Complexation of sulfonamides with β-cyclodextrin studied by experimental and theoretical methods

Article abstract of DOI:10.1002/jps.22062

The complex formation between three structurally related sulfonamides (sulfadiazine (SDZ), sulfamerazine (SMR), and sulfamethazine (SMT)) and β-cyclodextrin (β-CD) was studied, by exploring its structure affinity relationship. In all the cases, 1:1 stoich

Full text of DOI:10.1002/jps.22062

PHARMACEUTICAL TECHNOLOGY
Complexation of Sulfonamides With b-Cyclodextrin Studied by
Experimental and Theoretical Methods
ARIANA ZOPPI, MARIO A. QUEVEDO, ALICIA DELRIVO, MARCELA R. LONGHI
´
´
´
Departamento de Farmacia, Facultad de Ciencias Quımicas, Universidad Nacional de Cordoba, Cordoba, Ciudad Universitaria,
´
5000 Cordoba, Argentina
Received 20 August 2009; revised 10 November 2009; accepted 17 November 2009
Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.22062
ABSTRACT: The complex formation between three structurally related sulfonamides (sulfa-
diazine (SDZ), sulfamerazine (SMR), and sulfamethazine (SMT)) and b-cyclodextrin (b-CD) was
studied, by exploring its structure affinity relationship. In all the cases, 1:1 stoichiometries were
determined with different relative affinities found by phase solubility (SDZ:b-CD > SMR:b-
CD > SMT:b-CD) and nuclear magnetic resonance (NMR) (SMT:b-CD > SMR:b-CD > SDZ:b-
CD) studies. The spatial configurations determined by NMR were in agreement with those
obtained by molecular modeling, showing that SDZ included its aniline ring into b-CD, while
SMR and SMT included the substituted pyrimidine ring. Energetic analyses demonstrated that
hydrophobicity is the main driving force to complex formation. ß 2010 Wiley-Liss, Inc. and the
American Pharmacists Association J Pharm Sci 99:3166–3176, 2010
Keywords: sulfonamides; solubility; cyclodextrins; complexation; phase solubility studies;
NMR spectroscopy; molecular modeling
INTRODUCTION
Fig. 1) bond through a sulfonamide moiety to a
substituted pyrimidine ring (ring B, Fig. 1).
Sulfonamides comprise a large family of compounds
described first in 1935 to exhibit antibacterial activity
and being clinically used since 1968.1 From then
on, several types of additional pharmacological
applications have been reported, including antitu-
mor, anticarbonic anhydrase, diuretic, hypoglycemic,
antithyroid, or protease inhibitory activity,2 sulfona-
mides constituting a family of drugs widely used in
human and animal medicine. Although the thera-
peutic potential of such compounds is fully described,
suboptimal physicochemical properties, such as low
water solubility, represent an important concern as to
their therapeutic use which still needs to be further
addressed. Sulfadiazine, sulfamerazine, and sulfa-
methazine (SDZ, SMR, and SMT, respectively, Fig. 1)
exemplify the widespread use of sulfonamide drugs,
frequently administered as a ‘‘triple sulfa drug’’
formulation.3–5 These three molecules are structu-
rally closely related, with an aniline ring (ring A,
A strategy frequently adopted to enhance drug
solubility constitutes the complexation with cyclodex-
trins (CDs).6,7 b-cyclodextrin (b-CD, Fig. 2), formed
by seven units of glucopyranoses with a-1,4 bonds,
being the most widely used CD in the pharmaceutical
field. It exhibits a truncated-cone shape with a
hydrophobic inner and a hydrophilic outer surface,
capable of including molecules to form inclusion
complexes, and enhancing properties such as solubi-
lity, bioavailability and stability. Therefore, previous
reports demonstrated that the formation of complex
between several sulfonamides and CDs exhibited
improved aqueous solubility compared to that of the
free drugs,8–11 and thus may have implication on its
therapeutic applicability.
In this work, the complex formation between SDZ,
SMR, and SMT with b-CD was studied in detail with
the objective of understanding the basis of their
structure–affinity relationship. Hence, the affinity
and three-dimensional structures of the correspond-
ing complexes were determined by experimental
techniques such as phase solubility studies and
nuclear magnetic resonance. In addition, molecular
modeling techniques, including molecular docking
and molecular dynamics, were applied and correlated
with experimental results.
Correspondence to: Marcela R. Longhi (Telephone: 54-351-433-
4163 ext 107; fax: 54-351-433-4163 ext 115;
E-mail: mrlcor@fcq.unc.edu.ar)
Journal of Pharmaceutical Sciences, Vol. 99, 3166–3176 (2010)
ß 2010 Wiley-Liss, Inc. and the American Pharmacists Association
3166
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 7, JULY 2010

COMPLEXATION OF SULFONAMIDES WITH B-CYCLODEXTRIN
3167
1 h and placed in a 25.0 Æ 0.18C thermostatized water
bath for 72 h. After equilibrium was reached, the
suspensions were filtered through a 0.45 mm mem-
brane filter (Millipore) and analyzed by UV–Vis
spectrophotometry. Each experiment was repeated
at least three times and the results reported were the
mean values. The Kc values for the corresponding
guest:b-CD complex, were calculated from the slope of
the phase-solubility diagrams and S0 according to
Eq. (1):12
slope
Kc ¼
(1)
S0ð1 À slopeÞ
The quantitative determinations of each sulfona-
mide were performed spectrophotometrically (Shi-
madzu UV-Mini 1240 spectrophotometer) at 264, 263,
and 262 nm for SDZ, SMR, and SMT, respectively.
The stability of the three studied sulfonamides was
determined in water at 258C, no drug degradation
was found after 72 h of incubation.
Figure 1. Chemical structure of sulfadiazine (SDZ), sul-
famerazine (SMR), and sulfamethazine (SMT).
MATERIALS AND METHODS
Materials
NMR Studies
Pure sulfadiazine and sulfamerazine and sulfametha-
zine sodium salt were obtained from Parafarm
(Argentina), while sulfamethazine base was obtained
from neutralization with HCl and subsequent recrys-
talization. D2O 99.9 at.% D, used in spectroscopic
studies was purchased from Sigma1, while b-CD
(MW ¼ 1135) was kindly supplied by Roquette (Les-
trem, France). All other materials and solvents were
of analytical reagent grade. A Milli-Q Water Purifica-
tion System (Millipore1) (Bedford, Massachusetts)
generated the water used in solubility studies.
All experiments were performed on a Bruker Advance
II High Resolution Spectrometer, equipped with a
Broad Band Inverse probe (BBI) and a Variable
Temperature Unit (VTU). The spectra were measured
at 298 K and obtained at 400.16 MHz, with the
chemical shift of the residual solvent at 4.8 ppm used
as an internal reference. Dd in the 1H chemical shifts
for b-CD and each sulfonamide originated due to their
complexation were calculated using the following
equation:
Dd ¼ dcomplex À dfree
Solubility Studies
The effects of b-CD on the solubility of each
sulfonamide were studied as follows: an excess of
each sulfonamide was added to water containing
different amounts of b-CD (0–13.2 mM). The resulting
suspensions were sonicated in an ultrasonic bath for
The Kc of the inclusion complex in solution was
determined by applying Scott’s equation (Eq. 2):13
½b À CDŠ
Ddobs
½b À CDŠ
Ddc
1
t
t
¼
þ
(2)
KcDdc
Figure 2. b-cyclodextryn molecule: (a) chemical structure, (b) three-dimensional structure
showing the hydrophobic cavity and (c) hydrogen notation used for NMR studies.
DOI 10.1002/jps
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 7, JULY 2010

3168
ZOPPI ET AL.
where [b-CD]t is the total b-CD concentration; Ddobs is
the difference between the chemical shift of free drug
and the corresponding chemical shift of drug in
the presence of b-CD at each concentration; Ddc is the
difference between the chemical shift of a pure
sample of the complex and its free components. By
plotting [b-CD]t/Ddobs against [b-CD]t, Kc could be
calculated from the intercept obtained by a least-
squares linear analysis. In this experiment, the
concentration of each drug was kept constant at
1 mM, while b-CD concentration was varied from 5 to
10 mM for the D2O.
2D ROESY: Drug inclusion into the b-CD cavity
was studied by two-dimensional rotating frame
Overhauser experiments (2D ROESY), with spinlock
for mixing phase sensitive, using 180x 180 À x pulses
for polarization transfer. The spectra were measured
with a relaxation delay of 2 s, p15 pulse for ROESY
spinlock of 20 ms and l4, spinlock loop, (p15/
p25 Â 2) ¼ 400. Before Fourier transformation, the
matrix was zero filled to 4096 (F2) by 2048 (F1) and
Gaussian apodization functions were applied in both
dimensions. Working concentrations of each drug and
b-CD were 1 mM in D2O.
SDZ, SMR, SMT, and b-CD were identical to those
used for the molecular docking procedures described
above. The complexes obtained by molecular docking
were solvated with a preequilibrated TIP3P octahed-
ric water box, after which an initial minimization of
the solvent followed by a minimization of the whole
system was carried out. These minimized systems
were heated to 300 K for 20 ps, followed by a 100 ps
equilibration run. Production runs were performed
for 20 ns, using a timestep of 2 fs and under constant
pressure and temperature, with the SHAKE algo-
rithm being applied to constrain bonds involving
hydrogens.
Hydrogen bonds and energetic components were
analyzed using the Ptraj and Molecular Mechanics
Poisson-Bolzmann Surface Area (MM-PBSA) mod-
ules of Amber9, respectively.19 MM-PBSA analyses
were applied over the entire trajectory (20 ns), with
individual snapshots sampled every 10 frames. The
corresponding structures were visualized using VMD
v.1.8.6 software.20 Molecular dynamics simulations
were computed at the Fimm Cluster resource
(Parallab, Bergen Center for Computational Science,
University of Bergen, Norway).
Molecular Modeling Procedures
RESULTS AND DISCUSSION
Phase-Solubility Analysis
Initial structures of SDZ, SMR, and SMT were built
using Gabedit software,14 after which a conforma-
tional search was carried out in order to obtain the
minimum energy conformation. The systematic
search was conducted applying a semiempirical
method (AM1), with the final minimum energy
conformation being optimized using an ab initio
(HF-6-31GÃ) method. Conformational search and
optimization procedures were performed using Gaus-
sian98 software,15 with the results being processed
with Molden v.4.7 software.16 The initial structure of
b-CD was obtained from the Cambridge Structural
Database (code BCDEXD10).
The corresponding phase-solubility diagrams (PSD)
of each sulfonamide with b-CD in water are depicted
in Figure 3, with the corresponding affinity constant
(Kc), intrinsic solubility (S0) and maximum solubility
(Smax) values being calculated from each PSD
(Tab. 1). As it can be seen, the three sulfonamides
formed complexes with b-CD, demonstrated by the
marked improvement in their solubility when the
For the molecular docking procedure, restrained
electrostatic potential (RESP) fitted charges were
assigned to SDZ, SMR, and SMT, while Glycamm04
force field charges implemented in the Amber9
software package were assigned for b-CD.17 Autodock
v3.05 was used to accomplish the docking runs,18 by
applying a Lamarckian Genetic Algorithm (LGA) to
generate the population evaluated, using the follow-
ing parameters: a population of 150 individuals, a
maximum of 250,000,000 evaluations and 5000
generations as an end criterion. An elitism value of
1 was selected, with a probability of mutation and
crossing over of 0.02 and 0.8, respectively. Thirty
docking runs were assayed, with the lowest energy
cluster conformation being selected for subsequent
molecular dynamics assays.
Figure 3. Phase-solubility diagrams of the SDZ:b-CD
complex (&), SMR:b-CD complex (*), and SMT:b-CD
complex (~).
The Amber9 software package was used for the
molecular dynamics studies.17 Atomic charges for
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 7, JULY 2010
DOI 10.1002/jps

COMPLEXATION OF SULFONAMIDES WITH B-CYCLODEXTRIN
3169
Table 1. Data of the Phase-Solubility Studies of SDZ,
SMR and SMT With b-CD
exhibited displacements in their d in the presence of
the corresponding guest molecule, confirming the
formation of an inclusion complex. For the SDZ:b-CD
complex, downfield displacements were found for
both H3 and H5, suggesting the insertion of an
electronegative moiety into the b-CD hydrophobic
cavity, which produces the deshielding effects
observed. For the SMR:b-CD complex, a downfield
and an upfield displacement was found for H3 and
for H5, respectively, which may result from the partial
inclusion of an electronegative functional group, with
a deshielding effect only on H3. When the SMT:b-CD
complex was analyzed, both protons showed marked
upfield displacements, indicative of the inclusion of
an electron rich moiety rather than of an electro-
negative group. In addition, Dd corresponding to the
protons located in the outer surface of b-CD (H1, H2,
and H4) were also modified, which may be due to a
conformational rearrangement in the host mole-
cule.21–23
Kc (MÀ1
)
S0 (mg/mL)
Smax (mg/mL)
a
SDZ
SMR
SMT
282 Æ 15
198 Æ 22
101 Æ 4
0.07 Æ 0.01
0.26 Æ 0.01
0.42 Æ 0.02
0.33 Æ 0.02
0.83 Æ 0.02
0.93 Æ 0.03
aDrug solubility in b-CD (13.2 mM) solutions.
macromolecule was added to the solution. In all the
cases, AL type solubility diagrams were obtained, in
which a linear increase in solubility as a function of b-
CD concentration and a slope value less than unity
indicates a 1:1 stoichiometry.7 Comparing the solu-
bility parameters before (S0) and after (Smax) b-CD
addition, it can be seen that solubility increased to 4,
3.2, and 2.2 times for SDZ, SMR, and SMT,
respectively, indicating that the complexation with
b-CD can be an effective strategy to enhance the
aqueous solubility of these drugs.
When the Dd for the three sulfonamide molecules
were analyzed (Tab. 3, spectra not shown), it can be
seen that almost all proton resonances were modified
upon complexation, through which both upfield and
downfield displacements were found. For SDZ, down-
field displacements were observed for Hb and Hc,
probably due to van der Waals interactions between
SDZ and b-CD, suggesting the insertion of the central
portion of this molecule into the b-CD cavity. For
SMR, the most marked displacements corresponded
to protons located in ring B (Hc, Hd and HCH3 ), with
both downfield and upfield displacements suggesting
the establishment of van der Waals interactions and
the proximity of atoms rich in p electrons such as
glycosidic linkage oxygens, respectively, all of which
support the inclusion of ring B into the hydrophobic
cavity. From the Dd corresponding to the SMT:b-CD
complex, we founded that protons located in ring B as
well as those located in ring A exhibited significant
upfield displacements, it does not allow the identifi-
cation of the portion of the molecule inserted into the
b-CD cavity.
NMR Studies
Figure 4 shows the b-CD spectra obtained before and
after complex formation with the three sulfonamide
molecules, from which the corresponding chemical
shifts (d) and chemical shift displacements (Dd) were
determined (Tab. 2). In all the cases, protons lying in
the interior of the hydrophobic cavity (H3 and H5)
In order to calculate Kc values for each sulfonami-
de:b-CD complex, Scott’s plots (Fig. 5) were deter-
mined on the basis of the Dd of Hd. The corresponding
calculated values were 231, 368, and 826 MÀ1 for SDZ,
SMR, and SMT, respectively. When Kc obtained by
NMR studies were compared to those determined by
PSD, significant differences were found, with PSD
diagrams showing an increase in affinity in the order
SDZ > SMR > SMT, while NMR studies showed an
inverse fashion (SMT > SMR > SDZ). The discrepan-
cies between the Kc values determined by different
experimental methods have been reviewed exten-
sively by Loftsson et al.24 with experimental data
being inconclusive so as to unequivocally determine
the origin of this phenomena. Probably, the fact that
Figure 4. Spectra (a) SDZ:b-CD complex, (b) SMR:b-CD
complex, (c) SMT:b-CD complex, (d) b-CD free.
DOI 10.1002/jps
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 7, JULY 2010

3170
ZOPPI ET AL.
Table 2. Chemical Shifts for the Protons of b-Cyclodextrin in the Free State and in the Complex
Proton
b-CD Free (ppm)
SDZ:b-CDa (ppm)
Dd (ppm)
SMR:b-CDb (ppm)
Dd (ppm)
SMT:b-CDc (ppm)
Dd (ppm)
H1
H2
H3
H4
H5
H6
5.0765
3.6554
3.9729
3.5908
3.8579
3.8850
5.0965
3.6767
3.9839
3.6102
3.8633
3.9002
0.0200
0.0203
0.0110
0.0194
0.0054
0.0152
5.0956
3.6773
3.9747
3.6097
3.8482
3.9002
0.0191
0.0219
0.0018
0.0189
À0.0097
0.0152
5.0404
3.6200
3.9047
3.5509
3.7804
3.8486
À0.0361
À0.0354
À0.0682
À0.0399
À0.0775
À0.0364
a[SDZ]:[b-CD] ¼ 1:1.
b[SMR]:[b-CD] ¼ 1:1.
c[SMT]:[b-CD] ¼ 1:1.
PSD studies require the use of saturated drug
solution in contrast to NMR assays, where the drug
under study is completely dissolved, may explain the
differences observed.
position between the hydrogen bond to the nitrogen
atom and both oxygen atoms in the sulfonamide
moiety, which established intramolecular hydrogen
bonds responsible for the higher stability of these two
conformers. Similar results were recently reported for
SDZ when conformational properties were studied by
means of experimental and high-level theoretical
methods.25
2D ROESY experiments were carried out to confirm
the inclusion of each sulfonamide into the b-CD
cavity. Figure 6 shows partial contour plots of 2D
ROESY spectra for the three studied systems. For
SDZ:b-CD (Fig. 6a), it can be observed that Ha protons
of SDZ correlate with the inner protons of b-CD (H5),
indicating the formation of an inclusion complex in
which the ring A of SDZ is deeply inserted into the
hydrophobic cavity. For SMR:b-CD and SMT:b-CD
complexes (Fig. 6b and c, respectively), intermolecu-
lar cross-peaks between Hb protons of both guest
molecules and H3 protons of b-CD were found,
evidencing again the formation of an inclusion
complex but suggesting that the ring A of SMR and
SMT may not be so deeply inserted into the
hydrophobic cavity as for SDZ.
Molecular Docking
Considering that the minimum energy conformers
obtained are the most likely conformations in solu-
tion, docking procedures were applied by restraining
the rotation of the sulfonamide bond of the two
minimum energy conformers described previously,
allowing the rotation of all other rotatable bonds.
Figure 8 shows the three-dimensional structures of
the corresponding complexes obtained from docking
assays, in which ring B of the three sulfonamide
molecules is deeply inserted into the b-CD hydro-
phobic cavity. Similar docking poses and energy were
also observed for the two conformers analyzed.
Computational Studies
Further inspection of the docked conformations
shows that in all cases ring A is placed in a coplanar
position with respect to the b-CD cavity and
perpendicular to its axis, allowing the establishment
of hydrogen bonds between the hydroxyls of the
macromolecule wide rim and the primary amine and
sulfonamide moiety of the guest molecule. Hence, the
electrostatic component of the affinity seems to be
originated mainly in the interaction between the
guest molecule and the hydroxyls situated in the wide
Conformational Analysis
Three-dimensional structures of SDZ, SMR, and SMT
were built, after which energy minimizations were
performed. In order to assess the relative position of
ring A with respect to ring B, the dihedral angle
defined by the sulfonamide bond was rotated and the
resulting energy analyzed (Fig. 7). As shown, two
minimum energy conformers were found for each
molecule, both of them corresponding to the eclipsed
Table 3. Chemical Shifts for the Protons of SDZ, SMR, and SMT in the Free State and in the Complex
Proton SDZ (ppm) SDZ:b-CDa (ppm) Dd (ppm) SMR (ppm) SMR:b-CDb (ppm) Dd (ppm) SMT (ppm) SMT:b-CDc (ppm) Dd (ppm)
Ha
Hb
Hc
Hd
HCH
6.8317
7.6916
8.3406
6.8820
—
6.8302
7.7061
8.3515
6.8792
—
À0.0015
0.0145
0.0109
À0.0028
—
6.8595
7.7294
8.1681
6.8040
2.3853
6.8289
7.7383
8.2053
6.8071
2.4008
À0.0306
0.0089
0.0372
0.0031
0.0155
6.8380
7.7374
—
6.7181
2.3474
6.7602
7.7211
—
6.6902
2.3326
À0.0778
À0.0163
—
À0.0279
À0.0148
3
a[SDZ]:[b-CD] ¼ 1:1.
b[SMR]:[b-CD] ¼ 1:1.
c[SMT]:[b-CD] ¼ 1:1.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 7, JULY 2010
DOI 10.1002/jps

COMPLEXATION OF SULFONAMIDES WITH B-CYCLODEXTRIN
3171
Figure 5. Scott’s plots for proton Hd of SDZ (&,
r2 ¼ 0.969), SMR (*, r2 ¼ 0.979), SMT (~, r2 ¼ 0.969) with
increasing concentrations of b-CD.
rim of b-CD, whereas the hydrophobic contribution
results from the inclusion of ring B. This would
preliminarily suggest that SMT should exhibit the
highest affinity within the series of the compounds
studied due to the higher hydrophobicity of the
dimethylpyrymidine moiety compared to that of
methylpyrimidine and pyrimidine in SMR and
SDZ, respectively. When the docked energies
obtained were compared, it can be seen that the
complex SMR:b-CD is more stable than SDZ:b-CD
(À9.14 and À8.70 kcal/mol, respectively), in agree-
ment with SMR enhanced lipophilicity. However, for
SMT:b-CD (À8.86 kcal/mol), no additional contribu-
tion to the stability of the complex was noted after a
second methyl moiety was introduced into ring B.
Considering that b-CD is maintained rigid during the
docking procedure, the lower docked energy founded
for SMT:b-CD may be originate in a steric hindrance
when a second methyl group is introduced.
Figure 6. Partial contour plot of 2D ROESY spectrum of
(a) SDZ:b-CD complex, (b) SMR:b-CD complex, (c) SMT:b-
CD complex.
the bulk solvent, where hydrogen bonds are formed
with water molecules and maintained during the
entire simulation (mean frequency 46.4%). Simulta-
neously, part of ring A is buried into the cavity
through the wide rim, while ring B is partially
excluded from the b-CD in the opposite site of the
macromolecule (Fig. 9,1.a). In a second cluster of
conformations, ring A is deeply buried into the cavity,
with ring B almost completely excluded from the
hydrophobic cavity and the sulfonamide moiety
establishing hydrogen bonds with hydroxyls of the
narrow rim (Fig. 9,1.b). Thus, a dynamic equilibrium
between these two conformations is achieved, by
which the pattern of hydrogen bonds formed can be
observed (Tab. 4). For SDZ, and as evidenced by the
hydrogen bonding frequency, the conformation cor-
Molecular Dynamics
The complexes obtained by molecular docking were
solvated, heated, and equilibrated, after which
extensive simulation runs (20 ns) were performed.
In order to study the structure of the complex over
time, the establishment of intermolecular hydrogen
bonds was analyzed. Table 4 shows the most relevant
hydrogen bonds formed between SDZ and b-CD. It
should be noted that when the initial structure
obtained by molecular docking is solvated, heated,
and equilibrated, the guest molecule moves from
the initial docked position, by which the sulfonamide
moiety is inserted into the b-CD cavity early in the
simulation (Fig. 9,1.a). During this movement,
the amine function in ring A is oriented towards
DOI 10.1002/jps
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 7, JULY 2010

3172
ZOPPI ET AL.
Figure 8. Structure of the complexes between SDZ, SMR,
and SMT with b-CD as obtained by molecular docking
procedures.
dealing with the binding of SDZ to hydroxypropil-b-
CD also suggested the insertion of the ring A into the
hydrophobic cavity, in agreement with the complex
conformation properties described in this work.8
Table 5 summarizes the most relevant hydrogen
bonds detected for the SMR:b-CD complex. As found
for SDZ, during the heating and equilibration phase
the sulfonamide moiety in SMR is buried into b-CD
cavity (Fig. 9,2.a), with the primary amine moiety
establishing sustained hydrogen bonds with water
molecules throughout the trajectory (mean frequency
55.3%). A second cluster of conformations is formed
when ring B is reinserted into the b-CD cavity, with
the sulfonamide moiety and ring A being extruded
and exposed to the bulk solvent (Fig. 9,2.b). As
evidenced by the hydrogen bond analysis, an equili-
brium between these two spatial arrangements is
established, with the second cluster (Fig. 9,2.b) being
highly predominant. In this case, the insertion of ring
A into the hydrophobic cavity was not observed, since
this would encompass the extrusion of ring B on the
narrow rim of the macromolecule which, considering
its high hydrophobicity, would represent an unfavor-
Figure 7. Energetic profiles obtained for the conforma-
tional analyses of SDZ, SMR, and SMT.
responding to the first cluster (Fig. 9,1.a) is clearly
predominant. This model is consistent with the
downfield displacements of protons H3 and H5 found
in the corresponding 1H NMR spectra (Tab. 2, Fig. 3),
probably caused by the deshielding produced by the
electronegative sulfonamide moiety. Previous reports
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 7, JULY 2010
DOI 10.1002/jps

COMPLEXATION OF SULFONAMIDES WITH B-CYCLODEXTRIN
3173
Table 4. Hydrogen Bonds Between SDZ and b-CD
ns
Acceptor ! Donor
Frequency (%)
Distribution
2–3
O1 (SDZ) ! OH (CYC)
O2 (SDZ) ! OH (CYC)
O (CYC) ! NH (SDZ)
O (CYC) ! NH (SDZ)
O (CYC) ! NH(SDZ)
O (CYC) ! NH2 (SDZ)
O (CYC) ! NH(SDZ)
O (CYC) ! NH2 (SDZ)
O1 (SDZ) ! OH (CYC)
15.7
10.4
5.8
3–4
4–5
8–9
7.7
47.6
24.2
30.3
17.9
5.1
9–10
17–18
Ã
Frequency notation (%): . ¼ 5–20; - ¼ 20–40; o ¼ 40–60; Â ¼ 60–80; ¼ 80–95; @ ¼ 95–100.
able event in terms of energetic contribution. The
predominant cluster of conformations in which the
sulfonamide moiety is not deeply included is con-
sistent with the upfield and downfield displacement
observed for H5 and H3, respectively, since the
deshielding effect is produced on H3 while H5
experiments a shielding by the high electronic density
of the ring B.
Table 6 shows the hydrogen bond interactions as
founded in the trajectory for the SMT:b-CD complex.
In this case, after the initial heating and equilibra-
tion, hydrogen bonds between the sulfonamide and
the hydroxyls of the wide rim of b-CD are maintained
with high frequency, indicating that this moiety is not
deeply buried into the b-CD cavity. In this conforma-
tion, ring B is inserted into the b-CD hydrophobic
cavity, maximizing van der Waals interactions.
Additionally, a low frequency was observed for the
conformations in which the sulfonamide moiety is
included into the cavity and ring B is excluded
through the narrow rim, which may be due to the high
hydrophobicity of ring B. This model is in agreement
with the upfield displacements founded for H3 and H5,
which may be originate in the shielding produced by
ring B.
Energetic Analyses
Table 7 lists the energetic components as determined
for the three complexes studied using the MM-PBSA
methodology. As it can be seen, the major contribution
to the complex stability arises from the van der Waals
component, comparable to SDZ and SMR (À30.10 and
À30.02 kcal/mol, respectively) and slightly higher for
SMT (À31.82 kcal/mol). Significant differences were
observed in the electrostatic components, with its
contribution to affinity increasing in the order
SMT > SMR > SDZ (À10.84, À8.18, and À7.13 kcal/
mol, respectively).
The polar contribution to solvation (PBcal) is of
utmost importance in the formation of b-CD com-
plexes, and may be decisive in the overall affinity
observed.6,26 As it can be seen in Table 7, PBcal was
positive for the three complexes studied and
increased in the order SMT > SMR > SDZ, indicating
that the energy required to solvate the SMT:b-CD
complex is higher than that for SMR:b-CD and
SDZ:b-CD (29.16, 25.64, and 25.57 kcal/mol, respec-
tively). In contrast, the total interaction energy
(PBtot) was negative for the three cases, which
indicates that the formation of the corresponding
complexes is thermodynamically favorable. Thus, the
theoretically calculated affinity increased in the order
SMT:b-CD > SMR:b-CD > SDZ:b-CD, with energy
required for solvation being compensated by inter-
molecular van der Waals and electrostatic interac-
Figure 9. Hydrogen bonds and three-dimensional struc-
tures obtained for each sulfonamide:b-CD complexes.
DOI 10.1002/jps
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 7, JULY 2010

3174
ZOPPI ET AL.
Table 5. Hydrogen Bonds Between SMR and b-CD
ns
Acceptor ! Donor
Frequency (%)
Distribution
1–2
2–3
O1 (SMR) ! OH (CYC)
O2 (SMR) ! OH (CYC)
O1 (SMR) ! OH (CYC)
O1 (SMR) ! OH (CYC)
O2 (SMR) ! OH (CYC)
O1 (SMR) ! OH (CYC)
O2 (SMR) ! OH (CYC)
O1 (SMR) ! OH (CYC)
O2 (SMR) ! OH (CYC)
O1 (SMR) ! OH (CYC)
O2 (SMR) ! OH (CYC)
O2 (SMR) ! OH (CYC)
O1 (SMR) ! OH (CYC)
O2 (SMR) ! OH (CYC)
O1 (SMR) ! OH (CYC)
O2 (SMR) ! OH (CYC)
13.3
11.3
6.8
33.9
6.9
28.9
9.7
75
3–4
7–8
8–9
9–10
12
10–11
13–14
14–15
61.4
9.8
6.7
5.5
7.2
7.8
57
15–16
17–18
19–20
Ã
Frequency notation (%): . ¼ 5–20; - ¼ 20–40; o ¼ 40–60; Â ¼ 60–80; ¼ 80–95; @ ¼ 95–100.
tions. When the conclusions drawn from the energetic
analysis were compared with experimental results,
we observed that affinity predicted by PBtot was
consistent with Kc values obtained by NMR studies,
whereas affinities expected from the energetic payoff
to solvate the complex (PBcal) were in agreement with
Kc values found by PSD. As stated, discrepancies
between the affinities determined by different meth-
ods have been previously reported, and on the basis of
our experimental and theoretical results, it seems
feasible that differences in affinities calculated by
PSD and NMR were produced by a phenomenon
related to the solvation energies in the overall
complex formation process.
Table 6. Hydrogen Bonds between SMT and b-CD
ns
Acceptor ! Donor
Frequency (%)
Distribution
0–1
O1 (SMT) ! OH (CYC)
O2 (SMT) ! OH (CYC)
O1 (SMT) ! OH (CYC)
O2 (SMT) ! OH (CYC)
O1 (SMT) ! OH (CYC)
O2 (SMT) ! OH (CYC)
O1 (SMT) ! OH (CYC)
O2 (SMT) ! OH (CYC)
O1 (SMT) ! OH (CYC)
O1 (SMT) ! OH (CYC)
O1 (SMT) ! OH (CYC)
O2 (SMT) ! OH (CYC)
O1 (SMT) ! OH (CYC)
O2 (SMT) ! OH (CYC)
O1 (SMT) ! OH (CYC)
O2 (SMT) ! OH (CYC)
O1 (SMT) ! OH (CYC)
O2 (SMT) ! OH (CYC)
O1 (SMT) ! OH (CYC)
73.7
31.8
54.8
29.1
42.9
17.6
21.7
21.1
11
1–2
2–3
6–7
8–9
11–12
12–13
8.5
68.5
40.5
63.7
25.4
62.4
23.3
61.3
31.6
13.7
13–14
14–15
15–16
16–17
Ã
Frequency notation (%): . ¼ 5–20; - ¼ 20–40; o ¼ 40–60; Â ¼ 60–80; ¼ 80–95; @ ¼ 95–100.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 7, JULY 2010
DOI 10.1002/jps

COMPLEXATION OF SULFONAMIDES WITH B-CYCLODEXTRIN
3175
Table 7. Energetic Component Analysis Using the MM-
PBSA Methodology
sulfadiazine, sulfamerazine and sulfamethazine. Polyhedron
26:967–974.
4. Sanchez Pena M, Acedo MJ, Salinas F, Mahedero MC, Aaron
JJ. 1995. Analysis of sulfamethazine in the presence of sulfa-
merazine or sulfadiazine by first-derivative photochemically
induced fluorescence. J Pharm Biomed Anal 13:1107–1112.
5. Garcia-Alvarez-Coque MC, Simo-Alfonso EF, Ramis-Ramos G,
Esteve-Romero JS. 1995. High-performance micellar liquid
chromatography determination of sulphonamides in pharma-
ceuticals after azodye precolumn derivatization. J Pharm
Biomed Anal 13:237–245.
SDZ
SMR
SMT
EEle
EVdW
Egas
PBsur
PBcal
PBtot
À7.13
À30.10
À37.23
À3.63
À8.18
À30.02
À38.03
À3.73
À10.85
À31.82
À42.67
À4.00
25.57
25.64
29.16
À15.29
À16.11
À17.51
6. Fromming KH, Szejtli J. 1994. Cyclodextrins in
pharmacy. Dordrecht, The Netherlands: Kluwer Academic
Publishers.
7. Brewster ME, Loftsson T. 2007. Cyclodextrins as pharmaceu-
tical solubilizers. Adv Drug Deliv Rev 59:645–666.
CONCLUSIONS
The aqueous solubility of the three sulfonamides
assayed was improved by the formation of 1:1
inclusion complexes with b-CD. NMR studies and
molecular modeling techniques were in agreement
with the Kc values of the three compounds studied for
b-CD, indicating the following trend: SMT > SMR >
SDZ. This tendency could be attributed to the
hydrophobicity enhancement of ring B due to the
addition of the methyl groups, giving rise a molecule
with enhanced affinity. Hence, for the SDZ:b-CD
complex, the inclusion of ring A represented the
predominant conformation, while for both SMR:b-CD
and SMT:b-CD complexes, inclusion of ring B was
energetically favored. From this work, we can
conclude that the formation of the inclusion complex
between b-CD and SDZ, SMR, and SMT suggest an
effective strategy to enhance the solubility of the
three drugs.
´
´
8. De Araujo MVG, Vieira EKB, Silva Lazaro G, Conegero LS,
Almeida LE, Barreto LS, da Costa NB Jr, Gimenez IF. 2008.
Sulfadiazine/hydroxypropyl-b-cyclodextrin host-guest system:
Characterization, phase-solubility and molecular modeling.
Bioorg Med Chem 16:5788–5794.
9. Granero GE, Maitre MM, Garnero C, Longhi MR. 2008. Synth-
esis, characterization and in vitro release studies of a new
acetazolamide-HP-b-CD-TEA inclusion complex. Eur J Med
Chem 43:464–470.
10. Granero GE, Garnero C, Longhi MR. 2003. The effect of a basic
substance and pH on cyclodextrin complexation of sulfisoxa-
zole. Eur J Pharm Sci 20:285–293.
11. Pose-Vilarnovo B, Perdomo-Lopez I, Echezarreta-Lopez M,
Schroth-Pardo P, Estrada E, Torres-Labandeira JJ. 2001.
Improvement of water solubility of sulfamethizole through
its complexation with b-and hydroxypropyl-b-cyclodextrin—
Characterization of the interaction in solution and in solid
state. Eur J Pharm Sci 13:325–331.
12. Higuchi T, Connors K. 1965. Phase solubility techniques.
In: Reilly C, editor. Advances in analytical chemistry and
instrumentation. New York: Wiley Interscience. pp. 117–
212.
´
´
13. Ganza-Gonzalez A, Vila-Jato JL, Anguiano-Igea S, Otero-
Espinar FJ, Blanco-Mendez JJ. 1994. A proton nuclear mag-
netic resonance study of the inclusion complex of naproxen with
b-cyclodextrin. Int J Pharm 106:179–185.
ACKNOWLEDGMENTS
14. Allouche AR. Gabedit is a free Graphical User Interface for
computational chemistry packages. It is available from http://
gabedit.sourceforge.net/.
´
The authors thank Fondo para la Investigacion Cien-
´
´
´
tıfica y Tecnologica (FONCyT), prestamo BID 1728/
´
´
OC-AR PICT 1376, Secretarıa de Ciencia y Tecnica de
15. Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA,
Cheeseman JR, Zakrzewski VG, Montgomery JA, Jr., Strat-
mann RE, Burant JC, Dapprich S, Millam JM, Daniels AD,
Kudin KN, Strain MC, Farkas O, Tomasi J, Barone V, Cossi M,
Cammi R, Mennucci B, Pomelli C, Adamo C, Clifford S,
Ochterski J, Petersson GA, Ayala PY, Cui Q, Morokuma K,
Malick DK, Rabuck AD, Raghavachari K, Foresman JB,
Cioslowski J, Ortiz JV, Baboul AG, Stefanov BB, Liu G,
Liashenko AP, Piskorz I, Komaromi R, Gomperts RL, Fox
Martin DJ, Keith T, Al-Laham MA, Peng CY, Nanayakkara
A, Gonzalez C, Challacombe M, Gill PMW, Johnson B, Chen W,
Wong MW, Andres JL, Gonzalez C, Head-Gordon M, Replogle
ES, Pople JA. 1998. Software: GAUSSIAN 98. Pittsburgh, PA,
USA: Gaussian, Inc. www.gaussian.com.
16. Schaftenaar G, Noordik JH. 2000. Molden: A pre- and post-
processing program for molecular and electronic structures.
J Comput Aided Mol Design 14:123–134.
17. Case DA, Cheatham TE, Darden T, Gohlke H, Luo R, Merz KM,
Onufriev A, Jr., Simmerling C, Wang B, Woods RJ. 2005. The
Amber biomolecular simulation programs. Comput Chem 26:
1668–1688.
´
la Universidad Nacional de Cordoba (SECyT), and
´
Consejo Nacional de Investigaciones Cientıficas y
´
´
Tecnologicas de la Nacion (CONICET) for financial
support. We also thank Ferromet S.A. (agent of
Roquette in Argentina) for its donation of b-cyclodex-
trin, and also Dr. Petter Bjorstad (Research Director
at Bergen Center for Computational Science (BCCS))
for kindly providing our access to BCCS computing
resources.
REFERENCES
1. Connor EE. 1998. Sulfonamide antibiotics. Primary Care
Update Ob/Gyns 5:32–35.
2. Supuran CT, Casini A, Scozzafava A. 2003. Protease inhibitors
of the sulfonamide type: Anticancer, antiinflammatory, and
antiviral agents. Med Res Rev 23:535–558.
3. Golzar Hossaina GM, Amoroso AJ, Banua A, Malika KMA.
2007. Syntheses and characterisation of mercury complexes of
18. Morris GM, Goodsell DS, Halliday RS, Huey R, Hart WE, Belew
RK, Olson AJ. 1998. Automated docking using a Lamarckian
DOI 10.1002/jps
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 7, JULY 2010

3176
ZOPPI ET AL.
genetic algorithm and an empirical binding free energy func-
tion. J Comput Chem 19:1639–1662.
19. Kuhn B, Gerber P, Schulz-Gasch T, Stahl MJ. 2005. Validation
and use of the MM-PBSA approach for drug discovery. Med
Chem 48:4040–4048.
23. Mele A, Mendichi R, Selva A. 1998. Non-covalent associations
of cyclomaltooligosaccharides (cyclodextrins) with trans-b-
carotene in water: Evidence for the formation of large aggre-
gates by light scattering and NMR spectroscopy. Carbohydr
Res 310:261–267.
´
20. Humphrey W, Dalke A, Schulten K. 1996. VMD: Visual mole-
cular dynamics. J Mol Graph 14:33–38.
24. Loftsson T, Masson M, Brewster ME. 2004. Self-association of
cyclodextrins and cyclodextrin complexes. J Pharm Sci 93:
1091–1099.
`
21. Ventura CA, Giannone I, Paolino D, Pistara V, Corsaro A,
Puglisi G. 2005. Preparation of celecoxib-dimethyl-b-cyclodex-
trin inclusion complex: Characterization and in vitro permea-
tion study. Eur J Med Chem 40:624–631.
25. Ogruc-Ildiz G, Akyuz S, Ozel AE. 2009. Experimental, ab initio
and density functional theory studies on sulfadiazine. J Mol
Struct 924-926:514–522.
22. Ventura CA, Giannone I, Musumeci T, Pignatello R, Ragni L,
Landolfi C, Milanese C, Paolino D, Puglisi G. 2006. Physico-
chemical characterization of disoxaril-dimethyl-b-cyclodextrin
inclusion complex and in vitro permeation studies. Eur J Med
Chem 41:233–240.
26. Zoppi A, Quevedo MA, Longhi MR. 2008. Specific binding
capacity of b-cyclodextrin with cis and trans enalapril:
Physicochemical characterization and structural studies
by molecular modeling. Bioorg Med Chem 16:8403–8412.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 7, JULY 2010
DOI 10.1002/jps