Microsoft Word - Bhawani Datt Joshi et al _73-79_.doc Bhawani Datt Joshi et al./ BIBECHANA 8 (2012) 73-80 : BMHSS, p.73 BIBECHANA A Multidisciplinary Journal of Science, Technology and Mathematics ISSN 2091-0762 (online) Journal homepage: http://nepjol.info/index.php/BIBECHANA Molecular characterization of yohimbine hydrochloride using vibrational spectroscopy and quantum chemical calculations Bhawani Datt Joshi 1,3 , Poonam Tandon 1,* and Sudha Jain 2 1Department of Physics, University of Lucknow, Lucknow-226007, India 2Department of Chemistry, University of Lucknow, Lucknow-226007, India 3Department of Physics, Siddhanath Sc. Campus, Mahendranagar, Tribhuvan University, Nepal *Corresponding author. Tel.: +91 522 2782653; fax: +91 522 2740840. E-mail address: poonam_tandon@yahoo.co.uk, poonam_tandon@hotmail.com (P. Tandon) Article history: Received 22 June, 2011; Accepted 20 August, 2011 Abstract In this work, we have performed the extraction of yohimbine hydrochloride (C21H27ClN2O3) (YHCl). The optimized geometry, total energy, potential energy surface and vibrational wavenumbers of YHCl have been determined by using ab initio Hartree–Fock (HF) and density functional theory (DFT/B3LYP) method with 6-311++G(d,p) basis set. The calculated wavenumbers are scaled by a proper scaling factor. A selected number of vibrational assignment is provided for the observed Raman and IR spectra. Keywords: YHCl; vibrational spectroscopy; ESP; ab initio and DFT calculations 1. Introduction Yohimbine, an indole alkaloid is the active ingredient obtained from the bark of various tropical trees such as Pausinystalia yohimbe (formarly Corynanthe yohimbe), a tall evergreen tree indigenous to southern African countries, and from the Indian snake root Rauwolfia serpentina [1,2]. Yohimbine hydrochloride (17α – hydroxy –yohimbane 16α – carboxylic acid methyl ester hydrochloride), a standard form belongs to an orthorhombic system, probable space group P212121 having lattice parameters, a = 11.54Å, b = 24.88Å, c = 7.00Å and z = 4 [2]. It has been known as an aphrodisiac compound [2,3] and used traditionally since before the last century, even in the crude form by the African natives, and promoted for erectile dysfunction, weight loss (by releasing norepinephrine and blocking alpha-2 receptors) and depression (by blocking an enzyme called monoamine oxidase) [4-6]. It is used in tablets, capsule and tincture form for increased serumtesterone levels, muscle growth and strength, weight-loss, reduce fatigue in AIDS patients and libido enhancer [7]. Also it has been reported as traditional medicine to treat high blood pressure, chest pain, age- related cognitive disorders and obesity [8]. As the literature survey reveals neither Raman and IR spectra nor quantum chemical calculations for YHCl molecule have been reported so far, hence the present work was undertaken to study the vibrational spectra with quantum chemical calculation with greater accuracy. Although X-ray diffraction method is one of the most frequently applied techniques for structural characterization of pharmaceutical compounds but the use of vibrational spectroscopy is also gaining increasing attention. X-ray diffraction techniques are sensible to the long-range order while vibrational spectroscopy (IR [9] and Raman [10]) is applicable to the short-range structure of molecular solids. Bhawani Datt Joshi et al./ BIBECHANA 8 (2012) 73-80 : BMHSS, p.74 Fig. 1: Crystal structure of YHCl. Fig. 2: Optimized structure of YHCl. Bhawani Datt Joshi et al./ BIBECHANA 8 (2012) 73-80 : BMHSS, p.75 Furthermore, we interpreted the calculated spectra in terms of potential energy distributions (PED) and made the assignment of the experimental bands due to PED analysis results. Information about the geometry and structure of the molecule with their electrostatic potential surfaces should help in understanding the structure-activity relationship. Figure 1 shows the crystal structure of the YHCl molecule. 2. Materials and Methods 2.1 Experimental Methods The whole plants of Rauwolfia serpentine Benth. (Apocynaceae) were collected from Lucknow, Uttar Pradesh, India and identified by the Botany Division of Central Drug Research Institute, Lucknow, India. The plant material (2.4 Kg) was air-dried, powdered and percolated with ethyl alcohol (6 x 4 lt) at room temperature. The combined percolate was concentrated under reduced pressure below 40o to give the viscous mass. The material was partitioned between 2% tartaric acid and hexane. The aqueous solution was re-extracted with hexane (4 x 250 ml), basified with solid Na2CO3 (pH 7.5) and extracted with benzene (6 x 250 ml). The combined benzene layer was dried over anhydrous Na2SO4 and solvent removed under reduced pressure to give a residue - I. The aqueous solution left after extraction with benzene was further extracted with methylene chloride (5 x 250 ml). The combined methylene chloride layer was dried over anhydrous Na2SO4 and solvent removed under reduced pressure to give a residue -II. Both the residues I and II were mixed to afford crude alkaloidal mixture (4.0 g) which was subjected to column chromatography over neutral alumina. The column was successively eluted with hexane, hexane: benzene (20:80), benzene, benzene - ethyl acetate, v/v (98:2), (95:5), (90:10), (85:15), (50:50), (25:75), ethyl acetate, ethyl acetate-methanol (95:5) and methanol. Elution was monitored by thin layer chromatography (tlc). A total of 190 fractions, 100 ml each, were collected and mixed on the basis of tlc. The fractions 42 – 75 eluted from benzene-ethyl acetate (98:2), (95:5) and (90:10) were mixed and solvent removed. The crude product was subjected to preparative tlc (plates: SiO2 GF254; solvent: chloroform – methanol, 95:5; double run). The major band on the plates was scraped, extracted with chloroform – methanol (3:1), the solvent removed under reduced pressure to give a pure compound (8.4 mg), converted to its hydrochloride, crystallized from methanol - ether, m.p. 299.50 [3010][11,12]. The compound was identified as yohimbine by a direct comparison with an authentic sample procured from Sigma Chemical Company, USA. An excitation laser of wavelength 514 nm was emitted from an Argon ion laser source and a power of 12 mW was used to record the vibrational spectra using an efficient visible micro Raman setup at room temperature. The scattered Raman light was collected in a back scattering geometry using a microscope objective (ULW x50). The scattered light was dispersed using a monochromator with 1200 grooves/mm diffraction grating, and an entrance slit width of 200 micrometer. The Raman signals were detected using liquid nitrogen cooled charged coupled device (CCD) with an optimal sensitivity in the visible range. The total exposure time for each sample was 5 sec and averaged over five accumulations. Infrared spectra of YHCl were recorded on a Bruker TENSOR 27 FT-IR spectrometer with a spectral resolution of 4 cm-1 in the region 300-4000 cm-1. The KBr pellets of solid samples were prepared from mixtures of KBr and the sample in 200:1 ratio using a hydraulic press. Multi-tasking OPUS software was used for base line corrections. 2.2 Computational Methods Geometry optimization was performed as the first task of computational work for the YHCl molecule. The experimental geometric parameters from X-ray diffraction data [2] of YHCl molecule were used as the initial parameters for the theoretical calculations. The electronic structure and optimized geometry of the molecule were computed by ab initio Hartree-Fock (HF) and DFT (density functional Bhawani Datt Joshi et al./ BIBECHANA 8 (2012) 73-80 : BMHSS, p.76 theory) using Gaussian 09 [13] program package employing 6-311++G(d,p) basis set based on Becke’s three-parameter (local, non-local, Hartree-Fock) hybrid exchange functional with Lee-Yang- Parr correlation functional (B3LYP) [14].The basis set 6-311++G(d,p) augmented by d polarization functions on heavy atoms and p polarization functions on hydrogen atoms as well as diffuse functions for both hydrogen and heavy atoms were used. The optimized structural parameters were used to calculate the absolute Raman intensities and infrared absorption intensities in the harmonic approximation at the HF and DFT levels. The positive values of all the obtained 156 wavenumbers confirm the stability of optimized geometry. For analyzing the normal a complete set of 156 internal co-ordinates was defined using Pulay’s recommendations [15]. The vibrational assignments of the normal modes were proposed on the basis of the PED calculated using the program GAR2PED [16]. Raman and infrared spectra were simulated using a pure Lorentzian band profile (fwhm = 8 cm-1) using indigenously developed software. Visualization and confirmation of the calculated forms of the vibrations were done using the CHEMCRAFT program [17]. 3. Geometry Optimization and Energies Initial geometry taken from the X-ray diffraction data [2] of YHCl was minimized without any constraint to the potential energy surface and the optimized structural parameters were used in the vibrational frequency calculation to characterize all stationary points as minima. The equilibrium geometry has been determined by the energy minimization. The optimized structure of YHCl molecule is shown in Figure 2. The relative energies of the molecule are calculated employing ab initio HF and DFT functional (B3LYP). The DFT includes some electron correlation effects and hence the calculations with this method are better than the HF approach. The energy calculated by DFT (-1612.2632515 Hartree) is lower than the one calculated by HF (-1612.2607923 Hartree). The optimized structural parameters (bond lengths, bond angles, dihedral angles) of YHCl have been compared with the experimental one. The difference between experimental and calculated values of bond-lengths is not more than 0.04Å, both in DFT and HF methods. The bond angles differ by not more than 2.5o except the angles, C19-O2-C26 and O1-C13-C10 which differ by 4.82o/5.82o and 5.64o /5.64o in the DFT/HF respectively. Similarly the dihedral angles differ by not more than 4.9 o except the angles C6-C10-C19-O2 and C6-C10-C19-O3 differ by 6.83o/6.83o and 6.34o/6.34o in the DFT/HF respectively. 4. Results and Discussion 4.1 Molecular Electrostatic Potential The molecular electrostatic potential (ESP) of a molecule at a point in space around it gives an indication of the net electrostatic effect produced by the total charge distribution (electrons + nuclei) of the molecule. The molecular ESP of YHCl molecule is shown in Figure 3. It correlates the total charge distribution with dipole moments, partial charges, electro-negativity and site of chemical reactivity of a molecule. ESP provides a visual method to understand the relative polarity of a molecule and serves as a useful quantity to explain hydrogen bonding, reactivity and structure-activity relationship of molecules including biomolecules and drugs [18]. Different colours represent the different values of the electrostatic potential at the surface of a molecule. In general, red colour represents the regions of most negative electrostatic potential (possess electrophilic property), blue represents the regions of most positive electrostatic potential (possess nucleophilic property) and green represents the regions of zero potential. Potential increases in the order red < orange < yellow