1. hepatitis C virus activity, preferably for the

1. IntroductionThe human migraine is a severe headache often unilateral, commonly accompanied by nausea, vomiting, and extreme sensitivity to sound and light. Flunarizine dihydrochloride (FLN) is a large hydrophobic fluorinated piperazine derivative used in the prophylactic treatment of migraine, vertigo, occlusive peripheral vascular disease and epilepsy 1. FLN (Figure 1) is a di-fluorinated derivative of cinnarizine and is a poorly water-soluble drug. FLN is a selective calcium entry blocker with calmodulin binding properties and histamine H1 blocking activity. It is also known to prevent hepatitis C virus membrane fusion in a genotype dependent manner 2 and to suppress endothelial angiopoietin-2 in a calcium dependent fashion in sepsis 3. FLN is reportedly effective against hepatitis C virus activity, preferably for the genotype 2 viruses 4. Cyclodextrins (CDs) or cycloamyloses are truncated cone shaped macrocycles produced from starch through enzymatic degradation. CDs is a family of cyclic oligosaccharides and have been studied extensively as supramolecular hosts 5, 6. The three common CDs are crystalline, homogeneous, nonhygroscopic substances, consisting of six (?-), seven (?-), and eight (?-) D-glucose units, respectively, linked by ?-D-(1?4) glycosidic bonds (Figure 1) 5, 6. The glucose residue in CDs have 4C1 (chair) conformation 5. The primary hydroxyl groups (n) are located at the narrower rim whereas the wider rim is lined with secondary hydroxyl groups (2n). The outer surfaces of the CDs are highly hydrophilic due to the presence of large number of hydroxyl groups but the central cavities are relatively hydrophobic (Figure 1). The outer dimension of these three common CDs are constant at 0.78 nm but their inner dimensions are variable, being 0.57 nm for ?-, 0.78 nm for ?-, and 0.95 nm for ?-CD respectively. The H-3′ and H-5′ protons of these CDs are located in the hydrophobic central cavity whereas other protons (H-1′, H-2′, H-4′ and H-6′) are located at outer surface, which is relatively hydrophilic. These properties facilitate their aqueous solubility and ability to encapsulate hydrophobic moieties within their central cavities through non-covalent interactions. CDs form host-guest inclusion complexes upon penetration of guest molecule in the central cavity of host CDs.  CDs are extensively studied in various areas of chemistry including macrocyclic 7, supramolecular 8, 9, agro 10, click 11, analytical 12, chromatography 13, 14, sugar-based surfactants 15, foods 16, catalysis 17, 18, membranes 19, textiles 20, cosmetics 21, 22, fragrance and aromas 23, 24, enzyme technology 25, pharmacy and medicine 26-28, microencapsulation 29, nanotechnologies 30-33, remediation 34, decontamination 35 and biotechnology 36. The unique properties of CDs allow their various applications in many areas 37-39.  CDs are used to prepare inclusion complexes with pharmaceuticals for biomedical applications and biomedicine 22, 31, 36-38.  CDs are widely used in food industry as food additives, stabilizing flavors, to remove undesirable compounds such as cholesterol, and also as agents to avoid microbiological contaminations in the food 16. CDs can be used to enhance solubility, bioavailability and stability of pharmaceuticals 41-43. Upon complexation with pharmaceutical compounds, CDs form inclusion complexes with ability to alter the physiochemical properties of the complexed drug. Various drugs such as nimesulide, omeprazole, piroxicam, mitomycin, diclofenac sodium, indomethacin and others complexed with CDs are approved and are available in the market 42. Inclusion complexes with dimethyl-?-CD are used in the preparation of vaccine Deptacel (Sanofi Group, Pasteur) for protection against diphtheria, tetanus and pertussis. CDs are also used to stabilize sensitive substances to light or oxygen, proteins 44, nanoparticles 45, and add value addition of taste and colour of tooth paste 46.  Among various known spectroscopic methods such as Ultraviolet-visible (UV-Vis), Fourier-transform infrared spectroscopy (FTIR) for the studies of inclusion complexes of host CDs and guest molecules, Nuclear Magnetic Resonance (NMR) spectroscopy is considered as one of the most significant analytical tool for understanding the interaction between host and guest molecules 47. This technique provides not only the structural assignments of host and guest molecules but also data on the inclusion complex formation. Further NMR spectroscopy could also offer valuable information on chiral recognition or chiral discrimination or both 47-49. NMR spectroscopic titration data can be used to determine the stoichiometry and association constant of the host-guest complexes 50-52. Two-dimensional (2D) NMR method such as 1H-1H COSY (COrrelation SpectroscopY) is a useful technique, which provides information on the 1H signals arising from neighbouring protons connected through bonds and protons signals emerging from up to 4 bonds can be captured. 2D 1H-1H Rotating-frame Overhauser Effect SpectroscopY (ROESY) has been found to be very useful for the investigation of the interaction between CD and guest molecule as the Nuclear Overhausser Effect (NOE) cross peaks are observed between the protons that are close in space even if they are not bonded 47, 50-52. 2D 1H-1H ROESY provides useful information about the location and depth of inclusion of guest molecule into CD cavity 47, 50-52. The formation of an inclusion complex of a guest molecule with CDs results in the 1H chemical shift changes (??) in both the host and guest protons. Inclusion of a molecule inside the hydrophobic cavity of CD is mainly characterized by the chemical shift variation of the CD protons located inside the central cavity (H-3′ and H-5′), whereas other CD protons (H-1′ H-2′, H-4′ and H-6′) are less affected. During host-guest inclusion complex formation the guests molecules protons generally show downfield chemical shift changes but sometimes upfield chemical shift changes are also observed 47. These analytical procedures revealing the structural details of complexes are used in pharmaceutical industries for characterization. In order to understand correct inclusion architecture of interaction between guest FLN and host ?-CD, we report here a high-resolution NMR spectroscopic and computer based molecular docking study. We describe our results based on the 1H NMR spectral data with chemical shift changes, 2D 1H-1H COSY spectrum for assignment of protons and 1H-1H ROESY spectrum together with molecular docking approach thus elucidating the structure of the ?-CD-FLN inclusion complex.