Structural studies for specific binding capacity of β-cyclodextrin with ibuprofen

Article abstract of DOI:10.1002/jccs.201200071

Ibuprofen (Ibu) and β-cyclodextrin (βCD) and its derivative (hydroxypropyl-β-cyclodextrin, HPβCD) complexes spatial geometry information were studyed. Firstly, phase solubility experiment was carried out for S-(+)-ibuprofen (SIbu) and cyclodextrins comple

Full text of DOI:10.1002/jccs.201200071

JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Article
Structural Studies for Specific Binding Capacity of b-Cyclodextrin with Ibuprofen
Wei Liu,a,c* Yong Zhangb and Bing Zhaoc
aSchool of Pharmacy, Xinxiang Medical University, Eastern JinSui avenue, Xinxiang 453003, P. R. China
bInstitute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, P. R. China
cNew Drug Research and Development Center, School of Pharmaceutical Science, Zhengzhou University, No. 100,
KeXue avenue, Zhengzhou 450001, P. R. China
(Received: Feb. 8, 2012; Accepted: Apr. 10, 2012; Published Online: ??; DOI: 10.1002/jccs.201200071)
Ibuprofen (Ibu) and b-cyclodextrin (bCD) and its derivative (hydroxypropyl-b-cyclodextrin, HPbCD)
complexes spatial geometry information were studyed. Firstly, phase solubility experiment was carried
out for S-(+)-ibuprofen (SIbu) and cyclodextrins complex. The apparent stability constant (Kc) for 1:1
complexes are 1065 M-1 (bCD) and 1476 M-1 (HPbCD) respectively. Secondly, 1H NMR and two-di-
mensional rotating-frame overhauser effect spectroscopy (2D ROESY) were used for binding study, and
confirmed that benzene ring of Ibu is deeply included into the cavity and racemic Ibu (RSIbu) can be dis-
criminated by bCD or HPbCD. Finally, docking model was given by theoretical investigation. The model
with -4.77 kcal/mol binding energy matches experimental structure.
Keywords: NMR; ROESY; Ibuprofen; Molecular docking; Host-guest complex.
INTRODUCTION
Ibuprofen (Ibu) is a representative non-steroidal
and b-cyclodextrin (bCD) will no doubt be important for
pharmaceutical chemistry. The structure of bCD consists
seven a-D-glucose units. It is represented as a truncated
cone structure with a hydrophobic cavity and external
faces. The hydrophobic cavity offers the ability to form in-
clusion complexes by trapping foreign molecules (guest)
into the cavity (host).
anti-inflammatory drug (NSAID) that was first developed
as an antirheumatic drug in the 1960s.1 Ibu is approved as
an oral treatment for mild to moderate pain and for the re-
duction of fever in adults and in children.2,3 Because of its
safety, Ibu is used widely as a non-prescription analgesic.
Ibu is a drug molecule containing one single chiral
center. It is marketed as a racemic mixture though it is
known that SIbu inhibits cyclooxygenase enzymes and
subsequent synthesis of prostaglandins and related com-
pounds at peripheral sites within injured tissue.4,5 Al-
though, the R-(-)-Ibu (RIbu) can be converted to the active
SIbu in vivo at the presence of an isomerase, the research
still proved that half dosage SIbu is equal to racemic mix-
ture.5 More over, RIbu is harder to be cleaned out than SIbu
in vivo. The research of SIbu is more significant than
racemic Ibu (RSIbu).
Here, we study the spatial geometry information of
Ibu and bCD/HPbCD, obtaining apparent stability con-
stant, displaying bCD/HPbCD capability of RSIbu dis-
crimination.
EXPERIMENTAL
Chemical and instruments
Heavy water (D2O) was obtained from Aldrich (St.
Louis). HPbCD (degree of substitution is 6.64) and bCD
were purchased from Deli company (Xi’an, China). SIbu
and RSIbu were purchased from Furen company (Zheng-
zhou, China). All 1H NMR spectra were recorded at 400
MHz NMR spectrometer (Bruker AVANCE III). UV elec-
tronic absorption spectra were determined using UV-2500
(Shimadzu).
Chiral discrimination is one of the most interesting
phenomena and important domains in medicament chemis-
try. More than half of the drugs currently in use are chiral
compounds.6 Chiral cyclodextrins (CD), as the most prom-
inent host molecules used in supramolecular chemistry, has
been used widely as models for chiral molecular recogni-
tion. More over, CD can improve drugs solubility, chemical
stability and bioavailability.7,8,9 by forming inclusion com-
plexes. The study of interaction between optical isomers
UV experiment
A series of CD solution in different concentrations
from 1 to 6 mM were produced in dematerialized water. Su-
perfluous SIbu were dissolved in dematerialized water and
CD solutions. These solutions were ultrasonic dissolved
* Corresponding author. Tel: +86-0373-3831662; E-mail: liuw@iccas.ac.cn
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Article
Liu et al.
for 30 min and placed more than 8 hours in room tempera-
ture. Liquid supernatants were diluted 50 times after centri-
fuging (12000 rpm) 10 min. UV spectrometer was used to
detect absorbency of each solution.
that the absorptivity of Ibu increases with bCD or HPbCD
adding. Fig. 1 shows the relationship of SIbu solubility
along with bCD and HPbCD from 0 to 10 mM. The solubil-
ity of SIbu increased 7 or 9 times in bCD or HPbCD solu-
tions at 6 mM respectively. UV studies showed that the sol-
ubility of SIbu increased linearly, characteristic of a AL
type diagram.10 The linear increasing indicates that SIbu
and CD are combined in 1:1 stoichiometric ratio. The ap-
parent stability constant (Kc) for each complex was calcu-
lated based on the slope of straight-line, according to the
following equation (according to Higuchi and Connors):10,11
NMR experiment
The NMR spectrometer equips with a BBO probe and
a variable temperature unit (VTU). The Spectra were ob-
tained at 400.13 MHz. NMR experiment temperature was
all controlled at 298 K. The solution of RSIbu or SIbu with
bCD at 1:1 stoichiometric ratio was prepared and experi-
mentalized at former 1H NMR conditions.
Two-dimensional rotating-frame overhauser effect
spectroscopy (2D ROESY) was acquired with Bruker stan-
dard parameters (pulse program roesyphsw) for the geome-
try of the inclusion complex. Each spectrum consisted of a
matrix of 2K (F2) by 256 (F1). Size of FID covered a spec-
tral width of 4000 Hz. The spectra were measured with a
spin-lock mixing time (p15 pulse) of 200 ms, relaxation de-
lay 2s. Gaussian apodization functions were applied in both
dimensions.
slope
Kc =
S o (1-slope)
S0 is the intrinsic solubility of Ibu in the absence of
CD. The values of apparent stability constant for a 1:1 com-
plex are 1065 M-1 (bCD) and 1476 M-1 (HPbCD) respec-
tively. Kc values show that the process of the inclusion is
easier for HPbCD than bCD.
1H NMR characterization
Molecular docking
bCD has a hydrophobic cavity with H-3 and H-5 in-
side, which are near the narrower and wider rim respec-
tively, and H-2 and H-4 outside. H-6 is near the narrower
rim and H-1 is near the wider rim. CDs offer different
chemical and magnetic environment inside or outside the
hydrophobic cavity, and the guest inside the cavity can also
change the magnetic environment of H-3 and H-5 which
are inside the cavity. So that the analysis changing of chem-
ical shifts, including host and guest protons, can illuminate
the structure of complex.12,13,14 1H NMR spectra in D2O
have been obtained in order to evaluate the effect of Ibu in-
clusion on the chemical shifts. Table 1 shows the displace-
The initial structures of bCD and Ibu were con-
structed using GaussView3.0. First geometry optimiza-
tions were performed to both structures using Gaussian03
in B3LYP/3-21G level. Then molecular docking was car-
ried out in order to explain the detailed interaction mode
between bCD and Ibu using Autodock4.2. In the docking
process, bCD was thought as the rigid structure, and Ibu
was set to be flexible in which five rotatable bonds were de-
fined as active torsions. After grid point spacing (0.375 Å)
and local search probability (0.8) setting, genetic algorithm
and local search were used as conformational search
method to find the reasonable donor-acceptor location.
Semiempirical Autodock free energy force field, which in-
cluded Van der Waals, hydrogen bond, electrostatics and
desolvation, was used to estimate the interaction free en-
ergy. Docking results were analyzed, and the most possible
bCD-Ibu complex structure is found after considering
experimental data.
RESULTS AND DISCUSSION
Phase solubility
Phase solubility was studied in dynamic equilibrium
which reached up to 8 h after ultrasonic accelerating 30
min. UV method was used to study the phase solubility of
SIbu in different bCD or HPbCD concentrations. The solu-
bility value of SIbu increases with complex informing, so
Fig. 1. Phase solubility analysis for SIbu with bCD (n)
and HPbCD (l).
2
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JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Ibuprofen-cyclodextrin Complex Structural Study
Table 1. 1H chemical shifts of b-CD protons in the inclusion
Table 2. 1H chemical shifts of ibuprofen protons in the inclusion
complexes (Dd = dcomplex - dfree
)
complexes (Dd = dcomplex - dfree)
b-CD complex
Dd
SIbu complex
Dd
Protons of b-CD free
Protons SIbu free
(ppm)
(ppm)
(ppm)
(ppm)
b-CD
(ppm)
of Ibu
(ppm)
SIbu
RSIbu
SIbu
RSIbu
bCD
HPbCD
bCD
HPbCD
H-1
H-2
H-3
H-4
H-5
H-6
4.9451
3.5045
3.8604
3.4410
3.7890
3.7850
4.9298
3.4945
3.7771
3.4526
3.5783
3.7026
4.9330 -0.0153 -0.0121
3.4990 -0.0100 -0.0055
3.7824 -0.0833 -0.0780
H-a
H-b
H-e
H-f
H-h
H-i
H-j
H-k
1.3054
3.5288
7.1868
7.1296
2.3959
1.7556
0.7926
0.7926
1.3059
-
1.3122
-
0.0005
-
0.0068
-
7.1610
6.9849
2.3907
1.7529
0.8282
0.8252
7.1615
6.9840
2.4044
1.7870
0.8710
0.8642
-0.0258 -0.0253
-0.1447 -0.1456
-0.0052
-0.0027
0.0356
0.0326
3.4554
0.0116 0.0144
3.5857 -0.2107 -0.2033
3.7090 -0.0824 -0.0760
0.0085
0.0314
0.0784
0.0716
ments of the chemical shifts for free bCD protons and bCD
complex with SIbu or RSIbu. Upfield displacement for H-3
and 6, especially H-5, was observed clearly. H-1, 2 and 4
protons which are outside the cavity showed small chemi-
cal shifts. It suggests that aromatic ring of Ibu which is rich
in p electrons is included into bCD hydrophobic cavity.
In the presence of bCD, chemical shifts of all SIbu
protons, which serial number is showed in the insert of Fig.
3, were changed to upfield (H-e, f, h, i) or downfield (H-a,
j, k) compared to the pure drug (showed in Table 2). The
upfield displacement protons indicated that this moiety was
located in a rich electronic density environment that pro-
duced a shielding effect. It suggests that the aromatic ring
(H-e,f) may have been included into the bCD hydrophobic
cavity. A lower upfield displacement indicates that H-h and
H-i were still buried into the bCD cavity, but were closer to
the molecule torus rim. SIbu protons chemical shifts in
HPbCD were same to in bCD except H-h and i which were
downfield displacement. It means that H-h and i of SIbu in
bCD are richer in p electrons than in HPbCD.
plex. Free racemic mixture could not be discriminated (Fig.
2A) by NMR directly. However the use of chiral recogni-
tion agent such as CD can help to resolve the matter. H-a
proton of RSIbu is split in the present of bCD and HPbCD,
and H-e proton is split in present of bCD (Fig. 2D and E).
This phenomenon shows that SIbu and RIbu are different in
combination with bCD or HPbCD. H-a proton of SIbu and
RIbu are in different magnetic environment, while H-e pro-
ton of SIbu and RIbu are in the same magnetic environmen.
It provides theory proof for racemic mixture discrimination
and seperation by bCD or HPbCD. We also seen H-j and k
protons of SIbu been discriminated in the present of
HPbCD according to the peaks split. We thought the steric
effect that lead the H-j and k protons to spin slower and
caused the magnetic environment different, so that the
chemical shifts of H-j and k protons are discriminated. H-j
and k protons of SIbu are also in different magnetic envi-
Fig. 2 shows NMR signals of free Ibu and each com-
Fig. 3. Expansion 2D ROESY spectrum of SIbu-bCD
complex. F1 axis shows protons H-3, H-5 and
H-6 of bCD, and F2 axis shows protons H-e, f,
h, i, a, k and j of SIbu.
Fig. 2. 1H NMR for free Ibu and host-guest complexes.
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Article
Liu et al.
ronment in present of bCD. However discrimination capa-
bility is lower in present of bCD than HPbCD because the
peak of H-j and k of SIbu is only wider but not split.
2D ROESY experimental
CONCLUSIONS
Phase solubility experiment shows that SIbu forms
aqueous soluble 1:1 type complexes with bCD or HPbCD.
HPbCD solution brings solubility increasing higher than
bCD. The values of Kc for 1:1 complex are 1065 M-1 (bCD)
and 1476 M-1 (HPbCD), respectively. The different of Kc
More detail spatial interaction and corresponding
three-dimensional geometry information between host and
guest complex are given by ROESY spectra. The cross
peaks for protons, which the corresponding internuclear
distance is smaller than 3~4 Å, can be observed in 2D
ROESY. Fig. 3 shows the expansion of the ROESY spec-
trum. We do not study the H-b proton of SIbu interaction
with bCD because H-b proton is covered by bCD protons
peaks. Cross peaks between H-e and H-f protons of SIbu
and H-3, 5 and 6 protons of bCD were found, indicating
that aromatic protons of SIbu are deeply included into bCD
cavity. Cross peaks between H-h and i protons of SIbu and
H-3, 5 and 6 protons of bCD were also found, however the
cross peak intensity of H-h is larger than of H-i proton. This
suggests H-i proton is closer to the median line of cavity,
thus far away from H-3, 5 and 6, and H-h is closer to H-3, 5,
and 6. Cross peaks between H-j, k and H-3, 5 and 6 suggest
that methyl group is also inserted into the bCD cavity.
Cross peak between SIbu proton and H-6 suggest that H-e
to k are close to the narrower rim. None cross peak was
found between H-a and H-3, 5, and 6, that suggests H-a
proton is outside bCD cavity and near the wider rim.
Theoretical Investigation
1
values suggest the different inclusion process. The H
NMR shows different inclusion structure of SIbu-bCD and
SIbu-HPbCD complexes. 1H NMR also shows H-a and H-e
of RSIbu are split. This means RIbu and SIbu are discrimi-
nated by HPbCD or bCD. 1H NMR and ROESY show aro-
matic nucleus of Ibu is included into bCD cavity, and theo-
retical investigation manifest that SIbu-bCD docking
model with -4.77 kcal/mol binding energy matches experi-
mental structure.
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