Excimer formation in inclusion complexes of β-cyclodextrin with salbutamol, sotalol and atenolol: Spectral and molecular modeling studies

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 96 (2012) 95–107

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Excimer formation in inclusion complexes of b-cyclodextrin with salbutamol, sotalol and atenolol: Spectral and molecular modeling studies A. Antony Muthu Prabhu, V.K. Subramanian, N. Rajendiran ⇑ Department of Chemistry, Annamalai University, Annamalai Nagar 608 002, Tamil Nadu, India

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

" Addition of b-CD to aqueous

The energy minimized structures for the inclusion complexes between drugs and b-CD, (a) salbutamol, (b) sotalol and (c) atenolol.

"

"

"

"

solutions of salbutamol, sotalol, atenolol resulted in observation of excimer emission of each drug. The excimer emission is concluded to be due to a 1:2 b-CD:drug inclusion complex. Triexponential decay observed in nanosecond time-resolved fluorescence. Thermodynamic calculations confirm the better stability of the inclusion complex. Nanomaterial structural changes proved the formation of a inclusion complex.

a r t i c l e

i n f o

Article history: Received 1 February 2012 Received in revised form 20 March 2012 Accepted 7 April 2012 Available online 14 May 2012 Keywords: Drug delivery Excimer Cyclodextrin Encapsulation Molecular modeling

a b s t r a c t The inclusion complexation behavior of salbutamol, sotalol and atenolol drugs with b-cyclodextrin (b-CD) were investigated by UV–visible, fluorometry, time resolved fluorescence, FT-IR, 1H NMR, SEM and PM3 methods. The above drugs gave a single emission maximum in water where as dual emission in b-CD. In b-CD solutions the shorter wavelength fluorescence intensity was regularly decreased and longer wavelength fluorescence intensity increased. Addition of b-CD to aqueous solutions of drugs resulted into excimer emission. The excimer emission is concluded to be due to a 1:2 inclusion complex between b-CD and drug. Nanosecond time-resolved studies indicated that all drugs exhibited biexponential decay in solvents and triexponential decay in CD. Investigations of thermodynamic and electronic properties confirmed the stability of the inclusion complex. Ó 2012 Elsevier B.V. All rights reserved.

Introduction b-Cyclodextrin (b-CD) is a cyclic oligosaccharide consisting of seven glucose units [1] and like a bucket shaped structure. The 14 secondary-hydroxyls on C-2, C-3 are situated in the big orifice of the cone-shaped b-CD molecule and the seven hydroxyls of

⇑ Corresponding author. Mobile: +91 94866 28800; fax: +91 4144 238080. E-mail address: [email protected] (N. Rajendiran). 1386-1425/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2012.04.044

C-6 position are situated in the small orifice of the molecule. This makes the outer frame of the cone-shaped molecule hydrophilic. Although there are seven ether-like oxygen atoms in the indicant bonds of b-CD molecule, these oxygen atoms are shielded by C–H bonds in C-3(H) and C-5(H) of molecular inner side making the inner cavity hydrophobic. Due to the characteristic doughnut-like shape of b-CD, various kinds of organic compounds and biologically active materials are incorporated into the b-CD, forming an inclusion coordinated compound by intermolecular non-covalence force.

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The ability of b-CD to form inclusion coordinated compounds is highly affected by the size, shape and hydrophobic nature of the guest molecules. Usually, a single guest molecule is accommodated into the b-CD cavity, with the host/guest stoichiometry of 1:1. So bCD has been often used to enhance the fluorescence intensities of several organic compounds by a process of partial encapsulation or inclusion of the organic guest molecules, which isolates them and shields their excited singlet species from quenching reactions; it also prevents the non-radiative decay processes. Aqueous b-CD solution has been applied [2] as molecular ‘organizing’ media for both fluorimetric [3] and phosphorimetric [4] analysis and for the determination of several organic analytes. When a drug was included into the cavity, the fluorescence and selectivity with outer molecules increased. This lead to the wide application of b-CD in the fields of medicine, food, organic synthesis, environment protection, analytical chemistry, etc. [5–9]. The complexation of drugs with cyclodextrins (CDs) has been extensively studied in recent years due to its pharmaceutical interest [10–13]. CDs have been used as models for protein and enzymes, because they interact with many drugs in a manner similar to that of proteins and enzymes. The inclusion process of drug molecules with CDs leads to important modifications of pharmacokinetic properties of drug molecules; i.e., (i) increase in the solubility of insoluble drugs, (ii) improves the efficiency, chemical stability and bioavailability of poorly soluble drugs, (iii) reduces the drug toxicity by making the drug effective at lower doses, (iv) control the rate of release so on and so forth. Attempts to compare the respective specific efficiencies of the free drug and the inclusion complex require a careful determination of the stoichiometry and the binding constants. Therefore, it is essential to make a comprehensive study and understand the structure of the inclusion complex. In this regard, the present research was undertaken in an attempt to assess the encapsulation of drug molecules with bCD using its intrinsic fluorescence properties as well as via theoretical studies. In order to understand whether the encapsulation behavior of the drugs will be changed by the substituents or length of the aliphatic chain, in this paper, we studied the encapsulation characteristics of the following drugs: (i) salbutamol (-a-([t-butyl amino]methyl)-4-hydroxy-m-xylene-a,a0 -diol), (ii) sotalol (N-(4[1-hydroxy-2-(isopropylamino)ethyl] methane sulfonamide) and (iii) atenolol (4-(2-hydroxy-3-[(1-methyl ethyl)amino] propoxy)benzeneacetamide with b-CD. The spectral studies were also carried out in solvents of different polarity. Atenolol (adrenaline) is one of the most frequently used b-blockers in the treatment of cardiovascular diseases, because of its anti-hypertensic and antiarrhytmic properties. Salbutamol is used as an antiasthmatic and sotalol is used as an antihypertensive.

Experimental Reagents and methods Salbutamol, sotalol, atenolol and b-CD were obtained from Sigma–Aldrich and used as such. All used solvents of the highest grade (spectrograde) were commercially available. Solutions in the pH range 2.0–12.0 were prepared by adding the appropriate amount of phosphate buffer (NaOH and H3PO4). Triply distilled water was used for the preparation of aqueous solutions. The solutions were prepared just before taking measurements. The concentration of the drug solutions were the order of 4  104–4  105 M. The concentration of b-CD was varied from 1  103 to 10  103 M. The experiments were carried out at room temperature 303 K. The solid inclusion complexes were prepared by co-precipitation method.

Instruments Absorption measurements were carried out with a Shimadzu UV 1601 PC model UV–visible spectrophotometer and fluorescence measurements were made by using a Shimadzu spectrofluorimeter model RF-5301. The pH values in the range 2.5–12.0 were measured on an Elico pH meter (model Li-120). FT-IR spectra were obtained on Avatar-330 FT-IR spectroscopy using KBr pelleting in the range 500–4000 cm1. Microscopic morphological structure measurements were made with a JEOL JSM 5610LV scanning electron microscope. Bruker Advance DRX 400 MHz super conducting NMR spectrophotometer was used to record 1H NMR spectra. The fluorescence lifetime measurements were performed using a picoseconds laser and single photon counting setup from JobinVyon IBH. A diode pumped Millena CW laser (Spectra Analysis) 532 nm was used to pump the Ti-Sapphire rod in Tsunami picosecond mode locked laser system (Spectra physics Model No. 4690 M3S). The Ti-Sapphire rod is oriented at Brewster’s angle to the laser beam. The wavelength turning range was 280–540 nm, i.e., standard pico configuration. The fluorescence decay of the sample was analyzed using IBH data analysis software. The fluorescence decay profiles were fitted to the expression:

IðtÞ ¼ A1 exp IðtÞ ¼ A1 exp

  t

s1

  t

s1

þ A2 exp þ A2 exp

  t

ð1Þ

s2

  t

s2

þ A3 exp

  t

s3

ð2Þ

where s1, s2 and s3 are lifetimes of the three components, a1, a2 and a3 are the pre-exponential factors of the same and t is time. The average fluorescence lifetime was calculated using the equation:

hsi ¼

X

si ai :

ð3Þ

Molecular modeling The theoretical calculations were performed with Gaussian 03W package. The initial geometry of the drugs and b-CD were constructed with Spartan 08 and then optimized by PM3 (Parametric method 3). b-CD was fully optimized by PM3 without any symmetry constraint [14–18]. The glycosidic oxygen atoms of CD were placed onto the XY plane and their center was defined as the center of the coordination system. The primary hydroxyl groups were placed pointing toward the positive Z axis. The inclusion complexes were constructed from the PM3-optimized b-CD and guest molecules. The longer dimension of the guest molecule was initially placed onto the Z axis. The position of the guest was determined by the Z coordinate of one selected atom of the guest. The inclusion process was simulated by putting the guest in one end of b-CD and then letting it pass through the b-CD cavity. Since Density Functional Theory (DFT) calculations are expensive (cost and takes long time) in treating such large molecular systems, we used single point energy calculations to the PM3 optimized geometries using Hartree–Fock (HF) method as implemented in Gaussian 03W [14–18]. Results and discussion Effect of b-CD Table 1 and Figs. 1 and 2 depict the absorption and fluorescence maximum for salbutamol, sotalol and atenolol molecules (4  105 M) in different concentrations of b-CD. The inset Figs. 1 and 2 depict the changes for the absorbance and fluorescence intensities observed as a function of the concentration of b-CD added. Upon increasing the b-CD concentration (i) the absorbance

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0.6

0.30

Absorbance

Abs

a

0

10

15

1 0.2

300 250 275 Wavelength (nm)

225

Abs

0.30

1

325

350

λabs = 265 nm

0.20

0.4

0

5

10

[β-CD] × 10-3 M

7

15

0.2

200

220

240 260 280 Wavelength (nm)

0.6

0.30

7

Abs

Absorbance

5

[β-CD] × 10-3 M

b 0.6

c

λabs = 277 nm

0.20

7

0.4

200

Absorbance

of atenolol (kabs  270 nm) increased at the same wavelength, (ii) the absorbance decreased in sotalol whereas increased in salbutamol, (iii) the absorption maximum red shifted in sotalol (kabs  265–270 nm) and (iv) in contrast, it blue shifted in salbutamol (kabs  277–274 nm). The gradual change in the absorbance with the addition of b-CD was attributed to the enhanced dissolution of the drug molecules through the detergent action of b-CD indicating the formation of drug:b-CD inclusion complexes. The above results showed that, the drug molecules were transferred from more protic environments (bulk aqueous phase) to less protic b-CD cavity environments [19–25]. The absence of isosbestic point in the absorption spectra suggested these drugs are not formed 1:1 inclusion complex with bCD. The spectral shifts showed that different functional groups are encapsulated and these functional groups are interacted with the secondary hydroxyl groups of the b-CD. The obtained results indicated that the structure of these complexes is designable by appropriately selecting type, length and functional substituent group in the guest. Fig. 2 shows the emission spectra for these drugs (2  105 M) with varying concentrations of b-CD. The effect of b-CD on the emission spectra were more pronounced than the corresponding effect on the absorption spectra. In the absence of b-CD, single emission was observed whereas dual emission was noticed in bCD. With an increase in the b-CD concentration, the shorter wavelength (SW) emission intensity was gradually decreased with slight red shift, whereas the longer wavelength (LW) emission intensity was slightly increased at the same wavelength. The binding constant for the formation of inclusion complex were determined by analyzing the changes in the intensity of absorption and fluorescence maxima with the b-CD concentration. In order to determine the stoichiometry of the inclusion complex, the dependence on b-CD of the drugs absorbance and fluorescence were analyzed by using the Benesi–Hildebrand equation [26]. Plot of 1/I  I0 vs 1/[b-CD] (both absorption and fluorescence) gave nonlinear line (Figs. 3 and 4). This confirmed these drugs are not formed 1:1 inclusion complex with b-CD. The higher formation constant implied that these drugs are more tightly embedded in the b-CD cavity. The different K values in the b-CD were due to the difference in the length, size and presence of different substituents for these drugs. The different substituent in the aliphatic chain is responsible for the different association constants. Further, one should expect if aliphatic chain is present in the interior part of the b-CD cavity, due to space restriction, LW should appear in the drugs. With increase in the b-CD concentration, the LW emission enhancement for drugs is consistent with this speculation. On the basis of the above discussions, it is clear that the free rotation of the aliphatic chain is restricted as long as the aliphatic chain is present in the b-CD cavity.

0.10

300 250 275 Wavelength (nm)

225

5

10

15

[β-CD] × 10-3 M

1

200

320

λabs = 270 nm

0

0.3

300

325

350

Fig. 1. Absorption spectra of (a) salbutamol, (b) sotalol and (c) atenolol in different b-CD concentrations (M): (1) 0, (2) 0.001, (3) 0.002, (4) 0.004, (5) 0.006, (6) 0.008 and (7) 0.01. Inset figure: absorbance vs b-CD concentrations.

In b-CD solutions, a typical dual fluorescence was seen even though the emission quantum yield for the longer wavelength (LW) was extremely low. Upon addition of b-CD, the LW emission gradually enhanced but the SW emission decreased at the same wavelength. The fluorescence intensity ratio of the LW band to

Table 1 Absorption and fluorescence maxima (nm) of salbutamol, sotalol and atenolol at different concentrations of b-CD. No.

Concentration of b-CD (M)

1

Water (without b-CD)

2

0.002

3

0.006

4

0.010

5 6 7

Excitation wavelength (nm) Binding constant (M1) DG (kJ/mole)

Salbutamol

Sotalol

Atenolol

kabs

log e

kflu

kabs

log e

kflu

kabs

log e

kflu

277 227 276 226 274 222 274 222 270 219 13.58

4.20 4.44 4.28 4.48 4.34 4.53 4.41 4.57

307

265 226 265 226 265 225 270 225 260 578 16.02

4.21 3.76 4.12 3.93 4.04 4.07 3.74 4.18

304

270 224 270 224 270 224 270 215 270 578 16.02

3.11 3.90 3.46 3.98 3.78 4.13 4.00 4.03

299

307 451 307 452 309 453 852 13.70

305 450 307 450 308 450 746 16.66

300 430 300 430 301 430 763 16.72

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200

50

50 30

7

7

100 50

λemi = 450 nm 5 10 15 0

1/Δ ΔA

1

150

40

If

Fluorescence intensity

a

[β-CD] × 10-3 M

1

30 20 10 0

290 250

1

7

150

200

400

600 800 1/[β-CD] M-1

1000

1200

Fig. 3. Benesi–Hildebrand plot for the complexation of salbutamol (s), sotalol (4) and atenolol ( ) with b-CD. (a) Plot of 1/DA vs 1/[b-CD].

100

200

0

530

410 470 Wavelength (nm)

If 60

λemi = 436 nm 5 10 15

0

7

0.20

[β-CD] × 10-3 M

0.15 1/I-I0 × 10-2

Fluorescence intensity

b

35

1

100

290

350

530

410 470 Wavelength (nm)

0.10 0.05 0

300

0

80

1

If

Fluorescence intensity

c

40

225 7

150

7

λemi = 450 0

5

10

15

[β-CD] × 10 M -3

1

75

280

340

400 Wavelength (nm)

460

520

Fig. 2. Fluorescence spectra of (a) salbutamol, (b) sotalol and (c) atenolol in different b-CD concentrations (M): (1) 0, (2) 0.001, (3) 0.002, (4) 0.004, (5) 0.006, (6) 0.008 and (7) 0.01. Inset figure: fluorescence intensity vs b-CD concentrations.

the SW band (Ia/Ib) decreased; in other words, as the concentration of b-CD increased, Ia/Ib ratio is decreased. The enhancement of the LW emission in b-CD indicated that the energy barrier is not affected by the entrapment of drug in the non-polar b-CD cavity. More interestingly, at higher b-CD concentrations, the similar spectral shapes of the absorption and emission maxima suggested formation of similar type of inclusion complexes. On increasing the b-CD concentrations, the SW fluorescence intensity decreased at the same wavelength showing all the drug molecules were deeply encapsulated in the b-CD cavity. Since dipole–dipole interaction between the drugs and b-CD is lowered in the less polar environment (hydrophobic part) a decrease in the fluorescence intensity was noticed [19–25]. Both the absorption and emission maxima were regularly red shift in sotalol, indicating the polar substituent interacted with b-CD hydroxyl groups. Excimer emission The LW emission noticed in b-CD solutions are explained as follows: the presence of broad LW emission in b-CD solutions suggested the drugs are formed 1:2 inclusion complex with b-CD (i.e., b-CD:drug) [27,28]. With an increase in the b-CD concentration,

200

400

600 800 1/[β β-CD] M-1

1000

1200

Fig. 4. Benesi–Hildebrand plot for the complexation of salbutamol (s), sotalol (4) and atenolol ( ) with b-CD. Plot of 1/I – I0 vs 1/[b-CD].

intensities of the LW fluorescence band is enhanced accompanied by a sharpening of the bands. The peak position of the SW fluorescence band is decreased by the addition of b-CD. This broad emission is assignable to the excimer fluorescence of the drugs. The drug excimer fluorescence, which is due to a 1:2 b-CD–drug inclusion complex formed by the self-association of drug into b-CD cavity, is observed upon adding b-CD [27–29]. As in the case of drug, b-CD seems to form a 1:2 inclusion complex with drug that emits drug excimer fluorescence. When a dilute drug (2  106 M) solution containing b-CD was excited, no excimer fluorescence was observed (figure not shown), suggesting that the 1:1 b-CD–drug inclusion complexes self-associate at a higher concentration of drug. That is why, the SW fluorescence intensity is decreased in the presence of b-CD. Thus, from the fluorescence intensity change by the b-CD addition we can determine an equilibrium constant K1(b-CD) for the formation of a 1:1 b-CD–drug inclusion complex (b-CD–drug): K 1 ðb-CDÞ

drug þ b-CD ¢ drug—b-CD 1 1 1 ¼ þ ðIf  I0f Þ a ðaK 1 ðb-CDÞ½b-CD0

ð4Þ ð5Þ

where If and If0 are the fluorescence intensity in the presence and absence of b-CD, respectively, a is a constant and [b-CD]0 is the initial concentration of b-CD [27,28]. From a plot (Fig. 5) based on Eq. (5), a value of 1190 M is obtained as K1(b-CD). This K1(b-CD) value for drug is greater than that for drug, suggesting a stronger hydrophobicity of CD compared to drug. Taking into account the b-CD–drug system, in which a 1:2 inclusion complex is responsible for the excimer fluorescence, the excimer fluorescence in the b-CD–drug system is most likely

A. Antony Muthu Prabhu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 96 (2012) 95–107

to be due to the formation of the 1:2 b-CD–drug inclusion complex ((b-CD–drug)2):

2ðdrug—b-CDÞ

K 2 ðb-CDÞ

¢ ðdrug—b-CDÞ2

ð6Þ

where K2(b-CD) is the equilibrium constant for the formation of the 2:2 inclusion complex. When the above described scheme is true, the excimer fluorescence intensity should be proportional to the concentration of the 1:2 b-CD–drug inclusion complex under our experimental conditions. Thus, comparisons were made between the b-CD concentration dependence of the observed excimer fluorescence intensity and simulation curves for the (b-CD–drug)2 concentrations, which was calculated using the evaluated K2(b-CD) and an assumed K2(b-CD) value, according to the following equations:

½ðb-CD : drugÞ2  ¼ ð½drug0  ½drug  ½b-CD—½drugÞ=2

ð7Þ

2K 1 ðb-CDÞ2 K 2 ðb-CDÞ½b-CD20 ½drug2 þ ð1 þ K 1 ðb-CDÞ½b-CD0 Þ½drug  ½drug0 ¼ 0

ð8Þ

½b-CD—drug ¼ K 1 ðb-CDÞ½b-CD0 ½drug

ð9Þ

Here, [drug]0 and [drug] are the initial concentration of drug and the concentration of free drug, respectively. Fig. 5 shows the best fit curve of [(b-CD–drug)2] calculated with K2(b-CD) = 852, 746 and 763 M for salbutamol, atenalol and sotalol, respectively. The quality of the fit to the observed intensity data is satisfactory. There may be the possibility, however, that the excimer fluorescence is due to a 1:2 b-CD–drug inclusion complex (b-CD–(drug)2). Thus, to investigate this possibility, we compared observed data of the excimer fluorescence intensity with concentration curves calculated for the 1:2 b-CD–drug inclusion complex, as a function of b-CD concentration. Further, when an equilibrium constant for the formation of b-CD–(drug)2 from b-CD–drug and drug was varied from 102 to 105 M, the calculated curves could reproduce the b-CD concentration dependence of the observed excimer fluorescence intensity, evidently indicating that the 1:2 b-CD–drug inclusion complex is responsible for the excimer fluorescence. There is another method for identifying an excimer emitting species [27–29]. Above the pKa value of the secondary hydroxy group of b-CD, a 2:2 inclusion complex dissociates to two 1:1 inclusion complexes because of the electrostatic repulsion between negative charges on the secondary hydroxyl groups of associating b-CDs. Consequently, a drastic decrease in the excimer fluorescence intensity is expected above the pKa value when an excimer emitting species is truly a 2:2 inclusion complex. On the other hand, if a 1:2 b-CD–drug inclusion complex is an excimer emitting species, there is little or no pH dependence of the excimer

[β β-CD-(Drug)2] 0.002

0.004

0.006

0.008

0.010

Excimer intensity (If)

50

0.012

40 30 20 Salbutamol Sotalol Atenolol

10

99

fluorescence intensity. We, thus, attempted to examine the pH dependence of the excimer fluorescence intensity of the above drugs in b-CD (1  102 M) solution. The excimer fluorescence intensity of the above drugs was not considerable reduced above about pH 10 this finding supports our conclusion that the excimer fluorescence is due to the 1:2 inclusion complex. Further, the isosbestic points are not observed in the absorption spectra (Fig. 1) suggested the absence of the 2:2 inclusion complex. Effect of solvents To understand the excimer interactions between the drugs and b-CD, the solvent dependent changes in the absorption and emission spectra for the above drugs in polar and non-polar solvents were recorded. The absorption maxima, log e, fluorescence maxima and Stokes shifts of salbutamol, sotalol and atenolol in different solvents having different polarity are complied in Table 2. Due to very low solubility in cyclohexane, the absorption and fluorescence spectra for the above drugs were obtained using 2% diethyl ether solution of cyclohexane. The absorption maximum for the salbutamol and atenolol were slightly red shifted in polar solvents, whereas in sotalol it was blue shifted. The red shift increased in the following sequence: sotalol < atenolol = salbutamol. The above sequence indicated that the position of the substituent in the phenyl ring is the key factor for their absorption behavior. This is because, different electronic densities of the HOMO on each atom. Examination of these results revealed that the position and/or the molar extinction coefficient of this band were influenced by the nature of the polar substituent present in the phenyl ring. In all solvents, the absorption spectra of the drugs were characterized by two bands: longer wavelength band (LW) with a maximum around 265–278 nm and a shorter wavelength band (SW) at 220–228 nm. The LW band maximum was ascribed to p–p⁄ transition of the phenyl system because this maximum is close to that of phenol (water: kabs  270 nm; kflu  300 nm). The relative location of this LW band to the former one can be ascribed to the higher delocalization of p-electrons of the phenyl ring. Both the UV bands (p–p⁄) suffered a small shifts in solvents on changing the polar substituent attached to the phenyl moiety, which is characteristic behavior of the electronic transition corresponding to these bands. In all solvents, these drugs gave a single broad fluorescence spectrum. Emission maximum for the drugs in polar solvents were slightly red shifted than that of cyclohexane. The emission spectral maxima in water are given below: (atenolol: kflu  301 nm; sotalol: kflu  304 nm; salbutamol: kflu  308 nm). The results of the fluorescence maxima can be explained on the grounds that charge migration from the electron donating group towards the benzene ring increases on excitation. Generally, in the first excited singlet state, the polarity of the electron donating group is increased due to the increased charge transfer interaction from the electron donating group to the aromatic ring and hence the red shift was observed in water than cyclohexane. In sotalol, the presence of isoelectronic –SO2NH– group decreased the solvent interactions whereas in atenolol, CH2 group decreased the amide group interactions with the phenyl ring. Thus, the fluorescence maximum for sotalol and atenolol were blue shifted than that of salbutamol. In all the drugs, the small difference in the dipole moment values in the ground state indicated that the structural changes in the compounds were very small. Prototropic reactions in aqueous and b-CD medium

0 0

2

4

6 8 [β-CD]0 / 10-3 M

10

12

Fig. 5. Normalised concentration of salbutamol (), sotalol (j) and atenolol (N) with b-CD. Plot of [b-CD:(drug)2] vs [b-CD]0/103 M.

To know the effect of b-CD on the prototropic equilibrium between monocation, neutral and monoanion, the pH dependent changes in the absorption and emission spectra of both drugs in aqueous and b-CD medium were recorded. In aqueous medium,

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Table 2 Absorption and fluorescence spectral data (nm) observed for salbutamol, sotalol and atenolol in different solvents. No.

Solvents

1

Cyclohexane

2

1,4-Dioxane

3

Ethyl acetate

4

Acetonitrile

5

t-Butyl alcohol

6

2-Propanol

7

Methanol

8

Water (pH 6.5)

9 11

Onsager cavity radius (Å) ss Correlation coefficient ET ð30Þ vs Dm ss BK vs Dm

Salbutamol

Sotalol

Atenolol

kabs

log e

kflu

Stokes shift

kabs

log e

kflu

Stokes shift

kabs

log e

kflu

Stokes shift

272 220 274 225 275 225 275 225 276 227 277 225 278 227 278 227 5.55

3.80 4.21 3.96 4.27 3.95 4.41 3.97 4.43 3.98 4.42 3.96 4.46 3.90 4.36 3.88 4.20

303

3761

3722 4120

300

3274

305

3576

300

4402

301

3207

305

3314

304

4841

301

3141

306

3552

304

4699

301

3141

308

3568

305

4527

301

3408

308

3505

306

5056

301

3543

308

3505

306

5056

3.25 2.85 3.11 2.88 3.18 3.12 3.02 3.96 3.10 3.99 3.02 3.95 2.74 3.90 3.55 3.75

3635

300

271 225 274 225 274 225 275 226 275 225 273 224 272 224 270 224 5.22

300

3535

3.15 3.64 3.30 3.95 3.33 3.90 3.25 4.20 3.28 3.97 3.12 4.06 3.12 4.22 3.38 4.07

294

304

265 222 267 226 265 226 265 226 266 228 268 230 265 226 265 226 6.23

301

3815

0.9021 0.7212

the absorption and fluorescence spectra were studied in the H0/pH/ H range of 10 to 16.0. The results are summarized as below: With an increase in the proton concentration from pH 7 to H0 10, no significant absorption spectral shifts were observed. However, in the excited state the emission intensity was decreased from pH 3 and it was completely quenched at H0 0.80. This is due to the formation of proton induced quenching non-fluorescent monocation. When the pH was increased from 7 to 10, the absorption and emission maximum for salbutamol was red shifted (kabs  275 nm, 223 nm to 295, 244 nm; kflu  306–352 nm) indicating formation of hydroxyl monoanion. On further increase in the base concentration, a weak emission maximum appeared at longer wavelength suggesting deprotonation in the CHOH group. At pH 12.5 the absorption and emission spectrum of atenolol and sotalol were red shifted in comparison to the neutral species due to the anion formed by deprotonating the CHOH group [21,22]. This is because the pKa value for the deprotonation of aromatic alcohols falls in this range. In the excited state (i) at higher base concentration, emission intensity for atenolol was completely quenched; (ii) with an increase in base concentration, the neutral emission intensity was completely quenched and a weakly red shifted emission maximum was appeared in sotalol (sotalol: kflu  301–348 nm). The above results suggested that the deprotonation took place at the NH group of the sotalol. The absorption and emission maxima were studied in 8  103 M b-CD solutions in the pH range from 0.1 to 11. On comparison with aqueous and b-CD medium, no significant spectral shift was noticed in salbutamol, sotalol and atenolol neutral, monocation and monoanion maxima. Fluorescence lifetime To further analyze the b-CD induced changes in the fluorescence spectra of salbutamol, sotalol and atenolol we measured the emission decay of the normal (310 nm) and excimer emission (450 nm) in acetonitrile, water, 0.01 M b-CD were recorded. Fluorescence lifetimes, pre-exponential factors and average fluorescence lifetimes are presented in Table 3 and the decay curves are shown in Fig. 6. The decay curve of these molecules in acetonitrile and water fitted to a biexponential decay whereas b-CD fitted to triexponential showed existence of short and long lived species, i.e.,

0.9135 0.6881

0.9383 0.7022

monomer and excimer were obtained. The lifetime of both species increased with the increase of the polarity and protic nature of the solvents because charge transfer interaction was increased with bCD. The fluorescence decay of b-CD emission wavelength at 310 nm fitted to a biexponential function but the emission wavelength at 450 nm in b-CD followed triexponential decay. In b-CD, the decay pattern monitored at the LW emission (450 nm) differed considerably from that at SW emission (310 nm). This is because the LW emissions in b-CD confirmed the formation of excimer (two drugs are encapsulated into the b-CD cavity) which well suited our previous interpretation of steady-state emission data. In bCD, the lifetime of atenolol molecule was higher than sotalol and salbutamol. The above results indicated the tendency of complexation of b-CD, in other words atenolol had the high union with bCD. The decay time of the slow component (excimer) increases significantly from acetonitrile to b-CD solutions. The relative quantum yield of the excimer is increased upon addition of b-CD, while it is not observed in solvents. The monomer emission decay in the absence of b-CD exhibits (in acetonitrile and water) a very fast decay as a major decay component (monomer) while excimer emission is not observed. This decay behavior indicates in the presence of b-CD, the drugs are formed excimer. It is noteworthy that the decay time of the excimer is larger than that of monomer emission within experimental uncertainty. This indicates that the equilibrium is not achieved between the monomer and the excimer in water in a rather short period. However, this coincidence is baffled in the presence of b-CD, revealing that the equilibrium between the monomer and excimer states is modified by the formation of the bCD inclusion complexes. This is consistent with the enhancement of the excimer fluorescence in contrast it is not observed in water. One notes that a rise time of the excimer emission, which is different from the fast decay time of the normal emission, was increased as b-CD concentration increased, whereas it was not observed in water. This reflects that the excimer of the drug/ b-CD inclusion complex is quite different from that of the solvents. Thus, the above results conform the formation of the excimer becomes more favorable in the b-CD solution than in the water. Molecular modeling In order to gain more information about the inclusion complexes as described in the above experimental results, semiempir-

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A. Antony Muthu Prabhu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 96 (2012) 95–107 Table 3 Fluorescence decay parameters of salbutamol, sotalol and atenolol in acetonitrile, water and 0.01 M b-CD solution. Drug

Medium

Salbutamol

Acetonitrile (kemi: 310 nm) Water (kemi: 310 nm) b-CD (kemi: 310 nm) (kemi: 450 nm) Acetonitrile (kemi: 310 nm) Water (kemi: 310 nm) b-CD (kemi:305 nm) (kemi: 450 nm) Acetonitrile (kemi: 310 nm) Water (kemi: 310 nm) b-CD (kemi: 305 nm) (kemi: 450 nm)

Sotalol

Atenolol

Lifetime (ns)

hsi

Pre-exponential factor

s1

s2

0.115 0.130 0.072 0.488 0.281 0.257 0.136 0.597 1.851 1.910 0.271 0.348

2.329 1.86 2.15 2.185 2.950 3.304 2.785 2.359 3.588 5.077 4.677 3.942

s3

6.735

6.973

7.130

a1

a2

0.551 0.418 0.963 0.168 0.188 0.170 0.502 0.103 0.017 0.006 0.486 0.245

0.007 0.027 0.056 0.075 0.037 0.038 0.053 0.055 0.075 0.067 0.060 0.063

a3

0.020

0.012

0.018

0.079 0.104 0.188 0.380 0.161 0.169 0.215 0.274 0.300 0.351 0.412 0.460

kemi – emission wavelength.

out PM3 calculation on the ground state geometry of guest molecules in the vacuum phase. The optimized structure, bond distances, bond angles and most out of the ordinary dihedral angles in the drug molecule before and after complexation considered by PM3 method are presented in Tables 4 and 5. The optimized structure of stable inclusion complexes are shown in Fig. 7. From the evaluation of data the structure of free guest molecule was found completely altered after complexation. Considering the shape and dimensions of b-CD, the drugs cannot completely entrap in the b-CD cavity because the length of the drugs are greater than that of b-CD cavity. From the PM3 calculations, the conformation obtained had the phenyl ring of drug molecules along with the part of alkyl chain oriented toward the primary rim, while more polar groups were exposed to the external surface by the secondary rim of b-CD cavity. The complexation energy allowed us to evaluate the inclusion process and to find the most stable inclusion complex between the complexes studied. It was evaluated as

DEcomplexation ¼ Ecomplex  ðEb-CD  Edrug Þ

Fig. 6. Fluorescence decay curve of (a) salbutamol, (b) sotalol and (c) atenolol in acetonitrile, water and 0.01 M b-CD solution.

ical quantum mechanical calculations of the complexes were performed at PM3 level. These studies revealed that a preferred final relative orientation for all the complexes occurred in spite of the different initial configurations arbitrarily imposed. We also carried

ð10Þ

where Ecomplex, Eb-CD and Edrug represent the total energy of the complex, the free optimized b-CD and the free optimized drugs, respectively. The three complexation energies are negative which demonstrated that the inclusion processes of drugs in b-CD are thermodynamically favorable. The lowest values for complexation energy correspond to the most stable complex. Among the three inclusion complexes, sotalol–b-CD inclusion complex energy was lowest (22.47 Kcal mol1) than salbutamol (13.90 Kcal mol1) and atenolol (15.99 Kcal mol1) indicated that sotalol formed more stable inclusion complex. The binding energy (DE) of the isolated molecule and complex suggested that stability of complex was high compared to isolated molecule. From Table 4, the dipole moment of salbutamol complex was larger than atenolol complexes. The dipole moment of the salbutamol is 4.44 D, sotalol is 5.23 D and atenolol is 2.23 D. This was lower than that of the complex, salbutamol complex 13.3 D, sotalol complex 8.87 D and atenolol complex 11.73 D lower than the dipole moment for resident molecules. The above quantum mechanical computation values demonstrated a strong relationship with the complexation behavior. In addition, there was two intermolecular hydrogen bonding with the cavity in such a configuration. We noticed that the optimized structures in Fig. 7 represented two intermolecular hydrogen bonds respectively between hydrogen atom of hydroxyl group of drugs and oxygen atom of primary hydroxyl group of the CD with a dH  O distance less than 2.50 Å and between hydrogen atom of imine group and –OH group of CD with a distance of 1.88 Å. This justified the importance of both interaction energy between the drugs and b-CD necessary to ensure a bet-

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Table 4 HOMO–LUMO energies and thermodynamic parameters calculations for salbutamol (1), sotalol (2) and atenolol (3) the inclusion complexes using PM3 method. Parameters

1

2

3

b-CD

1:b-CD

2:b-CD

3:b-CD

EHOMO (eV) ELUMO (eV) EHOMO  ELUMO (eV)

9.05 0.10 8.95 4.57 4.45 2.34 0.22 4.44 133.86

9.07 0.53 8.54 4.80 4.00 2.88 0.25 5.23 109.00

9.34 0.12 9.22 4.73 4.61 2.42 0.21 2.23 121.05

10.35 1.23 11.58 4.56 5.79 1.79 0.17 12.29 1457.63

83.48

99.81

109.97

668.16

40.40

53.27

62.51

790.15

9.10 0.06 9.04 4.58 4.52 2.31 0.22 13.30 1605.39 13.9 596.64 155.00 745.98 84.57 0.490 0.063 947.55 0.00

8.94 0.26 8.68 4.66 4.34 2.43 0.23 8.88 1589.10 22.47 588.29 179.68 735.34 108.08 0.493 0.072 939.27 0.00

8.73 0.03 8.76 4.38 4.35 2.20 0.22 11.73 1594.67 15.99 571.28 206.85 719.48 133.18 0.497 0.071 961.52 0.00

l g x S Dipole (D) E (kcal mol1) DE (kcal mol1) H (kcal mol1) DH (kcal mol1) G (kcal mol1) DG (kcal mol1) S (kcal/mol K) DS (kcal/mol K) Zero point vibrational energy (kcal mol1) Mullikan properties

0.144 204.92 0.00

0.156

0.159

195.53 0.00

0.409

217.49 0.00

740.57

Table 5 Geometrical parameters of salbutamol (1), sotalol (2) and atenolol (3) before and after inclusion in b-CD for the most stable inclusion complexes. Bond atoms

1

1:b-CD

Bond length (Å) H20–C4 H7–H20 O1–O3 H7–N

2

2:b-CD

3

7.05 11.45 6.33 8.40

6.96 10.65 6.24 8.22

Bond angles (Å) C4–C8–C9 O2–C7–C2 C9–N–C10

109.18 114.54 114.52

Dihedral angle (Å) C4–C8–C9–N C9–N–C10–C13 C9–N–C10–C11

167.16 177.33 57.20

3:b-CD

H7–H20 H7–C9 C4–H20 H5–C12

13.48 8.37 6.59 6.93

12.65 8.12 6.48 6.85

H7–H22 N1–N2 O1–O2 O2–H22

13.92 10.76 7.22 7.06

12.73 9.71 7.22 7.10

108.63 116.38 115.97

C5–S–N2 C9–N1–C10 C8–C9–N1

101.32 115.47 112.37

100.10 112.23 112.11

O1–C8–N1 C4–O2–C9 C10–C11–N2

117.91 117.07 115.54

117.70 118.30 115.46

163.36 174.24 65.93

C7–S–N2–C1 C4–C8–C9–N1 O3–C8–C9–N1

98.79 74.77 49.72

96.12 70.28 59.00

N1–C8–C7– C1 C4–O2–C9–C10 C10–C11–N2–C12

66.76 176.41 80.61

66.08 107.38 81.92

1.792

HCHO  HO (2°-OH)

Hydrogen bond interactions (Å) SO  HO (2°-OH)

ter inclusion of the guest to the host. The above values were supported by the fact that the flexibility of the host molecule may be one of the structural requirements for inclusion complexes formation. Further the results in Table 5 confirmed that hydrogen bonding interactions also played major roles in the inclusion complexation process. The ‘K’ values are a reasonable measure of hydrogen bonding and the change in hydrogen bonding of the drugs are caused only by the hydrogen ion concentrations [30]. Since the drugs polar substituents are located near the wider end of the b-CD cavity and phenyl ring are located near the narrower range of the b-CD cavity, the ‘K’ values are proportional to the hydrogen bonding interactions. The energy involved in such hydrogen bond interaction is responsible for the higher/lower binding constants compared to those of the substituted/unsubstituted molecules. The difference in slope in Figs. 3 and 4 for these inclusion complexes indicated that the interactions of hydrogen atoms of the guests with b-CD were much stronger confirming the interactions were due to the hydrogen bonding. The Mullikan charge values are zero suggest no charge transfer interactions present between the guest and host molecules. The energetic features, thermodynamic characteristics and electronic properties of these structures are summarized in Table 4. HOMO as ionization energy (IE) and LUMO as electron affinity (EA) were used for calculating the electronic chemical potential (l) which is half of the energy of the HOMO and LUMO:

l ¼ ðEHOMO þ ELUMO Þ=2

2.491

ð11Þ

The hardness (g) as half of the gap energy between HOMO and LUMO was calculated using the following expression:

Gap ¼ EHOMO  ELUMO

ð12Þ

g ¼ ELUMO  EHOMO =2

ð13Þ

The electrophilicity of the components were calculated in HF method using the following equation:

x ¼ l2 =2g

ð14Þ

The HOMO–LUMO energy gap of salbutamol, sotalol and atenolol were calculated using PM3 and are shown in Table 4 and Fig. 8, which revealed that the energy gap reflected the chemical activity of the molecules. The LUMO as an electron acceptor represents the ability to obtain an electron and HOMO represents the ability to donate electron. Moreover, a lower HOMO–LUMO energy gap explained the eventual stability of the complex, i.e., isolated molecule had lower stability than complex molecule. Further the (EHOMO  ELUMO) gap is an important scale of stability [31] and chemicals with large (EHOMO  ELUMO) values tend to have higher stability. So we investigated the electronic structure of the complexes in with these considerations using PM3 method. The HOMO–LUMO value of salbutamol:b-CD complex is more negative than other two drug:b-CD complexes. This suggests salbutamol:b-CD inclusion complexes are more stable than sotalol:b-CD

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a

103

Side view

Upper view

b

c

Fig. 7. Axial and equatorial views of low energy structures calculated for the complexes between b-CD and (a) salbutamol, (b) sotalol and (c) atenolol.

and atenolol:b-CD inclusion complexes. In fact, with the increase of the (EHOMO  ELUMO) gap for the complexes, a new confirmation. However, the energy gap between HOMO and LUMO of each complex suggested that these will be no significant change in the electronic spectrum of the guest molecules driving molecular recognition and binding. We carried out statistical thermodynamic calculation at 1 atm and 303 K. From the results, we noticed that in vacuum, complexation reactions of these three drugs with b-CD are exothermic judged from the positive enthalpy changes which suggested that these inclusion processes are enthalpy driven in nature. The enthalpy changes for drugs/b-CD inclusion complex which was attributed to the more strong van der Waals interactions between drugs:b-CD. Experimentally, the free energy change was calculated from the formation constant (K):

DG ¼ RT ln K

ð15Þ

The values of thermodynamic parameter DG for the formation of the guest molecule to b-CD is given in Table 1. As can be seen from the Table 1, DG is negative which suggests that the inclusion proceeded simultaneously at 303 K. The experimental results indicated that the inclusion reactions were exothermic process. The entropy and Gibbs free energy changes were negative for complexation in all the molecules which implied that the inclusion process were spontaneous at room temperature. The differences between the experimental and theoretical values were due to solvent effect. The experiments were conducted in aqueous medium and the computational work was done at vacuum phase. We were enabling to do the computational work at the aqueous medium due to system limitation. Recently, some authors who encountered this discrepancy turned to experimental values to adjust their calculations. For example in the case of the complexes of both cis and trans isomers of Brooker’s mercyanine inserted within b-CD cavity, the author’s preliminary calculations of DG values predicted the complex

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Fig. 8. The optimized structures and HOMO–LUMO energy structures of salbutamol, sotalol and atenolol.

would not form spontaneously and the magnitude and the sign of DS and DG values were very different from the experiment values [32]. They argued that since experimentally the entropy of complexation depends on both the insertion of the dye molecule and the concurrent displacement of water molecules that are trapped with in the b-CD cavity, the water molecule should be included in the calculations and then they found thermodynamic values closer to the experimental results and the sign matched the reported values in all cases. Further Xing et al. [32] proposed a model to calculate DS of the inclusion complex in aqueous solution with the assumptions that the effect of water molecules on the entropy changes of the 2-hydroxy-5-methoxyacetophenone:b-CD system is mainly determined by the water molecules in the b-CD cavity and the effect of the H2O molecules out of the cavity is less important and thus can be negligible. To the best of our knowledge, the thermodynamic parameters (DG, DH, DS) that described the 1:1 drug b-CD complexation process were not characterized experimentally so we could not adjust our calculations for the moment. Further, from the semiempirical study we noticed that the dipole moment values of b-CD and free drugs increased when the hydrophobic guest entered into the CD cavity and the complex was formed which was an indication of the increase of the polarity. In Table 5 we report the selected bond distances, bond angles and the most interesting angles between the alkyl chain and the phenyl ring of the drug molecules before and after complexation as calculated by PM3 method for the most stable inclusion complex [32]. From comparison of free guest and the complex, it is clear that the geometrical structure of the drugs after complexation is completely altered. This alteration was achieved through the variation of the dihedral angles between the phenyl ring and the alkyl chain of these drugs which were subject to a distortion to adopt a specific conformation leading to the formation of a most stable complex.

In comparison to the structure of inclusion complex, we noticed that a part of the alkyl chain was left out of the hydrophobic cavity of b-CD and the OH group of the drug formed hydrogen bond with b-CD OH group. The non-bonded interaction between the drug phenyl ring and b-CD might be responsible for the difference in structure stability of the complex. Experiment thermo dynamical values will be helpful for numerical investigations in order to draw a conclusion about the effect of the solvent on the binding of the complexation.

Solid inclusion complex studies Further to confirm the drug:b-CD interactions, we also prepared the solid inclusion complexes and characterized by FTIR, 1H NMR and SEM methods.

FT-IR spectral studies Salbutamol The aromatic OH stretching frequency at 619 cm1 and alcoholic CHOH stretching frequency at 3380 cm1 were moved in the inclusion complex to 609 and 3372 cm1, respectively. The phenyl ring and C@C stretching frequency at 3365 and 1616 cm1 were moved in the inclusion complex to 3372 and 1644 cm1, respectively. The C–OH stretching frequency at 1112 cm1 was moved in the inclusion complex to 1156 cm1. NH–R and C–N stretching frequency at 3365 and 1198 cm1 were also moved in the inclusion complex to 3372 and 1252 cm1 respectively. C–CH3 stretching at 2981 cm1 and N–CH3 stretching at 2803 cm1 were shifted in the inclusion complex to 2923 cm1. The CH3 and CH2 deformation frequency at 1383 and 1444 cm1 were moved in the inclusion complex to 1367 and 1416 cm1, respectively.

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Sotalol The phenyl ring CH stretching frequency appears at 3078 cm1 was moved in the inclusion complex to 3382 cm1. The aromatic ring deformation frequency appeared at 689 cm1 was moved in the inclusion complex to 707 cm1. The C@C stretching frequency at 1613 cm1 was moved in the inclusion complex to 1649 cm1. The CHOH stretching frequency appeared at 3406 cm1 was largely shifted in the inclusion complex to shorter wave number at 3382 cm1. The C–OH stretching frequency at 1072 cm1 was also shifted in the inclusion complex to shorter wave number at 1027 cm1. The NH stretching frequency at 3406 cm1 and NH deformation vibration at 733 cm1 were also moved in the inclusion complex to 3382 and 756 cm1, respectively. CH3 group CH stretching

105

frequency at 2829 cm1 and CH3 deformation vibrations at 1394 cm1 were moved in the inclusion complex to 2926 and 368 cm1, respectively. The CH2 stretching frequency at 2788 cm1 was lost in the inclusion complex and CH2 deformation frequency at 1459 cm1 was moved in the inclusion complex to 1418 cm1. The SO2 stretching frequency at 1325 cm1 was moved in the inclusion complex at 1328 cm1. Atenolol The phenyl ring CH stretching frequency at 3100–3300 cm1 was moved in the inclusion complex to the shorter wave number. The aromatic C@C stretching frequency appears at 1613 cm1 was moved in the inclusion complexes to the longer wave numbers (1650 cm1). The amide stretching frequency appeared at 3330

Fig. 9. Scanning electron microscope photographs (Pt. coated) of (a) salbutamol, (b) salbutamol–b-CD, (c) sotalol, (d) sotalol–b-CD, (e) atenolol, (f) atenolol–b-CD; micrograph scale (a) 500 lm, (b) 3000 lm, (c–h) 5000 lm.

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and C@O stretching appeared at 1650–1700 were moved in the inclusion complexes to the longer wave number. The amide NH bending vibrations appears at 1580 cm1 and C–N stretching frequency appears at 1310 cm1 are moved in the inclusion complexes to 1510 and 1300 cm1, respectively. The methyl group stretching frequency appeared at 2964 cm1 and methyl deformation frequency appeared at 1380 cm1 were moved in the inclusion complex to 2926 and 1350 cm1, respectively. The CH2 deformation frequency appeared at 1417 cm1 was moved in the inclusion complex to the shorter wave number to 1411 cm1. The C–O–C stretching frequency at 1240 cm1 was also shifted in the inclusion complex to 1245 cm1. The above results confirmed that all the drugs were encapsulated in the b-CD nano cavity. NMR spectral studies NMR studies allowed us to distinguish between inclusion and other possible external interaction processes. In fact, NMR is the most powerful technique used to determine the inclusion of a guest molecule into the hydrophobic CD cavity, in solution. It is well known that the chemical shifts of the hydrogen atoms located in the interior of the CD cavity (H-3 and H-5) become shielded and usually show significant upfield shift in the presence of a guest molecule, whereas the hydrogen atoms on the outer surface (H1, H-2 and H-4) are not affected or experience only a marginal shift upon complexation. 1H NMR spectra of the salbutamol, sotalol and atenolol inclusion complexes were performed at 25 °C in D2O and compared with those of pure compounds. The variation of the chemical shifts of drug and the inclusion complex (in bracket) values are given below: Salbutamol and its inclusion complexes: C–CH3: 1.02 (1.01), CH2: 2.55 (2.58), CH–OH: 4.42 (4.45), CH2–OH: 4.47 (4.46), NH: 4.60 (4.64), Ar–H: 6.69 (6.73), Ar–H: 6.99 (7.02), Ar–H: 7.27 (7.32). Atenolol and its inclusion complexes: N–H2: 7.40 (7.40), Ar–C– H: 7.16 (7.17), Ar–C–H: 6.85 (6.90), N–H: 6.85 (6.85), CH2–C–O–H: 5.00 (5.02), CH2–C–H: 3.91 (3.93), O–CH2: 3.84 (3.87), CO–CH2: 3.29 (3.31), NH–CH2: 2.68 (2.68), NH–CH2: 2.54 (2.55), CH2–N–H: 1.50 (1.49), CH–CH3: 0.98 (0.97), CH–CH3: 0.97 (0.96). 1 H NMR spectra are one of the most direct evidence for the formation of the inclusion complex [33]. The changes of chemical shifts (Dd) in the complex were rather small, since there were no direct chemical bonds formed between b-CD and the drug molecules of the complex. The interactions between host and guest molecules are noncovalent bonds such as van der Waals forces, hydrophobic interactions and hydrogen bonds [34]. As shown in Fig. 8, H-3 and H-5 protons were located inside the cavity, H-3 closer to the wider rim and H-5 on the opposite. Due to the free rotation of the primary hydroxyl on the smaller rim leading to the steric effect, the most likely mode of complexation was insertion of the less polar moiety of the guest into the b-CD cavity from the wider rim resulting in obvious up field shifts in the signals for H-3 than H-5. The up field shifts is owning to the ring current effect of aromatic part of the guest [35]. The difference in Dd values of drug and the inclusion complex indicated that the molecule of the guest did not enter the b-CD cavity entirely. When the complex was formed, the b-CD H-3 proton located above the ring current of the phenyl ring made it to be shielded and upfield shift, while H-5 proton presented downfield shift owning to the deshielding zone of the ring current. On the contrary, signals for the guest protons will probably show downfield shift changes upon complexation with b-CD [34]. Signals for H-4, H-5 and H-9, 90 protons of these molecules showed downfield shift changes upon the complexation. As can be seen from the values, chemical shifts data for the inclusion complex were slightly different from the free compound. In particular, the resonance of the protons of b-CD located within

or near the cavity showed remarkable upfield shift (0.040 ppm) in the inclusion complex. In salbutamol and atenolol, the aromatic ring hydrogen were downfield shifted in the complex, which suggested that the aromatic ring was shielded largely in the complex and it must penetrate deeply into the cavity. Further, as can be seen from the above values, the resonance of salbutamol, sotalol and atenolol protons with in the b-CD cavity was shifted downfield when the inclusion complex was formed, which means that the salbutamol, sotalol and atenolol has preferred fixed orientation within the b-CD cavity. Nanomaterial structural observation In order to study the nano material structural changes, we recorded the images of the powdered form of the above drugs, b-CD and powdered form of the inclusion complex by scanning electron microscope (Fig. 9). These nano pictures clearly elucidated the difference between the drugs and the inclusion complexes. It is very clear from the SEM images that (i) b-CD is present in platted form, (ii) pure drugs are present in different form from their inclusion complexes. The difference in the structure of pure drugs and their inclusion complex supported the presence of solid inclusion complex. Nano material structural changes proved the formation of a new inclusion complex. Conclusion The present study showed that all the above drugs do not show any significant spectral shifts in the solvents. In aqueous solutions, b-CD has been found to be formed inclusion complexes with salbutamol, sotalol and atenolol. Addition of b-CD to aqueous solutions the above drugs has resulted in the observation of the excimer fluorescence of each compound. The excimer fluorescence concluded to be due to 1:2 b-CD–drug inclusion complex. Due to solvent effect the experimental results of the inclusion complexes were different from computational methods. However, both methods proved that these drugs were partially included in b-CD hydrophobic cavity with phenyl ring located near the primary rim and the alkyl chain near the secondary rim. The free energy, enthalpy and entropy changes suggested that the formation of drug:b-CD inclusion complex is an entropy driven process in vacuum phase and the driving force governing the complexation process was van der Waals interactions. Compared to the guest and host in the free state, complexation of drugs in b-CD showed an increase in the polarity. From the investigation of HOMO and LUMO frontier orbitals of the guest, before and after complexation, we concluded that no significant change in the electronic spectrum of these drugs will be observed upon complexation. Acknowledgments This work is supported by the CSIR (No. 01(2549)/12/EMR-II), UGC (No. F-351-98/2011 (S.R.) and DST, (No. SR/FTP/CS-14/2005). One of the authors A. Antony Muthu Prabhu is thankful to CSIR, New Delhi for the award of Senior Research Fellowship (ACK. No. 123029/2K9/1). The authors thank to Dr. P. Ramamurthy, Director, National Centre for Ultrafast Processes, Madras University for allowing the fluorescence lifetime measurements available for this work. References [1] J. Szejtli, Cyclodextrins and their Inclusion Complexes, Akademiai Kiado, Budapest, 1982. [2] W. Saenger, Angew. Chem. Int. Ed. Engl. 19 (1980) 344–362. [3] O. Jules, S. Scipinski, L.J. Cline Love, Anal. Chim. Acta 169 (1985) 355–360. [4] L.J. Cline Love, M.L. Grayesky, J. Novosky, Anal. Chim. Acta 170 (1985) 3–12.

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