Synthesis, Crystal Structures, and Antimicrobial and Antitumor Studies of Two Zinc(II) Complexes with Pyridine Thiazole Derivatives

Two pyridine thiazole derivatives, namely, 4-(pyridin-2-yl)-2-(2-(pyridin-2-ylmethylene)hydrazinyl)thiazole (L1) and 4-(pyridin-3-yl)-2-(2-(pyridin-4-ylmethylene)hydrazinyl)thiazole (L2), were afforded by a cyclization reaction between α-haloketone and thioamide, and their Zn(II) complexes were prepared by the reaction of ligands and corresponding metal salts, respectively, and characterized by X-ray diffraction and elemental analysis. Both crystals were obtained by ether diffusion and crystallized in a monoclinic system. The in vitro antimicrobial activity of the Zn(II) complexes and ligands was screened using the microplate reader method, and in vitro antitumor activities of the complexes were evaluated by MTT, with a view to developing new improved bioactive materials with novel properties. The biological activity studies of the compounds showed that the metal complexes were more active than the free ligands, and some compounds had absolute specificity for certain bacteria or cancer cell lines.


Introduction
iazole ring and pyridine ring are structural units with extensive biological activities, such as antibacterial [1], antitumor [2], antiviral [3], anti-inflammatory [4], and hypoglycemic [5] activities. Hydrazone compounds have also attracted much attention in various fields, especially in pesticide and pharmaceutical fields due to their unique physiological activity and strong coordination ability [6,7]. erefore, the introduction of pyridine hydrazone group into molecule containing thiazole is likely to produce stronger biological activity according to the synergistic effect of drug combination principles. Because of the particularity of the structures, thiazole ligands containing pyridine and hydrazone groups with multiple coordination sites often have good metal coordination ability which can form stable complexes with different transition metal [8,9]. Besides, the spatial conformation of the complex is determined by its coordinated modes, which have great influence on the biological properties of the compound [10][11][12]; the studies on transitional metal compounds of pyridine thiazole ligands have been of great significance. Co(III) complexes based on (1,3-selenazol-2-yl)-and (1,3-thiazol-2-yl)hydrazone were reported to have potent antimicrobial and antioxidant activity [13]. To further study the biological, physical, and chemical activities of these compounds and their Zn(II) complexes, two thiazole derivatives, L1 and L2, were synthesized by the cyclization reaction of pyridyl thiosemicarbazide and α-bromopyridine (Scheme 1). Complexes [Zn(L1) 2
e inhibitory activities of these compounds against seven pathogenic bacterial strains, namely, Escherichia coli (E.

Materials and Methods.
All the starting materials and reagents in this work were obtained commercially and used without further purification. Elemental analyses were performed on a vario EL analyzer (Elementar, Germany) and IR spectra on an Avatar 330 FT-IR spectrometer ( ermo Nicolet) with potassium bromide pellets. 1 H NMR spectra were determined by Bruker AVANCE-III 500 at 300-400 MHz, and MestReNova software was used for data analysis. Chemical shifts (δ values) and coupling constants (J values) are reported as ppm and Hz, respectively. e following abbreviations are used: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. Antimicrobial tests were performed on a SpectraMax ® ABS Absorbance Reader (Molecular Devices). Single-crystal X-ray diffraction was carried out by a Bruker Smart Apex-CCD and Agilent SuperNova diffractometer. e UV-Vis spectra were recorded on an Agilent Cary 60 spectrophotometer.

Synthesis of Ligands L1 and L2.
A mixture of 2-pyridine formaldehyde (10.7 g, 0.1 mol), 50 mL ethanol, 50 mL H 2 O, and 5 mL 36% concentrated hydrochloric acid was added to a 250 mL three-neck flask with magnetic agitator and reflex tube at 25°C. iosemicarbazide (10.9 g, 0.12 mol) in 10 mL ethanol was added slowly, and the solution immediately became yellow solid. After being stirred for 0.5 h and refluxed for 2 h, the solid was filtered and rinsed with ethanol to give the N-2-pyridine-methyl-thiosemicarbazide hydrochloride (20.2 g, yield 93.5%, Compound A) as a yellow acicular crystal [14].
2-Acetyl pyridine (12.1 g, 0.1 mol) was added to a solution of ice acetic acid (85 mL) and hydrobromic acid (40%, 13 mL), and the mixture was stirred for 10 min under ice bath. After being completely cooled, bromide (0.1 mol, 5.2 mL) was added three times slowly. en, the reaction mixture was stirred for 0.5 h at 0°C, 2 h at 40°C, and 2 h at 75°C, and the solution gradually changed from dark brown to light yellow with white crystal solid precipitation. After the reaction system was cooled to room temperature, 80 mL ethyl ether solution was added. en the mixture was stirred continuously for 10 min, filtered, and washed with dry ethyl ether three times to give bromo-2-acetylpyridine hydrobromide (19.5 g, yield 90.3%, Compound B) as a white crystal [15].

X-Ray Structure
Determination. Crystals were selected appropriately and mounted on a glass fiber in a random orientation. A single crystal of ligand L1 was mounted on a Bruker Smart Apex-CCD diffractometer with CuKα radiation (λ �1.54178Å) at 293 K, while the other crystals were carried out on an Agilent SuperNova diffractometer with CuKα radiation (λ �1.54178Å) and with MoKα radiation (λ � 0.71073Å) at 100K, respectively. e crystallographic data and structure refinement summary of ligands L1, L2 and complexes 1-2 are listed in Table 1. Using OLEX2 [16], the structure was solved by direct methods using the SHELXS program [17] and refined by full-matrix leastsquares techniques SHELXL-2014 [18] on F 2 . All nonhydrogen atoms were refined anisotropically. DIA-MOND was used for molecular graphics [19].

Antimicrobial Activity Studies.
Qualitative analysis for screening of antibacterial activity was carried out by the microplate reader method [20][21][22]. Mueller-Hinton agar plates were seeded with indicator bacteria, Escherichia coli All compounds were dissolved in DMSO (100 μg/mL as an initial concentration) and tested against the ten pathogenic bacteria strains for their inhibitory activity. e antimicrobial activity of the compounds was expressed as the bacteriostatic rate (%) and determined by the absorbance of the bacterial samples using an enzyme-labeled instrument. Subsequently, compounds with high inhibitory rate (+++) were selected for minimum inhibitory concentration (MIC) determination using double dilution method with concentrations ranging from 3.125 to 50 μg/mL. e MIC was recorded as the lowest concentration at which inhibitory rate was greater than 95%. As a blank group, only DMSO was added to the wells, and Ciprofloxacin was used as positive control for antibacterial activity. All tests and analyses were run in duplicate, and the results obtained were averaged. e percentage inhibition was calculated using the following equation: where Abs blank is the absorbance of DMSO + blank medium, Abs control is the absorbance of sample + blank medium, and Abs sample is the absorbance of sample (test samples/standard) + bacterial suspension in medium.   116.83 (18) e viability of the cell lines was tested using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [23,24]. e compounds were dissolved in DMSO and diluted with water to the required concentration (160 μg/mL, 80 μg/mL, 40 μg/mL, 20 μg/mL, 10 μg/mL, 5 μg/mL, 0 μg/mL). Human lung cancer cells (A549), human breast cancer cells (BT-20), human ovarian cell cells (MCF-7), and human osteosarcoma cells (U20S) cells were kindly provided by Stem Cell Bank, Chinese Academy of Sciences, and were plated in 96-multiwell plates (104 cells/well) for 24 hours before treatment with different concentrations of complexes to allow attachment of cell to the wall of the plates. en, the cells were cultured using MEM, DMEM, F-12K, and DMEM/F-12(GIBCO) culture medium containing 10% fetal bovine serum in a 5% (volume fraction) CO 2 , 37°C saturated humidity incubator for 48 hours, respectively. Cells were fixed, washed, and stained with MTT. Cell growth inhibition was determined by measuring the absorbance of each well at 570 nm with a Tecan Infinite M1000 Pro Microplate Reader (Männedorf, Switzerland). GraphPad Prism version 7.0 program was used for data processing, and IC50 was obtained by fitting the nonlinear regression model with S-shaped dose response in the program.

Solution Stability of Complexes 1-2.
e essential prerequisite for the biological evaluation of metal complexes is their stability in solution. In the light of this fact, complexes 1-2 were dissolved in DMSO and DMSO-d6 (a solvent used for the preparation of stock solutions for biological evaluation), and their UV-Vis and NMR spectra, respectively, were recorded immediately after dissolution, as well as after 48 h of standing in the dark at ambient temperature. From NMR measurements ( Figure S1), it can be concluded that the corresponding N-pyridine and N-thiazole remained coordinated to the Zn(II) ion during 48 h and that DMSO coordination did not occur. In the UV-Vis spectra ( Figure  S3), the shape of spectra and position of the absorption maxima (λ max � 255.0, 335.0 nm for complex 1; λ max � 260.4, 370.0 nm for complex 2) remained unmodified. Taken together, obtained spectroscopic data indicated the sufficient stability of complexes 1-2 in DMSO solution.

FTIR Spectral Elucidation.
e infrared absorption peaks of the main functional groups on ligands L1 and L2 and complexes 1-3 are listed in Table 2. Additionally, as    87.8 (2) shown in Figure S2,

X-Ray Crystal
Structure. Suitable single crystals of ligand L2 and complexes 1-2 for X-ray diffraction studies were obtained by the diffusion of diethyl ether into a DMF solution at room temperature. Ligand L2 was crystallized in a monoclinic system, and the spatial group was P2 1 /n. e selected bond lengths and bond angles of L2 are listed in Table 3. e N(3)-N(2) bond (1.360(2)Å) has the same length as the N(3)-C(7) bond. e molecular structure of L2 and its packing diagram are depicted in Figure 1. In the crystal of L2, the molecule is in the E-conformation, and the supramolecular arrangement is directed mainly by strong N(3)-H(3)···N(5) hydrogen bonds between two molecules (Table 4). e torsion angles of C(13)-C(9)-C(8)-N(4) (172.59°) and C(10)-C(9)-C(8)-C(14) (174.37°) indicate that the thiazole ring is almost coplanar with the neighboring pyridine ring.

Biological Activity.
e antibacterial activity of the ligands and complexes was presented as inhibition rate, and those with high inhibitory rate (+++) were selected for minimum inhibitory concentration (MIC) determination. As shown in Table 8, the complexes had a wider broad spectrum and stronger antibacterial property than the ligands, and such enhanced activity of metal chelates is due to faster diffusion of metal complexes as a whole through the cell membrane or due to combined activity effect of the metal and the ligand. To further measure the MIC, CIPRO was used as the positive control. Compared with the CIPRO, complex 2 had a better or the same inhibitory effect on E. coli, Sh. flexneri, and P. aeruginosa with the MIC of 3.13 μg·mL −1 , 3.13 μg·mL −1 , and 6.25 μg·mL −1 , respectively, while complex 1 held a stronger inhibitory effect on S. typhimurium and B. subtilis with the MIC of 6.25 μg·mL −1 and 3.13 μg·mL −1 , respectively (Table 9). ese results indicated that the complexes possess antibacterial activity inhibiting multiplication process of the microbes by blocking their active sites [26,27]. e in vitro antitumor activity of the compounds against human lung cancer cells (A549), human breast cancer cells    (BT-20), human ovarian cancer cells (MCF-7), and human osteosarcoma cells (U20S) was measured via MTT assay. In general, the complexes showed a stronger antitumor activity than the corresponding ligands (Table 10). Between the two complexes, complex 2 exerted a more effective antitumor activity with the lower IC 50 value. Among the four cancer cell lines, complex 2 had the most significant effect on A549 with the IC 50 value of 4.48 μg/mL. e result indicated the enhancement of the antitumor activity upon coordination. e enhancement of antitumor activity may be attributed to the fact that the positive charge of the metal increased the acidity of coordinated ligand that bears protons, leading to stronger hydrogen bonds which enhanced the biological activity [28]. Despite the conformed antitumor activity, a detailed molecular mechanism against cancer cell lines of the ligands and the complexes remains to be further elucidated.

Conclusion
In this paper, We have reported the preparation, characterization, and biological activities of new thiazole-hydrazone derivatives based on pyridine and their two Zn(II) complexes. Single-crystal X-ray diffraction analysis indicated that both ligand L2 and complexes 1-2 had mononuclear molecular structure. e in vitro antimicrobial activity of the complexes was screened using the microplate reader method, and in vitro antitumor activities of the complexes were evaluated by MTT. e results showed that the complexes coordinated with Zn 2+ had a better antimicrobial activity and antitumor activity than the corresponding ligands due to the lipophilic nature of the metal ions in complexes, which might provide valuable information for further designing and synthesizing new antimicrobial and antitumor agents.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.