Synthesis, Characterization, and BSA-Binding Studies of Novel Sulfonated Zinc-Triazine Complexes
Four Zn(II) complexes containing a pyridyl triazine core (L1 = 3-(2-pyridyl)-5,6-di(2-furyl)-1,2,4-triazine-5′,5″-disulfonic acid disodium salt and L2 = 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4′,4″-disulfonic acid sodium salt) were synthesized, and their chemical formulas were finalized as [Zn(L1)Cl2]·5H2O·ZnCl2 (1), [Zn(L1)2Cl2]·4H2O·2CH3OH (2), [Zn(L2)Cl2]·3H2O·CH3OH (3), and [Zn(L2)2Cl2] (4). The synthesized complexes are water soluble, making them good candidates for biological applications. All four complexes have been characterized by elemental analysis and 1H NMR, IR, and UV-Vis spectroscopy. The IR stretching frequency of N=N and C=N bonds of complexes 1–4 have shifted to lower frequencies in comparison with free ligands, and a bathochromic shift was observed in UV-Vis spectra of all four complexes. The binding studies of ligands and complexes 1–4 with bovine serum albumin (BSA) resulted binding constants (Kb) of 3.09 × 104 M−1, 12.30 × 104 M−1, and 16.84 × 104 M−1 for ferene, complex 1, and complex 2, respectively, indicating potent serum distribution via albumins.
The potential use of zinc complexes as antidiabetic insulin mimetics , antimicrobial , and anticancer agents  have garnered a renewed interest in such complexes among other applications, such as serving as tumor photo sensitizers , radioprotective agents , and antidandruff agents (Zn pyrithione-ZPT) . Our interest in sulfa drug moieties has been fueled by the fact that they possess a wide range of pharmaceutical applications . Of particular interest are 1,2,4-triazine derivatives because they have been reported to possess many biological activities such as kinase inhibition , antihypertensivity , antimicrobial , anticancer , anti-HIV [12, 13], and anti-inflammatory activities .
Novel polyanionic sulfonated aromatic synthetic platinum chelates were preliminarily evaluated for their HIV-1 virucidal activity, due to the presence of sulfonated aromatic groups and metals in the most active members [14, 15]. Furthermore, we recently reported that rhenium tricarbonyl complexes of ferene and ferrozine have exhibited the potential to be used as biological imaging agents . (Chemical structures of ferene and ferrozine are illustrated in Figure 1.) However, to the best of our knowledge, no reports exist of zinc complexes of sulfonated 1,2,4-triazine derivatives.
It is noteworthy that although zinc complexes bearing the 5,6-diphenyl-3, 2-pyridyl-1 2,4-triazine ligand were first synthesized more than a decade ago [17, 18], biological studies have been reported for only one such complex, albeit only recently . Therefore, our goal has been to synthesize zinc complexes bearing sulfonated pyridyl triazine derivatives and to assess their interaction with biological targets.
Being the most abundant protein in blood, serum albumin maintains the colloid osmotic pressure, while playing a major role in transport and sustained release of many biomolecules such as steroids, fatty acids, and hormones. Serum albumin also serves as a carrier protein for drug molecules . Small molecule interaction with serum albumin is thus exploited in pharmaceutical research, where affinity to albumins is indicative of drugs with high serum distribution . Through the interaction between bovine serum albumin (BSA), an analog of human serum albumin, and the novel compounds, we seek to investigate their pharmacokinetic associations.
Thus, we report here the synthesis and characterization of four novel metal complexes of the type MLnCl2 (Figure 2) (where M = Zn2+, L = 3-(2-pyridyl)-5,6-di(2-furyl)-1,2,4-triazine-5′,5″-disulfonic acid disodium salt/3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4′,4″-disulfonic acid sodium salt and n = 1/2) and their BSA-binding studies.
2.1. Materials and Methods
All chemicals (zinc chloride (3-(2-pyridyl)-5,6-di(2-furyl)-1,2,4-triazine-5′,5″-disulfonic acid disodium salt (ferene/L1) and 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4′,4″-disulfonic acid sodium salt (ferrozine/L2)), methanol, diethyl ether, ethanol, bovine serum albumin (BSA), tris-HCl buffer (tris(hydroxymethyl)-aminomethane), sodium chloride (NaCl), and analytical grade water) were obtained from Sigma-Aldrich. All the solvents and chemicals were of analytical grade and were used as received, without further purification.
2.2. NMR Measurements
1H NMR spectra were recorded in D2O on a Bruker 400 MHz spectrometer. Peak positions are relative to tetramethylsilane (TMS) as reference. All NMR data were processed with TopSpin 3.2 and Mestre-C software.
2.3. Elemental Analysis
CHNS elemental analysis was performed by PerkinElmer 2400 Series II CHNS/O Elemental analyzer at Atlantic Microlab, USA.
2.4. Melting Point Determination
Melting points were manually determined in open capillaries.
2.5. UV-Visible Spectroscopy
Electronic spectra for ligand and metal complex were obtained on Spectro UV-Vis auto version 3.10, UV-2602 spectrophotometer. The spectral range was 200–800 nm. Spectra were obtained in methanol with baseline correction. Spectral data were processed with UV WIN software.
2.6. FTIR Analysis
FTIR spectra were recorded on a Thermo Scientific NICOLET iS10 spectrophotometer. ATR spectra were obtained within the 4000–600 cm−1 spectral range. Spectral data were processed with OMNIC software.
2.6.1. Preparation of [Zn(L1)Cl2]·5H2O·ZnCl2 (1)
A solution of ferene (0.1 mmol, 0.0494 g) in methanol (4.0 cm3) was added to zinc chloride (0.1 mmol, 0.0140 g) in methanol (1.0 cm3). Then, the resulting mixture was stirred for 3 hours at 60–70°C . The yellow precipitate which was obtained was collected by filtration, washed with ethanol and diethyl ether, and dried. C16H8Cl2N4Na2O8S2Zn·5H2O·ZnCl2, yield 0.0517 g, 78%. Anal. calc. for C16H8Cl2N4Na2O8S2Zn·5H2O·ZnCl2: C, 22.54; H, 1.92; N, 6.68; S, 7.64. Found: C, 22.13; H, 1.89; N, 6.57; S, 7.70%; melting point: >315°C; UV-Vis (MeoH) (λmax (nm) (ε M−1·cm−1)): 210, 242, 331, 364; FTIR (ATR) (cm−1): 1504 (υ(N=N)),1582 (υ(C=N)); 1H NMR (D2O, δ ppm) 8.87 (d, H6), 8.79 (d, H3), 8.28 (t, H4), 7.87 (t, H5), 7.09–7.46 (m, 4H, furyl H).
2.6.2. Preparation of [Zn(L1)2Cl2]·4H2O·2CH3OH (2)
A solution of ferene (0.2 mmol, 0.0988 g) in methanol (4.0 cm3) was added to zinc chloride (0.1 mmol, 0.0140 g) in methanol (1.0 cm3) . Then, the resulting mixture was stirred for 6 hours at 60–70°C. The yellow precipitate which was obtained was collected by filtration, washed with ethanol and diethyl ether, and dried. C32H16Cl2N8Na4O16S4Zn·4H2O·2CH3OH, yield 0.0689 g, 61% based on zinc chloride. Anal. calc. for C32H16Cl2N8Na4O16S4Zn·4H2O·2CH3OH: C, 32.38; H, 2.56; N, 9.26; S, 10.57. Found: C, 32.5; H, 2.67; N, 9.63; S, 10.83%; melting point: >315°C; UV-Vis (MeoH) (λmax (nm) (ε M−1·cm−1)): 209, 244, 337, 370; FTIR (ATR) (cm−1): 1511 (υ(N=N)),1586 (υ(C=N)); 1H NMR (D2O, δ ppm) 8.86 (d, H6), 8.76 (d, H3), 8.27 (t, H4), 7.84 (t, H5), 7.15–7.41 (m, 8H, furyl H).
2.6.3. Preparation of [Zn(L2)Cl2]·3H2O·CH3OH (3)
A solution of ferrozine (0.1 mmol, 0.0492 g) in methanol (4.0 cm3) was added to zinc chloride (0.1 mmol, 0.0140 g) in methanol (1.0 cm3) . Then, the resulting mixture was stirred for 3 hours at 60–70°C. The light yellow precipitate which was obtained was collected by filtration, washed with ethanol and diethyl ether, and dried. C20H13Cl2N4NaO6S2Zn·3H2O·CH3OH, yield 0.0433 g, 69% based on zinc chloride. Anal. calc. for C20H13Cl2N4NaO6S2Zn·3H2O·CH3OH: C, 35.28; H, 4.24; N, 7.84; S, 8.97. Found: C, 35.27; H, 4.36; N, 7.89; S, 9.13%; melting point: >315°C; UV-Vis (MeoH) (λmax (nm) (ε M−1·cm−1)): 208, 240, 291, 323; FTIR (ATR) (cm−1): 1497 (υ(N=N)), 1599 (υ(C=N)); 1H NMR (D2O, δ ppm) 8.86 (d, H6), 8.76 (d, H3), 8.27 (t, H4), 7.84 (t, H5), 7.46–8.28 (m, 8H, phenyl H).
2.6.4. Preparation of [Zn(L2)2Cl2]·5H2O·CH3OH (4)
A solution of ferrozine (0.2 mmol, 0.0984 g) in methanol (4.0 cm3) was added to zinc chloride (0.1 mmol, 0.0140 g) in methanol (1.0 cm3) . Then, the resulting mixture was stirred for 6 hours at 60–70°C. The light yellow precipitate which was obtained was collected by filtration, washed with ethanol and diethyl ether, and dried. C41H40Cl2N8Na2O18S4Zn·5H2O·CH3OH, yield: 0.0579 g, 52% based on zinc chloride. Anal. calc. for C41H40Cl2N8Na2O18S4Zn·5H2O·CH3OH: C, 39.59; H, 3.24; N, 9.01; S, 10.32. Found: C, 38.93; H, 3.04; N, 8.84; S, 10.67%; melting point: >315°C; UV-Vis (MeoH) (λmax (nm) (ε M−1·cm−1)): 216, 242, 298, 336; FTIR (ATR) (cm−1): 1498 (υ(N=N)), 1596 (υ(C=N)); 1H NMR (D2O, δ ppm) 9.05 (d, H6), 8.99 (d, H3), 8.67 (t, H4), 8.67 (t, H5), 7.50–8.31 (m, 16H, phenyl H).
2.7. BSA-Binding Assay
A 6 µM BSA solution was prepared in a buffer containing 5 mM tris-HCl/50 mM NaCl by continuous stirring for 1 hr at room temperature. A 1 × 10−3 M stock solution of complexes and ligand was prepared in distilled water.
Absorption titration was carried out by keeping BSA concentration constant (6 µM) and varying the concentrations of the complexes and ligand (010 µM). After 10 min incubation at room temperature, absorbance was measured for each solution in the range of 250–300 nm wavelengths, and λmax was recorded at 280 nm. Then, the change of λmax was recorded for each solution. All absorbance measurements were triplicated and corrected for background absorbance by the compounds. The plot of 1/(A − A0) (where A0 is the initial absorbance of the free BSA at 280 nm and A is the absorbance of BSA in the presence of different concentrations of the complex) versus 1/[complex] is a linear curve, and the binding constant (Kb) can be obtained from the ratio of the intercept to slope .
3. Results and Discussion
In order to synthesize the metal complexes, zinc chloride and the relevant ligands in 1 : 1 and 1 : 2 ratios were used (Figure 2).
3.2. UV-Visible Spectroscopy
UV-visible spectra of ligands and complexes 1–4 recorded in methanol showed significant differences between the absorption peaks of ligands and their complexes (Table 1, Figures S1 and S2, Supporting Information). Since both ligands bear conjugated systems, π–π transition is possible. In all four complexes, the wavelengths have shifted towards the longer wavelength range (bathochromic shift) because of changes in the conjugated electron system due to formation of metal ligand bonds. These observations are in agreement with previously reported zinc pyridyl triazine derivatives  and copper pyridyl triazine derivatives , upon coordination of ligand to metal.
3.3. 1H NMR Analysis
The splitting pattern of the free ferene ligand can be observed for complexes 1 and 2. However, due to the donation of electrons from the nitrogen in pyridine and triazine rings to the metal, the electron density of the ferene ligand is reduced, and thus protons of the metal complex should appear more downfield than the ferene ligand. Furthermore, the downfield shift, which will hence be denoted as ∆δ, of the H6 signal is expected to be higher than that of other protons because it is closer to the pyridine N. In uncoordinated ferene ligand (L1), the pyridyl H6 signal (8.74 ppm, Table 2) is its most downfield doublet consistent with its close proximity to the pyridyl nitrogen atom. In a spectrum of complex 1, the H6 signal appears further downfield (8.87 ppm, Figure 3) confirming metal-pyridine N bond formation. However, the observed change in shift of H6 was small in both 1 (∆δ; +0.13 ppm at 8.87 ppm) and 2 ((∆δ; +0.12 ppm at 8.86 ppm) in comparison with the change in shift observed for the H3 proton (8.79 ppm and 8.76 ppm), which had the highest change in downfield shift ((∆δ; +0.25 and +0.22 ppm, resp.). The four doublets due to furyl ring protons (7.00–7.34 ppm range) also appear more downfield (7.15–7.45 ppm) upon metal bonding. Higher downfield shifts were observed for H3 versus H6 in 1H NMR spectra of Zn(dppt)Cl2·0·5H2O and Zn(dppt)2Cl2·2H2O (dppt = 5, 6 diphenyl-3-(2-pyridyl)-1,2,4- triazine) reported previously .
Although we expected similar observations for spectra of complexes 3 and 4, unusual upfield shifts of the peaks attributed to H6 (8.92 and 9.05 ppm), H5 (7.98 and 8.21 ppm), H4 (8.47 and 8.67 ppm), and H3 (8.89 and 8.99 ppm) (Figure 4) were observed in complexes 3 and 4, respectively, in comparison with that of the uncoordinated ferrozine ligand (H6: 9.15 ppm, H5: 8.27 ppm, H4: 8.81 ppm, and H3: 9.05 ppm). We attribute the observed upfield shift to possible π stacking of phenyl rings. Upfield shifts due to π stacking have been reported in previous studies on Zn(II) with mono- and dianionic pyrrole-2-imine complexes and zinc azadipyrromethene . Although the 1H NMR spectrum of ferrozine/L2 is comparatively more complicated due to the protons of the two phenyl rings, the pyridyl ring protons can be easily distinguished (Figure 4). Upfield shifts observed in complexes 3 and 4 were not observed in complexes 1 and 2, which had furyl rings (Figure 3).
3.4. FTIR Analysis
Literature data have been used where relevant to get assignment of ligands . The stretching frequency (ѵ) of the N=N bond in the triazine ring and C=N bond in the pyridine ring serves as important indicators of the formation of new metal ligand bonds . In all four complexes, νN=N and νC=N have shifted to lower frequencies (Table 3) due to the formation of new metal ligand bonds which in turn lowers the strength of the N=N and C=N bonds. This observation allowed us to confirm that the complex was formed via the donation of a lone pair of electrons each, from the triazine ring and from the pyridine ring, to zinc. For example, upon formation of complex 1, νC=N (1590 cm−1) and νN=N (1510 cm−1) appear at 1582 cm−1 and 1504 cm−1 (Table 3), respectively, due to the change in chemical environment. FTIR spectra show broad peaks at around 3400 cm−1 region due to OH vibration.
3.5. Elemental Analysis
Empirical formulas related to experimental values of the complexes (Experimental) give the exact molecular formulas and experimental values are closer to the expected values. Some deviate from the theoretical values due to the residual solvent (methanol) and water molecules.
Elemental analysis data suggest that complex 1 exists as [Zn(L1)Cl2]·5H2O·ZnCl2. The experimental data obtained from elemental analysis show a significant decrease in the carbon percentage of this complex than expected without the extra zinc ion, prompting us to include an associated zinc chloride molecule in 1. However, the exact binding mode or type of the interaction of this extra ZnCl2 molecule with ferene ligand cannot be explained with the obtained data. Complexes 2–4 have no such discrepancy, and the molecular formula were confirmed from elemental analysis to be [Zn(L1)2Cl2]·4H2O·2CH3OH, [Zn(L2)Cl2]·3H2O·CH3OH, and [Zn(L2)2Cl2]·5H2O·CH3OH, respectively.
3.6. BSA-Binding Assay
Anjomshoa and coworkers have previously investigated bovine serum albumin- (BSA-) binding properties of Zn(dppt)2Cl2·2H2O (dppt = 5,6-diphenyl-3,2-pyridyl-1 2,4-triazine) , which required an organic solvent such as dimethyl sulfoxide to be added to increase solubility. However, complexes 1–4 reported in this study are highly soluble in water which makes them compatible with biological systems.
Absorbance measurements at UV range are useful to identify the conformational changes in proteins. BSA has a maximum absorbance peak at 280 nm. Analyzing the absorbance spectra of BSA upon addition of ferene, complex 1, and complex 2 (Figure 5) clearly shows that the peak intensity at 280 nm increases upon addition of the compounds, indicating association with BSA causes a change in the polarity of the microenvironment of tryptophan and tyrosine residues in a concentration dependent manner.
With increasing concentrations of ferene ligand and complexes 1 and 2, the maximum absorbance was increased. Using the graph of 1/(A − A0) against 1/[C], binding constants were calculated (Figure 6) as 3.09 × 104 M−1, 12.30 × 104 M−1, and 16.84 × 104 M−1 for ferene, complex 1, and complex 2, respectively. These values are within the range of 104–106 M−1 as expected from a good BSA carrier activity in vivo . Such changes upon BSA addition were not observed in ferrozine and complexes 3 and 4.
In this study, we have described the synthesis and characterization of four novel zinc complexes by spectroscopic methods. According to the UV-Vis spectra, a bathochromic shift has been observed for all four complexes. FTIR data provide evidence that Zn-N bonds are formed via N atoms of the triazine and pyridine rings. Accordingly, the stretching frequency of N=N and C=N bonds of all four complexes have shifted to the low frequency range in comparison with the free ligands. Elemental analysis was used to determine the empirical formula of complexes 1–4.
It is our belief that the scaffolds reported herein may provide a novel platform for drug designing. We have demonstrated that such systems possess high affinity to serum albumin, indicating their potential to be distributed in serum . Sulfonated groups have aided to increase water solubility of the complexes, where aqueous solubility predicted by ChemAxon (https://disco.chemaxon.com) have also supported that the new complexes are soluble in water under biologically relevant pH ranges (data not shown). In vivo testing is warranted to explore the implications of such properties on cellular metabolism to delineate the function of the novel triazine complexes in biological systems.
Conflicts of Interest
The authors declare that they have no conflicts of interest regarding the publication of this paper.
Financial assistance by Grant no. ASP/01/RE/SCI/2015/19 of the University of Sri Jayewardenepura is gratefully acknowledged. The authors thank Professor Luigi Marzilli and Ms. Kokila Ranasinghe of Louisiana State University for obtaining NMR data and for useful discussions. Ms. Taniya Darshani is acknowledged for determination of melting points.
Figure S1. UV-visible spectra of ZnCl2, ferene, and complexes 1 and 2. Figure S2. UV-visible spectra of ZnCl2, ferrozine, and complexes 3 and 4. (Supplementary Materials)
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