1H NMR spectroscopic characterization of inclusion complexes of tolfenamic and flufenamic acids with β-cyclodextrin

Article abstract of DOI:10.1016/j.molstruc.2012.11.021

The complexation between the anionic forms of tolfenamic acid and flufenamic acid with β-cyclodextrin was investigated in solution by 1D and 2D proton NMR spectroscopy. The stoichiometry of the complexes was determined by the method of continuous variation using the chemical induced shifts of both the host and guest protons. An analysis of the spectroscopic data revealed that simultaneous inclusion of both rings of tolfenamic and flufenamic acids occur, giving rise each to two isomeric 1:1 complexes. The view of a bimodal binding between these two drugs and β-cyclodextrin was also supported by ROESY experiments. Using a rough approximation, we have estimated the association constants order of magnitude of the 1:1 complexes.

Full text of DOI:10.1016/j.molstruc.2012.11.021

Journal of Molecular Structure 1044 (2013) 72–78
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
1H NMR spectroscopic characterization of inclusion complexes of tolfenamic
and flufenamic acids with b-cyclodextrin
⇑
C.G. Floare, A. Pirnau, M. Bogdan
Department of Molecular and Biomolecular Physics, National Institute for Research and Development of Isotopic and Molecular Technologies, P.O. Box 700, 400293
Cluj-Napoca, Romania
h i g h l i g h t s
"
"
"
"
We report the complexation process between tolfenamic and flufenamic acid with b-CD.
Induced chemical shifts variation and ROESY spectra sustain a bimodal complexation.
The entrance of both aromatic rings occurs through the secondary rim of the b-CD.
The microscopic association constants order of magnitude was evaluated.
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 22 November 2012
The complexation between the anionic forms of tolfenamic acid and flufenamic acid with b-cyclodextrin
was investigated in solution by 1D and 2D proton NMR spectroscopy. The stoichiometry of the complexes
was determined by the method of continuous variation using the chemical induced shifts of both the host
and guest protons. An analysis of the spectroscopic data revealed that simultaneous inclusion of both
rings of tolfenamic and flufenamic acids occur, giving rise each to two isomeric 1:1 complexes. The view
of a bimodal binding between these two drugs and b-cyclodextrin was also supported by ROESY exper-
iments. Using a rough approximation, we have estimated the association constants order of magnitude of
the 1:1 complexes.
Keywords:
b-Cyclodextrin
Tolfenamic acid
Flufenamic acid
Inclusion complexes
Stoichiometry
Isomers
Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction
modified ones, may resolve this problem and may also reduce
the irritation or local damage of the gastrointestinal mucosa.
Tolfenamic acid N-(3-chloro-2-methyl-phenyl) anthranilic acid
(TA) and flufenamic acid N-(3-trifluoromethyl-phenyl) anthranilic
acid (FF), (see Fig. 1), are nonsteroidal anti-inflammatory drugs
(NSAIDs), which along with the other derivatives of anthranilic
acid (mefenamic and meclofenamic acid) are widely used as anal-
gesic, anti-inflammatory and antipyretic drugs. These agents inhi-
bit the biosynthesis of prostaglandins (PGs), as a consequence of
interfering within the arachidonic acid cascade, as well as the PG
receptors in certain tissues [1]. Like many NSAID drugs, TA and
FF are very sparingly soluble in water. Therefore aqueous solubility
and wettability of these fenamates gives rise to difficulties in phar-
maceutical formulations for oral or parenteral delivery, which may
lead to variable bioavailability.
Cyclodextrins are naturally occurring cyclic oligosaccharides
[2], known for their effect on stability, solubility and bioavailability
of various drugs, as well as for the reduction of drugs side-effects
[3]. Their unique macrocyclic structure is a result of the stable
chair conformation of the constituent
D-glucopyranosyl residues
and -1,4 glicosidic bonds linking the residues with each other.
a
The shape of naturally occurring cyclodextrins can be represented
as a toroidal hollow truncated cone having many primary and sec-
ondary hydroxyl groups crowning the narrower and wider rims of
the hollow cone, respectively. Methine protons and glycoxidic oxy-
gen atoms are located inside the hollow cone, colled the cavity.
Hence the interior is relatively apolar and hydrophobic, whereas
the exterior is relatively polar and hydrophilic. CDs are capable
of forming inclusion complexes with many drugs by taking up a
whole drug molecule, or some part of it, into the cavity. Such
molecular encapsulation will affect many of the physicochemical
properties of the drugs, such as their aqueous solubility and rate
of dissolution. During the drug–CD complex formation, no covalent
bonds are formed and the complex is readily dissociated and the
To overcome these drawbacks, increasing the aqueous solubility
of TA and FF is an important goal. The complexation of TA and FF
with naturally occurring
a, b and c-ciclodextrins, or chemically
⇑
Corresponding author. Fax: +40 264 420042.
E-mail address: mircea.bogdan@itim-cj.ro (M. Bogdan).
0022-2860/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molstruc.2012.11.021

C.G. Floare et al. / Journal of Molecular Structure 1044 (2013) 72–78
73
1H NMR spectroscopy is one of the most important methods to
quantitatively investigate the formation of CD inclusion complexes
in solution [9–11]. This paper describes the 1H NMR study of the
inclusion of TA and FF with b-CD. Firstly the stoichiometry of the
complexes was investigated using the current Job plot method.
The type of inclusion process of TA and FF in b-CD was confirmed
by 2D ROESY spectroscopy. Using a simplifying approximation, an
attempt was made in order to assess the association constants or-
der of magnitude for each 1:1 complex, based on the chemical shift
variation of some representative protons belonging to the drug
molecules.
HO
O
R1
H
N
10
R2
2
3
9
6
5
8
7
4
Fig. 1. TA and FF chemical structure and proton notations. TA: R1 = CH3, R2 = Cl; FF:
R1 = H, R2 = CF3.
2. Materials and methods
free drug molecules are in rapid equilibrium with the drug mole-
2.1. Materials
cules bound within the CD cavity. Among
a-, b- and c-CD, b-CD
is by far the most widely used compound owing to the optimal size
of its internal cavity for the encapsulation of NSAIDs.
Tolfenamic acid, flufenamic acid and b-cyclodextrin (water con-
tent 8 mol/mol) were purchased from Sigma–Aldrich Chemie
GmbH and used without further purification. The b-CD water con-
tent was taken into account in the calculation of solute concentra-
tion. The deuterium oxide (99.7% D) was obtained from Heavy
Water Plant Romag-Prod, Romania.
Various studies have been carried out on the inclusion of TA and
FF by naturally occurring CDs or chemically modified ones in aque-
ous media and in solid state. Several techniques have been pro-
posed for complex preparation such us freeze-drying [4],
kneading [5], or the development of a mucoadhesive film [6], based
on chitosan as polymer and glicerol as plasticizer, containing FF as
complex with HP-b-CD. During the physicochemical characteriza-
tion of the inclusion complexes by differential scanning calorime-
try and powder X-ray diffractometry, [5] or with spectroscopic
and chromatographic methods [7], significant ariations of some
experimental parameters were observed and attributed to the
complexation process. Thus, the TA was complexed with various
CDs (b-CD, HP-b-CD and Me-b-CD) and the stoichiometry of the
TA:Me- b-CD complex was determined to be 1:2, using the contin-
uous variation technique, [7]. A validated HPLC method was also
applied to obtain the binding constants of the complexes and to
evaluate the energetics of the system [8].
2.2. Sample preparation
In order to study the complexation process between TA, FF and
b-CD, three stock solutions in D2O, having 10 mM was prepared.
The TA and FF stock solutions were prepared containing 5 ll/ml
of NaOD 40% for dissolution of the drugs. Based on these equimolar
solutions, two series of samples containing b-CD and TA or FF were
prepared. This was accomplished by mixing two solutions to con-
stant volume at varying proportions so that a complete range
(0 < r < 1) of the mole fraction r = [X]/([G]+[H]) was sampled.
X = H or G and [H] and [G] are the concentrations of the host
[TA] = 10 mM
CH3
H6
H7
H2
H5
H3
H8
H4
2.15
7.7
7.6
7.5
7.4
7.3
7.2
7.1
7.0
6.9
6.8
ppm
ppm
[TA] = [ -CD] = 5mM
CH3
H8
H2
H6
H7
H3
H5
H4
7.8
7.7
7.6
7.5
7.4
7.3
7.2
7.1
7.0
6.9
2.30
ppm
ppm
Fig. 2. 1H NMR spectra of TA, in the absence as well in the presence of b-CD.

74
C.G. Floare et al. / Journal of Molecular Structure 1044 (2013) 72–78
H5
[FF] = 10 mM
H4
H10
H2
H3
H7
H6
H8
7.7
7.6
7.5
7.4
7.3
7.2
7.1
7.0
ppm
H3
H2
H10
[TA] = 7 mM; [ -CD] = 3
H5
H7
H4
H6
H8
7.8
7.7
7.6
7.5
7.4
7.3
7.2
7.1
7.0
ppm
Fig. 3. 1H NMR spectra of FF, in the absence as well in the presence of b-CD.
(b-CD) and guest (TA or FF), respectively. Thus the total concentra-
tion [H]t + [G]t = 10 mM was kept constant for each solution. The
two sets of samples were used both for determination of stoichi-
ometry and the evaluation of the association constants.
3.90
3.85
3.80
3.75
3.70
3.65
3.60
H3
H5
H6
2.3. Nuclear magnetic resonance measurements
All 1H NMR measurements were carried out on a Bruker
AVANCE III spectrometer operating at 500.13 MHz and equipped
with a broad band observe probe. The chemical shifts were refer-
enced to TMS. In all experiments the temperature was maintained
at 298 K and standard 5 mm NMR tubes were used. For each 1H
NMR experiment, 64 transients were collected into 32 K data
points over a 3000 Hz spectral window, using a 2 s relaxation de-
lay. The 2D ROESY spectra were acquired in the phase sensitive
mode and residual water suppression, using Bruker standard
parameters (pulse program roesyphpr). Each spectrum consisted
of a matrix of 4 K/4 K data points covering a spectral width of
4000 Hz. The spectra were obtained with a spin-lock mixing time
of 200 ms relaxation delay 3 s and 16 scans were recorded.
(a)
10
0
2
4
6
8
[β - CD] (mM)
7.4
3. Results and discussion
7.2
7.0
6.8
6.6
3.1. Determination of the stoichiometry
NMR is a technique, which provides the most evidence for the
inclusion of a guest into the hydrophobic b-CD cavity in solution.
Inclusion of TA and FF in b-CD cavity is put in evidence by the
change in chemical shifts of some of the guest and host protons,
in comparison with the chemical shifts of the same protons in
the free components. 1H NMR spectra of pure drugs and drug/b-
CD mixture are presented in Figs. 2 and 3.
H5
H4
H6
H8
R1
2.4
2.3
2.2
The 1H chemical shifts variation of some representative b-CD
and TA protons as well as b-CD and FF protons as a function of their
concentration is presented in Figs. 4 and 5, respectively. It can be
seen that in the presence of b-CD, almost all guest proton reso-
nances were affected. Furthermore, there is seemingly no prefer-
ence of inclusion of a particular aromatic ring, since similar
magnitude shifts were observed for the protons belonging to the
(b)
10
0
2
4
6
8
[TA] (mM)
Fig. 4. Chemical shifts variation of some representative: (a) b-CD and (b) TA
protons as a function of their concentration, ([b-CD] + [TA] = 10 mM).

C.G. Floare et al. / Journal of Molecular Structure 1044 (2013) 72–78
75
0.8
3.90
3.85
3.80
3.75
3.70
3.65
3.60
3.55
(a)
0.7
H3
H5
H6
H3
H5
H6
0.6
0.5
0.4
0.3
0.2
0.1
0.0
(a)
10
-0.1
0
2
4
6
8
0.0
0.2
0.4
0.6
0.8
1.0
[β-CD] (mM)
r1
7.9
7.8
7.7
7.6
7.5
7.4
7.3
7.2
7.1
7.0
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
-0.4
(b)
H5
H4
H2
H7
R1
H5
H6
R1
H8
(b)
0
2
4
6
8
10
0.0
0.2
0.4
0.6
0.8
1.0
[FF] (mM)
r2
Fig. 5. Chemical shifts variation of some representative: (a) b-CD and (b) FF protons
as a function of their concentration, ([b-CD] + [FF] = 10 mM).
Fig. 6. Job’s plots corresponding to the induced chemical shift variation of some: (a)
b-CD and (b) TA protons for the TA: b-CD system.
methyl-phenyl or benzoic groups of the TA or trifluorophenyl and
benzoic acid moiety in the case of FF.
For the shake of concision, only several protons (the most
markedly affected) have been selected and reported in Figs. 6
and 7. In all cases, Job’s plots show a maximum at 0.5, indicating
the existence of complexes with a 1:1 stoichiometry.
The aforementioned 1H NMR observations, for the two studied
systems, could imply two main possibilities: (a) the presence of
two types of 1:1 complexes with the inclusion of both aromatic
rings inside the b-CD cavity, which corresponds to a bimodal com-
plexation or, (b) the formation of a 1:2 drug: b-CD complex, where
the inclusion phenomena are simultaneously present for the two
rings. Considering the chemical structure of TA and FF, the simul-
taneous inclusion of one guest molecule into two b-CD molecules
seems to be sterically unfavorable, which avoids the formation of
1:2 complexes. In order to elucidate which of the two above possi-
bilities is correct the continuous variation method was employed
to establish the stoichiometry of the complexes. In our case, the
continuous variation method is based on the induced chemical
Thus a binary structure formed by one b-CD molecule surround-
ing one guest molecule can be considered reliable for both systems.
To confirm the hypothesis of bimodal inclusion complexation,
ROESY spectra were acquired for TA: b-CD and FF: b-CD complexes.
Due to the rapid dynamics of the complexation process, the ROESY
effects were only quantitatively used and no conclusions on inter-
molecular distances were extracted. The ROESY spectrum of TA:
b-CD complex is reported in Fig. 8. This spectrum allows us to
establish a spatial proximity between the TA protons and the inner
protons of b-CD.
The spectrum shows several cross-peaks between H3, H5 and
H6 protons of b-CD and protons of both aromatic rings of TA (H4,
H5, H6, H7 and H8), demonstrating the inclusion of these groups
in the hydrophobic cavity.
In the case of FF: b-CD system, (Fig. 9), no cross-peaks were ob-
served between H2 on the benzoic acid ring and H3, H5 and H6
protons of b-CD. All the other protons belonging to benzoic acid
moiety and trifluorophenyl moiety give rise to medium to strong
peaks with the inner protons of b-CD, except H3 which has a
cross-peak only with the corresponding H6 proton of the b-CD. It
is worth mentioning that we found cross-peaks only with the inner
b-CD protons, which establishes intra-cavity binding without evi-
dence for outside contributions. These results confirmed the
shift variation,
of the complex.
in the absence and in the presence of the other reactant. Thus if
a physical quantity, containing d, is plotted as a function of the
D
d, which is directly related to the concentration
D
d is defined as the difference in chemical shifts
D
mol fraction of the host or guest, r, (Job’s plot), its maximum value
will occur at r1 = m/(m + n) or r2 = n/(m + n), where m and n are,
respectively, the molar ratios of b-CD and drug in the (drug)n:(b-
CD)m complex. Under fast exchange conditions, for a signal belong-
ing to b-CD, for example, the calculated quantity
D
dꢀ[b-CD] is
proportional to the complex concentration and, can be plotted
against r1, [12]. The continuous variation method was applied for
all protons of the different molecules and yielded identical results.

76
C.G. Floare et al. / Journal of Molecular Structure 1044 (2013) 72–78
1.0
0.8
0.6
0.4
0.2
0.0
(a)
H3
H5
0.0
0.2
0.4
0.6
0.8
1.0
r1
0.8
0.6
(b)
H5
R1
Fig. 9. 2D ROESY spectrum of FF: b-CD system.
H8
H2
0.4
Similar results have been reported for other NSAIDs such as pir-
oxicam [13], naproxen [14], diclofenac [15], and mefenamic acid
[16] or nicardipine hydrochloride [17].
0.2
0.0
3.2. Evaluation of the binding constants
-0.2
-0.4
-0.6
As Connors pointed out [18], when a guest molecule possesses
two potential binding sites, (X and Y), a bimodal binding is possible
and two isomeric1:1 complexes (X0Y and XY0) can be formed. A
primed site indicates that a CD molecule is bound to that site.
The two isomers are characterized by two microscopic binding
0.0
0.2
0.4
0.6
0.8
1.0
r2
0
0
constants, KX Y and KXY and are related to the macroscopic equilib-
0
0
rium constant K11, by K11 = KX Y + KXY . Unfortunately, the determi-
nation of microscopic constants is not accessible through NMR,
and in some cases only the macroscopic constant can be deter-
Fig. 7. Job’s plots corresponding to the induced chemical shift variation of some: (a)
b-CD and (b) FF protons for the FF: b-CD system.
0
mined [19]. In order to evaluate the order of magnitude for KX Y
0
and KXY we made a rough approximation, assuming that we can
treat independently the two isomers by considering two separate
1:1 complexes, and neglecting [XY0] in the calculation of KX Y and
0
[X0Y] in the calculation of KXY , respectively. In this case, the associ-
0
ation constant, K, for a 1:1 complex can be determined according to
the following equation [20]:
8
<
9
=
"
#
1=2
ꢀ
ꢁ
2 ꢂ4½Hꢁ ðtiÞ½Gꢁ ðtiÞ
D
2½XꢁðtiÞ
dðcjÞ
1
K
1
K
þD
dði;jÞ
¼
½Gꢁ ðtiÞ þ½Hꢁ ðtiÞ
þ
ꢂ
½Gꢁ ðtiÞ þ½Hꢁ ðtiÞ
þ
:
;
ð1Þ
where i counts the sample number and j the studied proton. If the
studied proton belongs to the guest or host molecule, X = G or H
respectively. D
dðcjÞ represents the chemical shift difference (for a gi-
ven proton) between the free component and the pure inclusion
complex. Eq. (1) involves no approximations and correlates the total
concentrations of the guest and host molecules with the observed
deference in the chemical shift:
D
dði:jÞ ¼ dðjÞ ꢂ dði;jÞ
ð2Þ
free
obs
Fig. 8. 2D ROESY spectrum of TA: b-CD system.
We used a computer program [21] based on an iteration proce-
dure following specific algorithms in order to fit the experimental
values of
(i,j) to the appropriate equation. Each iteration step sets
up a quadratic function to determine the direction of search and
calculates the loss error function E:
hypothesis that two different 1:1 complexes are simultaneously
present in solution, each one involving the inclusion of both aro-
matic ends of TA and FF. A schematic representation of the bimodal
inclusion which may exist in solution is presented in Fig. 10.
D
d

C.G. Floare et al. / Journal of Molecular Structure 1044 (2013) 72–78
77
Fig. 10. Proposed model for the bimodal inclusion equilibria of TA and FF with b-CD.
constants values, we can conclude that FF is more tightly bound
to b-CD than TA, in spite of the fact that the structural differences
between these two drugs are small. The complete set of chemical
shifts in the free state and in the pure complex is reported in
Table 1.
Table 1
Chemical shifts of TA and FF protons in the free and complexed states.
values were obtained as a result of the fitting procedure.
D
dc = dfree ꢂ dc
Proton
TA
FF
dfree (ppm)
dc (ppm)
dfree (ppm)
dc (ppm)
Based on the complexation shifts, ROESY cross-peaks and the
magnitude of evaluated association constants, we conclude that
the inclusion process affects principally the methyl-phenyl and tri-
fluoromethyl-phenyl rings, which enters the b-CD cavity through
the secondary hydroxyl rim. In both cases, the interaction of b-
CD with the benzoic acid ring seems to be weaker, probably do
to the anionic form of this moiety.
2
4
5
6
7
8
R1
7.7572
7.2316
6.9134
7.1142
–
7.9018
7.3889
6.3822
7.3785
–
7.7495
7.3096
7.2713
7.2552
7.4239
7.3610
7.4757
7.8659
7.2699
7.0220
7.3558
7.5225
7.5200
7.1917
7.2142
2.2314
7.3902
2.4511
4. Conclusions
X
E ¼
ðD
d
ði;jÞ ꢂ
D
dðcia;jlÞcÞ
ð3Þ
2
The complexation between the anionic forms of tolfenamic acid
(TA) and flufenamic acid (FF) with b-cyclodextrin has been investi-
gated in solution by 1D and 2D 1H NMR spectroscopy. The con-
structed Job’s plots, based on the induced chemical shifts of the
b-CD, TA, and FF protons, confirm the existence of a bimodal bind-
ing in both cases. ROESY experiments reveal the correlation be-
tween the H3 and H5 protons of the b-CD cavity and both the
aromatic ring protons of TA and FF, supporting the bimodal binding
process. On the basis of these experimental data we conclude that
we have a simultaneous presence of two 1:1 complexes for TA as
well as FF, each one involving the inclusion in the b-CD cavity of
a different aromatic end. Based on the evaluated microscopic asso-
ciation constants, we believe that the complexation mainly affects
the methyl-phenyl or trifluoromethyl-phenyl ring, and to a lesser
extent, the benzoic acid moiety.
i;j
The treatment of the entire set of protons studied produces a
single K value for the entire process and a set of calculated
D
dðcjÞ val-
ues. The program is quite flexible as both the host and the guest
can be observed for spectroscopic perturbations, allowing up to
15 protons to be used in the fitting process. For TA: b-CD complex,
we applied Eq. (1) first for a set of protons consisting in H2, H4 and
H5 of TA and then for H6, H8 and R1 of TA. This means that we con-
sidered first the case when the benzoic acid ring is inserted in the
b-CD cavity and then the inclusion of the methyl- phenyl ring. The
association constants obtained, using the above described proce-
dure were KX Y = 144.1 Mꢂ1 with E = 1.53 ꢃ 10ꢂ3 and a correlation
0
factor r = 0.998, when the benzoic acid moiety is included in the
b-CD cavity and KXY = 151.9 Mꢂ1 with E = 8.41 ꢃ 10ꢂ4 and
0
r = 0.994 for the inclusion of methyl-phenyl moiety. With these
values, the macroscopic constant for the TA: b-CD complex has
the value K1:1 = 296 Mꢂ1. In the case of FF: b-CD complex, we ap-
plied Eq. (1) for H2, H4 and H5 protons of the benzoic acid moiety
and then for H6, H7, H8, and R1 protons belonging to trifluoro-
Acknowledgement
This work was financially supported by UEFISCDI Romania, Pro-
ject PCE-2011-3-0032.
methyl–phenyl ring. The obtained results were KX Y = 647.1 Mꢂ1
0
with E = 3.36 ꢃ 10ꢂ4 and a correlation factor r = 0.999, when the
benzoic acid moiety is included in the b-CD cavity and
Appendix A. Supplementary material
KXY = 802.6 Mꢂ1 with E = 1.5 ꢃ 10ꢂ3 and r = 0.998 for the inclusion
0
of trifluoromethyl-phenyl moiety. With these values, the macro-
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.molstruc.2012.11.
021.
scopic constant for the FF: b-CD complex has the value
K1:1 = 1449.7 Mꢂ1
.
Comparing the macroscopic association

78
C.G. Floare et al. / Journal of Molecular Structure 1044 (2013) 72–78
[10] K. Hirose, J. Incl. Phenom. Macrocycl. Chem. 39 (2001) 193–209.
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