Characterization and structural analysis of the potent antiparasitic and antiviral agent tizoxanide

Article abstract of DOI:10.1016/j.molstruc.2012.12.004

Tizoxanide [2-(hydroxy)-N-(5-nitro-2-thiazolyl)benzamide, TIZ] is a new potent anti-infective agent which may enhance current therapies for leishmaniasis, Chagas disease and viral hepatitis. The aim of this study was to identify the conformational prefere

Full text of DOI:10.1016/j.molstruc.2012.12.004

Journal of Molecular Structure 1036 (2013) 318–325
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Characterization and structural analysis of the potent antiparasitic
and antiviral agent tizoxanide
Flavia P. Bruno a, Mino R. Caira b, Eliseo Ceballos Martin a, Gustavo A. Monti c, Norma R. Sperandeo a,
⇑
a Departamento de Farmacia, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Haya de la Torre y Medina Allende, Ciudad Universitaria,
X5000HUA Córdoba, Argentina
b Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
c Facultad de Astronomía, Matemática y Física, Universidad Nacional de Córdoba and IFEG-CONICET, Ciudad Universitaria, X5000HUA Córdoba, Argentina
g r a p h i c a l a b s t r a c t
h i g h l i g h t s
Tizoxanide [2-(Hydroxy)-N-(5-nitro-thiazolyl)benzamide, TIZ],a new potent anti-infective agent, has
been structurally characterized by X-ray analysis, solid-state NMR, DRIFT, FTRaman and Thermal Analy-
sis. This modification of TIZ has relatively high thermal stability as a solid and a ‘graphitic’ structure, and
is composed of tightly packed layers of extensively hydrogen bonded molecules.
"
"
"
The crystal structure of unsolvated
tizoxanide (TIZ) was resolved.
This modification of TIZ has a
‘graphitic’ structure.
The various spectroscopic data were
consistent with the resolved
structure.
"
"
The behavior on heating of TIZ was
studied by DSC, TG and HSM.
TIZ decomposes above 270 °C and no
isolated melting event was detected.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 October 2012
Received in revised form 3 December 2012
Accepted 3 December 2012
Available online 10 December 2012
Tizoxanide [2-(hydroxy)-N-(5-nitro-2-thiazolyl)benzamide, TIZ] is a new potent anti-infective agent
which may enhance current therapies for leishmaniasis, Chagas disease and viral hepatitis. The aim of
this study was to identify the conformational preferences that may be related to the biological activity
of TIZ by resolving its crystal structure and characterizing various physicochemical properties, including
its experimental vibrational and 13C nuclear magnetic resonance properties, behavior on heating and sol-
ubility in several solvents at 25 °C. TIZ crystallizes from dimethylformamide as the carboxamide tauto-
mer in the triclinic system, space group P(ꢀ1) (No. 2) with the following unit cell parameters at
Keywords:
Crystal structure
13C CPMAS NMR
DRIFT
FT-Raman
Thermal analysis
Tizoxanide
173(2) K: a = 5.4110(3) Å, b = 7.3315(6) Å, c = 13.5293(9) Å,
a = 97.528(3), b = 95.390(4), c = 97.316(5),
V = 524.41(6) Å3, Z = 2, Dc = 1.680 g/cm3, R1 = 0.0482 and wR2 = 0.0911 for 2374 reflections. This modifica-
tion of TIZ has a ‘graphitic’ structure and is composed of tightly packed layers of extensively hydrogen-
bonded molecules. The various spectroscopic data [Diffuse Fourier transform infrared (DRIFT) and
FT-Raman, recorded in the range 3600–500 and 4000–200 cmꢀ1 respectively, and solid-state 13C NMR]
were consistent with the structure determined by X-ray crystallography. From DSC, TG and thermomi-
croscopy, it was concluded that TIZ is thermally stable as a solid and that melting is not an isolated event
from the one-step thermal decomposition that it undergoes above 270 °C. This modification of TIZ is prac-
tically insoluble in water and slightly soluble in polar aprotic solvents such as dimethylsulfoxide, dimeth-
ylformamide and dioxane.
Ó 2012 Elsevier B.V. All rights reserved.
⇑
Corresponding author.
E-mail address: nrscor@fcq.unc.edu.ar (N.R. Sperandeo).
0022-2860/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molstruc.2012.12.004

F.P. Bruno et al. / Journal of Molecular Structure 1036 (2013) 318–325
319
The importance of characterizing the solid-state properties and
the absolute configuration of a drug, which is a critical property in
biological systems as changes in this may alter the response of the
biologic system, has been well documented in the literature. To our
knowledge, the experimental data available in the pharmaceutical
literature on the solid-state properties of TIZ are the melting point
(with two different values reported, viz. 254 °C [9] and 279–281 °C
[10] and the FTIR spectrum (KBr), which was not thoroughly ana-
lyzed and assigned [9,11].
In this report, we describe for the first time, the crystal structure
of TIZ as determined by single crystal X-ray diffraction (SXRD). The
structural features of TIZ are also discussed in the context of its
vibrational [diffuse reflectance infrared Fourier transform (DRIFT)
and FT-Raman], spectroscopic [Solid-state (SSNMR) and solution
(S NMR) nuclear magnetic resonance spectroscopy] and thermal
[differential scanning calorimetry (DSC), thermogravimetry (TG)
and hot stage microscopy (HSM)] properties of this compound.
1. Introduction
Parasitic and viral diseases such as leishmaniasis, trypanosomia-
sis and hepatitis C represent a serious problem to the health and
economy of many countries. There are an estimated annual 1.5–
2.0 million new cases of leishmaniasis, of which approximately
500,000 belong to the potentially fatal visceral form [1]. The
American trypanosomiasis (Chagas disease) affects approximately
15–20 million people from Southern California to Argentina and
Chile; 2–3% of the Latin American (LA) population is infected,
encompassing a burden of about 670,000 Disability Adjusted Life
Years (DALYs), and the morbidity and mortality are more than one
order of magnitude higher than those found for malaria, schistoso-
miasis or leishmaniasis [2]. Current estimates indicate 200,000
new cases every year and an annual mortality of ꢁ50,000 [2]. On
the other hand, Hepatitis C virus (HCV) is a bloodborne infection that
affects nearly 2.2% of the world’s population, or 130 million people
[3]. Approximately 55–85% of newly infected cases progress to chro-
nicity; of those, 20–30% will develop liver fibrosis, cirrhosis, and li-
ver failure and 2–5% will advance to hepatocellular carcinoma [3].
Common chemotherapeutic agents currently used against leish-
maniasis and trypanosomiasis are often inadequate since they re-
quire long courses of administration, may have toxic side effects
or become less effective due to the emergence of resistant strains
[1]. On the other hand, the current standard treatment of care for
hepatitis C (peginterferon and ribavirin) is effective in about half
of all patients treated [4]. Therefore, new, effective and inexpensive
drugs that can be used to treat these diseases are urgently
required.
The thiazolides represent a novel class of anti-infective drugs,
with nitazoxanide [2-acetyloxy-N-(5-nitro-2-thiazolyl)benzamide,
AliniaÒ, NTZ] as the parent compound. NTZ is marketed in the US
and LA for treating diarrhea and enteritis caused by Cryptosporidi-
um spp. or Giardia lamblia in adults and children down to
12 months of age [5]. Following oral administration of a 500 mg
tablet, NTZ is partially absorbed from the gastrointestinal tract
and rapidly hydrolyzed in plasma to form its active circulating
metabolite, tizoxanide (TIZ, Fig. 1.), which is as effective as the par-
ent drug [6]. In fact, both NTZ and TIZ are active against Trypano-
soma cruzi (the causative agent of Chagas disease) and Leishmania
mexicana [1], and the ulcer-causing pathogen Helicobacter pylori
[7]. Also, both NTZ and TIZ are potent inhibitors of hepatitis B virus
(HBV), and in combination with other antiviral agents such as lam-
ivudine or adefovir show synergistic effects [5]. Additionally, NTZ
and TIZ are potent inhibitors of HCV in genotype 1a- and 1b-
derived replicon cells and genotype 2a-cell culture models, and
synergistic effects are observed when TIZ is combined with
interferon [8]. Thus, TIZ is a promising anti-infective agent that
may enhance current or future therapies for leishmaniasis, Chagas
disease and viral hepatitis caused by HBV or HCV.
2. Experimental
2.1. Materials
Tizoxanide, designated hereafter as TIZ-rp, was obtained by acid
hydrolysis (aqueous HCl 37% w/w, 50 °C, 24 h [8]) of NTZ commer-
cial raw powder (99.89% purity) and was used as such to record the
DRIFT, FT-Raman and NMR spectra, as well as the DSC and TG
curves. Single crystals were grown from a filtered hot saturated
solution of TIZ-rp in dimethylformamide. By slow recrystallization
at room temperature (25–30 °C), suitable crystals for X-ray analy-
sis were obtained. All other chemicals and solvents were of analyt-
ical reagent grade or spectroscopic quality.
2.2. Single-crystal X-ray structure determination
Table 1 lists crystal data and refinement parameters for TIZ.
During data-collection (program COLLECT [12]) the crystal was
cooled in a constant stream of nitrogen vapor (Oxford Cryostream,
UK). Program DENZO-SMN [13] was used for data-reduction and
unit cell refinement. Laue symmetry (ꢀ1) indicated the triclinic
crystal system and the value of hE ꢀ 1i = 0.984 indicated the centric
space group P(ꢀ1). The structure was solved by direct methods and
refined by full-matrix least-squares on F2 using programs in the
SHELX-suite [14]. Following isotropic and anisotropic refinement
of the non-hydrogen atoms, all H atoms were located in difference
electron density maps and were subsequently included in a riding
model with isotropic thermal displacement parameters Uiso equal
to 1.2 Ueq of their parent atoms.
2.3. Differential scanning calorimetry (DSC), thermogravimetry (TG)
and hot stage microscopy (HSM)
The DSC and TG measurements were recorded on MDSC 2920
and TG 2950 analyzers (TA Instruments Inc., USA) respectively, at
a heating rate of 10 °C/min under N2 (99.99% purity, flow rate
50 mL/min). For DSC measurements, closed and hermetically-
closed aluminum pans were used. The DSC and TG temperature
axes were calibrated with indium (99.99% purity, m.p. 156.6 °C)
and the Curie point of Ni (358.1 °C), respectively. Empty aluminum
pans were used as references. Samples with mass 1–2 mg were
employed. Data were treated with Universal Analysis 2000 soft-
ware (TA Instruments Inc.).
The physical and morphological changes that occurred during
the process of heating TIZ particles were observed through a
microscope fitted with a Kofler hot-stage (Leitz, Wetzlar, Germany)
at a constant rate of about 8 °C/min from about 25 °C.
Fig. 1. Chemical structure of tizoxanide (TIZ).

320
F.P. Bruno et al. / Journal of Molecular Structure 1036 (2013) 318–325
Table 1
8 Hz was used. The center of the solvent peak was used as an inter-
nal standard which was related to TMS. The processing of the data
was performed with a Bruker Standard program (TOPSPIN 2.0).
Crystal data and refinement parameters for tizoxanide (TIZ).
Chemical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
C10H7N3O4S
265.25
173(2)
0.71073, Mo K
2.5. Diffuse reflectance infrared Fourier transform (DRIFT) and Raman
spectroscopy
Triclinic
Space group
Unit cell parameters a, b, c
P(ꢀ1) (No. 2)
5.4110(3), 7.3315(6), 13.5293(9), 97.528(3),
DRIFT and Raman spectra were recorded on a Nicolet Avatar
360 FTIR spectrometer (Nicolet Instruments Corp., Madison, WI)
and a Horiba Jobin Ivon LabRaman HR instrument (Horiba, Kyoto,
Japan), respectively. For the DRIFT spectrum, a diffuse reflectance
accessory and macro-diffuse reflectance cups of 13-mm diameter
(ꢁ400 mg capacity) were used. For the preparation of the sam-
ple-KBr blend, dry KBr was ground for 2 min before mixing with
the sample (2.5% w/w) in an agate mortar with only light grinding
performed. The blend was then placed in the cup, and excess mate-
rial was removed by placement of a microscope slide against the
open cup in a rotary motion to leave a level but roughened surface,
that was scanned immediately. KBr scans were used as back-
ground. The Raman spectrum was acquired operating the instru-
ment at a resolution of 4 cmꢀ1, using a 632.8 nm excitation from
a He–Ne laser (at 10% of its power) in the range of 4000–
400 cmꢀ1. The data were acquired using back scattering with
1800 grating, 5 scans per 30 s (10 s) pulse and with 10ꢃ zoom.
The instrument was calibrated with a Si single crystal.
(Å),
a
, b,
c
(°)
95.390(4), 97.316(5)
524.41(6)
2
1.680
0.320
272
Volume (Å3)
Z
Calculated density (Mg mꢀ3
)
l
(mmꢀ1
F000
)
Crystal size (mm3)
Diffractometer/
0.13 ꢃ 0.16 ꢃ 0.20
Nonius Kappa CCD/x- and /-scans 1.0°
measurement method
Index ranges
h-range for data-collection
ꢀ7 6 h (7, ꢀ9 6 k 6 9, ꢀ17 6 l 6 17
1.00 6 h 6 27.48
(°)
Measured reflections
Independent/observed
reflections
4601
2374/1910
Rint
0.0204
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
R indices [I > 2
R indices (all data)
Full-matrix least-squares on F2
2374/0/164
1.055
r
(I)]
R1 = 0.0339, wR2 = 0.0846
R1 = 0.0482, wR2 = 0.0911
w = 1/[r2ðF2o Þ + (0.0502P)2 + 0.1025P],
Weighting scheme
P = ðF2o þ 2F2c Þ/3
ꢀ0.27, 0.33
2.6. X-ray powder diffraction (XRPD)
D
qmin, D )
qmax (e Åꢀ3
A Philips X’Pert PRO PANalytical powder diffractometer (Philips,
The Netherlands) was used. The measuring conditions were: 20–
25 °C, Cu Ka1 radiation (k = 1.54056 Å), 40 kV, 40 mA, step size of
2.4. NMR spectroscopy
0.02° (2h), time per step 5 s, time constant 0.03 s, angular range
3–35° 2h. A 25 mm diameter Si single crystal holder was used
and the sample (gently ground) was pressed by means of a clean
glass slide to ensure coplanarity of the powder surface with the
surface of the holder. Data were treated with the X’PERT Data
Viewer diffraction software.
13C SSNMR studies were performed using the ramp CPMAS se-
quence with proton decoupling during acquisition, at 25 °C in a
Bruker Avance II 300 spectrometer (Bruker, Germany), operating
frequency for carbons 75 MHz, equipped with a 4 mm MAS probe.
Spinning rate was 8 kHz. The recycling time was 20 s, the contact
time during CP was 2 ms, and 2048 scans were recorded. TPPM15
sequence was used for decoupling during acquisition with a proton
2.7. Solubility estimation
field H1H satisfying
were quoted with respect to tetramethylsilane (TMS) and mea-
sured via replacement with adamantane (29.50 and 38.56 ppm).
Interrupted decoupled experiments were conducted using
x1H/2p = cH H1H/2p = 50.0 kHz. Chemical shifts
The solubility of TIZ-rp at 25 °C was estimated according to a
previously reported procedure [16]. Briefly, an accurately weighed
portion of TIZ-rp (2–3 mg) was added to a 10 mL glass vial. Solvent
of known volume was added to the vial. The vial was shaken by
hand for 5 min. If solid remains, the procedure was repeated until
the solid was completely dissolved. Solubility was then bracketed
using the total volumes after the last two additions, and it was ex-
pressed as the weight of solid divided by volume of the solvent.
Solvents used in this study were acetone, acetonitrile, ammonium
hydroxide 1 M, benzene, chloroform, dimethylformamide, DMSO,
dioxane, ethanol, ethyl acetate, ethyl ether, n-hexane, hydrochloric
acid 1 M, iso-propyl alcohol, methanol, methylene chloride, n-pen-
tane, tetrahydrofuran, t-butanol, toluene and water.
a
40 s delay before acquisition. The complete signal assignments
l
of the spectrum were achieved by dipolar dephasing experiments
[which yields quaternary carbons only and the corresponding
non-quaternary suppression (NQS) spectrum] and by comparison
with one-dimensional solution 1H [spectral width of 20 ppm using
a 30° pulse (7.09 l
s) and 16 scans] and 13C (100 MHz using the fol-
lowing acquisition parameters: spectral width, 220 ppm; relaxa-
tion delay, 1.7 s; 2048 scans for sample, and a 30° excitation
pulse). NMR spectra (in DMSO-d6) of TIZ and NTZ [15] along with
two-dimensional COSY-45, HSQC and HMBC spectra [Bruker
Avance II 400 spectrometer current probe 5 mm BBI 1H/D-BB
Z-GRD Z8202/0349]. The COSY-45 spectrum (DMSO-d6) was ob-
tained using gradient pulses for selection, and the following acqui-
sition parameters: scans, 2; relaxation delay, 1 s; f1 channel 30°
3. Results and discussion
3.1. Molecular and crystal structure
high power pulse (7.09 ls) and sizes of fid of 256 (time domain
1) and 2048 (time domain 2). The HSQC spectrum (DMSO-d6)
was obtained using 4 scans, a relaxation delay of 1 s, a f1 channel
30° high power pulse of 7.09 ls, sizes of fid equal to those of the
COSY-45 spectrum, and a one-bond CAH coupling constant of
145 Hz. The HMBC spectrum (DMSO-d6) was obtained using acqui-
sition parameters identical to those of the HꢂSQC spectrum but
with 8 scans, and more than one-bond CAH coupling constant of
Selected molecular parameters for TIZ appear in Table 2. Hydro-
gen bonds are listed in Table 3. Fig. 2 (left) shows the molecular
structure of TIZ with the intramolecular NAHꢄ ꢄ ꢄO hydrogen bond
indicated by dotted lines. This interaction results in the 5- and 6-
membered rings being almost coplanar (angle of intersection of
their least-squares [LS] planes = 2.83(7)°). The maximum devia-
tions above and below the LS plane that includes all 18 non-H

F.P. Bruno et al. / Journal of Molecular Structure 1036 (2013) 318–325
321
atoms are those for atoms O18 [+0.067(1) Å] and O7 [ꢀ0.117(1) Å].
Common bond lengths and angles (Table 2) are in excellent
agreement with those reported previously for NTZ [15]. The latter
molecule is likewise constrained to be nearly planar by the
common intramolecular NAHꢄ ꢄ ꢄO hydrogen bond, but the overall
level of planarity is somewhat lower than in the TIZ molecule. This
can be attributed to the significantly weaker intramolecular
NAHꢄ ꢄ ꢄO(acetyloxy) bond in NTZ (Nꢄ ꢄ ꢄO = 2.700(2), 2.681(2) Å for
two crystallographically independent molecules) than the
NAHꢄ ꢄ ꢄO(hydroxyl) bond occurring in TIZ (Nꢄ ꢄ ꢄO = 2.646(2) Å).
The planarity of the TIZ molecule is further manifested in its
remarkable ability to form infinite layers via extensive intermolec-
ular OAHꢄ ꢄ ꢄN and CAHꢄ ꢄ ꢄO hydrogen bonding. In the representa-
tive portion of such a layer shown in Fig. 2 (right), the unique
hydrogen bonds are labelled 1–5 (as in Table 3) while individual
ring motifs are labelled a–e. H-bond 1 is the intramolecular inter-
action that results in the ring motif a, with graph-set notation S(6)
[17]. The remaining rings with their graph-set descriptors and the
H-bond combinations that generate them are as follows: ring b
[R44ð12Þ, containing two OAHꢄ ꢄ ꢄN and two NAHꢄꢄꢄO hydrogen
bonds], ring c [R22ð10Þ, containing two CAHꢄ ꢄ ꢄO(nitro) H-bonds],
ring d [R33ð12Þ, containing one OAHꢄ ꢄ ꢄN and two CAHꢄ ꢄ ꢄO(nitro)
H-bonds], and the large ring e [R44ð26Þ, formed by the combination
of two CAHꢄ ꢄ ꢄO(carbonyl) and two CAHꢄ ꢄ ꢄO(nitro) H-bonds]. The
layer is thus constructed from the co-operative effects of a series
of stable, hydrogen-bonded ring motifs of different topologies
and the TIZ molecules are closely packed within the layer. The
hydrogen bonding capacity of the TIZ molecule is fully utilized in
such layer formation and a stable crystal is formed by close stack-
Table 2
Selected bond distances, bond angles, and torsion
angles for TIZ.
Bond lengths (Å)
S12AC11
S12AC13
C13AC14
C14AN15
N15AC11
O7AC1
O9AC8
O17AN16
O18AN16
1.725(2)
1.723(2)
1.353(2)
1.364(2)
1.325(2)
1.363(2)
1.222(2)
1.234(2)
1.229(2)
Bond angles (°)
C11AS12AC13
S12AC13AC14
C13AC14AN15
C14AN15 -C11
N15AC11AS12
C11AN10AC8
N10AC8AC2
86.39(7)
113.2(1)
113.8(1)
109.8(1)
116.7(1)
122.1(1)
118.3(1)
123.8(1)
ing of such layers via strong p–p interactions between the phenyl
group of a TIZ molecule and the thiazolyl ring of a centrosymmet-
rically-related molecule. There are two unique interactions of this
type, with distances between the ring-centroids of 3.601(1) and
3.807(1) Å. The resulting ‘graphitic’ structure is illustrated in
Fig. 3, which shows three layers in both stick and space-filling
mode.
The refined single crystal X-ray data were used to compute the
idealized X-ray powder pattern [18] for this crystalline phase at
173 K (Fig. 4a). As expected, the peak of highest intensity (at
2h = 27.9°) corresponds to reflection of X-rays from the layers high-
lighted in Fig. 3, these layers coinciding with the crystal planes (1 2
0) whose interplanar spacing (d) is 3.2 Å at 173 K. We note that the
interlayer distance is therefore remarkably short and it is signifi-
cantly smaller than the distances cited above between the relevant
O17AN16AO18
Torsion angles (°)
C1AC2AC8AN10
C2AC8AN10AC11
C8AN10AC11AS12
S12AC13AN16AO17
5.5(2)
179.4(1)
ꢀ2.3(2)
1.3(2)
Table 3
Hydrogen bond parameters for TIZ.a
No.
Bond
Dꢄ ꢄ ꢄA (Å)
DAH (Å)
Hꢄ ꢄ ꢄA (Å)
ring centroids as a result of large offsets between the
p-stacked
phenyl and thiazolyl rings. The relative ‘simplicity’ of the crystal
structure of TIZ leads to only four very prominent peaks at 2h-val-
ues 6.6°, 16.6°, 24.7° and 27.9°, the remaining peaks having signif-
icantly lower intensities. The calculated pattern is in good
agreement with the experimental one (Fig. 4b), confirming that
there is no significant structural change when the crystal was
cooled from ambient laboratory temperature (294 K) to 173 K.
1
2
3
4
5
N10AH10ꢄ ꢄ ꢄO7
O7AH7ꢄ ꢄ ꢄN15i
C14AH14ꢄ ꢄ ꢄO18ii
C5AH5ꢄ ꢄ ꢄO17iii
C3AH3ꢄ ꢄ ꢄO9iv
2.646(2)
2.757(2)
3.371(2)
3.438(2)
3.448(2)
0.88
0.84
0.95
0.95
0.95
1.95
1.93
2.43
2.53
2.52
a
Symmetry operations: (i) ꢀx, 1 ꢀ y, 1 – z; (ii) ꢀx, 1 ꢀ y, ꢀz; (iii) x, y, 1 + z; (iv)
2 ꢀ x, 2 ꢀ y, 1 ꢀ z.
Fig. 2. Molecular structure and conformation of TIZ with non-H atoms shown as thermal ellipsoids drawn at the 50% probability level and H atoms shown as spheres of
arbitrary radii (left) and extensive hydrogen bonding within a layer of TIZ molecules (right).

322
F.P. Bruno et al. / Journal of Molecular Structure 1036 (2013) 318–325
Fig. 3. Portions of three stacked layers of TIZ represented in stick mode (left) and space-filling mode (right). The interlayer distance is ꢁ3.2 Å.
Fig. 5. Overlaid DSC and TG traces of TIZ-rp (10 °C/min, flowing N2 at 50 mL/min):
(a) DSC curve in crimped pan, (b) TG curve (open pan) and (c) DSC trace in
hermetically sealed pan. Insets: DSC peaks.
Fig. 4. (a) Computed XRPD trace of TIZ calculated at 173 K and (b) experimental
diffractogram of TIZ-rp.
by TG. On the other hand, the DSC trace of TIZ-rp obtained in a her-
metically-closed pan (Fig. 5c) showed only a sharp exothermic
peak at 276.7 °C (Tonset), which exhibited no baseline shift proba-
bly because the sample mass did not change, as volatile decompo-
sition products were not formed or they could not escape due to
the hermetic encapsulation. The absence of an endothermic peak
and the small upward shift of the exothermic one (from 273.4 to
276.7 °C) as the pan system changed from closed (Fig. 5a) to her-
metically-closed (Fig. 5b) evidently indicates that the thermal
decomposition of TIZ is affected by the sample encapsulation.
Taking into account that no sharp, isolated melting peak was
detected for TIZ-rp in either type of pan, observations under a
Kofler apparatus were made to identify whether or not there had
been melting, perhaps local and temporary, during decomposition.
On heating from 25 to 270 °C, the colorless transparent irregular
particles of TIZ-rp (Supplementary material) did not undergo
changes in shape, color or size, and no vapor condensation on
the cover slip was noted, discarding the occurrence of phase tran-
sitions or evaporation losses, and this was consistent with the DSC
and TG results. At about 275 °C, the crystalline particles of TIZ-rp
started to bubble, lose their shape and darken simultaneously.
Consequently, after cooling the slide, a gummy dark-brown residue
was obtained, confirming the thermal decomposition of the sam-
ple. According to these HSM results, melting of TIZ-rp did not occur
before its exothermal decomposition to yield a single coherent
droplet and an isolated DSC melting peak. Local and temporary
melting probably occurred, as indicated by the detection of bub-
bles and sintering, which is evidence that surface free-energy (li-
quid) forces control textural features resulting in surface area
minimization [21].
Subsequent to the above analysis, we were alerted to a recent
report of the X-ray crystal structure of the TIZ pyridine monosol-
vate [19] and we carried out a detailed comparison of the struc-
tures of the drug molecule in the crystal of pure TIZ and the
solvated species. In both crystals the TIZ molecule is essentially
planar, the co-planarity of the two rings being maintained by the
common intramolecular NAHꢄ ꢄ ꢄO bond. The latter is slightly stron-
ger in the solvated phase (Nꢄ ꢄ ꢄO 2.625(2) Å compared with
2.646(2) Å in the crystal of pure TIZ). Covalent bond distances in
the TIZ molecule do not, however, differ significantly in the two
phases. Finally, in the TIZ pyridine monosolvate structure, the drug
and included solvent molecules are linked by a OAHꢄ ꢄ ꢄN bond,
with the molecular planes of the TIZ molecule and the pyridine
molecule almost orthogonal, thus precluding the unusual
‘graphitic’-type structure reported here for pure TIZ.
3.2. Thermal, vibrational and spectroscopic data
The overlaid DSC and TG curves of TIZ-rp are shown in Fig. 5.
The DSC and TG curves obtained in closed and open pans respec-
tively (Fig. 5a and b) showed neither endothermic peaks nor TG
mass losses in the 25–240 °C temperature range, confirming the
absence of solvates or residual solvent, in accord with the SXRD
data that revealed that TIZ was unsolvated. Above 260 °C, the
DSC curve (Fig. 5a) exhibited a barely discernible endothermic
event superimposed on a sharp exothermic peak at 273.4 °C
(Extrapolated onset temperature, Tonset), ascribable to an exother-
mic decomposition process. The corresponding TG curve (Fig. 5b)
showed a mass loss of about 45% that coincided with the DSC
endo-exothermic events and it occurred in one-step between 230
and 300 °C, confirming the decomposition of TIZ. In addition, a
DSC baseline shift was noted after the exothermic peak, and this
was consistent with changes in the sample mass [20], as indicated
The 13C CPMAS and the NQS spectra of TIZ-rp are shown in
Fig. 6, while the carbon assignments and chemical shift values
are given in Table 4. The CPMAS spectrum (Fig. 6a) exhibited nine
signals for the ten carbons of the molecule, indicating that one of

F.P. Bruno et al. / Journal of Molecular Structure 1036 (2013) 318–325
323
them corresponded to two carbons, i.e. the signal at 163.1 which
was assigned to the C-8 carbonyl and the C-11 carbon. The NQS
spectrum (Fig. 6b) showed four signals, indicating that the peaks
observed in the CPMAS spectrum in the range 116–140 ppm corre-
sponded to the five CAH carbons of the molecule, i.e. the four ben-
zenoid carbons and C-14. The 13C SSNMR spectrum was also
compared with that recorded in DMSO-d6 solution and the data ap-
pear in Table 4. It is clear that the chemical shifts of five carbon
atoms of the molecule, C-4, C-5, C-6, C-11 and C-13, measured in
the solid-state have values close to those observed in solution
(the differences in chemical shifts were all less than 1.5 ppm),
indicating that the chemical environment around these carbons
is very similar in the solid-state and in solution. In contrast, the
C-1, C-2, C-3, C-8 and C-14 chemical shifts measured in the so-
lid-state are slightly different from those observed in DMSO-d6.
The chemical shifts of atoms C-1, C-2 and C-8 are shifted to a
possibly due to further rigidities of the nitro group and thiazole
ring in the solid state, as was also observed for NTZ [15].
The DRIFT and FT-Raman spectra (Fig. 7) were also compared to
obtain additional information regarding the intra- and intermolec-
ular interactions in TIZ-rp. The origins of the major peaks of both
spectra were assigned using published compilations and literature
studies [23–28]. As characteristic features, the DRIFT spectrum
(Fig. 7a) showed a strong NH stretch absorption centered at
3253.8 cmꢀ1, which is superimposed on a broad band ranging from
3500 to 2250 cmꢀ1. Neither band seems to have a counterpart in
the Raman spectrum (Fig. 7b). In the absence of H-bonding, it
would be reasonable to assume that the amide NAH stretching
mode would be observed above 3300–3500 cmꢀ1 [23]. The shift
to lower wavenumbers observed in the DRIFT spectrum indicates
that the NH group is associated through H-bonding, which is in
good agreement with the determined crystal structure of TIZ. In-
deed, in the crystal, the amide group hydrogen is involved in a
strong intramolecular hydrogen bonding interaction of the NHꢄ ꢄ ꢄO
type with the oxygen atom of the C1-OH group. The high intensity
of the NH vibration is additional evidence of such intramolecular
interaction [27], as well as for presence of a planar ACONH group
[28]. In fact, the FTIR spectra of various benzamides with ortho-
ring substituents that formed intramolecular H-bonds with polar
groups, like the phenolic C1-OH group of TIZ [27], and displayed
planar ACONH groups, did exhibit strong NH vibrations [28].
The absence of a separate AOH stretching vibration in the DRIFT
spectrum (Fig. 7a) also confirms the participation of the AOH pro-
ton in H-bonding since such interaction leads to a red shift of the
AOH stretching band and to a broad continuum in the 3200–
2500 cmꢀ1 range [29]. According to the SXRD data, a classical
OAHꢄ ꢄ ꢄN intermolecular H-bond is formed between the C1-OH
group of one molecule of TIZ and the thiazolic nitrogen of a sym-
metry-related molecule (Fig. 2), which is well evidenced in the
DRIFT spectrum by the broad AOH continuum in the range
3500–2300 cmꢀ1. On the other hand, the absence of the AOH
stretching band in the Raman spectrum (Fig. 7b) is characteristic
and offers a window to the weak CH stretching bands that are bur-
ied below the AOH continuum in the FTIR spectrum [29]. Thus,
both spectra exhibited bands at 3115.9 and 3071.1 cmꢀ1 (DRIFT)
and 3114.3 and 3072.2 cmꢀ1 (Raman), ascribable to the aromatic
CAH stretching vibrations that are normally found between 3100
and 3000 cmꢀ1 [23].
low frequency in the solid state (
D
solid-solution: ꢀ2.3, ꢀ3.5 and
ꢀ3.0 ppm, respectively), and since these carbons are close to the
intramolecular NAHꢄ ꢄ ꢄO bond, the shifts could be attributed to
the replacement of intramolecular H-bonding by intermolecular
H-bonds with DMSO-d6 molecules through the amide and/or OH
protons [22]. In contrast, the chemical shift of the C-3 carbon
was shifted to a high frequency in the solid-state, and this could
indicate a small change in the orientation of the amide group in
the solid-state with respect to the DMSO solution, as was also ob-
served for NTZ [15]. With regard to C-14, very similar chemical
shifts are observed for C-13 and C-14 in the 13C S NMR spectrum
(Table 4), indicating a chemical similarity of these carbons; how-
ever, two separate signals at 143.5 and 138.1 ppm with an accom-
panying low frequency shift of ꢀ3.8 ppm for the C-14 chemical
shift is observed for this carbon in the solid state, indicating that
the chemical environments are different for these carbon atoms,
Fig. 6. (a) 13C SS CPMAS and (b) NQS spectra of TIZ-rp.
Table 4
Comparison of the 13C NMR chemical shifts (in ppm) of TIZ-rp in the solid state
(CPMAS spectrum) and in solution (DMSO-d6).
C atom CPMAS spectrum DMSO-d6 (0.065 M) Difference (SS-DMSO-d6)
1
2
3
4
5
6
8
11
13
14
155.5
113.6
133.3
119.6
136.3
116.4
163.1
163.1
143.3
138.6
157.8
117.1
130.9
120.3
135.5
117.6
166.1
162.1
142.8
142.4
ꢀ2.3
ꢀ3.5
2.4
ꢀ0.7
0.8
ꢀ1.2
ꢀ3.0
1.0
0.5
ꢀ3.8
Fig. 7. (a) DRIFT (KBr) and (b) Raman (632.8 nm excitation, He–Ne laser) spectra of
TIZ-rp.
In the solid state (CPMAS spectrum) and in solution (DMSO-d6).

324
F.P. Bruno et al. / Journal of Molecular Structure 1036 (2013) 318–325
Table 5
Estimated solubility of TIZ in various solvents at 25 °C.
characteristics of TIZ. X-ray structural investigation of TIZ shows
that the analyzed modification has a ‘graphitic’ structure composed
of tightly packed layers of hydrogen bonded molecules that
are stacked on top of one another and held together by strong
Solvent
Solubility (mg/mL)
Acetone
0.38
p
-stacking interactions between the phenyl and thiazolyl rings of
Acetonitrile
Ammonium hydroxide 1 M
Benzene
Chloroform
Dimethylformamide
DMSO
Insoluble
Insoluble
Insoluble
Insoluble
3.00
inversion-related TIZ molecules. This is an exceptionally good
example of layer formation that involves the interlocking of a series
of stable ring structures created by extensive hydrogen bonding.
DRIFT and FT-Raman studies indicate that the vibrational spectra
of TIZ-rp exhibit features characteristic of a hydrogen bonded
molecule, confirming the intra- and intermolecular interactions as
revealed by the resolved crystal structure. Thermal analysis has
been used to further characterize TIZ-rp, and the information ob-
tained from the DSC and TG curves was that solid TIZ has relatively
high thermal stability, i.e. the Tonset is higher than 270 °C, and that
melting is not an isolated event from the one-step exothermic
decomposition that this modification undergoes.
4.50
2.00
Dioxane
Ethanol
Insoluble
Insoluble
Insoluble
Insoluble
Insoluble
Insoluble
Insoluble
Insoluble
Insoluble
Insoluble
Insoluble
Insoluble
Insoluble
Ethyl acetate
Ethyl ether
Hydrochloric acid 1 M
Iso-Propyl alcohol
Methanol
Methylene chloride
n-Hexane
n-Pentane
t-Butanol
Tetrahydrofuran
Toluene
Acknowledgements
Water
Financial support from SECyT-UNC and FONCyT of Argentina is
gratefully acknowledged. F.P. Bruno acknowledges financial sup-
port from FONCyT and CONICET fellowships. Helpful comments
on NMR solution spectra from Dra. G. Bonetto, who recorded the
spectra, are gratefully acknowledged. MRC thanks the University
of Cape Town and the National Research Foundation (Pretoria)
for research support. Thanks also go to Prof. Dr. E. Coronado
(Departamento de Fisicoquímica, FCQ, UNC) for the use of Micro-
Raman facilities.
Also present in the spectra were bands at 1673.9 (DRIFT) and
1665.9 cmꢀ1 (Raman) assigned to the amide carbonyl (8-C@O)
stretch, and 1539.6 cmꢀ1 in IR and at 1531.1 cmꢀ1 in the Raman
spectra, respectively, ascribed to the CA(NO2) asymmetric stretch-
ing of TIZ. The in-plane bending mode of the AOH group, normally
observed in the region 1250–1150 cmꢀ1, is observed at 1160.3
(DRIFT) and 1162.1 (Raman) cmꢀ1. The vibrations at 1359.3
(DRIFT) and 1361.9 cmꢀ1 (Raman) were assigned tentatively to
the amide CAN stretch overlapped with the symmetrical mode of
the NO2 group, and to the amide NAH bend and the asymmetrical
stretch of the NO2 group, respectively. Also visualized in the DRIFT
spectrum are medium bands at 897.2 and 813.4 cmꢀ1, which were
assigned to the aromatic CAH out-of-plane bending vibrations of
TIZ; whereas the bands at 672.8 and 655.4 (DRIFT) and
682.9 (Raman) cmꢀ1 were assigned to the CCC in-plane bending
mode [28].
Appendix A. Supplementary material
CCDC 857093 contains the supplementary crystallographic data
for this paper. These data can be obtained free of charge at
www.ccdc.cam.ac.uk/ retrieving.html [or from the Cambridge
Crystallographic Data Centre (CCDC), 12 Union Road, Cambridge
CB2 1EZ, UK; fax: +44(0)1223-336033; email: deposit@ccdc.cam.
ac.uk]. Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.
molstruc.2012.12.004.
3.3. Solubility estimation
References
The estimated solubility of TIZ rp in 21 solvents at 25 °C is
shown in Table 5. The obtained data showed that TIZ-rp is very
slightly soluble in acetone (0.38 mg/mL); slightly soluble in
dimethylformamide, DMSO and dioxane (3, 4.5 and 2 mg/mL
respectively), and practically insoluble in water and the rest of
the tested solvents, according to the USP solubility terms [30].
The insolubility of TIZ-rp in water is consistent with the crystal
and molecular structure of TIZ determined by SXRD since It is well
known that when a compound can assemble into a highly ordered
crystal it tends to have low solubility in aqueous media [31]. This
modification of TIZ has a ‘graphitic’ structure composed of tightly
packed layers of hydrogen bonded molecules that are stacked on
[1] M.J. Chan-Bacab, E. Hernández-Núñez, G. Navarrete-Vázquez, J. Antimicrob.
Chemother. 63 (2009) 1292.
[2] E.L. Romero, M.J. Morilla, Adv. Drug Deliv. Rev. 62 (2010) 576.
[3] S.S. El-Kamary, R. Jhaveri, M.D. Shardell, Clin. Infect Dis. 53 (2011) 150.
[4] R.P. Ward, M. Kugelmas, Am. Fam. Physician 72 (2005) 655.
[5] B.E. Korba, A.B. Montero, K. Farrar, K. Gaye, S. Mukerjee, M.S. Ayers, J.-F.
Rossignol, Antiviral Res. 77 (2008) 56.
[6] J.-F. Rossignol, J.E. Semple. Romark Laboratories, LC, United States Patent No.
8,124,632 B2, 2012.
[7] F. Mégraud, A. Occhialini, Antimicrob. Agents Chemother. 42 (1998) 2836.
[8] B.E. Korba, M. Elazar, P. Lui, J.-F. Rossignol, J.S. Glenn, Antimicrob. Agents
Chemother. 52 (2008) 4069.
[9] J.-F. Rossignol, F. Clearwater. Romark Laboratories, LC United States Patent
No. 6,020,353,2000 <http://www.ptodirect.com/Results/Patents?query=PN/
6020353>.
[10] Zhejiang NetSun Co., ChemNet.CAS: 173903-47-4 Tizoxanide. <http://
www.chemnet.com/cas/supplier.cgi?terms=tizoxanide&l=&exact=dict&f=plist&
mark=&submit.x=0&submit.y=0&submit=search>.
[11] M. Donadel Malesuik, H. Mazzotti Lopes Goncalves, C.V. Garcia, M. Rodrigues
Trein, N. Beyer Nardi, E.E. Scherman Schapoval, M. Steppe, Talanta 93 (2012)
206.
top of one another and held together by strong p-stacking interac-
tions, factors which evidently determine that TIZ-rp cannot be sol-
ubilized by aqueous media and polar protic solvents, at least at
25 °C.
[12] COLLECT, Nonius, Nonius BV, Delft, The Netherlands, 1998.
[13] Z. Otwinowski, W. Minor, in: M.G. Rossman, E. Arnold (Eds.), International
Tables for Crystallography, vol. F, Kluwer, Dordrecht, 2000.
[14] G.M. Sheldrick, Acta Crystallogr. A 64 (2008) 112.
4. Conclusions
[15] F.P. Bruno, M.R. Caira, G.A. Monti, D.E. Kassuha, N.R. Sperandeo, J. Mol. Struct.
984 (2010) 51.
[16] C.C. Sun, Int. J. Pharm. 319 (2006) 114.
[17] M.C. Etter, J.C. MacDonald, J. Bernstein, Acta Crystallogr. 46 (1990) 256.
[18] K. Yvon, W. Jeitschko, E. Parthe, J. Appl. Crystallogr. 10 (1977) 73.
By combining the results of the single crystal XRD analysis of TIZ
with those from 13C SS CPMAS, DRIFT and FT Raman spectra, DSC,
TG as well as estimated solubility data at 25 °C, it was possible to
characterize comprehensively for the first time the solid-state

F.P. Bruno et al. / Journal of Molecular Structure 1036 (2013) 318–325
325
[19] H. Zheng, H. Deng, Y. Chen, D. Li, Acta Crystallogr. E68 (2012) o1453.
[20] L.C. Thomas, TA-039: Interpreting Unexpected Events and Transitions in DSC
Results. TA Instruments, New Castle, DE. <http://www.tainst.com> (accessed
15.05.12).
[27] V. Arjunan, M. Kalaivani, P. Ravindran, S. Mohan, Spectrochim. Acta A 79
(2011) 1886.
[28] J.L. Castro, M.R. Lopez-Ramirez, J.F. Arenas, J. Soto, J.C. Otero, Langmuir 28
(2012) 8926.
[21] A.K. Galwey, J. Therm. Anal. 41 (1994) 267.
[22] M. Kondo, Bull. Chem. Soc. Jpn. 52 (1979) 521.
[29] P.E. Hansen, J. Spanget-Larsen, in: Zvi Rappoport (Ed.), NMR and IR
Spectroscopy of Phenols in the Chemistry of Phenols, John Wiley & Sons, NJ,
2004.
[30] The United States Pharmacopeia, 32th ed., United States Pharmacopeial
Convention, Inc., 2008.
[31] V. Hayden Thomas, S. Bhattachar, L. Hitchingham, P. Zocharski, M. Naath, N.
Surendran, C.L. Stoner, A. El-Kattan, Exp. Opin. Drug Metab. Toxicol. 2 (2006)
591.
[23] R.M. Silverstein, F.X. Webster, D.J. Kiemle, Spectrometric Identification of
Organic Compounds, seventh ed., John Wiley & Sons, Inc., NJ, 2005.
[24] C. Gobin, P. Marteau, J. Petitet, Spectrochim. Acta A 60 (2004) 329.
[25] Y. Akkaya, S. Akyuz, Vib. Spectrosc. 42 (2006) 292.
[26] H.T. Varghese, C. Yohannan Panicker, D. Philip, J.R. Mannekutla, S.R. Inamdar,
Spectrochim. Acta A 66 (2007) 959.