Chinese Chemical Letters  2016, Vol. 27 Issue (7): 1077-1082 PDF
Study on the inclusion behavior and solid inclusion complex of 5-amino-6-methyl-2-benzimidazolone with cyclodextrins
Sun Wei1, Wang Zhao-Hui1, She Meng-Yao1, Yang Zheng1,2, Jin Xi-Lang1, Wang Ya-Qi1, Shi Zhen1, Li Jian-Li1
a Ministry of Education Key Laboratory of Synthetic and Natural Functional Molecule Chemistry, College of Chemistry & Materials Science, Northwest University, Xi'an 710127, China ;
b School of Chemistry & Chemical Engineering, Xi'an University of Science and Technology, Xi'an 710054, China
Abstract: The inclusion behaviors of three native or modified CDs including β-CD, 2-hydroxypropyl-β-CD (2-Hp-β-CD) and 2, 6-dimethyl-β-CD (Me-β-CD) toward 5-amino-6-methyl-2-benzimidazolone (AMBI) were comparatively investigated by NMR and fluorescence titration in combination with IR spectra, X-ray diffractometry and scanning electron microphotographs. The experimental results jointly demonstrated that the phenyl ring of AMBI entered into the cavity of the CDs and located close to the narrow rims accompanied by the formation of the 1:1 inclusion complex with large stability constant in aqueous solution. The introduction of the hydroxypropyl unit to the host improved the solubility, ultimately effecting an obvious promoting in the fluorescence intensity and the stability constant.
Key words: Benzimidazolone     Cyclodextrin     Inclusion     Fluorescence     NMR
1. Introduction

In recent years, the construction of structurally well-defined host-guest systems for binding specific chemical and biological important functional heterocyclic compounds has evolved into the most attractive fields of supramolecular chemistry since the potential application in chemical sensing, materials science and chemical biology [1-3]. Relying on the non-bonding interactions like hydrogen bonds and van der Waals interactions, supramolecular host-guest investigation also has the potential to deepen our understanding of noncovalent association in complicated chemical and biological systems [4].

Among the most attractive hosts, cyclodextrins (CDs), which is a torus-shaped macrocycle oligosaccharides made up of 6-12 glucose units, has special ability in formation of inclusion water soluble complexes with a variety of water insoluble or poorly soluble functional organic compounds driven by the hydrophobic interaction between the CDs cavity and guest molecule, thus significantly improving the chemical and biological properties [5, 6]. Especially the modification of native CDs can particularly enhance the solubility, chemical stability and bioavailability remarkably (Fig. 1). As a result, significant interest has been acquired in the use of CDs as molecular carrier systems in a variety of fields including organic catalysis, materials science, agricultural pharmacology and pharmaceutics [7].

 Download: larger image Figure 1. Basic structures of β-CD and the inclusion of chemical and biological important guests

On the other hand, benzimidazole derivatives, which represent a predominant structural motif among many natural compounds, organic dyes and pharmaceuticals, have drawn tremendous attention due to the well biological activities that they have shown in drug development toward a lot of challenging diseases [8]. Furthermore, as electron rich ligands, they play important roles in maintaining the dimensionality of the structure and providing supramolecular recognition sites for π-π aromatic stacking interactions, which gains special importance in organometallic chemistry [9]. Additionally, benzimidazole derivatives have been used as pigments with a broad range of hues in watercolor painting and electrophotographic developer toner for over 30 years due to their endurance and light resistance [10]. As a consequence, many organic reactions have been carried out in the presence of inclusion formation by CDs and benzimidazole derivatives making it highly significance in understanding the inclusion process between CDs and benzimidazole derivatives [11].

Recently, much work has been published on the studying of supramolecular recognition and inclusion between CDs and important functional chemical and biological heterocyclic compounds [12]. For instance, Liu et al. [13, 14] studied the molecular induced aggregation of hepta-imidazoliumyl-β-CD toward anionic surfactant and the selective binding of bile salts by β-CD derivatives with appended quinolyl arms. Fan et al. [15] reported the structural analysis of the inclusion complex of β-CD with mnitrophenoxyacetic acid. However, few literatures focus on the investigation of inclusion properties of benzimidazole derivatives with CDs. At present, the research works related to this area are of great challenge and interest.

In this work, we focus on the study of the molecular association that takes place between 5-amino-6-methyl-2-benzimidazolone (AMBI) and three natural or modified CDs including β-CD, 2- hydroxypropyl-β-CD (2-Hp-β-CD) and 2, 6-dimethyl-β-CD (Me-β- CD) by fluorescent titration and two dimensional protons nuclear magnetic resonance (2D ROESY) spectroscopy in combination with infrared spectroscopy (IR), scanning electron microphotographs (SEM) and X-ray diffractometry (XRD).

2. Experimental 2.1. General reagents

β-CD (molecular weight = 1135), 2-Hp-β-CD (averaged molecular weight = 1542, DS = 5.5), Me-β-CD (averaged molecular weight = 1310, DS = 12) were supplied by Aoboxing Bio-tech Co., Ltd., and was purified by recrystallization from double-distilled water. The benzimidazole derivative AMBI was purchased from Aldrich Chemical Inc. D2O was purchased from CIL Chemical Company Inc. The Φ5 mm sample tubes were purchased from Norell Inc.

2.2. Preparation of solid complex of AMBI with the CDs

A solution of 1.0 mmol the CDs in 30 mL distilled water was prepared and added to a solution of 1.0 mmol AMBI in 10 mL methanol. This solution was continuously stirred for 12 h at 60 ℃ and for 12 h at room temperature. After cooling, a white precipitate was formed, then it was filtered off, washed with distilled water and dried at 60 ℃ for 4 h, white powdered products of inclusion complex were obtained.

2.3. Fluorescence measurement

Fluorescence measurements were performed by a Hitachi F-4500 spectrofluorometer using 1 cm quartz cell with 10 nm slit width, and fluorescence emission maximum of AMBI was obtained at 327 nm with 300 nm excitation. All experiments were carried out at 25 ℃. 1 mL 1.25 × 10-4 mol/L AMBI solution was added into 25 mL cuvette, then 1.25 × 10-2 mol/L the CDs solution from 0 to 6.00 mL was added into the cuvette dropwise, respectively, and diluted to 25 mL with doubly distilled water. The mixtures were enough oscillated at 25 ℃ for 12 h, then stood overnight.

2.4. NMR measurement

NMR spectra were recorded on the Bruker Avance Ⅲ-400 MHz spectrometer. The CDs and complex were dissolved in a F5mm sample tube with D2O and were characterized by 1H NMR, respectively. Chemical shift values were expressed in ppm downfield using sodium 2, 2-dimethyl-2-silapentane-5-sulfonate (DSS) as an internal standard. Then ROESY experimentswere carried out using a mixing time of 900 ms in the phase-sensitive mode.

2.5. IR measurement

IR spectra of free AMBI, the CDs, complexes and physical mixtures were performed on the Bruker Tensor 27 spectrometer in KBr disk.

2.6. XRD measurement

Powder X-ray diffractograms of free AMBI, the CDs, complexes and physical mixtures were obtained on the D/MAX-3C diffractometer using Cu${{\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{K}}_{\alpha }}$ (λ = 1.5406Å ) radiation. Diffractograms were collected under conditions of 35 kV, 25 mA. The instrument operated over the 2θ range of 5°-50° at a scanning rate of 48/min.

2.7. SEM measurement

Scanning electron microphotographs were performed on the Hitachi S-570 scanning electron microscope by taking at an excitation voltage of 20 kV and a magnification of 500× (Fig. 1).

3. Results and discussion

The inclusion complexations of AMBI with the CDs were first quantitatively investigated by fluorescent titration at 25 ℃ since the highly sensitive and simplicity of fluorimetric method. As shown in Fig. 2a, the fluorescence intensity of AMBI increased obvious upon combination with CDs in which the intensity of the inclusion complex between AMBI and 2-Hp-β-CD appeared to be the strongest. Thus, the inclusion process of AMBI and 2-Hp-β-CD was chosen to be particular discussed. The investigations of AMBI with β-CD and Me-β-CD were shown in the supporting information.

 Download: larger image Figure 2. (a) Fluorescence intensity of AMBI and the inclusion complex of AMBI with the CDs. (b) Fluorescent titration of AMBI (5 μmol/L) with different concentration of 2-Hp-β-CD (0–3 μmol/L). Inset: Fluorescence intensity of the inclusion complex of AMBI with 2-Hp-β-CD at 326 nm, lex = 300 nm. (c) Job's plot of AMBI with 2-Hp-β-CD. The total concentration was 2 μmol/L

Fig. 2b illustrates the fluorescence titration curves of 2-Hp-b- CD with AMBI. The emission intensity of AMBI gradually increases with the addition of varying amounts of 2-Hp-β-CD, suggesting the formation of the complex between 2-Hp-β-CD and AMBI since the 2-Hp-β-CD cavity provides an apolar environment for the AMBI molecule, which could increases the fluorescence intensity of AMBI via promoting the solubility, avoiding the inactivation collision and protecting the fluorescent singlet of AMBI from the external fluorescent quenching factors. In order to gain an insight of the binding stoichiometry of 2-Hp-β-CD and AMBI, Job's method was applied. The result suggested that 1:1 is the most possible stoichiometric ratio for the binding mode according to a maximum fluorescence emission at 326 nm when the molecular fraction of AMBI was close to 50% (Fig. 2c). Similarly, other cases of the inclusion between CDs and AMBI also suggested the 1:1 stoichiometry. Thus, the association constants (Ka) of the CDs to AMBI were obtained according to the 1:1 binding mode by nonlinear fitting of the fluorescent titration curve. The DG0 values were also calculated based on the Ka values. The results were listed in Table 1.

Table 1
Stability constants (Ka) and Gibbs free energy (ΔG0) for the inclusion complexation of AMBI with the CDs at 25 ℃.

High resolution nuclear magnetic resonance (NMR) was then used to study the modes and geometries of the inclusion process. Fig. 3A, (c) and (d) shows the 1H NMR spectra of 2-Hp-β-CD and 2-Hp-β-CD-AMBI in D2O solutions. Generally, AMBI can barely dissolved in water, thus no peaks of AMBI will be seen in the 1H NMR spectrum. As a result, the peaks at δ 6.916, δ 6.454 and δ 2.181 provided powerful evidence for the inclusion of AMBI with 2-Hpb- CD. In particular, a single peak appeared at δ 2.745 in the inclusion complex. This may be caused by anisotropic effect of the guest after the inclusion and thus resulted in the structure change of the host. Moreover, the H-5/H-6 signal of 2-Hp-β-CD moved upfield accompanied by the changing in the shape of the peak while other protons of the host showed no changing. The H3 signal moved 0.012 ppm upfield and H-5/H-6 signal moved 0.029 ppm upfield, indicating that the guest entered the host from the smaller entrance.

 Download: larger image Figure 3. (A) 1D NMR spectra of β-CD (a), 2-Hp-β-CD (c), 2, 6-dimethyl-β-CD (e) and the inclusion complex of β-CD (b), 2-Hp-β-CD (d), and 2, 6-dimethyl-β-CD (f) with AMBI, respectively, in D2O. (B) 2D ROESY spectrum of the inclusion complex of 2-Hp-β-CD with AMBI in D2O. (C) Possible structure of the inclusion complex of 2-Hp-β-CD with AMBI.

In order to gain the further information on the geometry of the inclusion complex, ROESY experiments were performed. According to the 1D 1H NMR spectrum, the protons on the phenyl ring of AMBI located in the low field while the protons of 2-Hp-β-CD located in the high field. In the 1H NMR spectrum of 2-Hp-β-CD, d 4.0 corresponding to H-3 which located at the broad rim and d 4.0 corresponding to H-5 which located at the narrow rim inside the cavity, respectively. As shown in Fig. 3B, cross-peaks were easily recognized between the protons on the phenyl ring of AMBI and the protons inside of 2-Hp-β-CD, indicating the entrance of AMBI into the cavity of 2-Hp-β-CD. Moreover, as the cross-peaks were corresponding to the H-4 and H-7 of AMBI with the H-5 of 2-Hp-b- CD and no cross-peaks between H-3 of 2-Hp-β-CD and the protons of AMBI were found, thus providing powerful evidences that the phenyl ring of AMBI located more close to the narrow rim of 2-Hpb- CD. The inclusion process was supposed most probably forced by the dimensional matching between the host and the guest as well as the formation of Van Edward force and hydrophobic force. The proposed structure of inclusion complex was presented in Fig. 3C.

The IR spectra of 2-Hp-β-CD, AMBI, the physical mixture and the inclusion complex were shown in Fig. 4. Both the vibration of the bound water at 1690 cm-1 and the C-C, C-O stretching vibration of the host at 1080 cm-1 and 1030 cm-1 decreased when the inclusion complex formation. In addition, the characteristic peaks of imidazole at 1495 cm-1, 1383 cm-1, 1357 cm-1 and 693 cm-1 as well as the peaks at 1695 cm-1, 1612 cm-1, 1495 cm-1 which corresponding to the frame vibration of the benzene ring all disappeared. The IR investigation demonstrated the formation of the inclusion complex between 2-Hp-β-CD and AMBI.

 Download: larger image Figure 4. IR analysis of (a) 2-Hp-β-CD, (b) AMBI, (c) 1:1 (mol proportion) physical mixture, and (d) inclusion complex in KBr disk.

Further evidence of complex formation is obtained by X-ray power diffraction. The diffractograms of pure AMBI exhibited obvious features of the crystal shape with characteristic diffraction peaks at 8.5, 12.7, 14.7, 24.1, 27.4 and 31.18 (2μ) (Fig. 5a). The diffractograms of 2-Hp-β-CD showed dispersive peaks. The parcelshaped at 10.48 and 18.78 indicated the microcrystal property of 2-Hp-β-CD (Fig. 5b). Fig. 5c corresponded to the spectrum of the physical mixture system, which is practically the superposition of the spectra of the single components. However, the spectrum of inclusion complex was quite different from those of AMBI, 2-Hpb- CD, and the physical mixture (Fig. 5d), which provide obvious evident for the formation of the inclusion complex. Compared with single AMBI, most of the characteristic diffraction peaks disappeared when the inclusion complex formed, indicating the rearrangement of AMBI along with the generation of an amorphous structure during the inclusion process, which may contribute to great significance in further application.

 Download: larger image Figure 5. PowderX-raydiffractograms of (a) AMBI, (b) 2-Hp-β-CD, (c) 1:1 (molproportion) physical mixture, and (d) inclusion complex.

We then observed the crystal formation of AMBI, 2-Hp-β-CD, inclusion complex and physical mixtures by scanning electron microscope. Pure AMBI was needlelike crystal particles (Fig. 6a) while 2-Hp-β-CD is spherical crystal with large dimensions (Fig. 6b). The physical mixture of AMBI and 2-Hp-β-CD just showed the simple mixing of the two substances that could be easily distinguished (Fig. 6c). However, the inclusion complex showed an irregular lamellar structure, which was different from the host or the guest, revealing the transformation of lattice orientation and the formation of the new crystal shape through inclusion.

 Download: larger image Figure 6. SEM of (a) AMBI, (b) 2-Hp-β-CD, (c) 1:1 (mol proportion) physical mixture, and (d) inclusion complex.

4. Conclusion

In conclusion, the formation of supramolecular association structures between AMBI and the CDs has been confirmed by fluorescent titration, NMR spectra, IR, XRD and SEM. The enhancement in the fluorescence intensity of AMBI in the presence of the CDs suggested that a certain fraction of AMBI located inside the cavity of the CDs due to the hydrophobic interaction in the formation of a stable 1:1 stoichiometric complex. 1H NMR data and 2D ROESY spectrometry indicate that the phenyl ring of AMBI entered into the cavity of the CDs and located close to the narrow rims. The hydroxypropyl modified β-CD was proven to be superior to the others, providing a meaningful method in the design and development of promising cyclodextrin host with prominent properties.

Acknowledgments

We are grateful for the support from the National Natural Science Foundation of China (Nos. 21572177, 21272184, 21103137 and J1210057), the Shaanxi Provincial Natural Science Fund Project (No. 2015JZ003), the Xi'an City Science and Technology Project (No. CXY1511 (3)), the Northwest University Science Foundation for Postgraduate Students (No. YZZ14052) and the Chinese National Innovation Experiment Program for University Students (No. 201510697004).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cclet.2016.03.009.

References
 [1] (a) X. Ma, Y.L. Zhao, Biomedical applications of supramolecular systems based on host-guest interactions, Chem. Rev. 115 (2015) 7794-7839; [2] (b) A. Harada, Y. Takashima, M. Nakahata, Supramolecular polymeric materials via cyclodextrin-guest interactions, Acc. Chem. Res. 47 (2014) 2128-2140. [3] (a) J.X. Zhang, P.X. Ma, Cyclodextrin-based supramolecular systems for drug delivery: recent progress and future perspective, Adv. Drug Deliv. Rev. 65 (2013) 1215-1233; [4] J. Szejtli. Introduction and general overview of cyclodextrin chemistry. Chem. Rev. 98 (1998) 1743–1754. DOI:10.1021/cr970022c [5] L. Szente, J. Szemá n. Cyclodextrins in analytical chemistry: host-guest type molecular recognition. Anal. Chem. 85 (2013) 8024–8030. DOI:10.1021/ac400639y [6] K.A. Connors. The stability of cyclodextrin complexes in solution. Chem. Rev. 97 (1997) 1325–1358. DOI:10.1021/cr960371r [7] (b) G. Ghale, W.M. Nau, Dynamically analyte-responsive macrocyclic host-fluorophore systems, Acc. Chem. Res. 47 (2014) 2150-2159. [8] J. Szejtli, Introduction and general overview of cyclodextrin chemistry, Chem. Rev. 98 (1998) 1743-1754. [9] L. Szente, J. Szemá n, Cyclodextrins in analytical chemistry: host-guest type molecular recognition, Anal. Chem. 85 (2013) 8024-8030. [10] K.A. Connors, The stability of cyclodextrin complexes in solution, Chem. Rev. 97 (1997) 1325-1358. [11] (a) S.M.N. Simões, A. Rey-Rico, A. Concheiro, C. Alvarez-Lorenzo, Supramolecular cyclodextrin-based drug nanocarriers, Chem. Commun. 51 (2015) 6275-6289; [12] (b) Y. Chen, Y. Liu, Cyclodextrin-based bioactive supramolecular assemblies, Chem. Soc. Rev. 39 (2010) 495-505; [13] D. Zhao, Y. Chen, Y. Liu. Comparative studies on molecular induced aggregation of hepta-imidazoliumyl-β-cyclodextrin towards anionic surfactants. Chin. Chem. Lett. 26 (2015) 829–833. DOI:10.1016/j.cclet.2014.11.028 [14] S.S. Zhai, Y. Chen, Y. Liu. Selective binding of bile salts by β-cyclodextrin derivatives with appended quinolyl arms. Chin. Chem. Lett. 24 (2013) 442–446. DOI:10.1016/j.cclet.2013.04.008 [15] C.H. Diao, Z. Xu, M.J. Guo, et al. , The structural analysis of the inclusion complex of β-cyclodextrin with m-nitrophenoxyacetic acid. Chin. Chem. Lett. 24 (2013) 487–490. DOI:10.1016/j.cclet.2013.03.047