Inclusion complex of 2-chlorobenzophenone with cyclomaltoheptaose (β-cyclodextrin): Temperature, solvent effects and molecular modeling

Article abstract of DOI:10.1016/j.carres.2011.05.002

A thermodynamic study of the inclusion process between 2-chlorobenzophenone (2ClBP) and cyclomaltoheptaose (β-cyclodextrin, β-CD) was performed using UV-vis spectroscopy, reversed-phase liquid chromatography (RP-HPLC), and molecular modeling (PM6). Spectrophotometric measurements in aqueous solutions were performed at different temperatures. The stoichiometry of the complex is 1:1 and its apparent formation constant (K^c) is 3846 M^-1 at 30 °C. Temperature dependence of K_c values revealed that both enthalpy (δH° = -10.58 kJ/mol) and entropy changes (δS° = 33.76 J/K mol) are favorable for the inclusion process in an aqueous medium. Encapsulation was also investigated using RP-HPLC (C18 column) with different mobile-phase compositions, to which β-CD was added. The apparent formation constants in MeOH-H₂O (K_F) were dependent of the proportion of the mobile phase employed (50:50, 55:45, 60:40 and 65:35, v/v). The K_F values were 419 M^-1 (50% MeOH) and 166 M ^-1 (65% MeOH) at 30 °C. The thermodynamic parameters of the complex in an aqueous MeOH medium indicated that this process is largely driven by enthalpy change (δH° = -27.25 kJ/mol and δS° = -45.12 J/K mol). The results of the study carried out with the PM6 semiempirical method showed that the energetically most favorable structure for the formation of the complex is the 'head up' orientation.

Full text of DOI:10.1016/j.carres.2011.05.002

Carbohydrate Research 346 (2011) 1978–1984
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Inclusion complex of 2-chlorobenzophenone with cyclomaltoheptaose
(b-cyclodextrin): temperature, solvent effects and molecular modeling
Matias I. Sancho a,b, Estela Gasull a, Sonia E. Blanco a,b, , Eduardo A. Castro c
⇑
a Área de Química Física, Facultad de Química Bioquímica y Farmacia, Universidad Nacional de San Luis, 5700 San Luis, Argentina
b Instituto Multidisciplinario de Investigaciones Biológicas (IMIBIO-SL) CONICET, 5700 San Luis, Argentina
c INIFTA, Dpto. de Química, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, 1900 Buenos Aires, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
A thermodynamic study of the inclusion process between 2-chlorobenzophenone (2ClBP) and cyclomal-
toheptaose (b-cyclodextrin, b-CD) was performed using UV–vis spectroscopy, reversed-phase liquid chro-
matography (RP-HPLC), and molecular modeling (PM6). Spectrophotometric measurements in aqueous
solutions were performed at different temperatures. The stoichiometry of the complex is 1:1 and its
apparent formation constant (Kc) is 3846 Mꢀ1 at 30 °C. Temperature dependence of Kc values revealed
Received 24 March 2011
Received in revised form 2 May 2011
Accepted 4 May 2011
Available online 10 May 2011
that both enthalpy (
D
H° = ꢀ10.58 kJ/mol) and entropy changes (
DS° = 33.76 J/K mol) are favorable for
Keywords:
b-Cyclodextrin
the inclusion process in an aqueous medium. Encapsulation was also investigated using RP-HPLC (C18
column) with different mobile-phase compositions, to which b-CD was added. The apparent formation
constants in MeOH–H2O (KF) were dependent of the proportion of the mobile phase employed (50:50,
55:45, 60:40 and 65:35, v/v). The KF values were 419 Mꢀ1 (50% MeOH) and 166 Mꢀ1 (65% MeOH) at
30 °C. The thermodynamic parameters of the complex in an aqueous MeOH medium indicated that this
2-Chlorobenzophenone
Solubility
Reversed-phase liquid chromatography
Apparent formation constants
Molecular modeling
process is largely driven by enthalpy change (
D
H° = ꢀ27.25 kJ/mol and
DS° = ꢀ45.12 J/K mol). The results
of the study carried out with the PM6 semiempirical method showed that the energetically most favor-
able structure for the formation of the complex is the ‘head up’ orientation.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
However, the poor aqueous solubility of BPs limits their phar-
macological uses due to their low dissolution rate and bioavailabil-
Benzophenones (BPs) are compounds present in a large number
of natural products,1–3 and they can also be obtained through syn-
thetic methods.4 They exhibit interesting physicochemical and bio-
logical properties. For example, due to their capacity to absorb and
dissipate UVA and UVB radiation (200–350 nm), a group of 12 BPs
is used in the cosmetics industry to produce sunscreens,5 and
numerous analytical methods have been used to perform quality
control on these products.6 BPs are also used in pharmacological
applications, such as the synthesis of tamoxifen7 and clomiphene
citrate.8 Benzodiazepines that are generally used as sedative, anx-
yolitic, anticonvulsant and muscle relaxant agents are derived from
chlorinated benzophenones. Some common examples are diaze-
pam and alprazolam (both derived from 5-chlorobenzophenone),
or clonazepam (derived from 2-chlorobenzophenone). Specifically,
ity. Different methods can be used to increase drug solubility, such
as the use of organic solvents,10 emulsions,11 liposomes,12 mi-
celles13 and the interaction of drugs with complexing agents.
Among these, a very frequently used method is the formation of
inclusion complexes with cyclomaltooligosaccharides (cyclodex-
trins, CDs).14–18 CDs are cyclic oligomers with a truncated cone
shape which are made up of six, seven or eight units of
a-(1?4)-
D
-glucose per molecule ( -, b- and - CD, respectively). The outer
a
c
layer of the molecule is hydrophilic, and organic molecules (drugs)
with an appropriate size and shape, and low solubility in water can
enter into its hydrophobic cavity, forming non-covalent inclusion
complexes as a result.19 This inclusion phenomenon may increase
the apparent solubility of the drug, and it can also modify its phys-
icochemical stability and its bioavailability. As a pharmaceutical
agent cyclomaltoheptaose (b-CD) appears particularly useful be-
cause of its complexing ability, cavity dimensions, low cost and
higher productive rate. Because of these applications new experi-
mental and theoretical techniques devoted to the determination
of the solution structure and the binding constants of the CD–drug
complexes are constantly under development.20
2-chlorobenzophenone is also
a reagent in the synthesis of
N-methyl-N-(1-methylpropyl)-1-(2-chlorophenyl)isoquinoline-3-
[
11C]carboxamide, which is used as a benzodiazepine receptor
ligand.9
To the best of our knowledge, there is no information in the
existing literature reporting the effect of b-CD on the solubility of
⇑
Corresponding author. Tel.: +54 2652 424689; fax: +54 2652 430224.
E-mail addresses: sblanco@unsl.edu.ar, sonia.e.blanco@gmail.com (S.E. Blanco).
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.05.002

M. I. Sancho et al. / Carbohydrate Research 346 (2011) 1978–1984
1979
2-chlorobenzophenone (2ClBP). For this reason, an experimental
and theoretical study on the interaction of 2ClBP with b-CD was
carried out. Determination of the stoichiometry and apparent for-
mation constant of the inclusion complex in an aqueous medium
at different temperatures was made by using the phase-solubility
method and UV–vis spectroscopy. The apparent formation con-
stant in MeOH–water of this inclusion complex was also deter-
mined by using a reversed-phase liquid chromatography method
(RP-HPLC), and the influence of mobile-phase composition and
temperature was analyzed. Additionally, a molecular modeling
study of the interaction between 2ClBP and b-CD was performed
in order to gain insight about the driving forces governing the
inclusion process. The PM6 semiempirical method was used to cal-
culate the formation constant of the inclusion complexes in the gas
phase. The geometries and interaction energies of the complexes
were also determined.
HPLC data were obtained as an average of two measurements
for each determination.
2.4. Data analysis
Processing of data for the fitting of the different equations was
done using the Scientist program Origin v. 8.0. Curve fittings were
achieved through least-squares regression and yielded optimized
values for the parameters. The curve-fitting procedures typically
gave good (>0.99) to excellent (>0.999) correlation coefficients.
2.5. Molecular modeling studies
Initial molecular geometries of b-CD, 2ClBP and inclusion com-
plexes were fully optimized using the PM6 method (Parametric
model 6).21 The corresponding frequencies were calculated to en-
sure that the obtained stationary points were true minima. All
the semiempirical calculations were performed using MOPAC
2009 (http://www.openmopac.net/; v9). Initial coordinates of b-
CD were obtained from crystallographic data.22
2. Materials and methods
2.1. Materials
For the inclusion process, the glycosidic oxygens of b-CD were
placed onto the x, y plane, and their center was defined as the cen-
ter of the coordination system. The b-CD was kept in this position,
while the guest molecule approached the b-CD cavity along the z-
axis. The relative position between the host and the guest molecule
was measured by the distance along the z-axis of the carbon atom
in the carbonyl group of 2ClBP to the coordination center. Inclusion
was emulated by manually moving the guest molecule from 6 Å to
ꢀ6 Å in stepwise units of 1 Å. For each step the geometry of the
complex was fully optimized by the semiempirical PM6 method.
Two possible orientations were considered for the inclusion pro-
cess: the ‘head down’ orientation, in which the 2ClBP initially
points toward the secondary hydroxyls of the b-CD, and the ‘head
up’ orientation, in which the 2ClBP initially points toward the pri-
mary hydroxyls of the b-CD cavity.
Anhydrous b-CD was purchased from MP Biomedicals and care-
fully dried in a vacuum at 60 °C for 24 h and stored in a container
with P2O5. 2ClBP was purchased from Sigma–Aldrich Chemical Co.
and was used without further purification. HPLC grade MeOH was
obtained from Merck, and double-distilled water was purified by
using a Super Q Millipore System, with conductivity lower than
1.8
l
S cmꢀ1. MeOH and water were also degassed and filtered with
m), before use.
Minisart RC filters (0.5
l
2.2. UV–vis measurements
Excess amounts (50 mg) of 2ClBP were added to volumetric
flasks containing 5 mL of aqueous solutions of different b-CD con-
centrations (0–1.2 mM). The flasks were placed in a water bath at
constant temperature (25.0, 30.0, 35.0 and 40.0 0.1 °C) and sha-
ken until equilibrium was reached (48 h). The solutions were then
filtered and adequately diluted. The concentrations of 2ClBP were
spectrophotometrically determined at 259 nm using a Varian Cary
50 spectrophotometer with temperature control. All studies were
carried out in duplicate. Baseline was established for each mea-
surement by placing an aqueous solution of b-CD in the reference
compartment at the same concentration of the sample.
3. Results and discussion
3.1. Spectrophotometric method for determining complexation
constant
The stoichiometric ratio and apparent formation constants (Kc)
were derived from the changes in the solubility of 2ClBP in an
aqueous medium at different temperatures, in the presence of
increasing amounts of b-CD. An increase of the absorption intensity
was observed, which indicates an increase in the solubility of the
analyzed complex.
2.3. HPLC measurements
Chromatographic experiments were performed using a Gilson
322 pump with a ThermaSphere TS-130 HPLC temperature con-
troller, a Rheodyne 7725i sample injector, a Gilson 152 UV–vis
detector and the UniPoint v 2.10 (Gilson) software. The analysis
was carried out with a Luna C18 (2) reversed-phase column
Figure 1 shows the phase-solubility diagram obtained when
representing the solubility of the drug against the b-CD concentra-
tion. The system presents a BS-type behavior, which means that
there is a linear variation of the solubility up to a certain concen-
tration level of b-CD (ꢂ1.2 mM); after that, a plateau appears indi-
cating that the complex has a limited solubility.23 This type of
behavior is frequently observed when b-CDs are used. The initial
ascending portion of Bs-type isotherm can be analyzed with the
same techniques used for the AL-type systems. In these diagrams,
slope values lower than the unity suggest the presence of a 1:1
complex. The slope value for the analyzed system was (0.548),
and the intercept (intrinsic drug solubility) was 0.26 mM. The stoi-
chiometry of complexes from Bs-type solubility isotherms can be
determined in several ways.24,25 In our case, very good results were
obtained when processing the experimental data of the ascending
linear segment with Scott’s equation:24
(5
l
m, 250 mm ꢁ 4.6 mm). In order to analyze the effect of the
mobile-phase composition, chromatographic studies were per-
formed with mobile phases of MeOH–H2O (50:50, 55:45, 60:40
and 65:35, v/v), and the temperature was maintained at 30 °C.
For the analysis of the temperature effect, the mobile-phase
composition was MeOH–H2O (60:40, v/v), and the temperature
was set at 25.5, 30.0, 35.0 and 40.0 0.1 °C. Several amounts of
b-CD (0–10 mM) were dissolved in the mobile phase. The con-
centration of 2ClBP in the injected solution was 8 mM, and the
volume of the injection was 20 lL. The mobile phase was
pumped at a flow rate of 1 mL/min, and the detector was set
at 259 nm. The capacity factor (k) was measured from chromato-
graphic data, k = (tR ꢀ to)/to. The mobile phase hold-up times
were measured by injecting a solution of K2Cr2O7 (1 mM). The
½BPtꢃ½b-CDtꢃL
1
1
cD
¼
½b-CDtꢃ þ
ð1Þ
D
A
D
e
K
e

1980
M. I. Sancho et al. / Carbohydrate Research 346 (2011) 1978–1984
Table 1
1.0
0.8
0.6
0.4
0.2
0.0
Apparent formation constants (Kc) of the inclusion complex between 2ClBP and b-CD
in water at different temperatures
T (°C)
Slope ꢁ103
3.191 (0.038)
2.630 (0.053)
3.241 (0.040)
2.705 (0.066)
Intercept ꢁ107
7.714 (0.271)
6.838 (0.384)
9.022 (0.286)
8.020 (0.488)
r
Kc (Mꢀ1
)
25.0
30.0
35.0
40.0
0.9993
0.9982
0.9993
0.9973
4137 217
3846 318
3592 158
3373 282
The slopes and intercepts reported correspond to Eq. 1 with the standard deviation
in parenthesis and r as the correlation coefficient of the linear fit.
8.5
ln Kc = 1272 (16) (1/T) + 4.06 (0.05)
r = 0.9998
0.0
0.5
1.0
1.5
2.0
2.5
[β-CD], mM
8.3
8.2
8.0
Figure 1. Phase-solubility diagram of 2ClBP in the presence of b-CD in water at
25 °C.
In this equation, [BPt] is the total concentration of the drug, [b-CDt]
is the total concentration of the solubilizer,
absorbance between the drug in the absence and presence of the
b-CD at a particular wavelength (259 nm), is the difference in
DA is the difference in
D
e
molar absorptivities between the free and the encapsulated drug,
and L is the path length. If the graphical representation of Eq. 1
yields a straight line, the stoichiometry of the complex is 1:1. Then,
the magnitude of Kc can be calculated from the slope and intercept.
Figure 2 shows the representation of the experimental data at
25.0 °C, according to Eq. 1. The Kc values obtained at different tem-
peratures through this method are presented in Table 1. As it can be
observed, the Kc values decrease as temperature increases.
3.2x10-3
3.3x10-3
1/T, K-1
3.4x10-3
Figure 3. van’t Hoff plot (ln Kc vs 1/T) for the inclusion complex of 2ClBP with b-CD
in water. Standard deviations of slope and intercept are in parenthesis.
at 298.15 K was calculated from these values and the results ob-
tained were:
Hꢄ ¼ ꢀ10:58 kJ=mol
Sꢄ ¼ 33:76 J=K mol
Gꢄ ¼ ꢀ20:65 kJ=mol
The thermodynamic parameters, Gibbs-free energy change
(DG°), enthalpy change (DH°), and entropy change (DS°) for the
inclusion complex 2ClBP–b-CD were obtained from the classical
D
D
D
van’t Hoff equation
D
Hꢄ
D
Sꢄ
R
ln Kc ¼ ꢀ
þ
ð2Þ
RT
H° and DS° of the complex formation were calculated from
Different types of forces have been analyzed for their role in driving
complex formation, including electrostatic interactions, van der
Waals contributions, hydrogen bonding, release of conformational
strain, exclusion of high energy water in the CD cavity and
charge–transfer interactions.28,29 Generally, the inclusion process
The
D
the slope and intercept by plotting ln Kc versus 1/T (Fig. 3). This
analysis assumes that CP is zero, which is not always true for this
kind of process. However, Figure 3 shows a linear relationship of
D
ln Kc versus 1/T, indicating the independence of
D
H° and
D
S° on T
G°
is associated with a relatively large negative value of
the
S° can be either positive or negative.30 The hydrophobic inter-
actions are related to a slightly positive H° and large positive S°,
indicating that the process is entropy driven. The S° positive val-
DH° while
(D
CP ꢅ 0) within the temperature range considered.26,27 The
D
D
D
D
D
4.0x10-6
3.0x10-6
2.0x10-6
1.0x10-6
ues are due to the breaking of highly ordered aqueous microenvi-
ronments surrounding the hydrophobic part of the guest
molecules upon binding the CD. In our case, the obtained results
indicate that the inclusion of 2ClBP into b-CD in aqueous solution
is favored by both the enthalpy (
terms. This can be seen in the G° value calculated at 298.15 K, in
which contributions of H° and T S° terms are almost 50% each
(Fig. 4). It can be concluded that the balance between the hydropho-
bic effects ( S° >0) and the van der Waals forces ( H° <0;
H° ꢂ 0;
S° <0) may be inclined in this particular case to the former. Daoud-
DH° <0) and entropy (DS° >0)
D
D
D
D
D
D
D
Mahammed et al. analyzed the inclusion complex between the
unsubstituted benzophenone (BP) and b-CD (BP–b-CD) in aqueous
medium.31 The thermodynamic magnitudes
D
H° (ꢀ16.77 kJ/mol)
S° (2.77 kJ/mol) were both favorable to the inclusion process,
but in this case the H° term is higher than T S°. The stoichiometry
4.0x10-4
8.0x10-4
1.2x10-3
and TD
0.0
D
D
[β-CD ], M
t
of the BP–b-CD complex was 1:1, and its apparent formation con-
stant determined by means of phase-solubility diagrams at 25 °C
was 2680 Mꢀ1. For the system 2ClBP–b-CD the apparent formation
Figure 2. Graphical representation of the solubility data of 2ClBP in water at 25 °C,
according to Eq. 1. Each data point is the mean of two replicates. Error bars of 1%.

M. I. Sancho et al. / Carbohydrate Research 346 (2011) 1978–1984
1981
(for MeOH, Km is equal to 0.32 Mꢀ1).32 All the analytical concentra-
tions of b-CD that had been used were corrected with this equation.
Figure 6 shows the overlaid chromatograms of 2ClBP obtained
in the presence of different concentrations of b-CD. As expected,
retention times decrease as the concentration of b-CD in the mo-
bile phase increases, due to the formation of the 2ClBP–b-CD com-
plex; this enhances the guest solubility in the mobile phase and
reduces its residency time in the column. The variation in the
retention times obtained by replication was in all cases lower than
1%. The capacity factors were calculated with the solute retention
time and the hold-up time. Then, the apparent formation constant
in MeOH–H2O (KF) of the complex was determined using the fol-
lowing expression:33
15
10
5
ΔHº
T ΔSº
ΔGº
0
-5
-10
-15
-20
-25
-30
Solubility
RP-HPLC
1
1
KF
¼
þ ½b-CDmꢃ
k0 k0
ð4Þ
k
Figure 4. Gibbs free energy (DG°), enthalpy (DH°) and entropy (TDS°) changes for
where k is the capacity factor of the solute at each b-CD concentra-
tion and k0 is the solute capacity factor in the absence of b-CD. For a
complex with a 1:1 stoichiometry, a plot of 1/k versus [b-CDm]
yields a straight line, and KF is obtained from the slope-to-intercept
ratio. Figure 7 shows the dependence of the capacity factor with the
[b-CDm] according to Eq. 4 for four percentages of MeOH in the mo-
bile phase. Table 2 reports the KF values obtained. For the lower per-
centage of MeOH in the mobile phase, KF is equal to 419 Mꢀ1. When
the concentration of MeOH increases from 50% to 65%, the decrease
in the polarity of the mobile phase favors the solubility of the
hydrophobic drug and, as expected, the affinity of the solute with
the b-CD cavity is reduced. KF values vary almost in a linear way
with the% MeOH (KF = ꢀ17.38% MeOH + 1287 (r = 0.995)) in the
interval analyzed.
The effect of temperature on KF of the 2ClBP–b-CD complex was
also analyzed. Experiments carried out at different temperatures
(25.5, 30.0, 35.0, and 40.0 °C), with a MeOH–H2O (60:40, v/v) mo-
bile phase showed that an increase in temperature enhances the
dissociation of the inclusion complex. Figure 8 illustrates the
changes of k with the effective concentration of b-CD in the mobile
phase at different temperatures. The KF values obtained from these
data are detailed in Table 3. The thermodynamic parameters were
calculated by means of Eq. 2, and Figure 9 shows the resulting van’t
Hoff plot. The following results were obtained from the fitted
equation:
the encapsulation of 2ClBP with b-CD determined by RP-HPLC and solubility study
at 298.15 K.
constant at 25.0 °C is greater (Kc = 4137 Mꢀ1). Then, the presence of
a chlorine atom in the ring of the BP could favor the formation of the
complex in its interaction with the cavity of the b-CD.
3.2. Chromatographic determination of apparent formation
constants
The apparent formation constants of the 2ClBP–b-CD complex
were also determined through RP-HPLC. In these types of studies,
the CDs can be part of the stationary phase (chemically bonded
to the silica gel), or they can be added as additives to the mobile
phase. As a result of host–guest interaction, the retention time
(tR) of the guest decreases when the inclusion takes place in the
mobile phase, and it increases if this happens in the stationary
phase. These variations in tR are related to the apparent formation
constant of the complex. Several equilibria take place in the chro-
matographic column when a solute is introduced, and a mobile
phase (MeOH–H2O, in this case) containing b-CD is used. As it is
represented in Figure 5, these equilibria comprise the solute parti-
tion between the mobile and stationary phases, and the solute
encapsulation with b-CD. However, the possible competition for
the CD cavity of the organic modifier (MeOH) should also be con-
sidered. Due to this competition, the available concentration of
b-CD used to interact with 2ClBP is not the total analytical concen-
tration ([CDt]), but rather the effective concentration ([CDm]),
which can be calculated with the following equation32
E
½CDtꢃ
Tr = 48.86
½CDmꢃ ¼
ð3Þ
1 þ Km½Mꢃ
D
where [M] is the MeOH concentration in the mobile phase and Km is
the association constant of the organic modifier: the b-CD complex
Tr = 52.99
C
Tr = 57.34
B
KF
Tr = 61.96
(2ClBP)m + ( β-CD)m
(Complex)m
+
M
A
Tr = 67.28
Km
(2ClBP)s
0
20
40
60
80
(β-CD-M)m
Time, min
Figure 5. Schematic representation of the equilibria proposed for the 2ClBP–b-CD
inclusion complex in an HPLC system with MeOH–H2O mobile phase. The
subscripts m and s, respectively represent the mobile and the stationary phases,
KF is the apparent formation constant of the complex, Km is the affinity constant of
the MeOH for the b-CD cavity, and M represents the MeOH molecules.
Figure 6. Decrease in the retention time of 2ClBP in the presence of increasing
concentrations of b-CD in the mobile phase (A = 0, B = 0.22, C = 0.45, D = 0.69 and
E = 0.95 mM) at 30 °C. Chromatographic conditions: column, Luna C18 (2), 5 lm,
250 mm ꢁ 4.6 mm ID; mobile phase, MeOH–H2O (50:50, v/v); flow rate: 1 mL/min;
detector wavelength, 259 nm.

1982
M. I. Sancho et al. / Carbohydrate Research 346 (2011) 1978–1984
5.6
0.24
0.20
0.16
0.12
0.08
0.04
50:50
55:45
60:40
65:35
ln KF = 3278 (313) (1/T) - 5.43 (1.03)
r = 0.9910
5.4
5.2
5.0
0.0
0.2
0.4
0.6
0.8
1.0
3.2x10-3
3.3x10-3
1/T, K-1
3.4x10-3
[β-CDm], mM
Figure 7. Plots of 1/k versus effective b-CD concentration ([b-CDm]) at 30 °C and
different mobile phase compositions of MeOH–H2O (v/v). Each data point is the
mean of two replicates.
Figure 9. van’t Hoff plot (ln KF vs 1/T) for the inclusion complex of 2ClBP with b-CD
in a MeOH–H2O mixture of (60:40, v/v). Standard deviations of slope and intercept
are in parenthesis.
Table 2
D
D
D
Hꢄ ¼ ꢀ27:25 kJ=mol
Sꢄ ¼ ꢀ45:12 J=K mol
Gꢄ ¼ ꢀ13:80 kJ=mol
Apparent formation constant (KF) of the 2ClBP–b-CD complex as a function of the
percentage of MeOH in MeOH–H2O mobile phase at 30 °C
%MeOH
Slope
Intercept ꢁ102
r
KF (Mꢀ1
)
The
MeOH–H2O is associated with a favorable enthalpic term (
and an unfavorable entropic term ( S° <0). Figure 4 shows that
D
G° reported refers to T = 298.15 K. The inclusion process in
50
55
60
65
16.80 (0.23)
25.07 (0.38)
28.42 (0.50)
35.18 (0.46)
4.005 (0.013)
7.426 (0.020)
12.51 (0.02)
21.22 (0.02)
0.9997
0.9996
0.9995
0.9997
419 14
337 13
227 11
166 6.5
D
H° <0)
D
the formation of the complex in both reaction media (H2O and
MeOH–H2O) is an exothermic process. Moreover, it can be seen that
The slopes and intercepts reported correspond to Eq. 4 with the standard deviation
in parenthesis and r as the correlation coefficient of the linear fit.
the
DS° presents a positive and a negative value in H2O and in
MeOH–H2O, respectively. When molecules with apolar moieties
are added to the medium (MeOH), the hydrophobic part of the guest
drug will be surrounded, in part, by these alcohol molecules. These
molecules replace the ordered aqueous microenvironment, which
0.17
25.5 ºC
30.0 ºC
35.0 ºC
results in a decrease of
D
S° in relation to the one obtained in a com-
G° values at 298.15 K are negative
pletely aqueous medium.34 The
D
in both cases. The transfer of the solute containing a hydrophobic
moiety, such as 2ClPB, from a polar solvent (H2O) to the hydropho-
40.0 ºC
0.15
bic b-CD cavity, produces
a large decrease in free energy
(D
G° = ꢀ20.65 kJ/mol) and favors encapsulation (Kc = 4149 Mꢀ1).
When polarity is reduced, in the case of MeOH–H2O (60:40, v/v),
the difference of polarity between the medium and the b-CD cavity
is lower. Then, the solute solubility in the eluent improves, and the
0.13
0.11
formation of the complex is less favored (
D
G° = ꢀ13.80 kJ/mol and
KF = 262 Mꢀ1).
3.3. Molecular modeling results
0.0
0.2
0.4
0.6
0.8
[β-CDm], mM
Quantum mechanics calculations were made in order to obtain
information related to the structural characteristics and thermo-
dynamic magnitudes of the 1:1 complex between 2ClBP and b-
CD. The values used in the calculation of these magnitudes are
the ones obtained in the frequency analysis carried out with the
Figure 8. Plots of 1/k versus effective b-CD concentration ([b-CDm]) at different
temperatures and a mobile phase of MeOH–H2O (60:40, v/v). Each data point is the
mean of two replicates.
Table 3
Apparent formation constant (KF) of the 2ClBP–b-CD complex at different temper-
atures in the MeOH–H2O (60:40, v/v) mobile phase
Table 4
Experimental and theoretical thermodynamic parameters of the 2ClBP–b-CD complex
T (C°)
Slope
Intercept ꢁ102
11.61 (0.03)
12.51 (0.02)
13.24 (0.03)
14.49 (0.02)
r
KF (Mꢀ1
)
PM6
‘head up’
PM6
‘head down’
UV–vis
spectroscopy
RP-HPLC
25.5
30.0
35.0
40.0
29.05 (0.82)
28.42 (0.50)
24.44 (0.69)
22.11 (0.46)
0.9992
0.9995
0.9992
0.9995
250 27
227 11
185 21
152 13
D
D
D
H° (kJ/mol)
S° (J/K mol)
G° (kJ/mol)
ꢀ88.24
ꢀ242.90
ꢀ15.82
ꢀ73.04
ꢀ240.49
ꢀ1.34
ꢀ10.58
33.76
ꢀ20.65
ꢀ27.25
ꢀ45.12
ꢀ13.80
The slopes and intercepts reported correspond to Eq. 4 with the standard deviation
in parenthesis and r as the correlation coefficient of the linear fit.
The values obtained by molecular simulation with the PM6 method include the two
considered orientations.

M. I. Sancho et al. / Carbohydrate Research 346 (2011) 1978–1984
1983
Figure 10. Molecular structures of the 2ClBP–b-CD complex with minimum energy obtained by PM6 calculations. Top left: ‘head down’ perpendicular to the cavity axis; top
right: ‘head down’ along the cavity axis; bottom left: ‘head up’ perpendicular to the cavity axis; bottom right: ‘head up’ along the cavity axis. The dotted lines indicate the
Clꢆ ꢆ ꢆH(3) interaction.
PM6 method. This analysis was performed on the structures of the
complex with the lowest energy which were obtained during the
simulation of the inclusion process in each one of the orientations
considered, as detailed in Section 2. The results obtained are
shown in Table 4. Experimental results are also displayed for com-
in vacuum, the previously mentioned effect is not considered. In
addition, van der Waals host–guest interactions are the most
important contribution to the stabilization of these complexes in
vacuum. These effects cause a decrease in translational and rota-
tional degrees of freedom of complexed molecules giving a more
parison purposes. The negative values of
indicate that the inclusion process is favorable, although the ‘head
up’ complex has a higher G° value (in absolute value) than the
‘head down’ complex. The theoretical formation constants (KT)
were calculated from the G° values. For the ‘head up’ orientation
D
G° in both orientations
ordered system.36 As a result, an underestimation of
D
S° in relation
to the values obtained by experimental methods is observed. In
this way, a positive S° value is observed in aqueous medium, a
D
D
small negative value in MeOH–H2O, and a large negative value
large in vacuum. As expected, this result indicates that as the
polarity of the medium decreases, the change in the standard en-
tropy of the reaction decreases.
The 2ClBP–b-CD molecular structures of minimum energy in
the ‘head up’ and ‘head down’ orientations are illustrated in
Figure 10. The chlorine atom is observed to be inside the CD cav-
ity in both orientations. Engeldinger et al. have reported that the
D
complex, KT = 591, and for the ‘head down’ orientation complex,
KT = 2. Thus, the numerical value for the constant obtained for
the former orientation is similar to those experimentally obtained
(RP-HPLC), while for the latter, KT is very small. The
DH° values
are negative, and the complex with ‘head up’ orientation presents
a higher value (in absolute value). Consequently, the ‘head up’ ori-
entation is clearly the preferred orientation for the formation of
the complex.
a
-CD cavity presents chlorophillic characteristics when inclusion
complexes with chlorides of transition metals are formed.37–39
These authors report a weak interaction between the chlorine
On the other hand, the
orientations considered. As compared to the
D
S° values are very similar for the two
S° values obtained
D
atom and the a-CD H(5) with a distance of 2.64 Å. For the
by experimental methods, those calculated by PM6 are large and
negative. In order to rationalize these results, it must be considered
that there is a gain in the system entropy due to the assimilation of
water molecules by the reaction medium after the inclusion of the
guest in the CD.35 Since the semiempirical calculations were made
2ClBP–b-CD, the ‘head up’ structure presents a Clꢆ ꢆ ꢆH(3) interac-
tion with a minimum distance of 2.87 Å, whereas the minimum
distance for the same interaction in the ‘head down’ structure is
2.73 Å. These results indicate that the b-CD also has some affinity
for chlorine.

1984
M. I. Sancho et al. / Carbohydrate Research 346 (2011) 1978–1984
4. Fu, Z.; Wang, M.; Dong, Y.; Liu, J.; Liu, Q. J. Org. Chem. 2009, 74, 6105–6110.
4. Conclusions
5. Nyeborg, M.; Pissavini, M.; Lemasson, Y.; Doucet, O. Int. J. Cosmet. Sci. 2010, 32,
47–53.
The formation of the inclusion complex of 2ClBP, a drug with
low aqueous solubility, with b-CD was analyzed using UV–vis
spectroscopy and HPLC techniques in various reaction media.
In all the cases studied, the complex was found to present a
1:1 stoichiometry. The formation constants of the complex in
purified water (Kc) and in MeOH–H2O (KF) were determined,
and it was observed that Kc > KF. Furthermore, it was determined
that KF decreases in an almost linear way in relation to the per-
centage of organic modifier in the interval of the MeOH concen-
6. Juang, L. J.; Wang, B. S.; Tai, H. M.; Hung, W. J.; Huang, M. H. J. Food Drug Anal.
2008, 16, 22–28.
7. Top, S.; Kaloun, E. B.; Vessières, A.; Leclercq, G.; Laïos, I.; Ourevitch, M.;
Deuschel, C.; McGlinchey, M. J.; Jaouen, G. ChemBioChem 2003, 4, 754–761.
8. Pakrasi, P. L.; Kumar, A. Curr. Sci. 2001, 80, 682–685.
9. Rahman, O.; Kihlberg, T.; Långström, B. J. Chem. Soc., Perkin Trans. 1 2002, 23,
2699–2703.
10. Li, Y.; Lu, X. Y. J. Chem. Eng. Data 2010, 55, 4019–4020.
11. Kitagawa, S.; Inoue, K.; Teraoka, R.; Morita, S. Y. Chem. Pharm. Bull. 2010, 58,
398–401.
12. Yang, X.; Zhao, X.; Phelps, M. A.; Piao, L.; Rozewski, D. M.; Liu, Q.; Lee, L. J.;
Marcucci, G.; Grever, M. R.; Byrd, J. C.; Dalton, J. T.; Lee, R. J. Int. J. Pharm. 2009,
365, 170–174.
trations used. The thermodynamic parameters,
DH°, DS° and
D
G°, were determined by analyzing the variation of ln Kc and
13. Zhang, Y.; Li, X.; Zhou, Y.; Wang, X.; Fan, Y.; Huang, Y.; Liu, Y. Nanoscale Res.
Lett. 2010, 5, 917–925.
ln KF with the inverse of the temperature. The results obtained
indicate that the forces driving the inclusion process change
according to the reaction medium. When the reaction takes
place in an aqueous medium, the encapsulation process is fa-
vored by both negative enthalpy and positive entropy changes.
However, the process is only enthalpically driven when the reac-
tion occurs in an MeOH–H2O medium, with a favorable enthalpic
term dominant over an unfavorable entropic term. These ther-
modynamic parameters were also calculated with the PM6 semi-
empirical method in vacuum. The results obtained are coherent
with the experimental results, since the absence of solvent mol-
14. Filippa, M.; Sancho, M. I.; Gasull, E. J. Pharm. Biomed. Anal. 2008, 48, 969–973.
15. Cannava, C.; Crupi, V.; Ficarra, P.; Guardo, M.; Majolino, D.; Mazzaglia, A.;
Stancanelli, R.; Venuti, V. J. Pharm. Biomed. Anal. 2010, 51, 1064–1068.
16. Cirri, M.; Maestrelli, F.; Mennini, N.; Mura, P. J. Pharm. Biomed. Anal. 2009, 50,
690–694.
17. Zhang, H.; An, W.; Liu, Z.; Hao, A.; Hao, J.; Shen, J.; Zhao, X.; Sun, H.; Sun, L.
Carbohydr. Res. 2010, 345, 87–96.
18. Martina, K.; Puntambekar, D. S.; Barge, A.; Gallarate, M.; Chirio, D.; Cravotto, G.
Carbohydr. Res. 2010, 345, 191–198.
19. Frömming, K. H.; Szejtly, J. Cyclodextrins in Pharmacy; Kluwer Academic
Publishers: Dordrecht, 1994.
20. Funasaki, N.; Ishikawa, S.; Neya, S. Pure Appl. Chem. 2008, 80, 1511–1524.
21. Stewart, J. J. P. J. Mol. Model. 2007, 13, 1173–1213.
22. Sharff, A. J.; Rodseth, L. E.; Quiocho, F. A. Biochemistry 1993, 32, 10553–10559.
23. Connors, K. A. Measurement of Cyclodextrin Complex Stability Constants In
Comprehensive Supramolecular Chemistry: Cyclodextrins; Szejtli, J., Osa, T., Eds.;
Elsevier: Oxford, 1996; Vol. 3, pp 189–203.
24. Brewster, M. E.; Loftsson, T. Adv. Drug Delivery Rev. 2007, 59, 645–666.
25. Higuchi, T.; Kristiansen, H. J. Pharm. Sci. 1970, 59, 1601–1608.
26. Canipelle, M.; Caron, L.; Christine, C.; Tilloy, S.; Monflier, E. Carbohydr. Res.
2002, 337, 281–287.
ecules yields an important reduction in the
DS° and large DH°
values (in absolute value). Two possible orientations for the
inclusion of the drug in the b-CD were considered, and the ‘head
up’ orientation was determined to be the most favorable one. Fi-
nally, it is important to note that the
DG° and DH° obtained pre-
sented negative values in all the cases, indicating that the
inclusion process is favorable and exothermic.
27. Canipelle, M.; Tilloy, S.; Ponchel, A.; Bricout, H.; Monflier, E. J. Inclusion Phenom.
Macrocycl. Chem. 2005, 51, 79–85.
28. Alvira, E. Chem. Phys. Lett. 2007, 439, 252–257.
29. Pinjari, R.; Joshi, K.; Gejji, S. J. Phys. Chem. A 2006, 110, 13073–13080.
30. Del Valle, E. M. M. Process Biochem. 2004, 39, 1033–1046.
31. Daoud-Mahammed, S.; Couvreur, P.; Bouchemal, K.; Chéron, M.; Lebas, G.;
Amiel, C.; Gref, R. Biomacromolecules 2009, 10, 547–554.
Acknowledgment
This work was supported by grants from National University of
San Luis (Argentine Republic).
~
32. Lo´pez-Nicola´s, J. M.; Nu´ nez-Delicado, E.; Pe´rez-Lo´pez, A. J.; Barrachina, A. C.;
Cuadra-Crespo, P. J. Chromatogr., A 2006, 1135, 158–165.
33. Claude, B.; Morin, P.; Lafosse, M.; Andre, P. J. Chromatogr., A 2004, 1049, 37–42.
34. Junquera, E.; Ruiz, D.; Aicart, E. J. Colloid Interface Sci. 1999, 216, 154–160.
35. Saenger, W. Angew. Chem., Int. Ed. Engl. 1980, 19, 344–362.
36. Junquera, E.; Mendicuti, F.; Aicart, E. Langmuir 1999, 15, 4472–4479.
37. Engeldinger, E.; Armspach, D.; Matt, D.; Jones, P. G.; Welter, R. Angew. Chem.,
Int. Ed. 2002, 41, 2593–2596.
38. Engeldinger, E.; Armspach, D.; Matt, D.; Jones, P. G. Chem. Eur. J. 2003, 9, 3091–
3105.
39. Engeldinger, E.; Armspach, D.; Matt, D. Chem. Rev. 2003, 103, 4147–4173.
References
1. Martins, F. T.; Assis, D. M.; dos Santos, M. H.; Camps, I.; Veloso, M. P.; Juliano, M.
A.; Alves, L. C.; Doriguetto, A. C. Eur. J. Med. Chem. 2009, 44, 1230–1239.
2. Wang, F. W.; Ye, Y. H.; Ding, H.; Chen, Y. X.; Tan, R. X.; Song, Y. C. Chem.
Biodiversity 2010, 7, 216–220.
3. Yang, H.; Figueroa, M.; To, S.; Baggett, S.; Jiang, B.; Basile, M. J.; Weinstein, I. B.;
Kennelly, E. J. J. Agric. Food Chem. 2010, 58, 4749–4755.