Studies on the inclusion behavior of 9-Aminoacridine into

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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 103 (2013) 18–24

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Studies on the inclusion behavior of 9-Aminoacridine into cyclodextrins: Spectroscopic and theoretical evidences C. Manivannan a, R. Vijay Solomon b, P. Venuvanalingam b,⇑, R. Renganathan a,⇑ a b

School of Chemistry, Bharathidasan University, Tiruchirappalli 620 024, India Theoretical & Computational Chemistry Laboratory, School of Chemistry, Bharathidasan University, Tiruchirappalli 620 024, 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

" 9-Aminoacridine is an active

chemotherapeutic agent. " 9-Aminoacridine incorporates into

the cavity of Cyclodextrins. " The inclusion phenomena were

analyzed using spectroscopic techniques and DFT calculations. " Cyclodextrins intact with drugs through their non-covalent interactions & hydrogen bonding. " The present work has great significance in pharmacology.

a r t i c l e

i n f o

Article history: Received 15 August 2012 Received in revised form 28 October 2012 Accepted 5 November 2012 Available online 16 November 2012 Keywords: 9-Aminoacridine b-CD Host-guest inclusion DFT calculations AIM analysis

a b s t r a c t 9-Aminoacridine (9-AA) is an important attractive pharmaceutical drug employed as chemotheraptic agent for wound dressings. However, 9-AA possesses limited solubility and rapid metabolic decomposition renders this potential drug to limit its applications. Here we propose Cyclodextrins (CDs) as a drug carrier to improve the bioavailability, solubility of 9-AA. The interaction between 9-AA and CDs (a-CD and b-CD) has been studied using UV–Vis absorption, steady state time resolved fluorescence, 1H NMR and FT-IR spectroscopy techniques. The spectroscopic measurements show that 9-AA does not form stable complex with a-CD and also confirmed by DFT calculations. On the other hand, 9-AA forms inclusion complex with b-CD in a 1:1 stoichiometry ratio. Our DFT results suggest that 9-AA stabilizes inside the CD environment through hydrogen bonding that has unambiguously confirmed by AIM analysis. Thus our studies provide a useful insights in the development of Aminoacridine based drugs & its delivery through a suitable carrier like CDs. Ó 2012 Elsevier B.V. All rights reserved.

Introduction Acridine and its derivatives have attracted much attention owing to their immense pharmacological importance [1–3]. 9-Aminoacridine (9-AA) and its salts are known for their excellent antitumor, cytotoxic, antibacterial activity and employed as potential chemotheraptic agents in recent years [4]. In modern pharmacology, the structure of host molecules contains cavity that can ⇑ Corresponding authors. Tel.: +91 431 2407053; fax: +91 431 2407045 (R. Renganathan), tel.: +91 431 2407053; fax: +91 431 2407045 (P. Venuvanalingam). E-mail addresses: [email protected] (P. Venuvanalingam), rren [email protected] (R. Renganathan). 1386-1425/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2012.11.014

accommodate the drugs and release it at a particular site in human body under desirable condition used as a plausible strategy. In recent years, there have been many host molecules reported to overcome such drug suitability issues by forming inclusion complexes. Literature is much flooded with many host molecules including calixarenes, cucurbiturils, crownethers and cyclodextrins. Most of the host molecules such as calixarenes, cucurbiturils possess limited solubility in aqueous solution [5]. In contrast to them, Cyclodextrins (CDs) are the most fascinating drug carrier and finds much attention in the various fields especially in pharmaceutical applications. Because the exterior hydrophilic nature renders to impart the water solubility of the complex formation and the hydrophobic cavities enables the

C. Manivannan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 103 (2013) 18–24

molecules to penetrate body tissues, and release biologically active compounds under specific conditions [6]. Understanding and rationalization of host–guest interactions has highly benefited from the development of theoretical methodologies and play a major role in drug development area [7]. Cyclodextrins (CDs) are torus shaped water soluble cyclic oligosaccharides composed of relatively non-polar internal cavity. The cyclic oligosaccharide contains six, seven, and eight glucopyranose units and they are called a-CD (cyclohexaamylose), b-CD (cycloheptaamylose) and c-CD (cyclooctaamylose) [8,9]. These interesting compounds are characterized by increasing their inner cavity diameter [10–12]. The cavity diameter enables CDs to form inclusion complexes selectively with wide variety of guest molecules of different sizes varying from organic compounds [13,14], drugs and pharmaceutical chemicals [15], aminoacids and their derivatives [16]. The chemical reactivity and spectroscopic properties of guest molecules are perturbed through formation of inclusion complex. Therefore it is interesting to study how CDs are suitable drug carrier for 9-AA and this could be perceptibly important from both industrial and pharmaceutical points of view. Hence, the present work is designed to investigate the complexation of 9-AA utilizing two different cyclodextrins (a-CD and b-CD) through UV–Vis, fluorimetry, time-resolved fluorescence, nuclear magnetic resonance (NMR) and infra red (IR) spectroscopy. To gain further insights, density functional theory (DFT) calculations have been performed and topological analysis has also been done to understand the nature of interactions between the 9-AA and cyclodextrin.

Experimental section Materials Cyclodextrins (a-CD and b-CD) were purchased from Sigma–Aldrich. 9-Aminoacridine hydrochloride was provided by Fluka. Water was triply distilled in all glass apparatus. The NMR samples were prepared with deuterated water (99.9 atom%D) purchased from Aldrich. All chemicals were used without further purification.

Physical measurements and instrumentation Absorption spectra were recorded using JASCO V-630 UV–Vis spectrophotometer. Fluorescence measurements were acquired by the use of a JASCO FP-6500 spectrofluorimeter. Each sample was deoxygenated by purging with nitrogen for 20 min prior to analysis. Fluorescence emission spectra were acquired with an excitation wavelength of 400 nm. Excitation and emission bandwidths were set at 3 nm. All measurements were performed at ambient temperature, unless otherwise indicated. Fluorescence lifetimes were measured using time-correlated single photon counting (TCSPC) spectrometer which comprised of a diode laser pumped milenia V (spectra physics) CW Nd-YVO4 laser that was used as the excitation source for a titanium-sapphire rod locked laser. The emitted photos were detected by a MCP–PMT (Hamamtsu R3809U) after passing through the monochromator (f/3). The laser source was operated at 4 MHz and the signal from the photodiode was used as a stop signal. The data analysis was carried out by the software provided by IBH (DAS-6). The kinetic trace was analysed by non-linear square fitting of mono exponential. The NMR spectra were recorded in D2O solvent with a Bruker-400 MHz instrument using TMS as the internal reference. Chemical shift are given in ppm referenced to solvents. The infrared spectra of compounds were recorded in KBr pellets with a Perkin-Elmer 597 spectrophotometer in the range 4000–400 cm1.

19

Methods Approximately 0.1 mM dye (9-AA) solution was prepared. From this 0.2 ml was added to different volumes of CDs and then made up to 5 mL with the triple distilled water. The sample were shaken with a wrist action shaker for 20 min, and allowed to equilibrate, prior to measurement. These samples were used for lifetime measurement as well. The blanks were prepared with appropriate concentrations of CDs and deionized water for absorbance measurements (corresponding concentrations of the CDs were used as a reference for the absorption measurements). Computational details Density functional theory (DFT) calculations have been performed to explore the possibility on the inclusion of 9-AA into acyclodextrin (a-CD) and b-cyclodextrin (b-CD). Geometry optimization of the a-CD, b-CD, 9-AA and host–guest complexes have been carried out at BP86/TZP using ADF 2007 program [17–19]. In order to gain further insights into the bonding and nonbonding interactions that exists between 9-AA and b-CD, topological analysis have been performed on the optimized 9AA/b-CD complex geometry using AIM2000 package [20] and the necessary wave function has been generated at B3LYP/6-31 g(d) level using Gaussian 09 W [21]. Result and discussion Evidences for the formation of 9-AA/CDs complex were obtained from UV–Vis spectrophotometry, steady-state fluorescence, time resolved, 1H NMR and FT-IR spectroscopies. Further DFT calculations were carried out to understand the inclusion process and nature of interactions between 9-AA and CDs. Effect of a-cyclodextrins (a-CD) In general the suitability of the host towards the guest molecule mainly depends on the size of the host cavity and the size of the incoming guest molecule. The schematic diagram of a-CD, b-CD & 9-AA along with their geometric features is shown in the Supplementary material. The experiments were carried out to study the feasibility of complexation between 9-AA and a-CD. The addition of a-CD, decreases the absorbance of 9-AA and the emission spectra remains unchanged. This leads to the conclusion that 9-AA is not suitable to form stable complexes with a-CD. Since the cavity size of a-CD (4.7–5.3 Å) is smaller than the 9-AA molecule, the a-CD restricts its interaction. This has been unambiguously confirmed by density functional theory (DFT) calculations which will be discussed in section ‘‘DFT calculations’’. Therefore our focus has been shifted to b-CD and its inclusion ability towards 9-AA. Effect of b-cyclodextrin on 9-AA An absorbance study was performed to investigate the ground state interaction of 9-AA with b-CD. Measurements of UV–Vis absorption spectra for all compounds were performed in aqueous media. 9-AA shows maximum absorbance at 400 nm. b-CD shows no characteristic absorbance. Fig. 1 depicts the absorption spectra of 9-AA in the absence and presence of b-CD in aqueous media. It is interesting to note from Fig. 1 that increasing the concentration of b-CD progressively increases the absorbance of 9-AA. The changes in the absorption spectrum of 9-AA in presence of b-CD indicates the formation of inclusion complex as reported earlier [22–24]. 9-AA shows emission maxima at 434 nm. Incremental addition of b-CD results in decrease of emission intensity of 9-AA

C. Manivannan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 103 (2013) 18–24

0.5

1.5

0.4

1.4

0.3

1.3

I 0 /I

Absorbance

20

0.2

1.2

0.1

1.1

0

1

380

390

400

410

420

430

0

440

1

2

Wavelength (nm) Fig. 1. Absorption spectrum of 9-Aminoacridine (4  105 M; Blue line) in the presence of b-CD (0, 3, 5, 7, 9 and 11  104 M; Pink line) in water. (The arrow indicates the absorbance increases with increasing concentration of b-CD). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

700 600 Intensity

500 400 300 200 100 0 410

460

510 Wavelength (nm)

560

Fig. 2. Fluorescence quenching of 9-Aminoacridine (4  106 M; Blue line) in the presence of b-CD (0, 1, 3, 5, 7, 9, 13 and 15  105 M; Pink line) in water. (The arrows indicate the decrease in fluorescence intensity with concomitant blue shift). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

accompanied with a concomitant hypsochromic shift of 3 nm as depicted in Fig. 2. The observation of blue shift in the fluorescence spectrum suggests the hydrophobic environment experienced by the 9-AA molecule with increase in concentration of the host molecule. The possible reason for experiencing such a hydrophobic environment suggest an encapsulation of 9-AA with b-CD and the hydrophobic nature of the host cavity is responsible for the hypsochromic shift of the fluorescence spectra of 9-AA and similar type of observation was already reported in literature [25–27]. Moreover it is interesting to discuss the mode of quenching mechanism resulting from the interaction of 9-AA with b-CD and the Stern–Volmer rate constant (KSV) was determined using the following equation:

I0 =I ¼ 1 þ K SV ½Q  ¼ 1 þ kq :s0

3

4 5 6 -5 [β -CD] x 10 M

7

8

9

10

Fig. 3. Stern–Volmer plots for the steady state fluorescence quenching of 9Aminoacridine by b-CD in water.

fluorophore and quencher [28]. Thus temperature study is performed to distinguish the modes of quenching. Upon increasing the temperature from 15 to 35 °C, the kq values are decreased. This suggests the existence of static quenching resulting from the formation of ground state complex between 9-AA and b-CD. Apart from inclusion process, the fluorescence quenching of 9AA can also result from the interaction with the saccharide units of b-CD. In order to examine this, the same set of experiments are carried out with D-(+)- glucose (noncyclic saccharide) instead of b-CD (see Supplementary material). It is worthy to note that the quenching rate constant for the noncyclic saccharide (9.12  109 M1s1) is 1–2 orders of magnitude lower than b-CD (9.038  1010 M1s1) in aqueous media. This unambiguously confirms the formation of inclusion complex rather than the mere interaction of 9-AA with saccharide unit. The results obtained agreed well with earlier reports [29]. Lifetime measurement The measurement of fluorescence lifetime is the sensitive technique to distinguish static and dynamic quenching [30]. Almost for all samples, two exponential fit to the fluorescence decay proved to be significantly better than the one exponential fit. The predominant (0.871 ± 0.005 relative amplitude) florescence lifetime of 9AA in water was found to be 15.8 ± 0.2 ns. However, there is also a minor component in the decay process (0.149 ± 0.0020 relative amplitude) with lifetime of 5.4 ± 0.5 ns. The obtained results found to be in agreement with the earlier reports [31]. The decay traces of 9-AA in absence and presence of b-CD is shown in the Supplementary material. Though the decay traces were actually plotted, however the lifetime remains same in both conditions. Hence the plot looks like single decay curve. This shows the fluorescence quenching was static in nature. Determination of the b-CD – 9-AA association constant

ð1Þ

where I0 is the fluorescence intensity of the fluorophore in the absence of quencher, I is the fluorescence intensity of the fluorophore in presence of quencher, KSV is the Stern–Volmer constant, s0 is the lifetime of the fluorophore in the absence of quencher, [Q] is the concentration of the quencher and kq is the bimolecular quenching rate constant. According to Eq. (1), Stern–Volmer (S–V) plot of I0/I against [b-CD] results in linear regression with a positive slope shown in Fig. 3. From the slope, KSV values are calculated. The mode of quenching is distinguished by their dependence on temperature and excited state lifetime measurement. Fluorescence quenching shall be dynamic resulting from collisional encounters between fluorophore and quencher or static resulting from the formation of ground state complex between

From the observed quenching behavior, it is crucial to understand the exact stoichiometry and association of 9-AA with b-CD. Benesi–Hilderbrand plots have been examined to characterize the stoichiometry and association of the complex formation [32]. The complex formation reaction between 9-AA and b-CD and the corresponding association constant (Ka) are given by Ka

9  AA þ b  CD 9AA    b  CD where Ka is the equilibrium constant of the above reaction and it can be expressed as

Ka ¼

½9  AA    b  CD ½9  AA½b  CD

ð2Þ

C. Manivannan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 103 (2013) 18–24

0.1

0.06

0

1/(F - F)

0.08

0.04 0.02 0 0

0.2

0.4

0.6 5

0.8

1

-1

1/[β -CD] x 10 (M ) Fig. 4. The Benesi–Hilderbrand plot of 1/(F0  F) vs 1/b-CD of 9-AA in the presence of b-CD.

where [9-AA  b-CD] and [9-AA] are the equilibrium concentration of the inclusion complex and free 9-AA respectively, for a given b-CD concentration.Based on Benesi–Hilderbrand method, plots for the 1:1 and 2:1 stoichiometry ratios are as follows:

1 : 1 complex : 2 : 1 complex :

1 ½F 0  F 1 ½F 0  F

¼

1 1 þ ½F  F 0  K a ½F 0  F½b  CD

ð3Þ

¼

1 1 þ ½F  F 0  K a ½F 0  F½b  CD2

ð4Þ

where Ka is the association constant, F0 is the initial fluorescence intensity of free 9-AA, F0 is the fluorescence intensity of the b-CD inclusion complex and F is the observed fluorescence intensity at its maximum. According to Eq. (3), a plot of 1/(F0  F) vs 1/[b-CD] shown in Fig. 4, indicates the linearity which reflects the formation of 1:1 complex [33]. The binding constant Ka was found to be 8.171  103 M1. Conversely, a plot of 1/(F0  F) vs 1/[b-CD]2 according to Eq. (4) reveals a regression having an upward curvature that the stoichiometry of the complex is not 2:1. The 1:1 association of b-CD with 9-AA reveals the highest degree of linearity, suggesting that the stoichiometry of b-CD with 9-AA is predominantly 1:1. Thermodynamic parameters The association constants (Ka) of the inclusion complexation of 9-AA with b-CD were determined at temperatures ranging from 15 to 35 °C using spectrofluorimeter titrations which are listed in Table 1. The free energy of reaction is derived from the equilibrium constant using the following relationship

DG ¼ DH  T DS ¼ RT ln K a ln K a ¼ DH=RT þ DS=R

ð5Þ ð6Þ

The plot of ln Ka vs 1/T gives a straight line (shown in the Supplementary material) from which the thermodynamic parameters can be assessed. The results obtained are also listed in Table 1. Inspection of Table 1 indicates the standard enthalpy and entropy changes for the formation of 9-AA/b-CD complex are negative.

Table 1 The bimolecular quenching rate constants (kq), association constants (Ka) and thermodynamic parameters of 9-AA/b-CD complex. Temperature (°C)

kq  1010 (M1 s1)

Ka  103 (M1)

DG 0 (kJ/ mol)

D H0 (kJ/ mol)

DS0 (J K1 mol1)

15 25 35

11.25 9.49 7.84

10.44 8.171 5.184

58.56 47.40 31.06

16.46

12.61

Error ±6%.

21

According to the theory of enthalpy–entropy compensation, the formation of inclusion compounds is achieved by the favorable gain in enthalpy change with the loss in entropy [34]. The negative enthalpy change reveals the presence of hydrophobic interaction between dye molecules with cyclodextrins. The negative entropy change observed in this work should result from the restricted rotation of complex once formed [35–38]. The observed negative enthalpy change in 9-AA reveals the presence of hydrophobic interaction in the 9-AA/b-CD system. The negative TDS values obtained are rationalized in terms of the decreased number of trapped water molecules that can be released upon complexation. When 9-AA moiety is included into the b-CD cavity the movement about the glycosidic linkage is restricted to rotation upon complexation and thus result in negative entropy change. 1

H NMR studies of b-CD/9-AA solutions

The investigation of the b-CD complexes with 9-AA using NMR technique is of obvious interest to understand the driving forces and binding modes of host–guest complexes [38–40]. Evidences on the inclusion of guest molecule inside the CD cavity can be accessed from 1H NMR spectroscopy that relies on the observation of selective line broadening, loss of resolution of 1H NMR signals observed upon addition of the guest/host molecule [22]. It is interesting to note the changes occurring in the aromatic region (7.3–6.6 ppm) for 9-AA in absence and presence of b-CD (shown in the Supplementary material). The assignments of 9-AA peaks are as follows. The H-1 and H-4 appear as doublets located at d 7.10 and 6.66; the H-2 and H-3 triplets located at d 7.32 and 6.89, respectively. The absence of –NH2 signals is due to the fast exchange of these protons with deuterium in solution. The chemical shifts of 9-AA are influenced by the b-CD. The downfield shifts of the H-1(0.3 ppm), H-2(0.2 ppm), H-3(0.23 ppm) and H-4(0.30 ppm) were observed. The observed downfield shifts of aromatic protons suggest that the aromatic ring of 9-AA form inclusion complex strongly with b-CD. Correspondingly, insertion of aromatic compounds in the host molecule produce significant shift which was found to be in semblance with the literature already reported for other aromatic compounds [41]. The NMR spectrum of b-CD consist six types of protons: the H-1 doublet at d 4.93, the H-3 triplet at d 3.827, a strong unresolved broad peak consisting of H-5 and H-6 at d 3.77–3.716. The H-2 signal appears as two doublets centered at d 3.523 and 3.497, while H-4 triplet appears at d 3.446. The chemical shift of b-CD protons reported by different authors was very close to those in this work ±0.05 ppm [38,42]. The existence of 9-AA into b-CD cavity, in fact causes a downfield shifted of b-CD as shown in the Supplementary material. More specifically, in presence of 9-AA, the H-5 and H-6 resonance appear somewhat resolved with respect to that in pure b-CD and downfield shifted by 0.02 ppm. H-3 signal shows downfield shift of 0.02 ppm. Similarly H-2 and H-4 signal shows downfield shifts of 0.015 and 0.014 ppm respectively. Since H-3 is located in the interior of the cavity, we suggest that H-5 signal shows similar amount of downfield shift because both are located inside the cavity and will suffer similar amount of shielding. The observed downfield shift of the signals of the internal protons signifies the formation of inclusion complex. FT-IR analysis of 9-AA/b-CD Evidence for the formation of 9-AA/b-CD complex was obtained by mixing saturated solution of 9-AA and b-CD in water. Amazingly, a white precipitate was formed which confirms the interaction and the complex formation between 9-AA and b-CD by IR spectroscopy. The IR spectrum has offered information about the

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Transmittance (%)

(a)

DFT calculations (b)

(c)

4000

the complex formation. The inclusion of guest molecule has been evidenced from the decrement in the intensity, change and shape of the signal indicates the formation of inclusion complex which was documented in the earlier report [44]. Interestingly, the intermolecular C–O–C stretching frequency (ether linkage of b-CD) at 1080 cm1 was not affected which indicates that 9-AA penetrates the b-CD cavity as shown in the Supplementary material. Aithal and his co-workers observed similar behavior for ciprofloxacin with b-CD [45].

3000

2000

1500

1000

400.0

Wavenumber (cm-1) Fig. 5. FT-IR spectra in KBr (a) 9-AA (b) b-CD and (c) 9-AA- b-CD complex.

complex formation in solid phase and emphasizes the functional groups responsible for the interaction when the complex was formed. The interaction established between 9-AA/b-CD in aqueous phase were mainly hydrophobic as previously evidenced, but some polar groups emerging from the cavity responsible to the complex stability probably by the involvement of hydrogen bonds, with outer rim hydroxyl groups. The observation of Fig. 5a–c shows the IR spectra of 9-AA, b-CD and 9-AA/b-CD. The obtained IR spectrum of b-CD resembles with the literature as reported earlier [43]. A comparison between FT-IR spectra of 9-AA, b-CD and 9-AA/b-CD, evidences from the significant changes in the shape and position of the absorbance bands of 9-AA and b-CD functional groups. From Fig. 5a, the sharp peaks appear at 3341 and 3422 cm1 assigned to –NH2 stretching vibrations of 9-AA. b-CD shows –OH stretching at 3391 cm1 as shown in Fig. 5b. The spectrum of the solid complex is quite convoluted at this region and shifted towards 3369 cm1. 9-AA shows aromatic C–H stretching vibrations which appear in the region of 2853–3143 cm1 disappeared in the 9-AA/b-CD complex which has been shown in Fig. 5c. The intense peak at 1656 cm1 assigned to the NH2 scissoring modes of 9-AA was shifted to 1659 cm1 followed with the decrease in the intensity and sharpness in the complex spectrum. Intensification of the C–C stretching, C–H bending, NH2 rocking, N–C pyridine stretching modes of 9-AA lies in the region of 1644, 1590, 1503, 1268 cm1 shows decrement in the intensity of the same band when complexed with b-CD. The aromatic out-of plane C–H bending modes of 9-AA in the region 656–800 cm1 shows decrease in its intensity in the complex spectrum revealing the interaction of the aromatic ring with the hydrophobic cavity of b-CD. All these findings reasonably confirm the formation of the inclusion complex between 9-AA and b-CD. The most interested signals are those due to the polar functional groups present in 9-AA and b-CD implying that polar interaction can act as stabilizing force in

The hydrogen bonding, van der Waals force or hydrophobic interactions are the main driving force responsible for the formation of inclusion complexes [46–53]. To gain further insights into the nature of interactions between 9-AA and CDs, density functional theory calculations were carried out. The guest molecule 9-AA can approach the host via vertically (approach (A)) or horizontally (approach (B)) (see Supplementary material). Due to the larger width of 9-AA compared to the wider rim of the CDs, approach (A) is not possible and therefore approach (B) alone is considered for computations. Here we adopt the rigid body approach to investigate the preferential inclusion geometry via vertical approach (B) where the center of CDs is fixed and the guest molecule 9-AA approaches the host along the Z axis (shown in the Supplementary material). Earlier reports show that the rigid body approach is one of the suitable methods to investigate such large host–guest inclusion complexes [51–53]. The distance between center of CDs and center of guest 9-AA is represented as distance (d) (see Supplementary material). It is modeled in such a way that, the guest molecule approaches the CDs from 10 Å distance from the center of the CDs in steps. As the distance (d) is reduced, the guest 9-AA approaches the CDs cavity and at each point, their energy is computed and an energy profile is plotted. The geometry with lowest energy is taken as a reference and the relative energies have been plotted. The obtained energy profiles for the inclusion of 9-AA into the a-CD & b-CD are given in Fig. 6a and b respectively. The guest 9-AA is gradually inserted into the a-CD and their corresponding energy is plotted against distance (d). As the guest 9-AA approaches the a-CD along Z-axis, the energy initially decreases and reaches the minimum and then it starts increasing steadily due to repulsion. The distance is varied from 10 Å to 4 Å and as moving from 10 Å to 9 Å, the energy is decreased (0.6 kcal/mol) and when it comes to 8 Å further 1.1 kcal/mol is reduced. The energy profile clearly reveals that minima is found to be at 7.1 Å and further inclusion is energetically disfavored as shown in Fig. 6a, indicating no inclusion complex is possible. For instance, 9-AA at 6 Å the repulsion predominates the interactions and resulting in higher energy. As discussed in section ‘‘Effect of a-cyclodextrins (a-CD)’’, the size of the cavity of a-CD is not suitable for the inclusion of 9-AA and is once again confirmed here. The van der Waals surfaces of free a-CD, b-CD and 9-AA/b-CD complex described in Fig. 7 and from the figure it is clear that the cavity of a-CD is not suitable for the guest 9-AA and at the same time the 9-AA perfectly fits into the cavity of the b-CD. Similarly, the inclusion of 9-AA in b-CD, the distance has been varied from 10 Å–2 Å and the energy profile has been obtained (Fig. 6b). From the figure, it is clear that the energy of the host– guest complex is gradually decreases till 4.6 Å and reaches a minimum. After that the energy of the inclusion complexes increases vigorously to reach the maximum as described in Fig. 6b. For instance, on moving from 3.5 Å to 3 Å the energy is increased tremendously from 12.5 kcal/mol to 37.7 kcal/mol respectively. The minimum energy geometry at 4.6 Å is optimized at BP86/TZP and is taken for further analysis. The optimized geometry of the 9-

C. Manivannan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 103 (2013) 18–24

23

Fig. 6. (a) The relative energy plotted against the host guest distance for the complexation of 9-AA into a-CD. (b) The relative energy profile for the complexation of 9-AA into b-CD.

Fig. 7. The vander waals surfaces of a-CD, b-CD and 9-AA/b-CD complex.

AA/b-CD complex along with important bond parameters is given in the Supplementary material. The computed geometrical parameters clearly tell that the guest 9-AA is stabilized inside the host b-CD through hydrogen bonding interactions. Earlier it was reported in literature that the hydroxyl groups of CDs are known to form hydrogen bonding with variety of guest molecules [54,55]. The hydrogen atoms of 9AA are present closer (2.15 Å) to the oxygen atom of hydroxyl group and are presumed to show hydrogen bond. Meanwhile, the other hydrogen atoms of C-H are also present at 2.74 Å and 2.72 Å distance from the rim oxygen atoms respectively. Similarly the nitrogen atom of –NH2 group present at 3.41 Å from the neighboring hydrogen of hydroxyl group of b-CD. This clearly indicates that 9-AA must have been involved in some sort of hydrogen bonding with the b-CD. The theory of atoms in molecules (AIM) is mainly based on electron density q(r) and it provides useful insights on the nature of bonding and non-bonding interactions present in a chemical system [49,50,46]. Earlier reports stand proof to the ability of AIM topological analysis in understanding the various stabilizing interactions in host–guest complexes [47,48]. According to Bader’s AIM theory, the presence of (3,-1) bond critical point (BCP) between any two atoms is accepted as a

criterion for the existence of interactions between them [49,50]. Further the q(r) at BCP describes the nature of interactions. The molecular graph of host–guest complex is drawn (shown in the Supplementary material) where the big spheres correspond to attractors attributed to positions of atoms and critical points such as (3, 1) BCP (red) and (3,+1) RCP (yellow) indicated by small spheres (shown in the Supplementary material). From the molecular graph, it is clear that the guest 9-AA show interactions with b-CD through hydrogen bonding. The hydrogen of amine group show interactions with the hydroxyl oxygen and at the same time, remaining 4 out of 3 hydrogen atoms inside the cavity of b-CD show interaction with neighboring oxygen atoms. Their topological parameters such as q(r), r2q, k1, k2, k3 and bond ellipticity have been gathered in Table 2. The negative value of r2q at BCPs implies that the interactions fall into closed shell category. From the molecular graph, it is obvious that hydrogen bonding interactions stabilizes the 9-AA inside the cavity of bCD. It is well known that the values of the electron density at the BCP should be within 0.002–0.035 a.u. for a typical hydrogen bond [40]. The q(r) values in Table 2 fall in this range and further support the presence of hydrogen bonding in this host–guest complex. Thus the hydrogen bonding plays a vital role in the stabilization of guest 9-AA inside the CD host environment.

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Table 2 Topological parameters of 9-AA-b-CD complex calculated at B3LYP/6-31 g(d) level. Interactions

q

r2 q

k1

k2

k3

Bond ellipticity (e)

NH- - -O CH- - -O CH- - -O CH- - -O

0.017633 0.006720 0.002862 0.006326

0.014642 0.006062 0.002698 0.005451

0.020627 0.005685 0.002442 0.006025

0.020274 0.002346 0.002036 0.005687

0.099471 0.032280 0.015271 0.033518

1.74277975e-002 1.42267488 e-00 1.99660449e-001 5.94537567e-002

Conclusions Herein we report the inclusion of 9-AA, an active chemotheraptic agent into the cavity of cyclodextrins by various spectroscopic methods and DFT calculations. Our results show that b-CD is a suitable drug carrier than a-CD due to their cavity size available for inclusion which is also confirmed by DFT calculations. b-CD forms 1:1 complex with 9-AA and the 9-AA stabilize inside the host environment through hydrophobic interaction and hydrogen bonding. The thermodynamic parameters for the inclusion complex formation were obtained and are useful in elucidating the complex formation. The formation of inclusion complex in solid phase between 9-AA and b-CD has been evidenced by the IR spectral studies and demonstrated that inclusion complex formation is due to the polar bonds which is well supported by AIM analysis. Overall the present study supports the complexation of b-CD with 9-AA which can be explored in future for pharmacological applications. Acknowledgements

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C.M thanks UGC-SAP fellowship for meritorious students. R.V.S. thanks the University Grants Commission, India, for financial support through the Maulana Azad National Fellowship (Ref. No. F.4017(C/M)/2009(SA-III/MANF). R.R thanks DST-FIST and UGC-SAP for spectrofluorimeter facility in the school of chemistry, Bharathidasan University, Trichy. Authors also thank Prof. P. Ramamoorthy, Director, NCUFP, University of Madras, Chennai for lifetime measurements. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2012.11.014.

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