 M^-1.

Figure 4

Expansion of a part of ¹H NMR data (500 MHz), showing β-CD protons in the presence as well as in the absence of FLZ.

Figure 5

Expansion of a part of ¹H NMR data (500 MHz), of FLZ protons showing deshielding in the presence of varying amount of β-CD.

Figure 6

2D COSY (500 MHz) spectra of FLZ:β-CD mixture (1:1) showing the cross peak between aromatic protons of FLZ.

Figure 7

Expanded region of 2D ROESY (500 MHz) spectra of FLZ:β-CD mixture (1:1) showing the intermolecular NOE's between β-CD protons and aromatic protons of FLZ.

Figure 8

FLZ docked with β-CD, FLZ is shown in Ball and Stick, β-CD in sticks (a) an all atom model with Connolly surface representation of FLZ, (b) FLZ and β-CD (side-view) and (c) FLZ and β-CD (top-view). Hydrogens are not shown in (b) and (c) for sake of clarity. All other atoms are shown in their elemental colour.