Investigation of the binding of roxatidine acetate hydrochloride with cyclomaltoheptaose (β-cyclodextrin) using IR and NMR spectroscopy

Article abstract of DOI:10.1016/j.carres.2011.07.003

NMR chemical shift changes of the cyclomaltoheptaose (β-cyclodextrin, β-CD) cavity protons as well as roxatidine acetate hydrochloride aromatic ring protons revealed the formation of a RAH-β-CD inclusion complex. Detailed FTIR and NMR spectroscopic (

Full text of DOI:10.1016/j.carres.2011.07.003

Carbohydrate Research 346 (2011) 1809–1813
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Carbohydrate Research
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Investigation of the binding of roxatidine acetate hydrochloride with
cyclomaltoheptaose (b-cyclodextrin) using IR and NMR spectroscopy
Arti Maheshwari a, , Manisha Sharma a, Deepak Sharma b
⇑
a Department of Chemistry, IET, Mangalayatan University, Beswan, Aligarh, India
b Department of Physics, IET, Mangalayatan University, Beswan, Aligarh, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 June 2011
Received in revised form 5 July 2011
Accepted 6 July 2011
Available online 18 July 2011
NMR chemical shift changes of the cyclomaltoheptaose (b-cyclodextrin, b-CD) cavity protons as well as
roxatidine acetate hydrochloride aromatic ring protons revealed the formation of a RAH–b-CD inclusion
complex. Detailed FTIR and NMR spectroscopic (1H NMR, COSY, NOESY, ROESY) studies have been done.
The stoichiometry of the complex was determined to be 1:1, and the overall binding constant was also
determined by Scott’s method. The NOESY spectrum confirmed the selective penetration of the aromatic
ring of RAH into the b-CD cavity in comparison to that of the piperidine ring. The mode of penetration of
the guest into the CD cavity and structure of the complex has been established.
Keywords:
Roxatidine acetate hydrochloride
b-Cyclodextrin
COSY
Ó 2011 Elsevier Ltd. All rights reserved.
NOESY
ROESY
1. Introduction
cyclodextrin. FTIR spectral studies give information regarding the
involvement of hydrogen in various functional groups. This gener-
Cyclomaltooligosaccharides (cyclodextrins, CDs) are oligosac-
charides composed of six to eight glucopyranose units bound by
ally shifts the absorbance bands to lower frequencies, increases the
intensity and widens the band caused by the stretching vibration
of the group involved in the formation of the hydrogen bonds.7
1H NMR spectroscopy is one of the most common useful tech-
niques for investigating the stability and stoichiometry of the CD
complexes, particularly in solution.8,9 2D NMR spectroscopy has
become an important tool for the investigation of the interactions
between CDs and guest molecules. According to the relative inten-
sities of the 2D NMR cross peaks, it is possible to estimate the ori-
entation of the guest molecule within the CD cavity.10,11
Roxatidine acetate hydrochloride (RAH), chemically known as
N-{3-[a-piperidino-m-tolyl)oxy]propyl}glycolamide acetate mono-
hydrochloride [93793-83-0], is a histamine H2 receptor antagon-
ist.12 The drug is recommended for the management of benign
and postoperative ulcers, as it does not appear to affect cyto-
chrome P450 and is therefore considered to have little effect on
the metabolism of other drugs.13
Although a small part of this study has already been published
in Pharmazie,14 we herein report the complexation of this drug
with b-CD again because in the previous study there were some
problems: due to poor resolution of the NMR spectra (200 MHz),
all the NMR signals of RAH could not be identified, especially in
the non-aromatic region. According to the previous study, it was
confirmed that RAH forms a 1:1 inclusion complex with b-CD,
but it could not be said with certainty, however, which part of
the guest is entering the b-CD cavity and also whether the penetra-
tion of the guest is from the wider or narrower rim side of the
a-(1?4) linkages that are commonly named a-, b-, and c-CD,
respectively. b-CD, in particular, has an internal cavity shaped like
a truncated cone. By virtue of their shape and the hydrophobic
nature of the cavity, CDS accommodate a variety of hydrophobic
molecules, or parts of them, inside their cavity through non-
covalent interactions to form inclusion complexes.1–3
Today, the study of the inclusion complexes of pharmaceuticals
with CDs is a subject of great interest because of their utility to im-
prove the solubility, dissolution rate and bioavailability of poorly
water-soluble drugs.4,5 Furthermore, in order to use CDs as drug
carrier systems, it is necessary to understand from the thermody-
namics and kinetics point of view how the drug molecule interacts
with the CD cavity and the dynamics of entry and exit of guest
molecules in the CD cavity.
Fourier-transform infrared (FTIR) spectroscopy is used to esti-
mate the interaction between cyclodextrin and the guest mole-
cules in the solid state.6 Cyclodextrin bands often change only
slightly upon complex formation, and if the fraction of the guest
molecules encapsulated in the complex is less than 25%, bands
which could be assigned to the included part of the guest
molecules are easily masked by the bands of the spectrum of
⇑
Corresponding author. Tel.: +91 09634184524.
E-mail addresses: arti_maheshwari2002@yahoomail.com, arti.chemistry@gmail.
com (A. Maheshwari).
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.07.003

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A. Maheshwari et al. / Carbohydrate Research 346 (2011) 1809–1813
work,15–17 we report herein our results of the complexation of
RAH with b-CD in solution as well as in the solid state.
2. Experimental
2.1. Material and instrumentation
FTIR spectra of the RAH–b-CD inclusion complex, b-CD, and RAH
were recorded on Perkin–Elmer model 377 spectrophotometer
using KBr pellets. The instrument was operated under dry air purge,
and the scans were collected at scanning speed 2 mm/s with a reso-
lution of 4 cmꢀ1 over the region of 4000–400 cmꢀ1. All the NMR
spectra were recorded on a 500 MHz instrument. The 1H NMR spec-
tra of RAH, in the absence as well as in the presence of b-CD, were re-
corded in D2O at room temperature. To determine the stoichiometry
and binding constant of the complex, the chemical shift change data
(Dd) for the b-CD protons were obtained by keeping the concentra-
tion of b-CD constant at 10 mM while varying the concentration of
RAH from 2.11 to 36.7 mM. The COSY spectrum was recorded for
pure RAH, while the ROESY and NOESY spectra were obtained for a
mixture of RAH and b-CD with a mixing time 200 ms and under spin
lock conditions. The COSY spectrum was recorded in DMSO with
0.2777588 s acquisition time, while the NOESY spectrum was re-
corded in D2O with 0.4981236 s acquisition time.
Figure 1. FTIR spectra of pure RAH (A), pure b-CD (B) and the RAH–b-CD complex (C).
Table 1
1H NMR chemical shift change data ( d) for b-CD protons in the presence of RAHa
D
[b-CD]/[RAH]
H-10
H-20
H-30
H-40
H-50
H-60
2.367
1.503
1.366
0.808
0.436
0.272
0.008
0.006
0.007
0.012
0.011
0.013
0.013
0.011
0.012
ꢀ0.014
ꢀ0.027
ꢀ0.032
ꢀ0.051
ꢀ0.069
ꢀ0.089
0.009
0.008
0.012
0.010
0.006
0.006
ꢀ0.004
ꢀ0.015
ꢀ0.018
ꢀ0.046
ꢀ0.062
ꢀ0.089
0.002
ꢀ0.001
ꢀ0.003
ꢀ0.008
ꢀ0.011
ꢀ0.023
0.004
2.2. Preparation of the RAH–b-CD inclusion complex
ꢀ0.001
ꢀ0.003
The RAH–b-CD complex was prepared by the co-precipitation
method. Roxatidine acetate hydrochloride (3.84 g) was dissolved
in a minimum quantity of water and was added dropwise to a solu-
tion of b-CD (11.35 g) in a minimum quantity of water at 25 °C
with continuous stirring. Stirring was maintained for 1 h at the
same temperature. Then, a precipitate of the complex was ob-
tained, which was filtered, dried and stored at room temperature.
a
Negative values indicate a high-field shift.
cavity. So, the structure for the RAH–b-CD complex could not be
confirmed. NMR spectra were taken at a frequency of 200 MHz,
and there was some element of doubt in the calculation of chemi-
cal shift changes. Due to these reasons, in continuation of our
Figure 2. Partial 1H NMR spectra (500 MHz) showing b-CD proton signals of the RAH–b-CD complex (A–E) in different [b-CD]/[RAH] molar ratios [A = 0.436, B = 0.808,
C = 1.366, D = 1.503, E = 2.367] in comparison to (F) pure b-CD.

A. Maheshwari et al. / Carbohydrate Research 346 (2011) 1809–1813
3. Results and discussion
1811
450
400
350
300
250
200
150
100
3.1. FTIR spectra
The infrared spectra of different samples (RAH, b-CD, RAH–b-CD
inclusion complexes) were recorded. We observed that the inten-
sity of bands of both pure samples (RAH and b-CD) was decreased
in the spectra of the RAH–b-CD complex. It can be observed that
this decrease in intensity is probably owing to a restriction of the
vibration due to the inclusion within the b-CD cavity.
The FTIR spectrum of the RAH–b-CD complex shows an absor-
bance atꢀ11272 cmꢀ1 for the aromatic carbon stretching vibration,
1742 cm , which is that of the aromatic ether portion of RAH,
1527 cmꢀ1 for the aromatic ring of RAH, 3265 cmꢀ1 for the N–H
stretching of RAH, and an intense band at 3300–3500 cmꢀ1 for
the oligomeric OH stretching for b-CD. The peak at 3354 cmꢀ1 from
pure b-CD shifted to 3456 cmꢀ1 in the RAH–b-CD complex due to
the intermolecular hydrogen bonding, which is not very significant
(Fig. 1). The FTIR spectrum of the RAH–b-CD complex did not show
any new peaks, indicating no chemical bond was created in the
formed complex. The remaining peaks of the pure drug do not
show any change after complexation. Thus, the FTIR spectra give
an indication that the RAH forms the inclusion complex with b-
CD under the conditions studied. These results were also corrobo-
rated by NMR experiments.
0
5
10
15
20
25
30
35
40
[RAH] 10-3
M
0
Figure 3. Scott’s plot of [RAH]/
the RAH–b-CD complex.
D
dH-5 versus [RAH] showing a 1:1 stoichiometry for
10
9
8
N
O
12
11
14
17
16
6
3
NH
O
1
5
18
3.2. Nuclear magnetic resonance (NMR) spectroscopy
O
15
13
19
O
2
4
3.2.1. 1H NMR chemical shift change data of b-CD
In the presence of RAH, the b-CD cavity protons positioned in-
Figure 4. Structure of roxatidine acetate hydrochloride.
side the b-CD cavity, namely H-30 and H-50, exhibited high-field
Figure 5. COSY spectrum (500 MHz) of pure RAH showing all the protons of RAH.

1812
A. Maheshwari et al. / Carbohydrate Research 346 (2011) 1809–1813
shift changes that increased with the increase in the concentration
of RAH, while the other protons remained more or less unaffected.
The chemical shift change data for all the b-CD cavity protons are
given in Table 1, and the gradual high-field shifts of the b-CD cavity
protons with the increase in concentration of RAH is shown in Fig-
ure 2. These shift changes in the H-30 and H-50 protons of b-CD can
only be explained in terms of the ring-current effect of the aro-
matic ring that is included in the cavity, thus confirming the forma-
tion of an inclusion complex between RAH and b-CD.
3.2.2. Stoichiometry of the RAH–b-CD complex
The stoichiometry of the RAH–b-CD complex was determined
by the Scott equation18 for the 1:1 complex
Gꢁ=
D
dobs ¼ ½Gꢁ=
D
dmax
þ
Ddmax=Ka
The 1H NMR chemical shift change data for the b-CD protons
Figure 6. Partial 1H NMR spectra (500 MHz) showing aromatic protons of RAH for
(A) a 2.367 [b-CD]/[RAH] mixture and (B) pure RAH.
were obtained in the presence of RAH by keeping the concentration
of b-CD constant while varying the concentration of RAH. The plot
0
0
of
gave a linear fit (Fig. 3) confirming the 1:1 stoichiometry for the
RAH–b-CD complex. The slope of the plot is thus equal to 1/ dmax
with the intercept with the vertical axis to dmax/Ka, allowing the
estimation of Ka to be 49 Mꢀ1
DdH-5 against [RAH] in the form of [RAH]/DdH-5 versus [RAH]
D
,
D
.
3.2.3. 1H NMR assignment of roxatidine acetate hydrochloride
An unambiguous 1H NMR spectral assignment is required to
establish the structure of the inclusion complex. The structure of
RAH is shown in Figure 4. The assignment of all the signals in
the 1H NMR spectrum of pure RAH was made with the help of a
COSY spectrum (Fig. 5). The aromatic protons were observed as a
singlet at d 6.980 (H-6), a multiplet of two protons at d 7.023 (H-
2, 4) and a triplet at d 7.362 (H-3). The signals for H-10 protons
are found as two multiplets centered at d 1.389 and 1.694, which
give a clear indication that the piperidine ring is in the chair con-
formation. The other proton assignments are shown in the COSY
spectra (Fig. 5).
3.2.4. 1H NMR chemical shift change data of roxatidine acetate
hydrochloride
In the presence of b-CD, changes in the chemical shifts and
shapes of the aromatic proton signals indicated the involvement
of the aromatic ring in complexation. The signal for H-6 exhibited
a high-field shift, while those for H-2, 4 and H-3 displayed down-
field shift changes. Figure 6 shows the changes in the aromatic re-
gion of the spectrum of RAH in the presence of b-CD compared to
that for pure RAH.
Figure 7. A partial NOESY spectrum (500 MHz) of a mixture of RAH and b-CD
showing dipolar interaction of aromatic protons of RAH and b-CD cavity protons.
10
9
8
N
O
12
11
14
3.2.5. NOESY/ROESY data for the RAH–b-CD complex
17
16
O
6
3
NH
O
5
4
1
2
A ROESY spectrum was recorded for a mixture of RAH and b-CD
which did not show any cross peak between aromatic protons of
RAH and the b-CD cavity protons. A NOESY spectrum (Fig. 7) of
the mixture of RAH–b-CD was recorded, which proved helpful in
the structural characterization of the complex. As expected the
H-2,4 protons of RAH displayed through-space interaction with
H-30 and H-60 of the b-CD cavity but not with H-50, proving the
shallow penetration of the aromatic ring into the b-CD cavity from
the wider side of the cavity. No aliphatic proton of RAH showed a
cross peak with the b-CD cavity protons.
18
O
15
13
19
Figure 8. Proposed structure for the 1:1 RAH–b-CD complex.
the structural orientation of the formed complex in solution. Infor-
mation gleaned through these studies offers quantitative informa-
tion on the strength of binding between RAH and b-CD. The study
proved the formation of the RAH–b-CD inclusion complex by the
penetration of aromatic ring of RAH into the b-CD cavity. The stoi-
chiometry of the complex was determined to be 1:1, and the
binding constant was estimated as 49 Mꢀ1 by Scott’s method. A
structure of the complex has been proposed which is well sup-
ported by NOESY spectral data.
The proposed structure for the complex formed between RAH
and b-CD in aqueous solution is shown in Figure 8.
4. Conclusions
This work provides evidence for the cavity inclusion of roxati-
dine acetate hydrochloride (RAH) and gives information about

A. Maheshwari et al. / Carbohydrate Research 346 (2011) 1809–1813
1813
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Roxatidine acetate hydrochloride and b-cyclodextrin were very
kindly provided by Dr. Reddy’s Laboratory, India, and Geertrui
Haest, Cerestar Cargill, Belgium, respectively. We are highly grate-
ful to Dr. Matthias Heydenreich, Universität Potsdam, Institut für
Chemie, for his help in obtaining some of the NMR data.
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