Iranian Journal of Pharmaceutical Research (2014), 13 (4): 1183-1190 Copyright © 2014 by School of Pharmacy Received: April 2013 Shaheed Beheshti University of Medical Sciences and Health Services Accepted: September 2013 Original Article Synthesis of Three Rimantadine Schiff Bases and Their Biological Effects on Serum Albumin Bing-Mi Liua, Ping Mab, Xin Wanga, Yu-Mei Konga, Li-Ping Zhanga and Bin Liua* aCollege of Pharmacy, Liaoning University, Shenyang 110036, P. R. China. bDepartment of Clinical Pharmacology, General Hospital of the Rocket Force, Beijing 100088, P.R.China. Abstract Three new rimantadine Schiff bases (RSBs) were prepared, and then the interaction of RSBs with bovine serum albumin (BSA) was investigated using fluorescence, synchronous fluorescence, UV-vis absorption spectroscopy under physiological conditions. The results showed that the three RSBs effectively quenched the intrinsic fluorescence of BSA via static quenching. Binding constant (Ka), number of binding sites (n), and the binding distance (r) between three RSBs and BSA were calculated by Stern-Volmer equation and Förster’s theory in this study. According to the results of displacement experiments of site probes, it was considered that the binding sites were located in hydrophobic cavities in sub-domains IIA of BSA. What is more, synchronous fluorescence studies indicated that the hydrophobicity around tryptophan residues was increased with the addition of rimantadine-o-vanillin (ROV) and rimantadine- 4-methoxy-salicylaldehyde (RMS), while there was no apparent change with the addition of rimantadine-salicylaldehyde (RS). Keywords: Rimantadine schiff base (RSB); Bovine serum albumin (BSA); Interaction; Fluorescence. Introduction viruses, in particular the influenza A virus (6,7). It has also been reported that rimantadine Schiff bases are a category of compounds hydrochloride has some effects on Parkinson’s containing C=N group. It is reported that many disease. Salicylaldehyde and its derivatives Schiff bases show a variety of interesting are a kind of important chemical-industrial biological actions, including antibacterial, raw material and organic synthesis midbody antifungal, anticancer and antiviral actions (1, (8). Also, they are often used in medicine 2). So, in recent years, some researches have chemical engineering (9) because of their been concentrated on them (3-5). biological activities, such as anti-inflammatory, Rimantadine hydrochloride (α-methyl- antibacterial and antiviral activities (10, 11). 1-adamantane methylamine hydrochloride), In recent years, the new pattern influenzal which exhibits equal efficacy and fewer adverse characteristic is often amphibianous. That is, the reactions than amantadine hydrochloride, has influenza is caused by viruses and germs at the been clinically used for therapy of infections same time (12, 13). Therefore, it is necessary caused by a broad range of RNA-containing to design the difunctional drugs, which have antiviral and antibacterial actions simultaneously * Corresponding author: (14). In this work, three new rimantadine Schiff E-mail: [email protected] (B. Liu) bases (RSBs), rimantadine-salicylaldehyde Liu B-M et al. / IJPR (2014), 13 (4): 1183-1190 Figure 1. Molecular structures of rimantadine-salicylaldehyde (RS), rimantadine-o-vanillin (ROV) and rimantadine-4-methoxy- salicylaldehyde (RMS) Schiff bases. (RS), rimantadine-o-vanillin (ROV) and Salicylaldehyde was purchased from Sinopharm rimantadine-4-methoxy-salicylaldehyde (RMS), Chemical Reagent Co., Ltd., China. The o-vanillin were synthesized which would display the better and 4- methoxy-salicylaldehyde were obtained biologic activities. The molecular structures of from Tianjing Tianhe Chemical Reagent Co., Ltd. three RSBs are shown in Figure 1. Bovine serum albumin (BSA, purity > 99.0 %) Protein plays an important role in life was purchased from Beijing Abxing Biological processes. Serum albumins are the most Technology Company. The BSA solution of 2.00 abundant proteins in the plasma and have many × 10-5 mol/L was prepared by dissolving 1.340 g physiological functions, the most outstanding BSA in 1 L buffer and kept in the dark at 4 °C. property of which is their ability to bind Buffer (pH 7.4) consisted of Tris (0.2 mol/L) reversibly a large variety of ligands, such as and HCl (0.1 mol/L), and the ion strength was cystein, glutathione, Schiff base ligands (15, 16) maintained by adding 0.05 mol/L NaCl. All other and so on. The binding ability of drug-albumin chemicals were of analytical reagent grade. in blood stream may have a significant impact on The structures of RSBs were analysed distribution, free concentration, and metabolism by an AV-600 NMR instrument (Bruker, of drug. Thus, it is important and necessary Germany) and a Fourier transform infrared to study the interaction of drug with serum spectrophotometer (Spectrum 100, PerkinElmer albumins at molecular level. Company, USA). Fluorescence emission spectra Spectroscopic method has been widely and synchronous spectra were collected on an applied in investigating drug binding with F-7000 spetrofluorophotometer (Hitachi, Japan). albumin because of its accuracy, sensitivity, The absorption spectra were recorded on a Cary rapidity and convenience of usage (17). Hence, 50 UV-vis spectrophotometer (Varian, USA). in this study, the interaction of RSBs (RS, ROV, RMS) with BSA was investigated via UV- Syntheses of rimantadine-salicylaldehyde vis absorption and fluorescence spectroscopy. (RS), rimantadine-o-vanillin (ROV) and Besides, the binding sites of RSBs to BSA rimantadine- 4-methoxy-salicylaldehyde (RMS) molecules were also explored by site probes 4.747 g (0.022 mol) rimantadine hydrochloride (ketoprofen and ibuprofen) (18). and 1.234 g (0.022 mol) KOH were added to 50 mL ethanol and then stirred for 2 h at room Experimental temperature. The precipitate was filtered off, and then the solution was transferred to a flask. Rimantadine hydrochloride was obtained from To this solution, 10 mL salicylaldehyde-ethanol Jinan Dachpharm Development Co., Ltd., China. solution (containing 0.02 mol salicylaldehyde) 1184 Synthesis of Three Rimantadine Schiff Bases and Their Biological Effects Table 1. Physical and Infrared spectral data of RS, ROV and RMS. Melting System Colour ν (N－H) (cm-1) ν (C=O) (cm-1) ν (C=N) (cm-1) Point (°C) Rimantadine 2902 Salicylaldehyde 1663 RS yellow 88-89 1630 o-Vanillin 1639 ROV yellow 86-88 1628 4-Methoxy- Salicylaldehyde 1659 RMS pale yellow 103-104 1622 was added drop-wise and stirred at 80 °C for 2 h. spectra of BSA with and without RSBs were After 2 h the reaction mixture was concentrated performed at 290 K in the range of 200-500 nm until RS (yellow precipitate) appeared on the upon excitation at 280 nm. The widths of both bottom. Naturally cooling to room temperature, the entrance and exit slit were set to 5 nm. The the solid mass formed was filtered and washed concentration of BSA firstly was fixed at 1.00 with ethanol three times and dried at 60 °C. × l0-5 mol/L, and then titrated with different According to the method above, ROV and RMS amount of RSBs’ stock solution (2.50 × 10-3 were also synthesized. The related data as follows: mol/L), which was prepared by dissolving the RS, m.p. 88–89 °C; 1H NMR (CDCl3) δ: 1.18 appropriate amount of RSBs with alcohol, and (d, 3H, CH3), 1.56 (s, 6H, CH2), 1.67 (dd, J = then diluting with Tris-HCl-NaCl buffer in 100 12.0, 42.6 Hz, 6H, CH2), 1.99 (s, 3H, CH), 2.83 mL volumetric flask. (q, 1H, N-CH), 6.86 (t, J = 7.8 Hz, 1H, Ph), 6.95 For every addition, the mixture solution must (d, J = 8.4 Hz, 1H, Ph), 7.24 (dd, J = 1.2, 7.2 Hz, be shaken and allowed to stand for 10 min. The 1H, Ph), 7.30 (m, 1H, Ph), 8.26 (s, 1H, CH=N), synchronous fluorescence spectra were recorded 13.91 (s,1H, Ar-OH). from 200 to 500 nm at ∆λ = 15 and ∆λ = 60 nm, ROV, m.p. 86–88 °C; 1H NMR (CDCl3) δ: respectively. Appropriate blanks corresponding 1.18 (d, 3H, CH3), 1.56 (s, 6H, CH2), 1.66 (dd, to the buffer were subtracted to correct the J = 12.0, 41.4 Hz, 6H, CH2), 1.97 (s, 3H, CH), fluorescence or absorption background. 2.87 (q, 1H, N-CH), 3.90 (s, 3H, OCH3), 6.76 (t, J = 7.8 Hz, 1H, Ph), 6.86 (dd, J = 1.2, 7.2 Hz, 1H, Results and Discussion Ph), 6.90 (dd, J = 1.2, 7.8 Hz, 1H, Ph), 8.23 (s, 1H, CH=N), 14.59 (s,1H, Ar-OH). Infrared spectra of RS, ROV and RMS RMS, m.p. 103–104 °C; 1H NMR (CDCl3) δ: Some physical data of three new RSBs and 1.19 (d, 3H, CH3), 1.55(s, 6H, CH2), 1.67 (dd, J = the most important IR peaks of the ligand are ν 12.0, 42.6 Hz, 6H, CH2), 1.99 (s, 3H, CH), 2.84 reported in Table 1. It can be seen that the (C=O) –1 (q, 1H, N-CH), 3.80 (s, 3H, OCH3), 6.33 (dd, J = of salicylaldehyde at 1663 cm disappears and 2.4, 8.4 Hz, 1H, Ph), 6.38 (d, J = 3.0 Hz, 1H, Ph), the ν(N－H) at 2902 cm–1 of rimantadine also 7.06 (d, J = 8.4 Hz, 1H, Ph), 8.02 (s, 1H, CH=N), disappears, then a new peak appears at 1630 cm– 14.40 (s,1H, Ar-OH). 1, which could be attributed to C=N vibrations At the same time, their infrared spectra were and support the formation of the RS. Similarly, also determined by using a Fourier transform new peaks observed at 1628 cm–1 for ROV and infrared spectrophotometer. The corresponding at 1622 cm–1 for RMS indicate the formation of results were offered in Table 1.