A new polymorph of norfloxacin
Journal of Thermal Analysis and Calorimetry, Vol. 89 (2007) 3, 687–692
A NEW POLYMORPH OF NORFLOXACIN
Complete characterization and relative stability of its trimorphic system
R. Barbas, R. Prohens* and Cristina Puigjaner**
Unitat de Química Fina, Serveis Científico-tÀcnics, Universitat de Barcelona, Josep Samitier 1-5, 08028 Barcelona, Spain
A new polymorphic form of Norfloxacin has been identified and fully characterized by a variety of methods including powderX-ray diffraction, vibrational spectroscopy (IR and Raman), thermal analysis (DSC and TG), SEM and solid-state NMR spectros-copy. The relationship between the new form C and the previously known forms A and B have been studied. Moreover, the crystalstructure of the known form A has been solved by single-crystal methods.
Keywords: crystal structure, fluoroquinolone, Norfloxacin, polymorphism, solvent mediated transformation, thermal analysis,
atmospheric pressure. When an organic compoundexhibits polymorphism of an enantiotropic type, the
Polymorphism is the ability of a substance to
knowledge of the different domains of thermo-
crystallize in different crystal modifications, each of
dynamic stability for every form is essential in order
them having the same chemical structure but different
to obtain the desired form by a robust crystallization
arrangements or conformations of the molecules in
process and to define the appropriate storage con-
the crystal lattice. Different polymorphs exhibit
different physicochemical properties such as solu-
bility, dissolution rate, bioavailability and chemical
oxo-7-(1-piperazinyl)-3-quinolinecarboxylic acid is a
and physical stabilities. So, polymorphism has
synthetic broad antibacterial compound discovered in
become a topic of great interest for both academic
the late nineteen-seventies, belonging to the group of
research and pharmaceutical industries as it has the
fluoroquinolones. In vitro, it is active against a broad
potential to significantly affect the physical properties
spectrum of gram-positive and gram-negative aerobic
bacteria. It is used in the treatment of gonorrhea,
prostate and urinary tract infections .
crystalline structure, either because of a different
Norfloxacin exists in several solid forms: two
arrangement of molecules in the lattice (packing
anhydrous polymorphs (form A and form B) and an
amorphous form , a methanol solvate  and
several hydrated forms [3, 6, 7]. In a recent study ,
(conformational polymorphism). Sometimes the most
we updated and corrected the previous knowledge of
stable polymorph is difficult to produce or a
this drug which was erroneous due to a wrong
metastable form has favorable properties. Regardless
identification of the system A and B as a monotropic
of which form is chosen for development, it is of
type. The results of solvent mediated transformation
greatest importance for the pharmaceutical industry to
ensure reliable and robust processes. The ability of a
endothermic solid-solid transition observed by DSC.
These evidences were enough to prove the enan-
determined by both thermodynamic and kinetic
tiotropic relationship between both polymorphs. This
factors. These factors must be well understood in
is important because many commercial samples of
order to explore and to control the polymorphic
Norfloxacin are provided as the metastable form at
behaviour of a substance. Hence, it is important, and
room temperature and then, undesirable trans-
even a regulatory requirement, to identify the possible
polymorphic forms of a product and to know whether
In the present study we describe the crystal
polymorphic modifications can transform reversibly
structure of the metastable form A by single-crystal
X-ray diffraction, as well as the discovery of a new
Author for correspondence: [email protected]
Author for correspondence: [email protected]
Akadémiai Kiadó, Budapest, Hungary
2007 Akadémiai Kiadó, Budapest
Springer, Dordrecht, The Netherlands
polymorphic form of Norfloxacin. Thermal analytical
0 and 115° in 2q, with a step size of 0.029° and a scan
spectroscopy (IR and Raman), SEM and solid-stateNMR spectroscopy have been applied in order to
characterize the new solid form C. The relativethermodynamic stability of the three anhydrous forms
Single-crystal X-ray diffraction data were collected on a
Bruker Smart CCD area detector with OxfordCryosystems low temperature system. Cell parameterswere refined from the setting angles of 5749 reflections
(q range 1.52<25.00°). Reflections were measured froma hemisphere of data collected of frames each covering
0.3 degrees in omega. Of the 58519 reflections mea-sured, all of which were corrected for Lorentz and polar-
ization effects and for absorption by semi-empirical
Norfloxacin was purchased from Sigma-Aldrich
methods based on symmetry-equivalent and repeated re-
flections (minimum and maximum transmission coeffi-cients 0.9627 and 0.9713), 4101 independent reflections
The structure was solved by direct methods and refined
Norfloxacin (100 mg) was suspended in 10 mL of ac-
by full matrix least squares methods on F2. Hydrogen
etone. The suspension was heated to reflux. Once the
atoms were placed geometrically and refined with a rid-
solid was dissolved, it was allowed to slowly cool to
ing model (including torsional freedom for methyl
room temperature. The solid obtained was isolated by
groups) and with U
iso constrained to be 1.2 (1.5 for
vacuum filtration and washed with cold pentane.
methyl groups) times U
eq of the carrier atom. Refine-
ment converged at a final R
2=0.1515, forall 5632 data, 415 parameters, mean and maximum d/s
0.000, 0.000) with allowance for the thermal anisotropyof all non-hydrogen atoms. Minimum and maximum fi-
nal electron density –0.482 and –0.919 e.–3. A weight-
Differential scanning calorimetry was carried out by
means of a Mettler-Toledo DSC-822e calorimeter.
where P=(Fo2+2*Fc2)/3 was used in the latter stages of
Experimental conditions: aluminum crucibles of
refinement. Complex scattering factors were taken from
40 mL volume, atmosphere of dry nitrogen with
the program package SHELXTLY as implemented on
50 mL min–1 flow rate, heating rate of 10°C min–1.
The calorimeter was calibrated with indium of99.99% purity.
Yvon T64000 instrument, with NIR excitation radia-
Thermogravimetric analyses were performed on a
tion at 514 nm and a liquid-nitrogen-cooled bidimen-
Mettler-Toledo TGA-851e thermobalance. Experi-
mental conditions: alumina crucibles of 70 mL vol-ume, atmosphere of dry nitrogen with 50 mL min–1
flow rate, heating rate of 10°C min–1.
FTIR spectra were recorded on a Bomem MB-120 IRspectrophotometer in KBr pellets, at 1 cm–1 resolu-
XRD patterns were obtained on two equipments:A) Panalytical X’Pert PRO powder diffractometer,
equipped with a CuKa source (l=1.54056 ) and a
Solid state 13C NMR spectra were collected on a
X’Celerator Detector, operating at 45 kV and 40 mA.
Varian Unity spectrometer operating at 75.4 MHz.
Each sample was scanned between 2 and 50° in 2q,
The samples were spun in a 7 mm zirconia rotor and
with a step size of 0.017° and a scan rate of 300 s/step.
registered at room temperature (22°C). High resolu-
B) Debye-Scherrer INEL CPS-120 diffractometer,
tion spectra were recorded using the CP/MAS method
equipped with a CuKa source (l=1.54056 ) and a
120° curved position sensitive detector, operating at
4000 rpm. A 2.5 s contact time and 3 s relaxation time
40 kV and 30 mA. Each sample was scanned between
was used. The resulting FIDs were processed with a
line broadening of 1 Hz. Samples were referenced to
Crystallographic data of form A
hexamethylbenzene (methyl: 17.3 ppm).
Solvent mediated transformation experiments
The general procedure consisted in suspending a mix-
ture of both solid forms (10–20 mg of each form) in
1.5 mL of the selected solvent and stirring with amagnetic bar under nitrogen atmosphere, during three
days at the temperature of study. The solid obtained
was investigated by XRPD and/or DSC analysis.
Results and discussion
Syngle crystal structure of form A
Here we report the first crystal structure solved for an
anhydrous form of Norfloxacin. Although no anhy-
drous crystal structures are available in the literature,
crystal structures are available for a dihydrate , amethanolate , a dihydrochloride monohydrate ,
[9, 11]. Single crystals of the metastable form A were
grown by vapor diffusion of MTBE into a DMF solu-
tion of Norfloxacin. The crystal structure of form A
was determined by single crystal X-ray diffraction.
Form A crystallizes in monoclinic centrosymmetric
space group P21/c. The ORTEP diagram and atomicnumbering are given in Fig. 1. Crystallographic data
The structure shows the molecule as a zwitteri-
onic form which crystallizes as a dimer (Fig. 2). Spe-cific intermolecular interactions (H-bonding) serve tostabilize this dimer as Norfloxacin has a single hydro-gen bond donor (protonated amine), and two good ac-ceptors (carbonylic oxygens).
Efforts to obtain single crystals of polymorphs B
and C suitable for an X-ray structure determinationwere unsuccessful.
Molecular structure of Norfloxacin observed in form A
Preparation and characterization of form C ofNorfloxacin
The new polymorph C of Norfloxacin was obtainedby crystallization in acetone and slowly cooling toroom temperature. Its observed morphology (SEM) isshown in Fig. 3 together with the observed morpho-logies of forms A and B.
The DSC curve of this sample shows an endother-
mic phenomenon at 207°C with a heat of fusion of130.1 J g–1, as shown in Fig. 4. Thermogravimetricanalysis of this sample shows no mass loss from room
ORTEP diagram and atomic numbering of the molecule
temperature to 230°C, showing that this product is nei-
ther a solvated, nor a hydrated form of Norfloxacin.
DSC curves of forms A, B and C of Norfloxacin carried
Observed morphologies (SEM) of the different crystal
The powder XRD pattern of polymorph C is dif-
X-ray powder diffraction patterns of forms A, B and C
ferent from the ones described for forms A and B and
allows a clear and fast identification of thispolymorph (Fig. 5). The most important positions and
The chemical shifts in the solid state NMR
relative intensities for the three modifications are
spectra of forms B and C are very similar. However,
some differences are observed when comparing
Moreover, form C of Norfloxacin has been charac-
resonances of polymorph A with B or C. The main
terized by means of FTIR and Raman spectroscopy (not
difference is the aromatic carbon (C3) a to the
shown). Only slight differences can be detected between
carboxylic group, which is shifted 9 ppm downfield in
polymorphs B and C, whereas significant differences
forms B and C relative to form A. As we reported in a
between the polymorphic forms A and C can be seen in
previous study , this shift can be attributed to the
the whole spectral region. The most striking differences
different orientation of the carboxylic group respect
can be found in the valence vibrations of the carboxylic
to the quinoline plane. Form A was supposed to have
group (A: 1731 cm–1; C: 1715 cm–1), the molecular
an out of plane orientation, which has been confirmed
vibrations (A: 1477 and 1249 cm–1; C: 1484 and
in the present paper by its crystal structure (23°
1254 cm–1) and the rocking vibrations of the alkyl
torsion angle, Fig. 2). Therefore, form C is likely to
chains (A: 749 cm–1; C: 737 cm–1) .
present an in plane conformation as form B does.
It is important to note that only X-ray diffraction
polymorph C together with resonances of forms A
and DSC analysis permit to distinguish between mod-
Most important two theta positions (2q/°) and relative intensities (I
) of the powder X-ray diffraction patterns of
Solid state 13C NMR peak assignments for Norfloxacin crystal forms
ifications B and C of Norfloxacin, whereas all afore-
Physicochemical data obtained from the thermal anal-
mentioned techniques are useful to differentiate be-
tween forms A and C. So, the method of first choicefor a clear distinction of the three crystal forms is
Relative stability of the different modifications
Norfloxacin exists at least in three polymorphic
A value for the heat of fusion of form B was
estimated, even though single melting DSC endotherms
forms. It is of practical interest to know the relative
thermodynamic stability of all forms. The main ques-tions to solve are whether two polymorphs are mono-
transition temperature of form B to form A. Therefore,
tropically (one form is more stable than the other at
we have estimated the transition temperatures between
any temperature) or enantiotropically (a transition
the two enantiotropic pairs by using the treatment of Yu
temperature exists, below and above which the stabil-
. This method needs the temperatures and enthalpies
ity order is reversed) related, and for an enantiotropic
of fusion to calculate the Gibbs free energy difference at
system, where transition temperature lies.
the melting temperature of the lower melting form and
Table 4 summarizes the physicochemical data of
to extrapolate to other temperatures.
the three modifications of Norfloxacin, obtained fromthe thermal analysis experiments. According to the Heat
of Fusion Rule of Burger and Ramberger , forms B
and C are enantiotropically related to form A due to the
lower enthalpy of fusion and the larger melting point of
this last form. Concerning the pair A and B, this rela-
A value of 0.003 was used for the factor k
tionship was also confirmed by the endothermic transi-
was empirically determined and allows a good ap-
tion of form B to form A at about 196°C observed in a
proximation of the heat capacity differences in the
DSC analysis . Also on the basis of the Heat of Fu-
majority of cases . Using this equation we calcu-
sion Rule, modifications B and C appear to be
lated values of 113°C for the pair C–A and 183°C for
monotropically related since the higher melting form B
has the higher enthalpy of fusion. Thus, form B is ther-
In order to confirm the enantiotropic transition
modynamically more stable than form C at all tempera-
temperature between polymorphs C and A, we have
tures up to the melting points of either polymorph.
used the so-called solvent mediated transformation
As mentioned above, polymorphs B and C are
method . This method is based on the relationship
enantiotropically related to form A. In the DSC
between solubility and stability of crystal forms, i.e.
experiments we could only determine the experimental
the less stable form will also be the most soluble at
Experimental conditions and results of the solvent
has been solved, showing a dimeric arrangement of the
mediated transformation experiments between A and
molecule as a zwitterionic form, stabilized by inter-
molecular hydrogen bonding. The relative thermody-
namic stability of the three forms has been fully deter-mined by means of thermal analysis and solvent
mediated transformation experiments. The data show an
enantiotropic relationship between A and C forms, as
well as a monotropic relationship between B and C
modifications. New form C shows a high similarity withform B regarding their molecular spectroscopy proper-ties (IR, Raman and NMR) but not with form A, sug-gesting conformational and spatial distribution differ-ences in the crystal lattice of form A compared to formsB and C. Unfortunately, it has not been possible to growsingle crystals of B and C forms suitable for X-ray struc-ture determination to confirm this assumption.
The authors thank the Unitat de Difracció de Raigs X, Unitatd’Espectroscàpia Molecular and Unitat de Resson´nciaMagnÀtica Nuclear of the Serveis Científico-tÀcnics (Univer-
Semi-schematic energy/temperature diagram of
sity of Barcelona) for their contributions to this work. We are
Norfloxacin, showing fusion temperatures of the three
also grateful to Harry Adams of the University of Sheffield
(UK) for single crystal X-ray diffraction data.
given conditions of temperature and pressure. Ifcrystals of both forms are mixed with a saturated
solution of the product, the most stable form willgrow at the expense of the less stable one. Mixtures of
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acin has been completed with the discovery of the newform C. Moreover, the single crystal structure of form A
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