Complexes Containing Gatifloxacin

Journal of Biomolecular Structure & Dynamics, ISSN 0739-1102 Volume 26, Issue Number 3, (2008) ©Adenine Press (2008)

Synthesis, Characterization, and DNA-binding Properties of Ln(ІІІ) Complexes Containing Gatifloxacin http://www.jbsdonline.com Abstract Four novel water-soluble complexes of Ln(Ⅲ) with gatifloxacin (HGA), [La(HGA)3Cl3]·2H2O, [Nd(HGA)3Cl3]·2H2O, [Eu(HGA)3Cl3]·2H2O, [Tb(HGA)3Cl3]·2H2O, have been synthesized and characterized by elemental analyses, molar conductivities, IR spectra, fluorescence spectra, and thermogravimetry/differential thermal analysis (TG-DTA). In addition, the DNAbinding properties of the ligand and its complexes have been investigated by absorption, fluorescence spectra, and viscosity measurements. The experimental results indicated that the complexes and ligand bind to DNA via groove binding mode. Key words: Gatifloxacin; Ln(ІІІ) complexes; DNA-binding.

Tonghuan Liu Huili Lu Pinxian Xi Xiaohui Liu Zhihong Xu Fengjuan Chen Zhengzhi Zeng* College of Chemistry and Chemical

Engineering and State Key Laboratory of Applied Organic Chemistry Lanzhou University

Lanzhou 730000, P. R. China

Introduction Quinolones (or quinolonecarboxylic acids or 4-quinolones) are a group of synthetic antibacterial agents containing a 4-oxo-1,4-dihydroquinoline skeleton (1). They can act as antibacterial drugs that effectively inhibit DNA replication and are commonly used for the treatment of many infections (2-5). Recent studies indicate an important role of metal ions in the mechanism of action of these drugs; from investigations of the structure and activity of certain quinolones and the interaction of their metal ions complexes on a DNA model, it was suggested that the intercalation of the quinolone complexed with a metal is an important step in these processes (6-8). Many complexes were reported to show stronger antimicrobial activity than ligand itself (9, 10). HGA as a member of this family, is active against Gram-negative bacteria, Grampositive cocci as well as anaerobes, and is thereby in common clinical use (11). However, investigation about the binding of HGA or its metal complex to DNA is few. In the present work, we synthesized and characterized four novel Ln(ІІІ) complexes of HGA. In addition, the DNA-binding properties with them have been studied with a view to evaluating their pharmaceutical activities. Experimental Reagents and Physical Measurements Tris-HCl buffer (10 mM Tris, 50 mM NaCl, pH 7.2) was used throughout our work. Nucleotides and DNA were purchased from Sigma and used without further purification. The stock solutions were stored at 4 ºC for short periods only. A solution of DNA in the buffer gave a ratio of UV absorbance at 260 and 280 nm of ca. 1.8-1.9 indicating that the DNA was sufficiently free of protein (12). DNA

* Phone: +86-931-8912541 Fax: +86-931-8912582 Email: [email protected]

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concentration per nucleotide was determined by absorption spectroscopy (ε260nm = 6600 M-1cm-1) (13). Other reagents used are of analytical grade. All solutions were prepared with double distilled H2O. Nitrogen, carbon, and hydrogen analyses were determined using a Vario EL elemental analyzer; Infrared spectra (4000-400 cm-1) were determined with KBr disks on a Therrno Mattson FTIR spectrometer. The UV-visible spectra were recorded on a Varian Cary 100 UV-Vis spectrophotometer (14, 15). The fluorescence spectra were recorded on a Hitachi RF-4500 spectrofluorophotometer (15, 16). Thermal analysis (TG-DTA) was carried out on a PCT-2A thermal balance. Condunctivity at 25 ºC was determined in double distilled water (1.0 × 103 M-1) using a DSS-11A molar conductivity meter. Syntheses of Complexes The solution of the HGA (0.31 mmol) in the acetone (20.0 ml) was added dropwise into EtOH (10 ml) containing (0.1 mmol) NdCl3·6H2O. A pale yellow colored precipitate appeared immediately. The mixture was stirred and refluxed for 2 h. The precipitate was centrifuged and washed with methanol, acetone and ether in turn, and then it was dried under vacuum over P2O5 for 48 h. Yield: 90%. [Nd(HGA)3Cl3]·2H2O, [Eu(HGA)3Cl3]·2H2O, [Tb(HGA)3Cl3]·2H2O were prepared as described above, and the yields are 79%, 88%, and 85%, respectively. DNA-binding Experiments All the DNA-binding experiments were performed at room temperature (25 ºC). The absorption titrations were carried out by keeping the concentration of compounds constant (8 μM) and varying DNA concentration. For fluorescent titrations of complexes and HGA with DNA/nucleotides, fixed amounts of compounds (4 μM) were titrated with increasing amounts of DNA/nucleotides. Excitation and emission wavelengths of HGA were 285 and 443 nm, and the complexes were 286 and 445 nm. Slit width was 5 nm. At the same condition, this experiment of HGA and complexes was also performed on Hitachi RF-4500 spectrofluorophotometer. Viscosity experiments were conducted on an Ubbelodhe viscometer thermostated at 25 (± 0.1) ºC in a constant temperature bath. Data are exhibited as (η/η0)1/3 vs. 1/Rt (Rt = [DNA]/[complex or HGA]), where η0 and η are the viscosity of DNA in absence and in presence of the compounds (17). Viscosity values were calculated from the observed flow time of DNA-containing solutions (t) corrected for the flow time of buffer alone (t0), η = t – t0 (18). Results and Discussion Characterization The analytical data and molar conductivities of complexes are shown in Table I. All complexes are soluble in water and DMSO, insoluble in acetone, ether, chloroform, and DMF. They are all stable in air. Thermal behaviors of the ligand and the Ln(III) complexes have been studied. Samples of about 10 mg were placed in a crucible, and heated up to 700 ºC at Table Analytical data and molar conductivities of complexes Complexes LaL3 NdL3 EuL3 TbL3

Analysis Found (Calc.)/% C H N 48.7(48.6) 5.05(5.01) 8.90(8.96) 48.3(48.5) 5.02(4.99) 8.86(8.92) 48.3(48.2) 5.00(4.97) 8.81(8.87) 48.0(48.1) 4.97(4.94) 8.77(8.83)

Molar Conductivity /s·cm2·mol-1 336 276 312 250

the rate of 10 ºC/min in an air atmosphere at ambient pressure, using α-Al2O3 as reference material. The DTA curves of present complexes have an endothermic peak at 73-92 ºC, the corresponding TG curves show that the weight losses are equal to two water molecular, suggesting that the presence of two lattice water in the complexes (19, 20). The result is accord with the elemental analyses study. In the range of 241536 ºC, there are three exothermic peaks appearing around for all complexes, this is because it has a decomposition process in the range of the temperature. While being heated to 700 ºC, the complexes become corresponding oxides (21). The residues are consistent with calculation (20). In Table II the most characteristic absorptions of IR spectra of HGA and complexes are listed. In the spectrum of free HGA, a broad peak appeared in the range 30002500 cm-1 is attributed to the vibration of the NH2+ group. The peaks at ca. 1638.6 and 1357.7 cm-1 are assigned to the νas and νs vibration of the COO- group. The results indicate the transfer of proton from carboxylic group to the terminal piperazinyl N atom (22). The broad band around 3410 cm-1 corresponds to the the O-H stretching vibrations of water molecules. Compared with that of HGA, the vibrations ν (COO-) as and ν (COO )s of carboxyl group and ν (CO)r of the complexes exhibited some shift by about 10-15 cm-1, indicating their involvement in coordination. The bands around 3000-2500 cm-1 (ν(NH2+)) were also found, which suggests that the complexes is similar in a neutral zwitterionic form as in free form (23). So we can infer that HGA coordinated with the lanthanide ions to form a stable six-membered ring through the ring carbonyl oxygen atom and one of the carboxylic oxygen atoms. Table II The main IR frequencies and assignments of HGA and its complexes (cm-1). Compounds HGA NaGA LaL3 NdL3 EuL3 TbL3

(C=O)r (on the ring) 1582.3 1582.6 1570.9 1570.3 1571.6 1572.6

as

(COO-)

1638.6 1621.0 1612.5 1612.6 1613.7 1614.9

s

(COO-)

1357.7 1365.7 1372.1 1371.8 1373.0 1371.4

On the basis of above evidence and analyses, the possible structure of the complexes is shown in Figure 1 and their formulae are Ln(HGA)3Cl3·2H2O (Ln = La, Nd, Eu, and Tb). The excitation and emission spectra, as well as the fluorescence intensity, were measured on dried and finely powdered samples at room temperature. The major fluorescence spectral data are summarized in Table III. The europium and terbium complexes exhibited characteristic fluorescence of europium and terbium ions (24). The emission spectrum of the europium complex is shown in Table IV. For EuL3, two visible bright emission peaks, which centered at 592 nm and 614 nm were

Figure 1: The tentative coordiation mode of HGA and lanthanide metals (Ln = La, Nd, Eu, Tb).

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observed (25) and assigned to 5D0 → 7F1 and 5D0 → 7F2, respectively. For TbL3, the weak emission at 545.6 nm [relative fluorescence intensity (RFI) = 84.73] is assigned to 5D4 → 7F5 transition. This can be rationalized as the fact that the energy transfer from ligands to the Tb(ІІІ) is uncompleted. Based on the theory of antenna effect (26, 27), the luminescence of Ln(ІІІ) chelates is related to the efficiency of the intramolecular energy transfer between the triple level of the ligand and the emitting level of the ions, which depends on the energy gap between the two levels. From the results, it can be found that the emission intensities of Eu(III) complex is stronger than the Tb(III) complex in solid state, This indicates that the ligand is a good organic chelator to absorb and transfer energy to metal ions. The lowest triplet state energy level of the ligand indicates that the triplet state energy level (T1) of the ligand matches better to the resonance level of Eu(III) than Tb(III). Table III Fluorescence data complex at room temperature. Complexe [Eu(HGA)3Cl3] · 2H2O [Tb(HGA)3Cl3] · 2H2O a

Ex/Em Slit (nm) 10

10

ex

(nm)

392 353

em

(nm)

593.6 614.0 545

RFI is relative fluorescence intensity. concentration : 2 10 b

RFIa 455.7 2450 84.73

Transition 5D0 5D0 5D4

7F1 7F2 7F5

4mol L-1.

Absorption Spectra The maximum absorption bands of HGA at 285 nm and complexes at 286 nm are monitored as a function of added DNA (Figure 2). In the case of HGA, a decrease in the absorption intensity was apparent. This change indicates that HGA can directly interact with DNA (28, 29). A 6.0~8.8% hypochromism and without a red shift at the ratio of Rt = 6 (Table IV) were observed for complexes after interaction with DNA. This result of the absorption spectra shows that the complexes interact with DNA through non-intercalating mode.

Figure 2: Absorption spectra of HGA (a) and EuL3 (b) upon introduction of DNA. [HGA, complexes] = 8 μM, Rt = 0, 1.2, 2.4, 3.6, 4.8, 6.0. Arrow indicates the absorbance changes upon increasing DNA concentration. Inset: plots of [DNA]/(εa – εf) vs. [DNA].

In order to compare quantitatively the binding strength of the complexes, the intrinsic binding constants Kb of the complexes with DNA were obtained by monitoring the changes in absorbance with increasing concentration of DNA using the following equation (30): [DNA]/(εa – εf) = [DNA]/(εb – εf) + 1/Kb(εb – εf)

[1]

where ε is the extinction coefficient, the subscripts b, f, and a denote bound, free, and total HGA or complex. [DNA] is the concentration of DNA in base pairs. In plots of [DNA]/(εa – εf) vs. [DNA], Kb is given by the ratio of the slop to the intercept. All results are listed in Table IV, it is clear that HGA interacts to DNA with moderate strength. Similar result was reported for the quinolone analogue norfloxacin (31, 32). In addition, the values of these complexes are higher than that of HGA. The result shows that these complexes interact with DNA much stronger

than HGA as non-intercalations, which is ascribed to the synergistic enhancement of the ligand activity upon Ln(ІІІ) conjugation. At the same time, the differences between the values of four complexes are not apparent, which suggests that the DNA-binding strengths are similar in different complexes.

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Salt Dependence of DNA-binding The absorption spectra of the complexes and HGA binding to DNA were also investigated in 10 mM NaCl Tris-HCl buffer. The results were assigned in Table IV. Obviously the Kb values and the hypochromism decreased with increasing NaCl concentration in buffer. This result implies that there are electrostatic interaction between the complexes and DNA (33). However, it is not the only reaction mode, since electrostatic interaction alone can not affect the absorption spectra of complexes at this low concentration of DNA. Table Binding constants and hypochromism of HGA and complexes to DNAa. Compounds HGA LaL3 NdL3 EuL3 TbL3 a

Kb ( 104 M-1)

[NaCl] = 10 mM 0.980 2.39 2.47 3.36 3.96

[NaCl] = 50 mM 1.00 1.97 2.33 3.02 3.53

Hypochromism (%)

[NaCl] = 10 mM 7.2 9.7 9.0 9.5 7.2

[NaCl] = 50 mM 7.6 7.9 6.5 8.8 6.0

Rt = 6.0.

Emission Spectra The fluorescence spectra of compounds and DNA also show the interactions between them. The plots of RFI (F0/F)/[DNA] were shown in Figure 3. As the DNA concentration increased, two different results were observed: (i) Nd(ІІІ), Eu(ІІІ), and Tb(ІІІ) complexes show an obvious enhancement. At the ratio of Rt = 10.7, the emission intensities of this complexes increase 2.4%, 22.5%, and 13.8%, respectively, compared with those in the absence of DNA. (ii) In contrast, the emission intensity of HGA decreased by 27.6%. This result agrees with the observation for norfloxacin reported by Shen et al. and Mendoza-Díaz (34). The fluorescence spectrum of La(ІІІ) complex also exhibits decrease by 28.1%. The fluorescence quenching data are analyzed by the Stern–Volmer equation: F0/F = 1 + Ksv[Q]

[2]

where F0 and F are the fluorescence intensity of compounds in absent and present nucleutides, respectively. Ksv is the Stern-Volmer quenching constant, and [Q] is the concentration of the quencher. The obtained Ksv of HGA and La(ІІІ) complex from Figure 4 are 2.90 × 103 M-1 and 3.50 × 103 M-1, respectively. The quenching mechanism is due to the electron transfer from the guanine base in the groove region to the compounds (35-37). When the HGA is excited by 285 nm UV light (LaL3 is excited by 286 nm UV light), HGA (La(ІІІ) complex) is in the excited state, has a stronger oxidizing. When DNA in the ground state (λmax = 260 nm), the guanine as a good electron donor is easily oxidized; thus, the electron transfer is easy. Especially the 4f electron shell of La(ІІІ) is empty, La(ІІІ) an accept electron from ligand, so it encourages the transfer process from guanine to ligand. This may be the reason why the efficiency of quenching on LaL3 is higher than that on HGA. For other complexes, the different results could be explained from the characterizations of 4f electron shell of Ln(ІІІ) ions. After binding to DNA, they can not easily

Figure 3: Plots of relative emission intensity (F0/F) of present complexes and HGA vs. the DNA concentration. [HGA, complexes] = 4 μM.

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Zeng et al. Figure 4: The Stern-Volmer plot of HGA (a) and LaL3 (b) quenched by DNA. [HGA, LaL3] = 4 μM.

accept electron from the guanine as La(ІІІ) complex does, which because the 4f electron shell of these central ions are filled with 3, 6, 8 electrons, respectively. From above binding properties in present ligand and complexes, the binding mode between them and DNA is groove binding mode (14, 16). Viscosity Measurements

Figure 5: Effect of increasing amounts of complexes or HGA on the relative viscosity of DNA at 25 (±0.1) ºC. The total concentration of DNA was 0.1 mM.

Hydrodynamic methods that are sensitive to length change (i.e., viscosity and sedimentation), which provides perhaps the most critical tests for a binding mode in the absence of crystallographic or NMR structural data (22). Intercalating agents are expected to elongate the double helix to accommodate the ligands in between the base leading to an increase in the viscosity of DNA. In contrast, complexes which bind exclusively in the DNA grooves by partial and/or non-classical intercalation, under the same conditions, typically cause less pronounced (positive or negative) or no change in DNA solution viscosity (38). The values of (η/η0)1/3 were plotted against [complex]/[DNA] (Figure 5). The results reveal that the complex effect relatively inapparent increase in DNA viscosity, which is consistent with DNA groove binding suggested above, which is also known to enhance DNA viscosity (22). Affinity of Complexes and HGA with Mononucleotides The interacting properties of these complexes with individual bases (adenine, guanine, cytosine, and thymine) should provide some insight into the binding behavior. So, the associations of complexes except Tb(ІІІ) complex [as it exhibits similar properties with Eu (ІІІ) complex in above experiments] and HGA with various nucleotides were tested by fluorescence spectroscopy. From the SternVolmer plots (Figure 6), the emission intensity of Ln(ІІІ) complex decrease in the presence of nucleotides, and similar behaviors were observed for HGA. The KSV are summarized in Table V. There is clear difference between pyrimidine and purine. In a word, both complexes and HGA binding to pyrimidines are apparently much preferred over the purines.

Figure 6: Increases in the fluorescence intensity ratio (F0/F) for LaL3 with increasing amounts of nucleotides. [LaL3] = 4 μM, Ex/Em = 286/445 nm, slit width: 5 nm.

Table The KSV of present compounds to nucleotides from the Stern-Volmer plot. Compounds LaL3 NdL3 EuL3 HGA

AMP

6.50 6.44 5.12 4.85

102 102 102 102

GMP

1.40 1.31 1.10 1.13

KSV/M-1

104 104 104 104

CMP

1.49 1.36 1.28 1.34

104 104 104 104

TMP

1.96 1.22 1.11 1.91

104 104 104 104

Conclusion The results show that HGA could interact with DNA directly. Spectroscopic and viscosity experiments demonstrate that the complexes and HGA interact to DNA mainly by groove binding mode. In addition, the binding strength of complexes

to DNA is stronger than that of HGA due to the synergistic enhancement of the ligand activity upon lanthanide ions conjugation. Shen proved that the binding affinity of the drug to DNA determines biological potency (39). The species of central ion has no significant effect on the binding affinities of complexes to DNA, but results in disparate properties in emission spectra due to the different characteristic of 4f electron shell of Ln(ІІІ). Furthermore, the solubility of HGA in water, methanol, and ethanol is poor, whereas its complexes can fairly solve in water, methanol, and ethanol. This increase of hydrotropy can enhance the ability of drug molecules in crossing the membrane of a cell, and hence raise the biological utilization ratio and activity of the drug. Acknowledgments We gratefully acknowledge to the National Nature Science Foundation of China for their financial support. References and Footnotes 1. I. Turel. Coord Chem Rev 232, 27-47 (2002). 2. D. C. Hooper, E. Rubinstein (Eds.) Quinolone Antimicrob. Agents, 3rd ed. ASM Press, Washington, DC (2003). 3. V. T. Andriole (Ed.) The Quinolones, 3rd ed. Academic Press, San Diego (2000). 4. D. Beermann, J. Kuhlmann, A. Dalhoff, H. J. Zeiler (Eds.) Quinolone Antibacterials. Springer-Verlag, Telos (1998). 5. D. E. King, R. Malone, and S. H. Lilley. Am. Fam. Physician 61, 2741-2748 (2000). 6. J. Robles, J. Martin Polo, L. Á lvarez Valtierra, L. Hinojosa, and G. Mendoza-Díaz. Metal Based Drugs 7, 301-311 (2000). 7. E. K. Efthimiadou, G. Psomas, Y. Sanakis, N. Katsaros, and A. Karaliota. J Inorg Biochem 101, 525-535 (2007). 8. E. Y. Bivián-Castro, F. Cervantes-Lee, G. Mendoza-Díaz. Inorg Chim Acta 357, 349353 (2004). 9. M. P. LópezGresa, R. Ortiz, L. Perelló, J. Latorre, M. LiuGonzález, S. García-Granda, M. PérezPriede, and E. Cantón. J Inorg Biochem 92, 65-74 (2002). 10. D. K. Saha, S. Padhye, C. E. Anson, and A. K. Powell. Inorg Chem Commun 5, 10221027 (2002). 11. H. M. Wexler, D. Molitoris, and S. M. Finegold. Anaerobe 7, 285-289 (2001). 12. J. Marmur. J Mol Biol 3, 208-218 (1961). 13. M. E. Reichmann, S. A. Rice, C. A. Thomas, and P. Doty. J Am Chem Soc 76, 30473053 (1954). 14. V. G. Vaidyanathan, B. U. Nair. J Inorg Biochem 94, 121-126 (2003). 15. R. Vijayalakshmi, M. Kanthimathi, V. Subramanian, B. U. Nair. Biochim Biophys Acta 1475, 157-162 (2000) 16. A. G. Krishna, D. V. Kumar, B. M. Khan, S. K. Rawal, K. N. Ganesh. Biochim Biophys Acta 1381, 104-112 (1998). 17. S. Satyanarayana, J. C. Dabrowiak, J. B. Chaires. Biochem 31, 9319-9324 (1992). 18. M. Eriksson, M. Leijon, C. Hiort, B. Norden, and A. Gradsland. Biochem 33, 50315040 (1994). 19. J. A. Kim, H. Park, J. C. Kim, A. J. Lough, S. Y. Pyun, J. Roh, B. M. Lee. Inorg Chim Acta 361, 2087-2093 (2008). 20. H. J. Zhang, G. Li, L. Yan, R. D. Yang. J Luminescence 127, 316-320 (2007). 21. H. Park, A. J. Lough, J. C. Kim, M. H. Jeong, Y. S. Kang. Inorg Chim Acta 360, 28192823 (2007). 22. I. Turel. Coord Chem Rev 232, 27-47 (2002). 23. N. Jiménez-Garrido, L. Perelló, R. Ortiz, G. Alzuet, M. González-Álvarez, E. Cantón, M. Liu-González, S. García-Granda, M. Pérez-Priede. J Inorg Biochem 99, 677-689 (2005). 24. X. L Tang, W. Dou, S. W. Chen, F. F. Dang, W. S. Liu. Spectrochim Acta A 68, 349-353 (2007). 25. V. Patroniak, Z. Hnatejko, A. M. Grochowska, A. R. Stefankiewicz. Spectrochim Acta A 64, 830-834 (2006) 26. J. M. Lehn. Angew Chem Int Ed Engl 29, 1304-1319(1990). 27. M. Larva, H. Takalo, K. Simberg, and J. Kankare. J Chem Soc Perkin Trans 2, 995 (1995). 28. Z. Q. Liu, Y. T. Li, Z. Y. Wu, S. F. Zhang. Inorg Chim Acta (2008). 29. J. K. Barton, A. T. Danishefsky, J. M. Goldberg. J Am Chem Soc 106, 2172-2176 (1984). 30. A. Wolfe, G. H. Shimer, and T. Meehan. Biochem 26, 6392-6396 (1987). 31. E. K. Efthimiadou, H. Thomadaki, Y. Sanakis, C. P. Raptopoulou, N. Katsaros, A. Scorilas, A. Karaliota, G. Psomas. J Inorg Biochem 101, 64-73 (2007). 32. E. K. Efthimiadou, N. Katsaros, A. Karaliota, G. Psomas. Inorg Chim Acta 360, 40934102 (2007).

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Date Received: January 10, 2008

Communicated by the Editor R. Sowdhamini