Inclusion of Paracetamol into β-cyclodextrin nanocavities in solution and in the solid state

Article abstract of DOI:10.1016/j.saa.2011.05.084

We report on steady-state UV-visible absorption and emission characteristics of Paracetamol, drug used as antipyretic agent, in water and within cyclodextrins (CDs): β-CD, 2-hydroxypropyl-β-CD (HP-β-CD) and 2,6-dimethyl-β-CD (Me-β-CD). The results reveal

Full text of DOI:10.1016/j.saa.2011.05.084

Spectrochimica Acta Part A 79 (2011) 1904–1908
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Inclusion of Paracetamol into ˇ-cyclodextrin nanocavities in solution and in the
solid state
Maged El-Kemarya,∗, Saffaa Sobhya, Samy El-Dalyb, Ayman Abdel-Shafic
a Nanochemistry Laboratory, Chemistry Department, Faculty of Science, Kafrelsheikh University, 33516 Kafr ElSheikh, Egypt
b Chemistry Department, Faculty of Science, Tanta University, 31527 Tanta, Egypt
c Department of Chemistry, Faculty of Science, Ain Shams University, 115611 Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
We report on steady-state UV–visible absorption and emission characteristics of Paracetamol, drug used
as antipyretic agent, in water and within cyclodextrins (CDs): ˇ-CD, 2-hydroxypropyl-ˇ-CD (HP-ˇ-CD)
and 2,6-dimethyl-ˇ-CD (Me-ˇ-CD). The results reveal that Paracetamol forms a 1:1 inclusion complex
with CD. Upon encapsulation, the emission intensity enhances, indicating a confinement effect of the
nanocages on the photophysical behavior of the drug. Due to its methyl groups, the Me-ˇ-CD shows
the largest effect for the drug. The observed binding constant showing the following trend: Me-ˇ-
CD > HP-ˇ-CD > ˇ-CD. The less complexing effectiveness of HP-ˇ-CD is due to the steric effect of the
hydroxypropyl-substituents, which can hamper the inclusion of the guest molecules. The solid state
inclusion complex was prepared by co-precipitation method and its characterization was investigated
by Fourier transform infrared spectroscopy, 1H NMR and X-ray diffractometry. These approaches indi-
cated that Paracetamol was able to form an inclusion complex with CDs, and the inclusion compounds
exhibited different spectroscopic features and properties from Paracetamol.
Received 25 January 2011
Received in revised form 19 May 2011
Accepted 25 May 2011
Keywords:
Paracetamol
ˇ-Cyclodextrin
Hydroxypropyl-ˇ-cyclodextrin
Methyl-ˇ-CD
Inclusion complex
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
Recently, the spectroscopy and photophysical properties of
Paracetamol was reported in ␣-, ␤- and ␥-cyclodextrin cavities and
Cyclodextrin (CD) nanocavities are potential candidates for drug
delivery systems through the formation of inclusion complexes
(Scheme 1) on the basis of noncovalent interaction [1], due to their
effect on solubility, dissolution rate, chemical stability and absorp-
tion of drugs. The hydrophobic environment of a CD nanocavity can
lead to the alteration of physical, chemical, and biological proper-
ties of guest molecules [2–8]. As already reported for a large variety
of systems, a CD cavity is able to influence both the fate of the reac-
tion intermediates and the deactivation pathways of the excited
caged drugs [9,10]. This may lead to a decrease in phototoxicity.
Paracetamol (N-acetyl-4-aminophenol, Scheme 2) is a popular
antipyretic agent which also possessed analgesic (pain-relieving)
properties. In several countries, it is most commonly used by people
who are sensitive or allergic to aspirin. Although, it is not effective
against inflammation, compared to Aspirin or Ibuprofen; it is well
tolerated by most people, including children. Paracetamol rarely
causes side effects as long as it is taken as directed. However, pro-
longed or habitual use of Paracetamol may lead to liver damage or
failure.
micellar medium [11]. The systematic study of some modified ˇ-CD
as HP-ˇ-CD and Me-ˇ-CD in solution and in the solid state is also
important. Most CD derivatives are highly water-soluble products.
Among the chemically modified ˇ-CDs, methylated and hydrox-
yalkylated CDs have received considerable attention because their
physicochemical properties and aqueous solubility are significantly
changed and the inclusion behavior is largely magnified, depending
on the degree of substitution [12].
In this paper we present the influence of native and modified ˇ-
CD on the complexation of Paracetamol by the determination of the
corresponding stability constants in the hope of improving some
of the pharmaceutical properties of Paracetamol using UV–visible
absorption and fluorescence spectroscopy. Solid inclusion com-
plexes containing Paracetamol and cyclodextrins were prepared by
co-precipitation method and characterized by Fourier-transform
infrared and X-ray diffractometry techniques.
2. Experimental
2.1. Materials
∗
Paracetamol 99% (Aldrich), diethylether (Sigma–Aldrich), ˇ-CD
(Fluka), HP-ˇ-CD (Fluka) and Me-ˇ-CD (Fluka) were analytical
Corresponding author. Tel.: +20 473 215 174; fax: +20 473 215 175.
E-mail address: elkemary@yahoo.com (M. El-Kemary).
1386-1425/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2011.05.084

M. El-Kemary et al. / Spectrochimica Acta Part A 79 (2011) 1904–1908
1905
2.3. Preparation of the physical mixture
Appropriate amounts of drug and CD to give 1:1 molar ratio were
mixed thoroughly in a mortar by geometric dilution technique [13].
The physical mixture was sieved and stored in airtight container.
2.4. Preparation of solid inclusion complexes
The solid inclusion complexes used in this investigation were
prepared by the Co-precipitation method. Amounts of the drug and
CD with 1:1 molar ratios were accurately weighed. Drug was dis-
solved in 20 ml of ether and CD was dissolved in 100 ml of water.
The two solutions were mixed, agitated for 24 h at 28 ◦C and then
cooled to 2 ◦C in a refrigerator. The mixture was filtered, washed
with ether and dried at 25 ◦C under vacuum for 24 h.
Scheme 1. Schematic view of the process of drug cyclodextrin inclusion.
OH
O
3. Results and discussions
CH
N
H
3
3.1. Steady-state absorption and fluorescence spectra
Fig. 1A displays the UV–visible absorption spectra of Paraceta-
mol drug in neutral water and in the presence of Me-ˇ-CD. A very
small change in the spectra was detected upon addition of Me-ˇ-
CD, suggesting a weak interaction between CD and drug or a small
change in the molar absorption coefficient of the drug. Fig. 1B shows
the steady-state emission spectra of neutral aqueous solution of
Paracetamol and in the presence of different Me-ˇ-CD concentra-
tions, upon excitation at 275 nm. In water, Paracetamol exhibits a
band with a maximum at 302 nm. Upon addition of Me-ˇ-CD, the
emission intensity is enhanced with a small red shift of the band,
suggesting the formation of complex between Paracetamol and Me-
ˇ-CD (Scheme 1). For ˇ-CD and HP-ˇ-CD solutions, the increase in
the emission intensity is relatively weaker.
The fluorescence band maximum of Paracetamol displays also
insignificant shift on going from protic polar water (302 nm) to rela-
tively aprotic non-polar cyclohexane (304 nm; not shown in figure).
A similar spectral change was also observed on the absorption spec-
tra of Paracetamol, reflecting non-polar neutral structure of both
ground and excited states of Paracetamol.
Scheme 2. Paracetamol (N-acetyl-4-aminophenol).
reagent grades and used without further purification. Double dis-
tilled water was used for the preparation of all solutions.
2.2. Apparatus and methods
The absorption and fluorescence spectra were recorded on a Shi-
madzu 2450 UV–visible spectrophotometer and a Shimadzu 5301
PC spectrofluorometer, respectively. Fourier-transform infrared
(FT-IR) spectra were measured with a JASCO spectrometer 4100.
The powder X-ray diffraction (XRD) analysis was made on a Rigaku
2550D/max VB/PC X-ray diffractometer using Cu K␣ radiation
(ꢀ = 0.154056 nm).
NMR spectra were recorded in D2O on a Varian Mercury VX-300
spectrometer. 1H NMR data analysis was carried out using MestReC
4.9.9 software. Chemical shifts have been expressed in parts per
million (ppm) relative to HDO signal as a reference at 4.80 ppm.
For 1H NMR measurements, the concentrations of Paracetamol and
ˇ-CD were about 2 × 10−3 M and 0.02 M, respectively.
The geometry of the molecule was optimized using AM1 of the
MOPAC 97 program.
This result indicates that binding of Paracetamol to CD changes
the local solvation environment (water) in a way similar to chang-
ing the solvent from protic to aprotic medium. Since Paracetamol
has three hydrogen-bonding sites (amino nitrogen, phenolic oxy-
gen as well as carbonyl group, Scheme 2). Upon encapsulation,
Paracetamol experiences a reduced H-bonding interaction with
water.
1.0
A
B
[Me-β-CD] / mM
300
8
7
6
5
4
0.8
[Me-β–CD]/ mM
0.6
8
200
7
3
2
1
6
0.4
5
4
100
0
3
0.2
2
0
0.0
0
280
300
320
340
360
380
400
250
300
350
400
Wavelength / nm
Wavelength / nm
Fig. 1. (A) Absorption and (B) fluorescence spectra of Paracetamol (10−4 M) in water in absence and presence of Me-ˇ-CD at 298 K.

1906
M. El-Kemary et al. / Spectrochimica Acta Part A 79 (2011) 1904–1908
3.2. Association constants
tion of substrate binding, without causing any structural hindrance
to the drug inclusion [19]. On the contrary, some effect of steric
blocking, due to the presence of the hydroxypropyl-substituents,
which can hamper the inclusion of the guest molecules, could
explain the less complexing effectiveness exhibited by HP-ˇ-CD.
It should be noted that the hydrogen-bonding ability of the
alcoholic hydrogens in ˇ-CD is low compared to that of HP-ˇ-
CD because, in the former, the secondary alcoholic –OH groups
at the 2- and 3-positions of the adjacent glucopyranose rings
are engaged in intramolecular hydrogen-bonding with each other.
In the case of Me-ˇ-CD, however, this intramolecular hydrogen-
bonding is destroyed due to substitution of the alcoholic proton
at the 2-position with a methyl group. This structure enhances
the intermolecular hydrogen-bonding ability of the alcoholic –OH
group at the 3-position with water. Although the methylation
makes the host molecule incapable of forming intramolecular
hydrogen bonds, the stability constant with Me-ˇ-CD is higher than
that with ˇ-CD, therefore the role of the hydrogen binding seems
to be less important [20].
However, for HP-ˇ-CD the hydroxyl groups and the hydrox-
ypropyl groups on the exterior of the molecule can interact with
water to provide the increased aqueous solubility of HP-ˇ-CD and
complexes made with it. These groups may also account for the
stronger hydrogen bonding between Paracetamol and HP-ˇ-CD
than the ˇ-CD. On the other hand, hydrophobic bonding may be
another important contribution to the interactions between Parac-
etamol and HP-ˇ-CD. This would account for the higher value of
association constant for Paracetamol/HP-ˇ-CD complex than for
Paracetamol/ˇ-CD complex.
The equilibrium constant K assuming the formation of a
1:1 Paracetamol/CD can be calculated based on the following
Benesi–Hildebrand [14] plots using fluorescence data of Fig. 1A:
1
I − I0
1
1
1
=
+
(1)
I1 − I0
K(I1 − I0) [CD]0
where [CD]0 represents the initial concentration of CD, I0 and I
are the fluorescence intensities in the absence and presence of CD,
respectively, and I1 is the limiting intensity of fluorescence.
A plot of 1/(I − I0) versus 1/[CD]0 give a straight line, indicating
the formation of 1:1 inclusion complex between Paracetamol and
CD. The K values were obtained from the slope and intercept of the
plots. The Benesi–Hildebrand plots (Fig. 2) show excellent linear
regression (r = 0.99), supporting the assumed 1:1 Paracetamol/Me-
ˇ-CD inclusion complex. From the plot, K is evaluated. We obtained
K values were 1.48 × 102 M−1, 1.89 × 102 M−1 and 2.10 × 102 M−1
for the Paracetamol complexes with ˇ-CD, HP-ˇ-CD and Me-ˇ-CD,
respectively.
The Benesi–Hildebrand plots assuming 1:2 stoichiometry can
be represented by Eq. (2):
1
I − I0
1
1
1
=
+
(2)
2
I1 − I0
K2(I1 − I0)
([CD]0)
where K2 represents the association constant of 1:2 complex. The
plots of 1/(I − I0) versus 1/([CD])2 according to Eq. (2) are curved as
shown in Fig. 2B, confirming thereby the previous result of a 1:1
stoichiometric ratio.
The results suggest that the size of CD cavity plays a role in
the formation and stability of the formed complex. The dimen-
sions of Paracetamol geometry of the ground-state were optimized
by using AM1 (MOPAC 97 program). This calculation revealed that
the molecular length of Paracetamol (distance between atoms 15
It is readily seen that the magnitude of inclusion equilibrium
constant increases in the order Me-ˇ-CD > HP-ˇ-CD > ˇ-CD. For Me-
ˇ-CD, the K value is almost larger than that for ˇ-CD and HP-ˇ-CD.
A significant increase in the stability constants of many drugs in the
presence of Me-ˇ-CD in comparison with ˇ-CD has been reported
[4,12–16]. Both ˇ-CD and Me-ˇ-CD have the same cavity diameter.
˚
and 17) is ∼8 A, and its width (distance between atoms 18 and 19)
˚
˚
is ∼5 A, Scheme 3. The internal diameter of the ˇ-CD is approxi-
However, the unsubstituted ˇ-CD has height (∼8 A), therefore part
˚
˚
mately 6–6.5 A and its height is 7.8 A [21,22]. Due to the methyl
of the drug could still be outside the nanocage (vide infra). Due to
˚
˚
groups, the height of Me-ˇ-CD cavity is longer (∼11 A) [17]. The
the methyl groups, the height of Me-ˇ-CD cavity is longer (∼11 A)
resulting structures show that the Paracetamol drug is included in
the cavities with the aliphatic part and the stability constant value
of drug–cyclodextrin complexes is a useful index of the binding
strength of the complex. This conclusion has been confirmed by
using 1H NMR spectroscopy as shown in next section.
[17]. The hydrophobic part of Me-ˇ-CD provided by the methyl
groups, at the 2- and 6-positions increases the hydrophobicity of
the cavity [17,18] and leads to a greater efficiency towards the drug
compared to that with ˇ-CD and enhances more penetration of the
drug inside the cavity of Me-ˇ-CD.
From the above results we concluded that the drug is well fitted
and deeply included into the deeper Me-ˇ-CD cavity than that of
ˇ-CD because many methyl groups are located at both ends of the
cavity and the whole Paracetamol molecule, is buried in the host.
However, the observed better performance of Me-ˇ-CD com-
pared to HP-ˇ-CD has also been attributed to the presence of the
methyl groups that increased the hydrophobic region of the macro
molecule by capping the edge of the cavity and expanding the loca-
6
6
A
B
5
4
3
2
1
5
4
3
2
1
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
-6
1 /[Me- -CD]2 x10 M-2
1 / [Me- -CD] x 10-3 M-1
Fig. 2. Benesi–Hildebrand plot of data from Fig. 1 for (A) 1:1 and (B) 1:2 inclusion complex.

M. El-Kemary et al. / Spectrochimica Acta Part A 79 (2011) 1904–1908
1907
Scheme 3. Optimized geometry of the ground-state Paracetamol by using AM1.
This may explain the higher stability constant of Paracetamol with
Me-ˇ-CD in comparison with ˇ-CD.
300
200
100
0
A
3.3. Solid state inclusion complex
Fig. 3 shows the X-ray diffraction patterns of Paracetamol, ˇ-CD,
the physical mixture and inclusion complex. The diffraction pattern
of physical mixture (1:1) is simply the superposition of signals of
the two components, while that of inclusion mixture is different
and its peaks change apparently contrasted with those of Paraceta-
mol and ˇ-CD. A diffraction pattern that is clearly distinct and not
a superposition of each of the components of the binary system are
indicative of formation of a true inclusion complex. The diffraction
pattern of Paracetamol shows intense and sharp peaks indicating
the crystalline nature of the inclusion complex.
B
C
D
On the other hand, the X-ray diffractogram of HP-ˇ-CD physical
mixture corresponded to superimposition of the individual com-
ponents, whereas that of complex consisted fundamentally of a
single very broad band, Fig. 3B. These results suggest the amor-
phous nature of the caged drug in the nanocavity [23].
4000 3600 3200 2800 2400 2000 1600 1200 800
400
-1
Wavenumber / cm
Fig. 4. FT-IR of (A) Paracetamol, (B) ˇ-CD, (C) their physical mixture and (D) their
inclusion complex.
To confirm our observations on the inclusion complex, we per-
formed series of FTIR measurements on the drug with different CDs.
No significant variations were observed in the absorption spectra
corresponding to the physical mixtures which resulted from the
overlapping of the simple spectra of the drug and CD. On the other
hand, a shift and attenuation in the absorption band corresponding
to the carbonyl of drug were observed when the complex is formed.
In Fig. 4 we show the FTIR spectrum of the inclusion complex
drug/ˇ-CD as representatives. The spectrum shows specific absorp-
20000
20000
A
B
16000
15000
Physical mixture
Physical mixture
12000
10000
Inclusion complex
Inclusion complex
8000
5000
4000
0
Paracetamol
Paracetamol
HP -CD
-CD
0
10
20
30
40
50
60
10
20
30
40
50
60
2
Degree)
2
Degree)
Fig. 3. X-ray diffraction patterns of Paracetamol: (A) ˇ-CD and (B) HP-ˇ-CD, their physical mixture and inclusion complexes.

1908
M. El-Kemary et al. / Spectrochimica Acta Part A 79 (2011) 1904–1908
H'-2,H'-6
for Hꢀ-3 and Hꢀ-5 is about 0.03. Methyl group protons overlapped
H-6
H'-3,H'-5
with H-2 and H-4 ˇ-CD protons with approximate upfield shift of
0.01 ppm. Therefore, we can conclude that the upfield shift of Parac-
etamol protons together with ˇ-CD protons confirm tight inclusion
of Paracetamol inside ˇ-CD cavity.
P.... -CD
CD
P
4. Conclusions
6.9
7.0
7.1
7.2
7.3
The Paracetamol molecule forms inclusion complexes with CDs
(ˇ-CD, Me-ˇ-CD and HP-ˇ-CD). The stoichiometry of the complexes
is 1:1. The stability constants determined by spectrofluorometric
method. The stability constant with Me-ˇ-CD is higher than with ˇ-
CD and HP-ˇ-CD owing to the extension of the hydrophobic depth
of the cavity. The difference in the stability constants between HP-
ˇ-CD and ˇ-CD may be ascribed to the stronger hydrogen bonding
between Paracetamol and HP-ˇ-CD than the ˇ-CD. Solid inclusion
complexes with ˇ-CD or HP-ˇ-CD and drug can be obtained by co-
precipitation and their formation has been probed by using FTIR
1H NMR and X-ray diffraction techniques.
H-1
H-3
H-5
H-4
H-2
3.75
3.50
4.00
5.00
5.25 ppm
Fig. 5. 1H NMR spectra of ˇ-cyclodextrin (ˇ-CD), Paracetamol (P) and their mixture
(P. ˇ-CD) in D2O.
Acknowledgement
tion peaks at 3402 cm−1 (OH stretching H-bonded), 2929 cm−1 (OH
stretching), 1648 cm−1 (OH bending), 1353 cm−1 (OH deforma-
tion), 1243 cm−1 (CH bending), 1160 cm−1 (COC stretching and OH
bending), 1079 and 1027 cm−1 (COC stretching) characteristic for
ˇ-CD. The band at 3388 cm−1 from pure drug shifted to 3402 cm−1
in drug/ˇ-CD inclusion complex due to the intermolecular hydro-
gen bonding.
The chemical shift of ˇ-CD protons reported by different authors
was very close to those in this work 0.05 ppm [7,8 and references
therein]. It is well known that the H-3 and H-5 protons are located
in the interior of the ˇ-CD’s cavity and it is, therefore, likely that
the inclusion of Paracetamol with ˇ-CD will specifically affect the
chemical shifts of these two protons. The addition of Paracetamol
to ˇ-CD causes an upfield shift of ˇ-CD spectrum (Fig. 5). The 1H
NMR spectrum of Paracetamol–ˇ-CD shows that the signals due
to H-1, H-2 and H-4 are upfield shifted by about 0.007, 0.005 and
0.02 ppm, respectively, with similar amount of shifts as observed
for other systems [7,8]. The signal due to H-3 is upfield shifted by
about 0.05 ppm while the unresolved band becomes resolved with
a maximum for H-6 is slightly shifted by less than 0.009 ppm while
that for H-5 is upfield shifted by about 0.07 ppm. That assignment
for H-5 protons is based on the fact that H-3 and H-5 are located in
the interior of the cavity and therefore is expected to suffer similar
amount of shielding. The upfield shift observed for H-3 and H-5
protons confirm the inclusion inside the cavity.
The authors acknowledge the Kafrelsheikh University for a sup-
porting research grant.
References
[1] M. Davis, M. Brewster, Nat. Rev. Drug Discov. 3 (2004) 1023–1035.
[2] S. Monti, S. Sortino, Chem. Soc. Rev. 31 (2002) 287–300.
[3] N. Nandi, K. Bhattacharyya, B. Bagchi, Chem. Rev. 100 (2000) 2013–2045.
[4] M. El-Kemary, J. Organero, L. Santos, A. Douhal, J. Phys. Chem. B 110 (2006)
14128–14134.
[5] M. El-Kemary, I. El-Mehasseb, Talanta 62 (2004) 317–322.
[6] M. El-Kemary, H. El-Gezawy, H. El-Baradie, R. Issa, Spectrochim. Acta A 58
(2002) 495–502.
[7] A. Abdel-Shafi, Spectrochim. Acta A 66 (2007) 732–738.
[8] A.A. Abdel-Shafi, Spectrochim. Acta A 57 (2001) 1819–1828.
[9] M. El-Kemary, A. Douhal, A. Photochemistry and photophysics of cyclodex-
trin caged drugs, in: A. Douhal (Ed.), Cyclodextrin Materials Photochemistry,
Photophysics and Photobiology, Elsevier, 2006 (Chapter 4).
[10] P. Bortolus, S. Monti, Adv. Photochem. 21 (1996) 1–133.
[11] N.S. Moyon, T.S. Singh, S. Mitra, Biophys. Chem. 138 (2008) 55–62.
[12] K. Uekama, T. Irie, in: D. Duchêne (Ed.), Cyclodextrins and Their Industrial Uses,
Editions de Santé, Paris, 1987, pp. 395–439.
[13] C. Rosel, J. Carreno, M. Rodriguez-Baeza, J. Alderetr, Quim. Nova 23 (2000)
749–752.
[14] H. Benesi, J. Hildebrand, J. Am. Chem. Soc. 71 (1949) 2703–2707.
[15] A. Green, E. Miller, J. Guillory, J. Pharm. Sci. (1991) 186–189.
[16] S.K. Mondal, D. Roy, K. Sahu, P. Sen, K. Bhattacharyya, J. Photochem. Photobiol.
A 173 (2005) 334–339.
[17] T. Steiner, W. Saenger, Carbohydr. Res. 275 (1995) 73–82.
[18] C.J. Easton, S.F. Lincoln, Modified Cyclodextrins, Scaffolds and Templates for
Supramolecular Chemistry, Imperial College Press, London, 1999.
[19] P. Mura, N. Zerrouck, M.T. Faucci, F. Maestrelli, C. Chemtob, Eur. J. Pharm. Bio-
pharm. 54 (2002) 181–191.
[20] O. Abou-Zied, A. Al-Hinai, J. Phys. Chem. A 110 (2006) 7835–7840.
[21] J.E. Hansen, E. Pines, G.R. Fleming, J. Phys. Chem. 96 (1992) 6010–6904.
[22] A. Mun˜oz de la Pen˜a, T. Ndou, J.B. Zung, I.M. Warner, J. Phys. Chem. 95 (1991)
3330–3334.
The 1H NMR spectrum of Paracetamol is shown in Fig. 5 and
reveals that Paracetamol has three types of protons: two doublets
for (Hꢀ-2 and Hꢀ-6) at 7.27 ppm and (Hꢀ-3 and Hꢀ-5) at 6.95 ppm
and singlet of CH3 group is found at 3.61 ppm (Fig. 5). The chemical
shift values given by the Chem-Draw version 8 NMR calculations
show that (Hꢀ-2 and Hꢀ-6) > (Hꢀ-3 and Hꢀ-5). The upfield chemical
shift observed for Hꢀ-2 and Hꢀ-6 doublets is about 0.02 while that
[23] S. Baboota, M. Dhaliwal, K. Kohli, AAPS Pharm. Sci. Technol. 6 (2005) 83–89.