FT IR SPECTROSCOPY AND MOLECULAR MODELLING OF

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1 FT IR SPECTROSCOPY AND MOLECULAR MODELLING OF BENZOCAINE / β-cyclodextrin INCLUSION COMPLEX I. Bratu 1, A. Hernanz 2, Alexandra Olaru 3 1 National Institute for R&D of Isotopic and Molecular Technologies, P.O Box 700, R Cluj-Napoca, Romania 2 UNED, Depto de CC y TT Físico Quimicas Senda del Rey 9, Madrid, Spain 3 "Babes-Bolyai" University, Faculty of Physics, 1 Kogalniceanu st., Cluj-Napoca, Romania Abstract: Benzocaine is a local anesthetic used for different pain treatments. Including it in various cyclodextrins one can increase the solubility of this drug. Several inclusion compounds of β-cyclodextrin (β-cd) with benzocaine, prepared by kneading and coprecipitation methods, were investigated. In order to understand the inclusion process mechanisms FT IR spectroscopic investigation and molecular mechanics calculations were used. The intensity changes and frequency shifts for several molecular vibrations indicate the molecular groups implied in the complexation process. The aromatic ring enters inside cyclodextrin cone, its motional freedom being restricted. The hydrogen bonds play an important role in the inclusion process. MM+ molecular mechanics calculations established the spatial geometry of this inclusion complex. The para-amino group is located near the primary O-H groups (the narrow rim) of the cyclodextrin whereas the ester group is situated near the secondary O-H groups (the larger rim) belonging to the CD truncated cone. Keywords: IR spectroscopy, inclusion complexes, cyclodextrin, benzocaine INTRODUCTION Benzocaine (BZC, molecular formula C9H11NO2, CAS Registry Number ) (Synonyms: 4-aminobenzoic acid ethyl ester; ethyl 4- aminobenzoate; ethyl p-aminobenzoate; ethyl p-aminophenylcarboxylate; p- aminobenzoic acid ethyl ester) is a topical anesthetic. It numbs wherever it is applied. It is widely used in first aid creams and sunburn remedies. Benzocain is an ester, a compound made from p-amino benzoic acid (PABA) and ethanol: 15

2 BZC cross-reacts with other benzoic acid derived local anaesthetics (both topical and injectable forms). It also has the potential to cross-react with p-phenylenediamine found in permanent hair dyes, sulfonamides, sulfonylureas, PABA-based sunscreens, and thiazide-related diuretics. It is used in pharmaceutical preparations and rarely cosmetics. Doctors and dentists use BZC preparations, especially on mucosal surfaces such as the mouth, to prepare or 'numb' a site for injection. BZC eardrops are used for temporary relief of ear pain. BZC will not treat an ear infection. Antibiotics are still needed if ear pain is due to an infection. It is used also for topical anaesthesia in a wide variety of clinical situations including mucous membrane anesthesia prior to endoscopic examination or instrumentation, gag reflex suppression, and anorectal disorders and various pain syndromes. It is available in many dosage forms including gels, creams, ointments, lotions, aerosols, and lozenges. Many nonprescription products are available for the temporary relief of dental or oral pain. BZC and tetracaine are more likely to cause contact sensitization than are other local anesthetics. BZC interferes with the ability of certain nerves to conduct electrical signals, which blocks the transmission of nerve impulses that carry pain messages. BZC works by stopping the sodium entering the nerve ending at the site of the pain. This prevents an electrical signal building up and passing along the nerve fibres to the brain. In this way benzocaine causes numbness and relieves pain at the area it is applied to. In order to increase the water solubility, BZC was included [1] in various cyclodextrins - cyclic oligosaccharides containing six (α-cd), seven (β-cd) or eight (γ-cd) α-1,4-linked glucopiranose units. Crystallographic studies on β-cd (cyclomaltoheptaose or cycloheptaamylose) inclusion complex with BZC were published [2]. Dimeric arrangement of cyclodextrins in crystal 16

3 state was demonstrated [3]. It is important to determine the structure and the forces implied in the inclusion complex formed between cyclodextrin and BZC molecules. Some theoretical investigations (free energy prediction) of several organic molecules (among them BZC) with α and β-cd inclusion complexes were published [4]. The aim of the present paper was to establish the molecular groups implied in the inclusion complex of BZC with β-cd based on FT IR spectral analysis and to compare these results with molecular mechanics calculations. EXPERIMENTAL Inclusion complexes of BZC with β-cd by physical mixture (pm), kneading (kn) and coprecipitation (co), respectively were prepared. The so obtained complexes were recorded in the cm -1 spectral domains with a JASCO FT IR-610 spectrometer using the KBr pellet technique. Molecular mechanics In order to optimize the geometry in vacuum, molecular mechanics computations have been done with the HyperChem software [5]; the details of the algorithms are given elsewhere [6]. The well-known MM+ (with the Polak- Ribière conjugate gradient) method was used to energy-minimize the structures until a RMS gradient lower than kcal mol -1 Å -1 was obtained. RESULTS AND DISCUSSION Infrared absorptions The following IR bands, observed in the FT IR spectra of BZC and its β-cd complex, see Fig. 1, were assigned: O-H stretching band (between 3200 and 3600 cm -1, broad band), N-H stretching band ( cm -1 ), aliphatic C-H stretching bands ( cm -1 ), aromatic C-H stretching bands ( cm -1 ). 17

4 Absorbance solid BZC dash -pm 3341 dot -kn dash dot -co 3222 short dash β-cd Wavenumber / cm -1 Fig. 1.- FT IR spectra, cm -1 The absorption band at 1685 cm -1, see Fig. 2, is the characteristic for the ester group. 18

5 2,0 1, solid BZC dash pm dot kn dash dot co short dash β-cd 1514 Absorbance 1,0 0, , W avenumber / cm -1 Fig. 2.- FT IR spectra, cm solid BZC dash pm dot kn dash dot co short dash β-c D Absorbance Fig. 3.- FT IR spectra, cm W avenum ber / cm -1 Fig.3 19

6 1,2 1,0 0,8 772 solid BZC dash pm dot kn 701 dash dot co short dash β-cd Absorbance 0,6 0,4 0,2 0, W avenumber / cm -1 Fig. 4.- FT IR spectra, cm -1 The C-O stretching can be observed at 1280 cm -1, see Fig. 3. Other amine vibrations, N-H bending and out-of-plane N-H bending are located at 1640~1560 cm -1 and 900~600 cm -1, see Figs.2 and 4, respectively. By comparing the FT IR spectra of the pure BZC and β-cd with the spectra of pm, kn and co samples, the following changes may be noticed: - The band at cm -1 remains unchanged in complexes as well as the band located at 1574 cm The characteristic aromatic absorption of the BZC, cm -1, is shifted to cm -1 for kn and co. - The band at 1514 cm -1 for BZC pure compound is shifted to 1518 cm -1 for kn and co complexes. Possibly, the hydrogen bonds are broken out. 20

7 - The bands at 701 and 775 cm -1, characteristic for the aromatic out-ofplane bending, decreases in intensity, i.e. the motional freedom of the aromatic ring protons is restricted. - In the high frequency region, between 4000 and 2500 cm -1 one can identify the O-H stretching band at 3380 cm -1 for β-cd. This band is shifted to 3366 cm -1 (co) and 3373 cm -1 (kn), respectively. Hydrogen bonding provides an important contribution to the formation of the inclusion complex and it can explain this frequency shift. The N-H stretching bands ( cm -1 ) may be identified perfectly in the pm spectrum whereas for the really obtained inclusion complexes (kn and co) these bands are smeared out, being observed as shoulders of the O-H stretching envelope. Because the high frequency side of this band for co spectrum has a base line of lower intensity than the corresponding ones for pm and kn spectra, one cannot exclude the expelling of the inner cyclodextrin water molecules during the inclusion process [7]. Molecular mechanics In order to find a theoretical support for the previous interpretations of the observed spectral changes, molecular mechanics calculations have been done. The CDs act as complexing agents with different molecules, which enter inside CD torus, and interact with the OH groups of CD by Van der Waals, electrostatic forces and hydrogen bonding [8]. The models have been built starting by locating the BZC molecules at the larger side of the β-cd cavity. The results of the geometry optimisation in vacuum through MM+ molecular mechanics of a β-cd molecule and BZC molecules is presented in Fig

8 Fig. 5: Spatial geometry of BZC / β-cd system after MM+ molecular mechanics computations. X-ray diffraction studies confirm this supramolecular structure [2], i.e. the para-amino group is located near the primary O-H groups (the narrow rim) of the cyclodextrin whereas the ester group is situated near the secondary O-H groups (the larger rim) belonging to the CD truncated cone. CONCLUSIONS True inclusion complexes of BZC with β-cd were obtained by using kneading and coprecipitation methods. They were investigated by FT IR spectroscopy showing the molecular groups involved in the inclusion process. Hydrogen bonding is one of the important contributions in this process. The intensity decrease of the aromatic vibrations suggests its motional hindering, i.e. the aromatic ring is included inside cyclodextrin cone. The expelling of the water molecules during the inclusion process cannot be neglected, also. The spatial structure of this supramolecular assembly was obtained by MM+ molecular mechanics computations and confirmed by X-ray diffraction. 22

9 REFERENCES [1] J.P. Moldenhauer, Ger. Offen. (2002) CODEN GWXXBX DE A Preparation of inclusion complex of benzocaine with γ-cd and its use as local anesthetics. [2] J.A. Hamilton, M.N Sabesan, Carbohydr. Res., 1982, [3] J.A. Hamilton, M.N. Sabesan, L.K. Steinrauf, Carbohydr. Res., 1981, [4] T. Suzuki, J. Chem. Inf. Comput. Sci., 2001, 41(5) [5] HyperChem TM Release 4, Hypercube, Inc. (1994), Waterloo, Ontario N2l 3X2, Canada. [6] Computational Chemistry, Hypercube, Inc. Waterloo, Ontario N2l 3X2, Canada (1994). [7] Jose M. Gavira, Antonio Hernanz, Ioan Bratu, Vibr. Spectrosc., 2003,. 32, Issue 2, [8] Lei Liu, Qing-Xiang Guo, Journal of Inclusion Phenom. and Macrocyclic Chem. 2002, 42,