Download the structural characterization of a new form of clenbuterol, a

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.
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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.
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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).
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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).
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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.
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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.
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