NMR study of the inclusion complexes of β-cyclodextrin with diphenhydramine, clonidine and tolperisone

Forming complexes with β-cyclodextrin can enhance stability, dissolution rate, solubility, and bioavailability of an active pharmaceutical ingredient. In this study, the inclusion behavior between β-cyclodextrin (β-CD) and diphenhydramine, clonidine, and tolperisone in DMSO-d6 was investigated using NMR spectroscopy. 1H, 13C, COSY, HMQC, and ROESY data were applied to determine the structure of inclusion complexes, and molecular docking analysis was engaged to identify the most favorable host–guest interactions in the inclusion complexes. Complexation of β-CD with diphenhydramine, clonidine, and tolperisone is accompanied by the insertion of a molecular fragment of the guest molecule, one molecule of diphenhydramine and tolperisone, and two molecules of clonidine, into the inner sphere of one host molecule. The reported study provides useful information for the potential application of the complexation of β-CD with diphenhydramine, clonidine, and tolperisone. This may be a good strategy for the development of solid pharmaceutical dosage forms based on β-CDs as a drug delivery system. The inclusion complexes of β-CD and diphenhydramine, clonidine, and tolperisone were synthesized and analyzed using 1Н, 13С, COSY, HMQC, and ROESY spectroscopy. Diphenhydramine, clonidine, and tolperisone interact with β-CD with the formation of stable 1:1 stoichiometric complexes for β-CD:diphenhydramine and β-CD:tolperisone, and 1:2 stoichiometric complex for β-CD:clonidine. Possible structures of the inclusion complexes between β-CD and diphenhydramine, clonidine, and tolperisone were determined using molecular docking in the software AutoDock 4.0. The inclusion complexes of β-CD and diphenhydramine, clonidine, and tolperisone were synthesized and analyzed using 1Н, 13С, COSY, HMQC, and ROESY spectroscopy. Diphenhydramine, clonidine, and tolperisone interact with β-CD with the formation of stable 1:1 stoichiometric complexes for β-CD:diphenhydramine and β-CD:tolperisone, and 1:2 stoichiometric complex for β-CD:clonidine. Possible structures of the inclusion complexes between β-CD and diphenhydramine, clonidine, and tolperisone were determined using molecular docking in the software AutoDock 4.0.


Introduction
Currently, due to the intensive development of the pharmaceutical industry, the search for new forms of drugs has great significance [1]. In the modern pharmaceutical industry, great prospects are associated with the encapsulation of drugs with effective receptors, which make it possible to obtain solid dosage forms from liquid ones, help to stabilize active pharmaceutical ingredients (APIs) toward the action of light and heat, increase the solubility of the drug, improve its bioavailability, and mask unwanted odors and taste [2]. The encapsulation of pharmaceuticals allows obtaining drugs with prolonged action and increases the possibility of targeted drug transport in the body directly to the site of its action. In this regard, the development of supramolecular forms of APIs diphenhydramine 1, clonidine 2, and tolperisone 3 with β-cyclodextrin (β-CD) and the investigation of their structure is an urgent task of modern chemistry and medicine.
The choice of APIs diphenhydramine, clonidine, and tolperisone can be explained by their high pharmaceutical activity and the importance of searching for longterm action forms of these APIs [3][4][5]. Diphenhydramine, an antihystamine with a bitter taste, is mainly used to relieve symptoms of allergy, insomnia, and fever, to treat tremor and nausea. This drug has a bitter taste due to the amino groups. Studies [6] have shown that the inclusion of diphenhydramine inside the cavity of β-CD masks the bitter taste of the drug due to the interaction between the amino groups of diphenhydramine and the hydrogen atom of β-CD. Clonidine is known as an adjuvant to local anesthetics, which prolongs their action, reducing the dosage required for anesthesia. In vitro studies have shown that complexation of clonidine with β-CD increases clonidine's adjuvant effect of clonidine without changing the intrinsic toxicity of clonidine [7]. Tolperisone is a centrally acting muscle relaxant, which is used to treat increased muscle tone caused by neurological diseases. Side effects include body weakness, nausea, dizziness, increase in liver enzymes and muscle pain. According to the studies [6,7], the inclusion of the APIs into the β-CD cavity may result in reduced side effects of the APIs. Presumably, the same effect may be obtained in the complex between tolperisone and β-CD (Fig. 1).
As for the host molecule, β-CD has been chosen because among the currently known encapsulating receptors for APIs, it stands out in several remarkable properties due to its structure [8]. β-CD is a cyclic oligosaccharide composed of seven D-glucopyranose units. The β-CD molecule has a shape of a truncated cone. Protons H-3 and H-5 are located in the inner hydrophobic bonding surface, and H-2 and H-4 are located in the outer one. The most important feature of β-CD is its ability to bind the guest molecule in its cavity in the aqueous environment (Fig. 2).
One of the main methods of studying supramolecular inclusion complexes is NMR spectroscopy [8,9]. We used this method to study new complexes of APIs diphenhydramine, clonidine, and tolperisone with β-CD.
According to [9], the 1 H NMR spectrum of β-CD, obtained in DMSO-d 6 , consists of six groups of signals in the range 3.23-3.32; 3.45-3.53; 3.56-3.60; 4.47-4.49; 4.77-4.78; 5.66-5.73 ppm. Herein, we study the formation of inclusion complexes of β-CD with APIs 1-3 by determining the difference in the values of 1 H and 13 C chemical shifts of substrates (1-3) and the receptor (β-CD) in the free state and as a part of complexes due to the intermolecular interaction. By the magnitude of the chemical shifts of the internal or external protons of β-CD, it is possible to reveal the formation of, respectively, internal, external, or mixed complexes. Changes in the 1 H and 13 C chemical shifts in the spectra of the substrate make it possible to determine the direction of entry of the latter into the β-CD cavity or interaction with the outer segment of the cavity [12,13].
Firstly, we explored the NMR data for compounds 1-3.
Analysis of the 1 H NMR spectra of compound 1 and supramolecular complex 4 showed ( Table 1)   is observed for two methyl-group protons H-12, H-19, and aliphatic protons H-10. Due to the superposition of the chemical shifts of the H-9 protons with more intense signals from the cyclodextrin molecule, aliphatic protons could not be detected in the supramolecular complex. All the aromatic protons undergo a significant shift to the upfield region (− 0.06 − (− 0.08) ppm) as a result of supramolecular interaction. The smallest change in the chemical shifts of the spectra is recorded for the tertiary proton H-7.
The supramolecular interaction of β-CD with 1 was accompanied by a shift of 5 of 6 considered cyclodextrin signals to the weak field region (0.01-0.03 ppm). The greatest difference (− 0.06 ppm) in the values of chemical shifts of protons was observed for the intracavitary proton H-3, located in the middle of the cyclodextrin cone. A significant change in the chemical shifts of the intracavitary H-5 proton located in the narrow side of the cyclodextrin rim, as well as the position of adjacent H-6 protons, confirms the formation of inclusion complexes. It can be assumed that the formation of inclusion complexes is accompanied by the entry of two hydrophobic phenyl radicals into the β-CD cavity. Changes in the chemical shifts of the intracavity protons of β-CD can also occur when methyl and methylene aliphatic protons of dimethylaminoethoxy fragment enter its cavity. The supramolecular interaction of 1 with external protons of β-CD is accompanied by a change in the chemical shifts of the latter and the formation of external complexes. Comparison of the integral intensities of the signals of protons 1 and β-CD in the composition of supramolecular complex 4 showed that supramolecular ensembles are mainly formed with the composition of one molecule 1 per one molecule of β-CD.
The study of two-dimensional NMR spectra COZY ( 1 H-1 H) and HMQC ( 1 H-13 C) shows correlations in molecule 2 shown in Fig. 2. In the 1 H-1 H COZY spectra of the compound, spin-spin correlations are observed through three bonds of protons of neighboring methine groups H9 and H11 with H10 of the benzene ring by cross-peaks with coordinates at 7.23, 6.80 and 6.80, 7.23 ppm. Heteronuclear interactions of protons with carbon atoms through one bond were established using 1 H-13 C HMQC spectroscopy for the pairs present in the compound: H2 and H3 with C2 and C3 (3.48, 42.58) and H9 and H11 with C9 and C11 (7.22, 128.41) ppm (Fig. 4).
The supramolecular interaction of β-CD with 2 was accompanied by a shift of all considered cyclodextrin 6 signals to the weak field region (0.01-0.03 ppm), and the greatest shift of proton signals is observed in both external (H-4, H-6) and internal (H-3, H-5) cyclodextrin protons. Significant changes in chemical shifts in complex 5 compared to molecule 2 occurred for the amine protons H-4 and H-6. The hydrophilic nature of the pyrazole fragment of molecule 2 allows us to conclude that this fragment cannot enter the inner cavity of β-CD. Therefore, there is a high probability of the formation of external supramolecular complexes of molecules 2 with β-CD. Changes in chemical shifts in the process of supramolecular complexation of aromatic protons H-9 and H-11 of 2 are much less noticeable than those of the analogous aromatic protons of molecule 1 in complex 4. This may indicate a lower complexation activity of the dichlorophenyl fragment during the formation of an internal complex. Comparison of the integral intensities of the signals of protons 2 and β-CD in the composition of supramolecular complex 5 showed that predominantly supramolecular ensembles with the composition of two molecules of 2 per one molecule of β-CD are formed.
The structure of compound 3 was also confirmed by two-dimensional NMR spectroscopy COZY ( 1 H-1 H) and HMQC ( 1 H-13 C). The observed correlations in the molecule are shown in Fig. 5    In order to construct the inclusion complexes, molecular docking was used. The binding mode of a guest to its host was investigated by the software AutoDock 4.0 [14]. The 3D structure of the host (β-CD molecule) was downloaded from the Protein Data Bank (PDB ID: 2V8L) [15]. In the downloaded.pdb file β-CD molecule is located in the "A" chain, the chain "B" was deleted. 3D structures of guest molecules (ligands)-diphenhydramine [16], clonidine [17], and tolperisone [18] were downloaded from PubChem databases in.sdf format and then converted into.pdbqt format in Open Babel software [19]. Gasteiger method was used to assign partial charges of host and guests molecules. The grid spacing was set at 0.0375 nm in each dimension, and each grid map consisted of 40^40^40 grid points. To find a globally optimized conformation and model the interaction between docked molecules (guest and host), the Lamarckian genetic algorithm (LGA) was implied. Fifteen runs were performed during each docking experiment. As for the inclusion complex between β-CD and clonidine (1:2), two docking processes were run consequently. Cluster analysis to docking results was performed to select a complex as a representative binding mode. Complexes were selected by the lowest docking energy.

Experimental part
99% purity β-CD (manufactured by Fluka) was used in the experiment. 1 H and 13 C NMR spectra of substrates (1-3) and β-CD and their supramolecular complexes (4-6) were recorded in DMSO-d 6 and d-chloroform on a JNM-ECA 400 spectrometer (400 MHz for 1 H and 100 MHz for 13 C nuclei) by Jeol (manufactured in Japan). Chemical shifts were measured relative to residual protons or carbons of deuterated dimethyl sulfoxide. In all experiments, the temperature was maintained at 298 K, and standard 5 mm NMR tubes were used. To obtain inclusion complexes of 1-3 with β-CD, the method of coprecipitation from a water-ethanol solution was implied. An ethanol solution of API (0.5 g) was added dropwise to an aqueous solution of β-CD (1:1 mol ratio) at 40-50 °C, at a dropping rate of 1 drop per minute, the mixture was stirred for 20-30 min and then left to evaporate naturally to produce inclusion complexes.

Conclusion
In this study, we have shown that diphenhydramine, clonidine, and tolperisone interact with β-CD with the formation of stable 1:1 stoichiometric complexes for β-CD:diphenhydramine and β-CD:tolperisone, and 1:2 stoichiometric complex for β-CD:clonidine. The chemical shifts in the 1 H and 13 C NMR spectra of the inclusion complexes showed similar characteristics with slight differences compared to their parent molecules. This study provides the idea to support the application of β-CD as a key tool to enhance the pharmaceutical and pharmacological aspects of diphenhydramine, clonidine, and tolperisone and to turn these widely available APIs into more effective drug forms. Data availability All data generated or analysed during this study are included in this published article.

Conflict of interest
The authors declare that they have no conflict of interest.
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