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Copyright ©JCPDS-International Centre for Diffraction Data 2013 ISSN 1097-0002 THE STRUCTURAL CHARACTERIZATION OF A NEW FORM OF CLENBUTEROL, A WELL KNOWN DECONGESTANT AND BRONCHODILATOR ALSO USED AS A PERFORMANCE-ENHANCING DRUG R. Toro,1 J. Bruno-Colmenárez,2 G. Díaz de Delgado,1 and J.M. Delgado1 1 Laboratorio de Cristalografía-LNDRX, Departamento de Química, Universidad de Los Andes, Mérida, Venezuela. 2 Unidad de Caracterización Estructural de Materiales, Instituto Zuliano de Investigaciones Tecnológicas (INZIT), La Cañada, Zulia, Venezuela. ABSTRACT Clenbuterol hydrochloride is an active pharmaceutical ingredient usually prescribed for the treatment of respiratory diseases due to its activity as a decongestant and bronchodilator. It has also been used as a performance-enhancing drug. In the PDF-4/Organics 2012 database there are six entries related to this compound: three for its hydrochloride phase calculated using single crystal data, two for a MeOH and a DMSO solvate of two Cu-clenbuterol complexes, and one experimental unindexed pattern. In this contribution the powder diffraction pattern and the crystal structure, determined using single crystal X-ray diffraction techniques of Clenbuterol hemihydrate, C12H18Cl2N2O·0.5H2O, an unreported phase, is presented. INTRODUCTION Most pharmaceutical materials, the so-called active pharmaceutical ingredients (APIs) and the excipients, are in solid phase. Additionally, the most common drug products, tablets or capsules, are solids as well. The choice of the form of the drug depends on several physicochemical factors associated with its stability, solubility, bioavailability, efficacy, and manufacturing. Different polymorphs and compositions (solvates, anhydrates, etc.) of a given API can display different physicochemical and mechanical properties. Therefore, it is important to properly characterize all the materials, APIs and excipients, involved in the manufacture of a drug in order to ensure the quality, safety, and efficacy of a product (Zhang et al., 2004; Cui, 2007). Clenbuterol (Figure 1), usually marketed as a hydrochloride, is an API used as a bronchodilator in the treatment of reversible obstructions in the respiratory system, as in asthma and in other chronic obstructive pulmonary diseases. Clenbuterol has also been used to promote weight gain in animals raised for human consumption. This drug has been used in sports for its anabolic effects. It is a stimulant of the central nervous system and produces an increase in the aerobic capacity, blood pressure, and oxygen transportation (Sweetman, 2009). In recent years, athletes from different disciplines have been penalized, suspended from competition, and even stripped of their titles for testing positive for clenbuterol. In several cases, the athletes have claimed that they have consumed the drug inadvertently, due to food contamination. 110 This document was presented at the Denver X-ray Conference (DXC) on Applications of X-ray Analysis. Sponsored by the International Centre for Diffraction Data (ICDD). This document is provided by ICDD in cooperation with the authors and presenters of the DXC for the express purpose of educating the scientific community. All copyrights for the document are retained by ICDD. Usage is restricted for the purposes of education and scientific research. DXC Website – www.dxcicdd.com ICDD Website - www.icdd.com Copyright ©JCPDS-International Centre for Diffraction Data 2013 ISSN 1097-0002 Figure 1. Chemical structure of clenbuterol. A search of the Cambridge Structural Database, V 5.33 (Allen, 2002), of the clenbuterol skeleton (omitting hydrogen atoms) resulted in five (5) entries. These entries have their calculated powder diffraction pattern incorporated in the PDF-4/Organics database. Three of them correspond to its hydrochloride phase: the racemic mixture (PDF 02-060-0184, CSD Refcode ACBUET), the S isomer (PDF 02-089-4160, Refcode SAZRUH), and the R isomer (PDF 02-089-4161, Refcode SAZSAO). Two additional entries correspond to Cu-clenbuterol solvated complexes: with MeOH (Refcode QAXFUR) and DMSO (Refcode QAXFOL), respectively. The PDF-4/Organics also contains an entry with an experimental unindexed pattern (PDF 00-057-1640). As part of the work being carried out in our laboratory to characterize common active pharmaceutical ingredients and to examine the possible formation of polymorphs under different crystallization conditions, a study by FT-IR spectroscopy, thermal analysis (TGA-DSC), and Xray diffraction of a new form of Clenbuterol was performed. EXPERIMENTAL Crystallization of Clenbuterol A sample of clenbuterol hydrochloride, a white solid with a melting point of 173 ºC, was dissolved in water. The pH of the solution was adjusted using NH4OH. Several extractions with ether were carried out and the extracts were allowed to evaporate at room temperature. Melting points were determined using a Digital Melting point Apparatus (Model 9100, Electrothermal Engineering LTD. IR spectroscopy and Thermal Analysis The FT-IR spectra were recorded in KBr pellets, using a Perkin-Elmer PE-1600X spectrophotometer with IRDM software. Thermogravimetric analysis (TGA/DTG) and Differential Scanning Calorimetry measurements (DSC) were performed in a Thermal Analyzer SDT Q600 V3 using 6.0670 mg of sample heated to 350 ºC, at a rate of 10 ºC/min, under a dynamic nitrogen atmosphere at 120 mL/min. 111 Copyright ©JCPDS-International Centre for Diffraction Data 2013 ISSN 1097-0002 X-Ray Powder Diffraction Data Collection Powder diffraction patterns were recorded at room temperature on a Bruker D8 Advance diffractometer working in the Bragg-Brentano geometry using CuK radiation (=1.54187 Å), operating at 50 kV and 30 mA. The pattern was recorded in steps of 0.01526° (2), from 5 to 60° at 2 sec/step. The diffractometer was equipped with a secondary monochromator and a LynxEye detector. Single Crystal X-ray Data Collection Intensity data was collected at room temperature on a colorless plate with dimensions 0.49x0.27x0.13 mm using a Bruker APEX Duo Diffractometer with Mo-K radiation (=0.71073 Å) and a graphite monochromator. Data collection and unit cell refinement were carried out with SMART and data reduction with SAINT, both Bruker proprietary software. A total of 3770 unique reflections were obtained from 22654 reflections measured (Rint=0.0165, R=0.0114). RESULTS The diffraction pattern recorded for raw clenbuterol hydrochloride is similar to the unindexed pattern reported in entry PDF 00-057-1640 of the PDF-4/Organics (Figure 2) and to the pattern calculated (Figure 3) for the racemic mixture of this material (PDF 02-060-0184, ACBUET). However, the pattern for the crystals obtained after recrystallization is different from the experimental pattern of the raw material and all the entries in the PDF (Figure 4). Figure 2. Comparison of the powder diffraction pattern of Clenbuterol_raw (blue) with PDF 00-057-1640 (red). Figure 3. Comparison of the powder diffraction pattern of Clenbuterol_raw (blue) with PDF 02-060-0184 (red). 112 Copyright ©JCPDS-International Centre for Diffraction Data 2013 ISSN 1097-0002 The indexing of this pattern carried out using DICVOL06 (Boultif and Louër, 2004) produced a monoclinic unit cell with parameters: a=16.256(3) Å, b=11.276(3) Å, c=15.819(3) Å, â=91.82(2)° and V=2898.34 Å3. The figures of merit associated with the indexing were M20 = 25.9, F30 = 48.2 (0.0090, 69). The fitting of the whole pattern, using the above mentioned unit cell parameters, with the Le Bail algorithm implemented in FULLPROF (Rodriguez-Carvajal, 1990) accounts for all the diffraction maxima recorded. The comparison of the powder diffraction pattern (PDP) of this phase with the pattern calculated using the single crystal data (SCD) obtained in the structural analysis is shown in Figure 5. Both analyses clearly indicate the formation of a new clenbuterol phase. Figure 4. Comparison of the PDP of raw Clenbuterol with the pattern of the recrystallized material. Figure 5. Comparison of the PDP of recrystallized Clenbuterol with the pattern calculated using the SCD data obtained. The FT-IR spectrum of raw Clenbuterol indicates that the amino group is protonated (–OH stretch: í=3401.00 cm-1 and í=2628.30 cm-1 for the –NH+ stretch). In contrast, the spectrum for the recrystallized Clenbuterol form indicates that the amino group is deprotonated (–OH stretch: í=3469.80 cm-1). The new Clenbuterol phase melts and immediately decomposes at ~125 ºC. This is a different behavior from raw Clenbuterol hydrochloride for which the melting and decomposition begins at 179 °C, according to the DSC analysis. The TGA curve of the new phase shows that the material is stable up to 80 ºC and undergoes a weight loss of 3.795 % between 81.8 and 202.53 ºC corresponding to the loss of half a molecule of water, followed by melting at 120-126 °C. A weight loss of 87.01 %, attributed to the total decomposition of the material, occurs at a peak temperature of 263.05 ºC. The single crystal structural study reveals that this compound crystallizes in space group C2/c with unit cell parameters a=16.2685(9) Å, b=11.2647(6) Å, c=15.8089(9) Å, â=91.822(1)°, V=2895.67(29) Å3, Z=4. The asymmetric unit contains the formula C12H18Cl2N2O·0.5H2O, consistent with the TGA results. The structure was solved by Direct Methods and refined by least-squares methods with SHELXS and SHELXL, respectively (Sheldrick, 2008). The graphical user interface, ShelXle, was used in the refinement process (Hübschle et al., 2011). 113 Copyright ©JCPDS-International Centre for Diffraction Data 2013 ISSN 1097-0002 Hydrogen atoms were placed in calculated positions and refined using a riding model with their displacement parameters equal to 1.2Uiso of the non-hydrogen atom to which they are attached. The refinement converged to R=0.0386, wR2=0.1139, S=1.03. A complete account of the structure refinement results will be published elsewhere. The structure of Clenbuterol·0.5H2O can be described in terms of hydrogen bonded dimers (see Figure 6). The hydrogen bonding pattern observed can be represented by the graph set symbol ܴଶଶ ሺͳͲሻ (Etter et al., 1990). The water molecules present in the structure, occupying a special position, connect these dimers to other dimers to form chains approximately parallel to the caxis. Figure 7 shows a view of the unit cell down the c-axis. Figure 6. Hydrogen bonded dimers with graph set symbol ܴଶଶ ሺͳͲሻ in Clenbuterol·0.5H2O. Figure 7. A view down the c-axis of the structure of Clenbuterol·0.5H2O. 114 Copyright ©JCPDS-International Centre for Diffraction Data 2013 ISSN 1097-0002 ACKNOWLEDGEMENTS This work was possible thanks to Grant LAB-97000821 from FONACIT (Universidad de Los Andes) and Grant LOCTI-2007-0003 (INZIT). REFERENCES Allen, F. H. (2002). “The Cambridge Structural Database: a quarter of a million crystal structures and rising”, Acta Crystallogr., Sect. B: Structural Science, 58, 380-388. Boultif, A. and Louër, D. (2004). “Powder pattern indexing with the dichotomy method” J. Appl. Crystallogr., 37, 724-731. Cui, Y. (2007). “A material science perspective of pharmaceutical solids”, Inter. J. Pharmaceutics, 339, 3-18. Etter, M. C., MacDonald, J. C., Bernstein, J. (1990). “Graph-set analysis of hydrogen-bond patterns in organic crystals”, Acta Crystallogr. Sect. B: Structural Science, 46, 256-62. Hübschle, C. B., Sheldrick, G. M., and Dittrich, B. (2011). “ShelXle: a Qt graphical user interface for SHELXL”, J. Appl. Crystallogr., 44, 1281-1284. British Pharmacopeia (2009), The Stationery Office Books, London, England. Rodriguez-Carvajal, J. (1990). “FULLPROF: A Program for Rietveld Refinement and Pattern Matching Analysis”, Abstracts of the Satellite Meeting on Powder Diffraction of the XV Congress of the IUCr, Toulouse, France, 127. Sheldrick, G.M. (2008). “A short history of SHELX”, Acta Crystallogr. Sect. A: Foundations of Crystallography, 64, 112-122. Sweetman, S. C. (Ed.) (2009). Martindale. The Complete Drug Reference. (Pharmaceutical Press, London). 36th ed., p. 1120. Zhang, G. G. Z., Law, D., Schmitt, E. A., Qiu, Y. (2004). “Phase transformation considerations during process development and manufacture of solid oral dosage forms”, Advanced Drug Delivery Reviews, 56, 371-390. 115