Proparacaine complexation with β-cyclodextrin and p-sulfonic acid calix[6]arene, as evaluated by varied 1H-NMR approaches

Article abstract of DOI:10.1002/mrc.2460

This study focused on the use of NMR techniques as a tool for the investigation of complex formation between proparacaine and cyclodextrins (CDs) or p-sulfonic acid calix[6]arene. The pHdependence of the complexation of proparacainewith β-CD and p-sulfoni

Full text of DOI:10.1002/mrc.2460

Research Article
Received: 18 January 2009
Revised: 29 April 2009
Accepted: 5 May 2009
Published online in Wiley Interscience: 25 June 2009
(www.interscience.com) DOI 10.1002/mrc.2460
Proparacaine complexation with
β-cyclodextrin and p-sulfonic acid
calix[6]arene, as evaluated by varied 1H-NMR
approaches
a
c
b
´
Lucas Micqueias Arantes, Camilla Scarelli, Anita Jocelyne Marsaioli,
Eneida de Paula,c and Sergio Antonio Fernandesa∗
This study focused on the use of NMR techniques as a tool for the investigation of complex formation between proparacaine
and cyclodextrins (CDs) or p-sulfonic acid calix[6]arene. The pH dependence of the complexation of proparacaine with β-CD and
p-sulfonic acid calix[6]arene was studied and binding constants were determined by 1H NMR spectroscopy [diffusion-ordered
spectroscopy (DOSY)] for the charged and uncharged forms of the local anesthetic in β-CD and p-sulfonic acid calix[6]arene.
The stoichiometries of the complexes was determined and rotating frame Overhauser enhancement spectroscopy (ROESY) 1D
experiments revealed details of the molecular insertion of proparacaine into the β-CD and p-sulfonic acid calix[6]arene cavities.
The results unambiguously demonstrate that pH is an important factor for the development of supramolecular architectures
based on β-CD and p-sulfonic acid calix[6]arene as the host molecules. Such host–guest complexes were investigated in view
of their potential use as new therapeutic formulations, designed to increase the bioavailability and/or to decrease the systemic
c
toxicity of proparacaine in anesthesia procedures. Copyright ꢀ 2009 John Wiley & Sons, Ltd.
Keywords: proparacaine; cyclodextrin; calixarenes; NMR
Introduction
p-sulfonic acid calix[6]arene 3 (Scheme 1) provides important
information to optimize its possible future use, which requires a
betterknowledgeofthemolecularpropertiesofthecomplexes.[31]
Crucial information for the characterization of these complexes in-
clude the stoichiometry, the complexed population (%pbound), the
formation/dissociation constant (Ka) and the relative positioning
of the carrier/guest inclusion complex, which can be determined
from pulsed field gradient spin-echo(PGSE)[32–36] and nuclear
Overhauser effect (NOE)[37] experiments, respectively.
The main objective of this investigation was the analysis of
the complexation by β-CDs 2 or p-sulfonic acid calix[6]arene 3
(Scheme 1) of the local anesthetic proparacaine 1 in an aqueous
medium under various experimental conditions (pH 5 and pH
10). Using the Job plot method, PGSE and NOE NMR approaches,
we obtained large variety of information about the molecular
interactions that drive the complexation process of proparacaine.
The formulation of drugs is an important topic in pharmaceutical
sciences. Many therapeutically active molecules are chemically
and biologically fragile; so they need to be encapsulated in a
drug carrying system to improve their physicochemical stability.
The desirable features of drug carriers in drug-delivery systems
include controlled release, targeting and absorption enhancing
abilities. From the safety point of view, bio-adaptability is an
important requirement for drug carrier systems, as well as
high quality and low cost.[1,2] These carriers protect the loaded
drug against degradation, the guest molecule being transported
effectivelyinbiologicalmedia.[3] Thedevelopmentofsuchsystems
include cyclodextrins (CDs),[4–9] calix[n]arenes,[10–15] micelles,[16]
liposomes,[17,18] micro-[19] and nanoparticles[20] and solid lipid
nanoparticles[21–23] as drug carriers.
CD was one of the first molecular receptors (‘host’ molecule)
whose ability to bind organic (guest) compounds was recognized
andextensivelystudiedbyvariousexperimentaltechniques.[24–26]
Consequently, CDs have pharmaceutical applications as high per-
formance biomaterials in drug-delivery systems due to their ability
to change physical, chemical and biological properties of guest
molecules through the formation of inclusion complexes.[27–29]
Surprisingly, calix[n]arenes,[30] organic macrocyclic host
molecules formed by the ortho-condensation of para-substituted
phenols and formaldehyde, are well investigated in supramolecu-
lar chemistry but less studied with respect to their drug-delivery
properties.[10]
∗
Correspondence to: Sergio Antonio Fernandes, Departamento de Química,
´
Universidade Federal de Vic¸osa (UFV), Campus Universitario, Avenida P.H.
Rolfs, s/n, Vic¸osa, MG, 36570-000, Brazil. E-mail: santonio@ufv.br
´
a
b
c
Grupo de Química Supramolecular e Biomimetica (GQSB), Departamento de
Química, Universidade Federal de Vic¸osa (UFV), Vic¸osa, 36570-000, MG, Brazil
ˆ
Departamento de Química Organica, Instituto de Química, Universidade
Estadual de Campinas, Brazil
The characterization of the delivery systems formed by com-
plexation of the local anesthetic proparacaine 1, in β-CDs 2 or
Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de
Campinas, Brazil
c
Magn. Reson. Chem. 2009, 47, 757–763
Copyright ꢀ 2009 John Wiley & Sons, Ltd.

L. M. Arantes et al.
OH
O
O
HO
O
OH
HO
OH
O
OH
SO3H
SO3H
SO3H
SO3H
HO
SO3H
OH
HO
SO3H
O
OH
O
6
3'
O
O
N
7'
O
5
1
2
OH
6'
4'
10
8
HO
O
OH
O
OH
OH
OH
9
OH
O
OH
OH
NH2
OH
3
OH
OH
1
O
O
OH
OH
O
O
OH
HO
O
OH
2
Scheme 1. Chemical structure of proparacaine (1), -cyclodextrin (2) and p-sulfonic acid calix[6]arene (3).
Results and Discussion
determined (D1 (pH 5) = 5.30 × 10−10, D2 (pH 5) = 3.27 × 10−10
,
=
D3 (pH 5) = 3.05 × 10−10, D1 (pH 10) = 4.72 × 10−10, D2 (pH 10)
3.16 × 10−10 and D3 (pH 10) = 3.08 × 10−10m2 s−1, respectively,
We determined the ionization constant of proparacaine by the
pH-titration method as 8.33. From that we designed experiments
at pH 5 and 10 to evaluate the complexation of charged and
uncharged proparacaine 1 species, with β-CDs 2 or p-sulfonic acid
calix[6]arene 3. Bothanestheticformsseemtobeimportantforthe
anesthesia mechanism, with the uncharged species binding more
strongly to the membranes while the protonated form has the
necessary water solubility for the anesthetic to cross the biological
compartments and reach the targeted axonal membranes in
sufficient concentration.[38–40]
Table 3). In the presence of 2 or 3, compound 1 showed a
significantreductioninitsdiffusionrate(D1/2 (pH 5) = 4.73×10−10
,
D1/3 (pH 5) = 3.32 × 10−10, D1/2 (pH 10) = 4.13 × 10−10 and
D1/3 (pH 10) = 4.36×10−10 m2 s−1, Table 3), indicating that1forms
host–guest complexes with either 2 or 3, at both pH. Moreover, the
diffusion rate values (Table 3) of 1 in the 1/2 complex at pH 10 is in
agreementwith the assumption of a strong association. In the case
of the 1/3 complex a more pronounced variation was observed in
the diffusion coefficient of compound 1 at pH 5 (Table 3).
We started our investigation by analyzing the complexation-
induced hydrogen chemical shifts (ꢀδ) in the 1/2 and 1/3
complexes, relative to free 1.
Taking into account that we are dealing with a system under fast
equilibrium on the NMR scale, both chemical shifts and diffusion
coefficients of 1 are the average values of the free and bound
species. From these diffusion coefficients, and applying a well
established methodology[44] we have calculated the complexed
population (%pbound) and apparent binding constants (Ka) of the
complexes (Table 3). %pbound was found to be 22 and 29% for 1/2,
and 73 and 14% for 1/3 at pH 5 and pH 10,−r1espectively. The values
of Ka at both conditions (181 and 288 M for 1/2, respectively)
confirmed that more association took place between the neutral
form of proparacaine (pH 10) and β-CD than between charged 1
and 2 (pH 5). In an earlier work, conducted at pH 7, an association
constant (Ka = 208 M−1) has been reported for the 1/2 complex,
in good agreement with the values found in this study.[18] Studies
conducted for 1/3 revealed a strong association between the
charged proparacaine species 1 and p-sulfonic acid calix[6]arene
3 (Ka = 5007 M−1 at pH 5) and a diminished association between
the uncharged proparacaine 1 and 3 (Ka = 11 M−1). The results
obtainedfrommeasurementsinanacidicmedium(pH5)showthat
the protonated proparacaine molecule forms strong complexes
with p-sulfonic acid calix[6]arene 3, as opposed to the association
of charged 1 with β-CD 2.
On the other hand, neutral molecules of proparacaine 1 (pH 10)
showed a higher affinity for the β-CD cavity than those bearing
formal charges. Moreover, the ester group of 1 became fully
protected from nucleophilic attack of HO−, the complexed drug
being unreactive to alkaline hydrolysis (not shown) as observed
earlier for another ester-type local anesthetic, tetracaine.[10]
Further details on the 1/2 (pH 10) and 1/3 (pH 5) molecular
complexation were obtained from rotating frame Overhauser
enhancement spectroscopy (ROESY)-NMR experiments.
The hydrogens of 1 displayed discrete chemical shift variations
(≤0.04 ppm) in the presence of 2 complex (1/2) in pH 5 (Table 1).
When the (1/2) complex was prepared in pH 10, the hydrogen
chemical shift variations of 1 were more pronounced (up to
0.12 ppm, Table 2). This fact can be rationalized by the smaller
water solubility of the uncharged proparacaine 1 species at pH
10, which favors the inclusion of 1 into the hydrophobic cavity of
β-CD 2 at alkaline but not at acidic pH.
Complexation between 1 and 3 at acidic pH induced large
shielding effects in all hydrogens of 1, mainly H-3ꢁ, H-4ꢁ, H-6ꢁ and
H-7ꢁ (ꢀδ = 0.74, 0.38, 0.84 and 0.42 ppm, respectively – Table 1),
revealing interactions between the charged ammonium group of
1 (pH 5) and the SO3H group of 3 (Table 1).[10,41]
As expected, smaller hydrogen chemical shifts changes were
observed between free 1 and 1/3 complex at pH 10, when
proparacaine is in its neutral form (Table 2) than at pH 5.
We further established the stoichiometries for the 1/2 and 1/3
complexes (pH 5 and pH 10), using the Job plot method.[42,43] The
plots obtained from the NMR analyses indicated the predominant
formation of 1 : 1 complexes, for 1/2 and 1/3, at both pH. A
representative Job plot experiment is illustrated in Fig. 1.
Diffusion-ordered spectroscopy (DOSY) NMR experiments were
pivotal to demonstrate that 1/2 and 1/3 form stable complexes.
These experiments allow distinguishing compounds or complexes
by their differences in diffusion coefficients.[14] Representative
spectra are given in Figs 2 and 3 for pure 1 and for the 1/2
complex (2 mmol l−1 samples, 298 K, respectively). The diffusion
coefficients of pure 1, 2 and 3 (at pH 5 and 10) were first
c
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Copyright ꢀ 2009 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2009, 47, 757–763

NMR in proparacaine complexation
Table 1. 1H NMR: Chemical shifts (δ) and chemical shift differences (ꢀδ = δ1 free − δ1 complex) of pure 1 and its complexes with β-CD 1/2 or p-sulfonic
acid calix[6]arene 1/3, at pH 5 (2 mmol l−1 samples, 298 K)
1
1/2
1/2
1/3
1/3
Hydrogen
δ
δ
ꢀδ = δ1 free − δ1 complex
δ
ꢀδ = δ1 free − δ1 complex
H-10
H-7ꢁ
H-9
H-6ꢁ
H-3ꢁ
H-8
H-4ꢁ
H-5
H-2
H-6
0.92
1.24
1.73
3.25
3.54
4.02
4.55
6.93
7.38
7.46
0.92
1.24
1.73
3.25
3.55
4.02
4.56
6.89
7.38
7.44
0.00
0.00
0.00
0.00
0.01
0.00
0.01
0.04
0.00
0.02
0.65
0.83
1.61
2.41
2.80
3.87
4.17
6.83
7.39
7.46
0.27
0.42
0.12
0.84
0.74
0.15
0.38
0.10
−0.01
0.00
Table 2. 1H NMR: Chemical shifts (δ) and chemical shift differences (ꢀδ = δ1 free − δ1 complex) of pure 1 and its complexes with β-CD 1/2 or p-sulfonic
acid calix[6]arene 1/3, at pH 10 (2 mmol l−1 samples, 298 K)
1
1/2
1/2
1/3
1/3
Hydrogen
δ
δ
ꢀδ = δ1 free − δ1 complex
δ
ꢀδ = δ1 free − δ1 complex
H-10
H-7ꢁ
H-9
H-6ꢁ
H-3ꢁ
H-8
H-4ꢁ
H-5
H-2
H-6
0.91
1.01
1.72
2.68
2.95
4.01
4.35
6.92
7.37
7.44
0.92
1.02
1.73
2.65
2.91
4.02
4.35
6.85
7.32
7.32
−0.01
−0.01
−0.01
0.03
0.74
0.89
1.68
2.38
2.75
3.96
4.22
6.86
7.33
7.40
0.17
0.12
0.04
0.30
0.20
0.05
0.13
0.06
0.04
0.04
0.04
−0.01
−0.00
0.07
0.05
0.12
respectively) of β-CD 2. At pH 5, the same increments were
observed in the rOe between the hydrogens of proparacaine 1
and β-CD 2, but with smaller intensities. We therefore suggested
the presence of two species, A and B (Fig. 4) on the basis of
molecular models and the nuclear Overhauser enhancements.
The proposed topologies are in agreement with a recent work
published by Marsaioli and collaborators.[18]
30
28
26
24
22
20
18
16
The signal enhancements in 1/3 (rOe) between H-6ꢁ and H-7ꢁ
of proparacaine 1 (1.27 and 0.52%, respectively, at pH 5) and
H-Ar of the p-sulfonic acid calix[6]arene 3 indicate the binding of
the ammonium group of 1 with the SO3H group of 3. This was
confirmed by experiments performed at pH 10.0, which revealed
that the association constant between 1 and 3 decreased for
−1
Ka = 11 M in relation to that in pH 5 (Ka = 5007 M−1). The two
proposed topologies, C and D (Fig. 5) for the 1/3 complexes at pH
5 were established using ROESY 1D.
0.2
0.3
0.4
0.5
[1]/([2]+[1])
0.6
0.7
0.8
Figure 1. Representative Job plot for the complex formed between
proparacaine1andβ-CD2.ChemicalShiftsweremeasuredat499.885 MHz
in D2O, at 298 K and pH 10, in 2 mmol l−1 samples).
Conclusion
Several noncovalent weak forces, including electrostatic, hy-
drophobic, π-π interaction and van der Waals, cooperatively
contribute to the formation of the supramolecular complexes.
Among these, electrostatic and hydrophobic interactions as well
as structural matching effect are thought to play principal roles in
forming the supramolecular systems 1/2 and 1/3.
Specific rOe signals were observed between H-2/H-5, H-6 and
H-8 of 1 (pH 10), and H-3 (enhancement of 0.57, 1.36, 0.47%,
respectively) and H-5 (0.80, 1.73, 0.36% of signal enhancement
c
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L. M. Arantes et al.
Figure 2. Representative 1H DOSY NMR experiment of pure 1 (499.885 MHz, D2O, 298 K, pH 10, 2 mmol l−1).
Figure 3. Representative 1H DOSY NMR experiment for the 1/2 complex (499.885 MHz, D2O, 298 K, pH 10, 2 mmol l−1).
c
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NMR in proparacaine complexation
Table 3. Diffusion coefficients of pure 1, 2, 3 and 1/2 and 1/3 complexes in D2O (2 mmol l−1 samples, 298 K) at pH 5 and 10
O
2
−1
)
Complex
–
Compounds
Condition (pH)
D (10−10 m2 s−1
)
D/DH
%p
Ka (M
1
1
2
2
3
3
1
2
1
3
1
2
1
3
5
10
5
5.30 0.04
4.72 0.03
3.27 0.03
3.16 0.02
3.05 0.02
3.08 0.01
4.73 0.05
3.30 0.01
3.32 0.03
2.85 0.02
4.13 0.03
3.08 0.02
4.36 0.07
3.13 0.04
0.24
0.22
0.16
0.15
0.14
0.15
0.22
0.15
0.16
0.13
0.20
0.15
0.21
0.15
–
–
–
–
–
–
–
–
–
–
–
10
5
–
–
10
5
–
1/2
1/3
1/2
1/3
22
181
5
73
29
14
5007
288
11
10
10
N
O
O
O
H
H2N
H
5
3
3
3
3
H
H
H
5
5
H
5
H
H
6
6
6
6
NH2
O
O
HOH2C
HOH2C
CH2OH
CH2OH
O
N
B
A
Figure 4. Proposed topology for the uncharged proparacaine/β-CD (1/2 complex at pH 10), based on 1H NMR evidences.
In summary, complexes possessing different degrees of com-
pactness were constructed by the complexation of 2 and 3 with
proparacaine 1, at pH 5 and pH 10. Increasing the alkalinity of the
medium with carbonate buffer favored the complexation of 1 in
2 (Ka = 288 M−1) in relation to that in pH 5 (Ka = 181 M−1) and
prevented the alkaline hydrolysis of 1. However the complexation
of 1 and 3 at pH 10 (Ka = 11 M−1) is weaker than that observed at
pH 5 (Ka = 5007 M−1), which is mainly a result of the protonation
of the amine groups of 1, and binding of such +NHR2 group of
1 to the SO3H groups of 3. These observations unambiguously
demonstrate that the ionization state of the guest molecule is a
crucial factor in the design of supramolecular architectures based
on 2 and 3 as the host molecules.
govern complexation are different for 1/2 and 1/3: hydrophobic
interactions for the 1/2 complex and ionic-pair formation for the
1/3 complex.
Experimental
Chemicals and reagents
Proparacaine hydrochloride 1 (99%), β-CD 2 (99%), and D2O
(99.75%) were purchased from Aldrich, Acros Organics and Merck,
respectively. All other reagents were of analytical grade. p-sulfonic
acid calix[6]arene 3 was synthesized in our laboratory following
literature procedures.[45–47]
The topology of the complexes (Figs 4 and 5) was determined
from the 1D ROESY NMR results, revealing that the forces that
c
Magn. Reson. Chem. 2009, 47, 757–763
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L. M. Arantes et al.
O
H2N
O
O
HO3S
HO3S
HO3S
HO3S
SO3H
SO3H
SO3H
SO3H
N
SO3H
H-3
SO3H
H-3
HO3S
HO3S
H
O
H
N
O
CH2
CH2
HO
HO
OH
OH
HO
HO
OH
OH
H2N
OH
OH
HO
HO
D
O
C
Figure 5. Proposed topologies for the charged proparacaine/p-sulfonic acid calix[6]arene (1/3 complex, pH 5) in fast equilibrium, according to 1H NMR
evidences.
Preparation of solid inclusion complexes
Routine 1D
1H experiments were acquired either with an INOVA-500 Varian
spectrometer operating at 499.885 MHz for 1H (64 k data points,
30◦ excitation pulse with duration of 2.2 µs, spectral width of
6 kHz, acquisition time of 3.3 s and relaxation delay of 10 ms) in a
5-mm probe with inverse detection mode at room temperature
unless stated otherwise.
Inclusion complexes (1/2 or 1/3) with 1 : 1 molar ratios were
prepared by shaking appropriate amounts of 1 and 2 or 3, e.g.
2 mmol l−1, in deionized water at room temperature (298 1 K)
for 1 h. Kinetic experiments revealed that equilibrium was reached
after 40 min (data not shown).
After reaching equilibrium, the solution was freeze-dried in a
Labconco Freeze-dry system (Freezone 4.5) and stored at 253 K
until further use.
NOE measurements
The ROESY 1D experiments were obtained with a selective 180◦
and a nonselective 90◦ pulse, with a mixing time of 0.5 s during
the spin-lock. The selective pulses were generated by a waveform
generator which automatically attenuates the shape, power, and
pulse duration to obtain the required selectivity. The subtraction
of the on- and off-resonance acquisition furnished the ROESY 1D
experiment. All spectra were acquired with a 5-mm inverse probe
at 298 K in 5-mm tubes.
NMR Spectroscopy
All experiments were performed at 298 K in D2O. For the
experiments with uncharged 1, the pH value of the solutions
was adjusted by addition of lyophilized 0.02 mol l−1 carbonate
buffer resuspended in D2O.
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NMR in proparacaine complexation
HR-DOSY experiments were carried out by carefully choosing
the correct pulse sequence and gradients for the experiments.
The measurements were made using: (i) 5 mm inverse probe with
z-gradient coil; (ii) the Gradient Compensated Stimulated Echo
Spin Lock (GCSTESL) HR-DOSY sequence; (iii) amplitudes of the
gradient pulses ranging from 0.000685 to 0.003427 T cm−1, where
anapproximately90–95%decreaseintheresonanceintensitywas
achieved at the largest gradient amplitudes. For all experiments,
25 different gradient amplitudes were used. The baselines of all
spectra were corrected prior to data processing. The processing
program (the DOSY macro in a Varian instrument) involves the
determination of the peak heights of all signals above a pre-
established threshold and the fitting of the decay curve for each
peak to an exponential decay. The DOSY macro was run with
data transformed using fn = 64 K. Very crowded spectra were
processed in sections due to the limitation of handling only 512
lines at a time. The results of the DOSY method of analysis are
pseudo two-dimensional spectra with NMR chemical shifts along
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Acknowledgments
ThisworkwassupportedbyFAPEMIG(grantsEDT-346/07), FAPESP
(Proc. 2005/00602-4) and CNPq (E.P. fellowship). The authors also
thank Prof. Carol Collins for English text revision.
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