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Diorganotin(IV) complexes derived from N-terminal methylation of Triapine: synthesis, characterization and antibacterial activity evaluation
Journal Pre-proof Diorganotin(IV) complexes derived from N-terminal methylation of Triapine: synthesis, characterization and antibacterial activity evaluation Cuili Xing, Yan Fang, Lei Jiang, Yahong Zhang, Mingxue Li PII:
S0022-328X(20)30054-1
DOI:
https://doi.org/10.1016/j.jorganchem.2020.121153
Reference:
JOM 121153
To appear in:
Journal of Organometallic Chemistry
Received Date: 25 December 2019 Revised Date:
15 January 2020
Accepted Date: 1 February 2020
Please cite this article as: C. Xing, Y. Fang, L. Jiang, Y. Zhang, M. Li, Diorganotin(IV) complexes derived from N-terminal methylation of Triapine: synthesis, characterization and antibacterial activity evaluation, Journal of Organometallic Chemistry (2020), doi: https://doi.org/10.1016/j.jorganchem.2020.121153. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.
Diorganotin(IV) complexes derived from N-terminal methylation of Triapine: synthesis, characterization and antibacterial activity evaluation Cuili Xinga, Yan Fanga, Lei Jianga, Yahong Zhangb*, Mingxue Lia* a
Henan Key Laboratory of Polyoxometalates, Institute of Molecular and Crystal Engineering, College of Chemistry and Chemical Engineering, Henan University, Kaifeng 475004, PR China，Tel/Fax: +86 371 23881589 b Key Laboratory of Natural Medicines and Immune Engineering, Henan University, Kaifeng 475004, PR China E–mail address: [email protected] (Y. H. Zhang), [email protected](M.X. Li) Abstract The reaction of diorganotin(IV) with ligands (HL1 = 3-aminopyridine-2-carbaldehyde N(4) methylthiosemicarbazone, HL2 = 3-aminopyridine-2-carbaldehyde N(4)-dimethylthiosemicarbazone) afforded two compounds [PhSn(L1)Cl2] (1) and [(Ph)2Sn(L2)Cl] (2), respectively. The compounds were characterized by elemental analysis, infrared spectrum, UV-vis spectrometry, and X-ray crystallography. The ligands and corresponding complexes were assayed for their in vitro pharmaceutical activity against different pathogenic strains of bacteria by disk diffusion method. The results demonstrate that 1–2 have remarkable antibacterial activity against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). 1 exhibit higher antibacterial properties against S. aurenu (MIC = 0.00095 µg/mL) and E. coil (MIC = 0.0019 µg/mL). In the same experimental conditions, the two complexes display more potent activity relative to standard antibiotics Kanamycin and Ampicillin.
Keywords: Thiosemicarbazones; Diorganotin(IV); Antibacterial activity; Mechanism analysis 1. Introduction To treat infectious diseases caused by bacteria is still an urgent and challenging public health concern. Moreover, common pathogens and new pathogenic species treated with current antibiotics develop drug resistance [1,2]. The drug resistance of bacteria leads to a reduction in the effect of some common antibiotics and may result in higher mortality and morbidity [2]. Therefore, it is inevitable to fabricate new antibacterial agents, in particular the synthesis of metal complexes with bioactive ligands. Thiosemicarbazones containing a significant class of N, S donor are pre-eminent transition metallic chelators, having infinite interest in the coordination chemistry [3-5]. Thiosemicarbazones are also an important class of the carbonyl derivatives and
have been proved to own versatile medicinal applications, for instance, antibacterial [6], antitumor [7], antihypertensive [8], anticonvulsant [9], antitrypanosomal [10], and antiviral agents [11]. Additionally, the function of N-terminal methylation of thiosemicarbazone complexes is crucial in the biological activities [12]. A solid example is Dp44mT (di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone) and Dp4mT (di-2-pyridylketone-4-methyl-3-thiosemicarbazone). These chelators belong to the dipyridyl thiosemicarbazone (DpT) class of ligands and show different selectivity against tumor cells [13,14]. As antitumor agent, the currently most prominent representative in this family is the 3-aminopyridine-2-carbaldehyde thiosemicarbazone (Triapine), which has been investigated in clinical phase I and II studies [15-18]. Furthermore, a variety of new derivatives have been prefabricated in recent years for the sake of improving thiosemicarbazone-based therapy. Organotin (IV) derivatives have been widely investigated owing to their structural diversity and biological activity. Organotin (IV) compounds exhibit specificity in synthesis and structure, and their broad-spectrum applications can be attributed to ligands connected in the central tin metal atom [19]. Moreover, organotin(IV) compounds display multifunctional applications from industry, catalysis to therapeutics due to their diverse as well as fascinating structural potential [20]. Nowadays, organotin(IV) complexes formed by coordination with ligand are appealing scaffolds for new type drug research on account of their spectacular bioactivities. In recent years, many organotin(IV) complexes have been synthesized and investigated to be more remarkable than traditional metal drugs in antitumor activity [21-23]. Based on literatures, the biological properties are reliant on numbers and properties of organic groups, framework and ligancy of tin atom as well as the intrinsic performance of donor ligand connecting with tin metal atom [24,25]. Furthermore, some organotin(IV) compounds of Schiff bases containing a pyridine group display prominent antibacterial activities [26-28]. Significantly, organotin(IV) compounds may hold lower toxicity, better excretion from the body and less side effects along with no drug resistance formation compared to cisplatin analogues [29,30]. As a part of our research of Schiff based diorganotin(IV) compounds [31], herein we designed two organotin(IV) complexes, derived from HL1 and HL2 (Scheme 1). The biological activities of complexes 1 and 2, free ligands and metal precursors were evaluated by antibacterial test against two strains of E. coli and S. aureus. Antibacterial results indicated that 1 and 2 show obvious sterilizing effect. Most notably, 1 has more potent biological activity than 2 in promoting bacterial cell death and the MIC values are lower than common antibiotics Kanamycin and Ampicillin. Hence 1 as antibacterial agent has greater potential to be applied to reality.
Scheme 1 Synthesis of 1-2 2. Results and discussion 2.1 Chemistry In this paper, two complexes were synthesized by solvent evaporation method. The crystal structures of two complexes are shown in Fig. S1 and the crystal data are collected in Table S1. Bond length and bond angles are given in Table S2. These data are displayed in supplementary information. The synthesized material was confirmed by elemental analysis, infrared spectrum (IR) and UV-vis absorption spectrum, and the fluorescence emission spectra of the two products were tested. The characteristic bands observed in the region of 3178, 3376 and 3235 cm−1 are assigned to ʋ(N–H) of parent ligands (Fig. 1). These characteristic peaks also appear in the spectrum of the complexes. In the IR spectra of complexes, adsorption band of ʋ(C=N) was shifted to lower frequency by 16-52 cm−1, which indicated that the coordination via azomethine nitrogen atoms [32]. Furthermore, ʋ(N-N) increased from 1221 to 1227 cm−1 and 1202 to 1256 cm−1 in the spectra 1 and 2, respectively, which showed coordination through imine nitrogen [33]. 795 and 798 cm−1 observed for free ligand can be assigned to the ʋ(C=S) vibration. These bands shifted to 728 and 734 cm−1 in compounds 1 and 2, respectively, indicating the coordination of the sulfur atom [34]. Some new bands that emerging in the region 448-454 cm−1 and 694-695 cm−1 are assigned to ʋ(Sn-N) and ʋ(Sn-C) respectively, indicating chelation between the ligand and metal atom [35].
Fig. 1 Infrared spectra of HL1, HL2, 1–2
As shown in Fig. 2, UV-vis spectrum of both HL1 and HL2 show two strong bands at 292 and 372, 292 and 380 nm in methanol, respectively. The former may be due to π–π* transition of pyridine ring and the later may be attributed to n–π* transition of imine and thiocarbonyl moiety [36]. Coordination with Sn atom increased the maximum absorption wavelength of compounds. 1–2 have a great red shift from 372 to 440 nm and 380 to 448 nm respectively due to the n–π* transition of ligand-to-metal charge transfer (LMCT) [36].
Fig. 2 UV-Vis spectrums of ligands and compounds in methanol solution (10−4 mol / L)
To observe the influence for the luminescent properties through organic group exchange, the fluorescence emission spectrum was shown in Fig. 3. The compound 1 displayed a strong photoluminescence with maximum emission at 444 nm whereas the ligand showed maximum emission at 413 nm. This variation indicated the coordination of ligands with metal central atom. For 2, two strong photoluminescence with maximum emission at 413 and 531 nm were observed as indicated in Fig. 3B. Compared with the free ligand, strong fluorescence intensity of complexes 1–2 are observed, illustrating that fluorescent properties has been affected remarkably by introducing the Sn(IV) ions [37]. The fluorescence emission spectra prove that the two complexes have fluorescence properties, which enable them to be used in the field of photochemistry.
Fig. 3 Fluorescence emission spectra in 50% DMSO (10−5 mol / L). λex = 366 nm
2.2 Antibacterial activity The free ligands, metal precursor and tin(IV) compounds were evaluated for their antibacterial activity against E. coli (G-) and S. aureus (G+). The MIC values were determined and listed in Table 1. The optical images of zone of inhibition (zoi) were
shown in Fig. 4. The concentration was 1000, 500, 250 and 125 µg/mL, respectively. In this concentration gradient, the diameters of inhibitory zone of compounds 1–2, HL1, HL2, Ph2SnCl2, solvent (DMSO) and standard antibiotics Kanamycin and Ampicillin against E. coli and S. aureus were listed in Table 2. The results showed that solvent DMSO had no effect on the experimental bacterial strains. The metal chelates had higher activity than the free ligands as well as metal precursors. 1–2 exhibit a broad and effective antimicrobial activity against the tested bacteria. Additionally, the MIC values of 1 against two strains of tested bacteria E. coli (0.0019 µg/mL) and S. aureus (0.00095 µg/mL), which were lower than 2 (0.24 and 3.9 µg/mL). A possible explanation of enhanced activity of compounds relative to ligands is that better membrane penetrating ability endowed by the intensive lipophilicity via complexation. The coordination of ligand to tin result in electron delocalization and increase the lipophilic character and effectively disperse the metal complexes into bacterial cells [38]. The coordination with metal exhibits synergetic effect on antimicrobial activity. Table 1 MIC (µg/mL) of ligands, complexes and controls MIC values (µg/mL) E. coli S.aureus
1 0.0019 0.00095
̵ No inhibition
2 0.24 3.9
HL1 7.81 7.81
HL2 31.2 125
Ph2SnCl2 7.81 3.9
DMSO ̵ ̵
Amp 1.95 0.12
Kan 7.81 3.90
Fig. 4 Optical images of zone of inhibition against (A) E. coli and (B) S. aureus, respectively
Table 2 Antibacterial activity data showing zone of inhibition (mm) of the tested substances Inhibition zone diameter /mm (concentration in µg/mL)
1 2 HL1 HL2 Ph2SnCl2 DMSO Kanamycin Ampicillin
1000 25 21 20 24 15 ̵ 21 23
E. coli 500 250 23 24 20 22 17 15 20 17 14 13 ̵ ̵ 20 19 22 21
125 21 19 14 14 12 ̵ 17 19
1000 20 20 20 24 18 ̵ 28 29
S. aureus 500 250 21 21 21 20 19 14 19 13 16 15 ̵ ̵ 23 22 27 25
125 22 22 13 ̵ 14 ̵ 20 23
̵ No inhibition zone 2.3 Overviews of the mechanism of action In order to better explain the way of antibacterial action, test was expanded from the following aspects. In this paper, 1 was taken as an example to explore its antibacterial mechanism. To some extent, the damage to the cell membrane is caused by the interaction between antibacterial drugs and bacteria. The breakup of the membrane can cause some small molecular mass such as RNA, DNA, K+ and other substance (polysaccharide) to leak out. Intracellular components, such as DNA and RNA show specific absorbance at 260 nm [39]. Fig. 5 illustrated the damage degree of the cell membrane in bacteria. As can be seen from the test data in Table 4, the absorbance of intracellular components is about 0.01 at 0 minute. After 15 minutes, the absorbance value treated with the sample is 0.38. Simultaneously, the absorbance value without sample is 0.07. The changes of absorbance indicate that the cell membrane of bacteria is destroyed.
Fig 5 The absorbance of Intracellular components at OD260 nm The destruction of the integrity of bacterial cell membrane can lead to the release of intracellular proteins, hence another indicator of cell membrane damage is protein leakage. Moreover, coomassie brilliant blue G-250 determines that protein content belongs to a dye binding method. Red in the free state and the maximum light absorption is 488 nm, but it becomes cyan and the protein-pigment conjugate has the maximum light absorption at the wavelength of 595 nm when it binds to the protein. Its light absorption value is proportional to the protein content. Therefore, the absorbance of the bacterial solution treated with the sample was determined at 595 nm [40]. The results for different time interval are shown in Table 3. The results also illustrated the destruction of the cell membrane of the bacteria. It can be seen from Table 3 that the absorbance of the experimental group was higher than that of the control group after 15 minutes. With the increase of incubation time, the absorbance value of the control group at 595 nm was kept at about 0.6, while the absorbance of the experimental group was increased slowly, reaching 1.008 after 90 minutes. The increase in absorbance indicates that the protein is constantly leaking and also demonstrates the destruction of the cell membrane. Table 3 Intramolecular protein leakage at different time interval
Control group test group
15 min
30 min
45 min
60 min
90 min
0.54 ± 0.0002
0.54 ± 0.0001
0.55 ± 0.0005
0.56 ± 0.0002
0.58 ± 0.0004
0.877 ± 0.001
0.935 ± 0.003
0.937 ± 0.003
0.954 ± 0.001
1.008 ± 0.0004
Normal operation of bacterial electron transfer chain has enormously significant effect to generate ATP [41]. Consequently, abnormal activity of respiratory chain dehydrogenase may be one of reasons for antibacterial action to some extent. Colorless iodine nitrotetrazole chloride (INT) can be reduced to a dark red water-insoluble iodonitrotetrazolium formazan (INF) by respiratory chain dehydrogenase in living cells [42]. Thus, the dehydrogenase activity can be
determined by the change of the spectrophotometric value of INF. Because it is a common method to determine the activity of respiratory chain dehydrogenase to characterize the metabolic activity of microorganisms, the dehydrogenase activity can be used as a detection index to assay sterilizing effect. The enzyme of bacteria boiled for 20 minutes is completely inactivated. The data of negative control was used as a reference for the enzyme inactivation of the bacteria treated with the sample. From Table 4, obvious data changes can be observed. After 15 minutes, the spectrophotometric value of INF in the experimental group was 0.0666, while that of the positive control group was 0.4997 and the negative control group was 0.0032. The results showed that the sample could destroy respiratory chain dehydrogenase and inhibit the respiration of cells. GSH protect cell from damage caused by oxidation stress. Ellman’assay has been used to measure ROS independent oxidizing stress GSH oxidation [43]. Glutathion (GSH) can be oxidized to Glutathion disulfide (GSSG). Therefore, the disulfide formation can be utilized to determine loss of GSH. As a result, positive control 1mM H2O2 induced almost 90.19% loss of GSH, which is agreement with literature [44], indicating that the experimental conditions will not lead to the oxidation of glutathione. Similarly, the compounds 1 and 2 induced almost 83.99% and 89.81% loss of GSH at the concentration of 1 mg/mL, respectively. The results indicated that synthetic complex converted directly GSH into GSSG or induced ROS generation to make GSH transform into GSSG. Ultimately, depletion of Glutathion leads to bacterial damage. In brief, the antibacterial effect may not be explained by a single substance, but by the multiple interactions of several components, which eventually lead to the death of bacterial cells. All results were presented in Table 4. The antibacterial mechanism can be explained in three aspects (Fig. 6). On the one hand, the addition of 1 can obviously inhibit bacteria growth (Fig. 4 A1 and Fig. 4 B1), so that the bacteria cannot proliferate normally and the normal physiological activity is affected, for example, the activity of the respiratory chain dehydrogenase is significantly reduced. On the other hand, 1 can destroy the cell membrane structure of bacteria, which leads to the acceleration of cell content leakage and bacteria death. On the last aspect, the complex can induce the electron donor on the cell surface to react with oxygen molecules to generate ROS and lead to bacterial death. Table 4 Experimental data of multiple bacterial death factors Cell membrane integrity (OD260 nm) control group(0 min)
0.016 ± 0.002
test group(0 min)
0.015 ± 0.001
control group(15 min)
0.070 ±0.0001
test group(15 min)
0.380 ± 0.002
enzymatic activity (OD490 nm)
quantitative detection of Thiol (OD412 nm)
0.0038 ± 0.0001(-) 0.0842 ± 0.0006(+) 0.0086 ± 0.0003 0.0032 ± 0.0001(-) 0.4997 ± 0.0003(+) 0.0666 ± 0.0002
2.3955 ± 0.0002(-) ̶ 2.3960 ± 0.0003 2.3946 ± 0.0002(-) 0.2350 ± 0.0005(+) 0.3834 ± 0.0002
Fig 6 The scheme of proposed antibacterial mechanism of 1 against E. coli 3.Conclusion In summary, HL1, HL2, compounds 1 and 2 are successfully synthesized and characterized. Both the ligands and its complexes exhibit good fluorescence properties. Ligands and Ph2SnCl2 exhibit desirable antibacterial activity. Meanwhile, the coordination to Sn(IV) metal atom increased antibacterial activity. 1 shows a more efficient antibacterial action than standard antibiotics Kanamycin and Ampicillin. Compound 2 has more significant bactericidal effect on E. coli. In addition to sterilization, the possible antibacterial mechanism is also discussed. The results show that the cause of bacterial death is attributed to a variety of factors such as membranolysis, respiratory dehydrogenase inactivation and glutathion depletion. 4. Experimental procedures 4.1 Materials and physical measurements All chemicals and reagents were analytical grade and used as received without further purification. Elemental analyses of C, H and N were conducted on Perkin-Elmer 24000-II elemental analyzer. Infrared spectra (IR) were recorded on American Nicolet 170 Fourier infrared spectrometer. Hitachi U-4100 spectrophotometer was employed to measure the UV-vis absorption spectra. Photofluorescent spectroscopy was obtained with F-7000 Fluorescence spectrometer. X-ray crystallography was acquired with Bruker ApeX-II CCD single-crystal diffractometer. 4.2 Synthesis of ligands and corresponding complexes The ligands were synthesized according to the literature [45,12] and authenticated by physicochemicl measurements. Complexes 1–2 were synthesized by solvent evaporation method. To synthetize 1, 3-aminopyridine-2-carbaldehyde N(4) methylthiosemicarbazone (0.049 g, 0.2 mmol) and sodium acetate (0.016 g, 0.2 mmol) was dissolved in methanol (20 mL). Then the methanolic solution of Ph2SnCl2 (0.07g, 0.2mmol) was added. After stirring and refluxing 1 hour, the subsidence was filtered. Yellow single crystals suitable for X-ray measurement were acquired through evaporation filtrate. Yield: 0.0950 g, 80%. C14H15Cl2N5SSn (474.96): calcd. C, 35.40; H, 3.18; N, 14.74. Found: C, 35.21; H, 3.03; N, 14.41. IR (KBr, cm−1): 1529 (C=N), 1227 (N–N), 795 (C=S). UV-vis [λ(nm), CH3OH ]: 292, 440. Synthesis of 2 was the same as that of 1, only the ligand was replaced by 3-aminopyridine-2-carbaldehyde N(4)-dimethylthiosemicarbazone (0.045 g, 0.2 mmol). Similarly, suitable yellow
single crystals are obtained. Yield: 0.0807 g (76%). Anal. calc. for C21H22ClN5SnS (530.64): C, 47.53; H, 4.18; N, 13.20. Found: C, 47.50; H, 4.12; N, 13.17. IR (KBr, cm−1): 1506 (C=N), 1256 (N–N), 734 (C=S). UV-vis [λ(nm), CH3OH ]: 296, 448. 4.3 Evaluation of antibacterial activity Antibacterial activity was assessed by agar-well diffusion to evaluate minimal inhibitory concentration (MIC). Antibacterial activities of ligands and complexes were investigated by the method reported in the literature [46]. A series of concentrations were obtained on the basis of 1 mg/mL using a double step-by-step dilution method, respectively, from which 100 microliters were added to the agar well to obtain the respective MIC according to the size of the bacteriostatic ring. In addition, results were explicated by the diameter of inhibition zone (mm). Ampicillin and Kanamycin were used as positive control. Solvent DMSO was used as blank control. Each experiment was carried out in triplicate under sterile conditions and the reported results were obtained from three independent measurements. 4.4 Antibacterial mechanism The excellent performance can always prompt us to further explore its mechanism of action. In view of the documents reported, the way of antibacterial action is the synergistic effect of multiple influencing factors. Up to date, a large number of research results have explained the mechanism of bacterial death including injuries of cell membrane, cell structure change, protein leakage and oxidative stress and so on. These aspects will be tested in this paper and the conclusions will be drawn. Taking sample 1 as an example, the mechanism was investigated. E. coli was used as a test strain. Detection of cell membrane integrity of bacteria, protein leakage test, determination of respiratory chain dehydrogenase activity and thioalcohol oxidation and quantitative determination were carried out according to the literature [39]. A colony was selected to be cultured in a 37oC with 150 rpm for 16 hours. The filtrate obtained by centrifugation at 4000 rpm was washed three times with phosphate buffer (PBS), and then suspended in sodium chloride, and a bacterial solution of optical density at 600 nm (OD600 = 0.5) was obtained by dilution, this concentration of bacteria was applied to the following procedure. 4.4.1 Bacterial cell membrane integrity detection The rupture of cell membrane will lead to the leakage of intracellular components of bacteria. The integrity of bacterial cell membrane was judged by the leakage of intracellular components, which was intuitively proved by measuring the absorbance at 260 nm. The group without sample was used as the control group. The bacterial suspension mixed with 4 mg complex 1 was cultured at 37 oC for 15 minutes, then filtered with a filter membrane of 0.22 microns and measured by UV-vis spectrophotometer. 4.4.2 Protein leakage test The damage of bacterial cell membrane leads to the leakage of intracellular components such as RNA, protein, potassium ion and so on, so the leakage of protein can also be used as an index to evaluate the integrity of cell membrane [47]. 1 mL bacterial suspension and 4 mg 1 were added into EP tube, then 5 mL Coomaslan
solution also was added. The mixture was shaken well and reacted for two minutes, and the absorbance at 595 nm was measured [48]. 4.4.3 Detection of respiratory chain dehydrogenase activity The activity of respiratory chain dehydrogenase has a great effect on the survival of bacteria [39]. The untreated bacteria were used as positive control. Bacteria boiled for 20 min were regarded as negative control. 0.1 mL (0.5%) INT solution and 4 mg complex 1 were added to 3 mL bacterial suspension. The mixture was incubated for 15 minutes at a dark condition of 37 oC. Then 50 µL formaldehyde was added to stop the reaction and the product in the tube was collected by centrifugation. The INF solution was extracted twice in a mixture of acetone and ethanol with a volume ratio of 1 : 1. The supernatant was mixed together, and the absorbance at 490 nm was measured. 4.4.4 Oxidation and quantitative detection of Glutathion Glutathion plays an important role in biological system, the thiol group in GSH is responsible for its biological activity [49] and its detection is completed according to literature [39]. The concentration of thiols in GSH was quantified by the Ellman's assay [50]. Bicarbonate buffer (pH 8.6) was used as reaction solution and all samples were prepared in triplicate. GSH solution without 1 was used as a negative control. GSH (0.8 mM) oxidization by H2O2 (1 mM) was used as a positive control. 1 (4 mg) was added into GSH (0.8 mM, 1mL) solution and the mixture was incubated with a speed of 150 rpm at 37 oC for 2 hours. After incubation, 3.5 mL Tris-HCl and 70 µL DNTB (Ellman's reagent, 5,50-dithio-bis-(2-nitrobenzoic acid) were added and the mixture became yellow solution. The yellow product was filtrated with 0.22 µm microporous filtering film. Finally, their absorbance at 412 nm was measured. The loss of GSH was calculated by the following formula: loss of GSH % = (absorbance of negative control - absorbance of sample)/absorbance of negative control͢ × 100%. Conflict of interest The authors declare no competing interests. Acknowledgements This work was supported by the National Natural Science Foundation of China (21671055) and the Program for Innovation Teams in Science and Technology in Universities of Henan Province [20IRTSTHN004]. Appendix A. Supplementary material CCDC numbers 1820173–1820174 contain the supplementary crystallogphic datas of 1–2. These data can be obtained free of charge from the Cambridge Crystallographic Centre via www.ccdc.cam.ac.uk/data_ request/cif. References [1] K. E. Jones, N. G. Patel, M. A. Levy, A. Storeygard, D. Balk, J. L. Gittleman, P. Daszak, Nature. 451 (2008) 990. [2] C. Huedo, F. Zani, A. Mendiola, S. Pradhan, C. Sinha, E. López‐Torres. Appl. Organomet.
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Highlights • 1 and 2 were successfully synthesized and characterized. • The compounds 1 and 2 have good biological activity, and 1 shows a more efficient antibacterial action than standard antibacterial substances. • The antibacterial performances of two compounds are different, which can be attributed to the difference of the ligand N-terminal substituents. • The possible antibacterial mechanism of 1 was explored systematically.
The authors declare no competing interests.
S0022-328X(20)30054-1
DOI:
https://doi.org/10.1016/j.jorganchem.2020.121153
Reference:
JOM 121153
To appear in:
Journal of Organometallic Chemistry
Received Date: 25 December 2019 Revised Date:
15 January 2020
Accepted Date: 1 February 2020
Please cite this article as: C. Xing, Y. Fang, L. Jiang, Y. Zhang, M. Li, Diorganotin(IV) complexes derived from N-terminal methylation of Triapine: synthesis, characterization and antibacterial activity evaluation, Journal of Organometallic Chemistry (2020), doi: https://doi.org/10.1016/j.jorganchem.2020.121153. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.
Diorganotin(IV) complexes derived from N-terminal methylation of Triapine: synthesis, characterization and antibacterial activity evaluation Cuili Xinga, Yan Fanga, Lei Jianga, Yahong Zhangb*, Mingxue Lia* a
Henan Key Laboratory of Polyoxometalates, Institute of Molecular and Crystal Engineering, College of Chemistry and Chemical Engineering, Henan University, Kaifeng 475004, PR China，Tel/Fax: +86 371 23881589 b Key Laboratory of Natural Medicines and Immune Engineering, Henan University, Kaifeng 475004, PR China E–mail address: [email protected] (Y. H. Zhang), [email protected](M.X. Li) Abstract The reaction of diorganotin(IV) with ligands (HL1 = 3-aminopyridine-2-carbaldehyde N(4) methylthiosemicarbazone, HL2 = 3-aminopyridine-2-carbaldehyde N(4)-dimethylthiosemicarbazone) afforded two compounds [PhSn(L1)Cl2] (1) and [(Ph)2Sn(L2)Cl] (2), respectively. The compounds were characterized by elemental analysis, infrared spectrum, UV-vis spectrometry, and X-ray crystallography. The ligands and corresponding complexes were assayed for their in vitro pharmaceutical activity against different pathogenic strains of bacteria by disk diffusion method. The results demonstrate that 1–2 have remarkable antibacterial activity against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). 1 exhibit higher antibacterial properties against S. aurenu (MIC = 0.00095 µg/mL) and E. coil (MIC = 0.0019 µg/mL). In the same experimental conditions, the two complexes display more potent activity relative to standard antibiotics Kanamycin and Ampicillin.
Keywords: Thiosemicarbazones; Diorganotin(IV); Antibacterial activity; Mechanism analysis 1. Introduction To treat infectious diseases caused by bacteria is still an urgent and challenging public health concern. Moreover, common pathogens and new pathogenic species treated with current antibiotics develop drug resistance [1,2]. The drug resistance of bacteria leads to a reduction in the effect of some common antibiotics and may result in higher mortality and morbidity [2]. Therefore, it is inevitable to fabricate new antibacterial agents, in particular the synthesis of metal complexes with bioactive ligands. Thiosemicarbazones containing a significant class of N, S donor are pre-eminent transition metallic chelators, having infinite interest in the coordination chemistry [3-5]. Thiosemicarbazones are also an important class of the carbonyl derivatives and
have been proved to own versatile medicinal applications, for instance, antibacterial [6], antitumor [7], antihypertensive [8], anticonvulsant [9], antitrypanosomal [10], and antiviral agents [11]. Additionally, the function of N-terminal methylation of thiosemicarbazone complexes is crucial in the biological activities [12]. A solid example is Dp44mT (di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone) and Dp4mT (di-2-pyridylketone-4-methyl-3-thiosemicarbazone). These chelators belong to the dipyridyl thiosemicarbazone (DpT) class of ligands and show different selectivity against tumor cells [13,14]. As antitumor agent, the currently most prominent representative in this family is the 3-aminopyridine-2-carbaldehyde thiosemicarbazone (Triapine), which has been investigated in clinical phase I and II studies [15-18]. Furthermore, a variety of new derivatives have been prefabricated in recent years for the sake of improving thiosemicarbazone-based therapy. Organotin (IV) derivatives have been widely investigated owing to their structural diversity and biological activity. Organotin (IV) compounds exhibit specificity in synthesis and structure, and their broad-spectrum applications can be attributed to ligands connected in the central tin metal atom [19]. Moreover, organotin(IV) compounds display multifunctional applications from industry, catalysis to therapeutics due to their diverse as well as fascinating structural potential [20]. Nowadays, organotin(IV) complexes formed by coordination with ligand are appealing scaffolds for new type drug research on account of their spectacular bioactivities. In recent years, many organotin(IV) complexes have been synthesized and investigated to be more remarkable than traditional metal drugs in antitumor activity [21-23]. Based on literatures, the biological properties are reliant on numbers and properties of organic groups, framework and ligancy of tin atom as well as the intrinsic performance of donor ligand connecting with tin metal atom [24,25]. Furthermore, some organotin(IV) compounds of Schiff bases containing a pyridine group display prominent antibacterial activities [26-28]. Significantly, organotin(IV) compounds may hold lower toxicity, better excretion from the body and less side effects along with no drug resistance formation compared to cisplatin analogues [29,30]. As a part of our research of Schiff based diorganotin(IV) compounds [31], herein we designed two organotin(IV) complexes, derived from HL1 and HL2 (Scheme 1). The biological activities of complexes 1 and 2, free ligands and metal precursors were evaluated by antibacterial test against two strains of E. coli and S. aureus. Antibacterial results indicated that 1 and 2 show obvious sterilizing effect. Most notably, 1 has more potent biological activity than 2 in promoting bacterial cell death and the MIC values are lower than common antibiotics Kanamycin and Ampicillin. Hence 1 as antibacterial agent has greater potential to be applied to reality.
Scheme 1 Synthesis of 1-2 2. Results and discussion 2.1 Chemistry In this paper, two complexes were synthesized by solvent evaporation method. The crystal structures of two complexes are shown in Fig. S1 and the crystal data are collected in Table S1. Bond length and bond angles are given in Table S2. These data are displayed in supplementary information. The synthesized material was confirmed by elemental analysis, infrared spectrum (IR) and UV-vis absorption spectrum, and the fluorescence emission spectra of the two products were tested. The characteristic bands observed in the region of 3178, 3376 and 3235 cm−1 are assigned to ʋ(N–H) of parent ligands (Fig. 1). These characteristic peaks also appear in the spectrum of the complexes. In the IR spectra of complexes, adsorption band of ʋ(C=N) was shifted to lower frequency by 16-52 cm−1, which indicated that the coordination via azomethine nitrogen atoms [32]. Furthermore, ʋ(N-N) increased from 1221 to 1227 cm−1 and 1202 to 1256 cm−1 in the spectra 1 and 2, respectively, which showed coordination through imine nitrogen [33]. 795 and 798 cm−1 observed for free ligand can be assigned to the ʋ(C=S) vibration. These bands shifted to 728 and 734 cm−1 in compounds 1 and 2, respectively, indicating the coordination of the sulfur atom [34]. Some new bands that emerging in the region 448-454 cm−1 and 694-695 cm−1 are assigned to ʋ(Sn-N) and ʋ(Sn-C) respectively, indicating chelation between the ligand and metal atom [35].
Fig. 1 Infrared spectra of HL1, HL2, 1–2
As shown in Fig. 2, UV-vis spectrum of both HL1 and HL2 show two strong bands at 292 and 372, 292 and 380 nm in methanol, respectively. The former may be due to π–π* transition of pyridine ring and the later may be attributed to n–π* transition of imine and thiocarbonyl moiety [36]. Coordination with Sn atom increased the maximum absorption wavelength of compounds. 1–2 have a great red shift from 372 to 440 nm and 380 to 448 nm respectively due to the n–π* transition of ligand-to-metal charge transfer (LMCT) [36].
Fig. 2 UV-Vis spectrums of ligands and compounds in methanol solution (10−4 mol / L)
To observe the influence for the luminescent properties through organic group exchange, the fluorescence emission spectrum was shown in Fig. 3. The compound 1 displayed a strong photoluminescence with maximum emission at 444 nm whereas the ligand showed maximum emission at 413 nm. This variation indicated the coordination of ligands with metal central atom. For 2, two strong photoluminescence with maximum emission at 413 and 531 nm were observed as indicated in Fig. 3B. Compared with the free ligand, strong fluorescence intensity of complexes 1–2 are observed, illustrating that fluorescent properties has been affected remarkably by introducing the Sn(IV) ions [37]. The fluorescence emission spectra prove that the two complexes have fluorescence properties, which enable them to be used in the field of photochemistry.
Fig. 3 Fluorescence emission spectra in 50% DMSO (10−5 mol / L). λex = 366 nm
2.2 Antibacterial activity The free ligands, metal precursor and tin(IV) compounds were evaluated for their antibacterial activity against E. coli (G-) and S. aureus (G+). The MIC values were determined and listed in Table 1. The optical images of zone of inhibition (zoi) were
shown in Fig. 4. The concentration was 1000, 500, 250 and 125 µg/mL, respectively. In this concentration gradient, the diameters of inhibitory zone of compounds 1–2, HL1, HL2, Ph2SnCl2, solvent (DMSO) and standard antibiotics Kanamycin and Ampicillin against E. coli and S. aureus were listed in Table 2. The results showed that solvent DMSO had no effect on the experimental bacterial strains. The metal chelates had higher activity than the free ligands as well as metal precursors. 1–2 exhibit a broad and effective antimicrobial activity against the tested bacteria. Additionally, the MIC values of 1 against two strains of tested bacteria E. coli (0.0019 µg/mL) and S. aureus (0.00095 µg/mL), which were lower than 2 (0.24 and 3.9 µg/mL). A possible explanation of enhanced activity of compounds relative to ligands is that better membrane penetrating ability endowed by the intensive lipophilicity via complexation. The coordination of ligand to tin result in electron delocalization and increase the lipophilic character and effectively disperse the metal complexes into bacterial cells [38]. The coordination with metal exhibits synergetic effect on antimicrobial activity. Table 1 MIC (µg/mL) of ligands, complexes and controls MIC values (µg/mL) E. coli S.aureus
1 0.0019 0.00095
̵ No inhibition
2 0.24 3.9
HL1 7.81 7.81
HL2 31.2 125
Ph2SnCl2 7.81 3.9
DMSO ̵ ̵
Amp 1.95 0.12
Kan 7.81 3.90
Fig. 4 Optical images of zone of inhibition against (A) E. coli and (B) S. aureus, respectively
Table 2 Antibacterial activity data showing zone of inhibition (mm) of the tested substances Inhibition zone diameter /mm (concentration in µg/mL)
1 2 HL1 HL2 Ph2SnCl2 DMSO Kanamycin Ampicillin
1000 25 21 20 24 15 ̵ 21 23
E. coli 500 250 23 24 20 22 17 15 20 17 14 13 ̵ ̵ 20 19 22 21
125 21 19 14 14 12 ̵ 17 19
1000 20 20 20 24 18 ̵ 28 29
S. aureus 500 250 21 21 21 20 19 14 19 13 16 15 ̵ ̵ 23 22 27 25
125 22 22 13 ̵ 14 ̵ 20 23
̵ No inhibition zone 2.3 Overviews of the mechanism of action In order to better explain the way of antibacterial action, test was expanded from the following aspects. In this paper, 1 was taken as an example to explore its antibacterial mechanism. To some extent, the damage to the cell membrane is caused by the interaction between antibacterial drugs and bacteria. The breakup of the membrane can cause some small molecular mass such as RNA, DNA, K+ and other substance (polysaccharide) to leak out. Intracellular components, such as DNA and RNA show specific absorbance at 260 nm [39]. Fig. 5 illustrated the damage degree of the cell membrane in bacteria. As can be seen from the test data in Table 4, the absorbance of intracellular components is about 0.01 at 0 minute. After 15 minutes, the absorbance value treated with the sample is 0.38. Simultaneously, the absorbance value without sample is 0.07. The changes of absorbance indicate that the cell membrane of bacteria is destroyed.
Fig 5 The absorbance of Intracellular components at OD260 nm The destruction of the integrity of bacterial cell membrane can lead to the release of intracellular proteins, hence another indicator of cell membrane damage is protein leakage. Moreover, coomassie brilliant blue G-250 determines that protein content belongs to a dye binding method. Red in the free state and the maximum light absorption is 488 nm, but it becomes cyan and the protein-pigment conjugate has the maximum light absorption at the wavelength of 595 nm when it binds to the protein. Its light absorption value is proportional to the protein content. Therefore, the absorbance of the bacterial solution treated with the sample was determined at 595 nm [40]. The results for different time interval are shown in Table 3. The results also illustrated the destruction of the cell membrane of the bacteria. It can be seen from Table 3 that the absorbance of the experimental group was higher than that of the control group after 15 minutes. With the increase of incubation time, the absorbance value of the control group at 595 nm was kept at about 0.6, while the absorbance of the experimental group was increased slowly, reaching 1.008 after 90 minutes. The increase in absorbance indicates that the protein is constantly leaking and also demonstrates the destruction of the cell membrane. Table 3 Intramolecular protein leakage at different time interval
Control group test group
15 min
30 min
45 min
60 min
90 min
0.54 ± 0.0002
0.54 ± 0.0001
0.55 ± 0.0005
0.56 ± 0.0002
0.58 ± 0.0004
0.877 ± 0.001
0.935 ± 0.003
0.937 ± 0.003
0.954 ± 0.001
1.008 ± 0.0004
Normal operation of bacterial electron transfer chain has enormously significant effect to generate ATP [41]. Consequently, abnormal activity of respiratory chain dehydrogenase may be one of reasons for antibacterial action to some extent. Colorless iodine nitrotetrazole chloride (INT) can be reduced to a dark red water-insoluble iodonitrotetrazolium formazan (INF) by respiratory chain dehydrogenase in living cells [42]. Thus, the dehydrogenase activity can be
determined by the change of the spectrophotometric value of INF. Because it is a common method to determine the activity of respiratory chain dehydrogenase to characterize the metabolic activity of microorganisms, the dehydrogenase activity can be used as a detection index to assay sterilizing effect. The enzyme of bacteria boiled for 20 minutes is completely inactivated. The data of negative control was used as a reference for the enzyme inactivation of the bacteria treated with the sample. From Table 4, obvious data changes can be observed. After 15 minutes, the spectrophotometric value of INF in the experimental group was 0.0666, while that of the positive control group was 0.4997 and the negative control group was 0.0032. The results showed that the sample could destroy respiratory chain dehydrogenase and inhibit the respiration of cells. GSH protect cell from damage caused by oxidation stress. Ellman’assay has been used to measure ROS independent oxidizing stress GSH oxidation [43]. Glutathion (GSH) can be oxidized to Glutathion disulfide (GSSG). Therefore, the disulfide formation can be utilized to determine loss of GSH. As a result, positive control 1mM H2O2 induced almost 90.19% loss of GSH, which is agreement with literature [44], indicating that the experimental conditions will not lead to the oxidation of glutathione. Similarly, the compounds 1 and 2 induced almost 83.99% and 89.81% loss of GSH at the concentration of 1 mg/mL, respectively. The results indicated that synthetic complex converted directly GSH into GSSG or induced ROS generation to make GSH transform into GSSG. Ultimately, depletion of Glutathion leads to bacterial damage. In brief, the antibacterial effect may not be explained by a single substance, but by the multiple interactions of several components, which eventually lead to the death of bacterial cells. All results were presented in Table 4. The antibacterial mechanism can be explained in three aspects (Fig. 6). On the one hand, the addition of 1 can obviously inhibit bacteria growth (Fig. 4 A1 and Fig. 4 B1), so that the bacteria cannot proliferate normally and the normal physiological activity is affected, for example, the activity of the respiratory chain dehydrogenase is significantly reduced. On the other hand, 1 can destroy the cell membrane structure of bacteria, which leads to the acceleration of cell content leakage and bacteria death. On the last aspect, the complex can induce the electron donor on the cell surface to react with oxygen molecules to generate ROS and lead to bacterial death. Table 4 Experimental data of multiple bacterial death factors Cell membrane integrity (OD260 nm) control group(0 min)
0.016 ± 0.002
test group(0 min)
0.015 ± 0.001
control group(15 min)
0.070 ±0.0001
test group(15 min)
0.380 ± 0.002
enzymatic activity (OD490 nm)
quantitative detection of Thiol (OD412 nm)
0.0038 ± 0.0001(-) 0.0842 ± 0.0006(+) 0.0086 ± 0.0003 0.0032 ± 0.0001(-) 0.4997 ± 0.0003(+) 0.0666 ± 0.0002
2.3955 ± 0.0002(-) ̶ 2.3960 ± 0.0003 2.3946 ± 0.0002(-) 0.2350 ± 0.0005(+) 0.3834 ± 0.0002
Fig 6 The scheme of proposed antibacterial mechanism of 1 against E. coli 3.Conclusion In summary, HL1, HL2, compounds 1 and 2 are successfully synthesized and characterized. Both the ligands and its complexes exhibit good fluorescence properties. Ligands and Ph2SnCl2 exhibit desirable antibacterial activity. Meanwhile, the coordination to Sn(IV) metal atom increased antibacterial activity. 1 shows a more efficient antibacterial action than standard antibiotics Kanamycin and Ampicillin. Compound 2 has more significant bactericidal effect on E. coli. In addition to sterilization, the possible antibacterial mechanism is also discussed. The results show that the cause of bacterial death is attributed to a variety of factors such as membranolysis, respiratory dehydrogenase inactivation and glutathion depletion. 4. Experimental procedures 4.1 Materials and physical measurements All chemicals and reagents were analytical grade and used as received without further purification. Elemental analyses of C, H and N were conducted on Perkin-Elmer 24000-II elemental analyzer. Infrared spectra (IR) were recorded on American Nicolet 170 Fourier infrared spectrometer. Hitachi U-4100 spectrophotometer was employed to measure the UV-vis absorption spectra. Photofluorescent spectroscopy was obtained with F-7000 Fluorescence spectrometer. X-ray crystallography was acquired with Bruker ApeX-II CCD single-crystal diffractometer. 4.2 Synthesis of ligands and corresponding complexes The ligands were synthesized according to the literature [45,12] and authenticated by physicochemicl measurements. Complexes 1–2 were synthesized by solvent evaporation method. To synthetize 1, 3-aminopyridine-2-carbaldehyde N(4) methylthiosemicarbazone (0.049 g, 0.2 mmol) and sodium acetate (0.016 g, 0.2 mmol) was dissolved in methanol (20 mL). Then the methanolic solution of Ph2SnCl2 (0.07g, 0.2mmol) was added. After stirring and refluxing 1 hour, the subsidence was filtered. Yellow single crystals suitable for X-ray measurement were acquired through evaporation filtrate. Yield: 0.0950 g, 80%. C14H15Cl2N5SSn (474.96): calcd. C, 35.40; H, 3.18; N, 14.74. Found: C, 35.21; H, 3.03; N, 14.41. IR (KBr, cm−1): 1529 (C=N), 1227 (N–N), 795 (C=S). UV-vis [λ(nm), CH3OH ]: 292, 440. Synthesis of 2 was the same as that of 1, only the ligand was replaced by 3-aminopyridine-2-carbaldehyde N(4)-dimethylthiosemicarbazone (0.045 g, 0.2 mmol). Similarly, suitable yellow
single crystals are obtained. Yield: 0.0807 g (76%). Anal. calc. for C21H22ClN5SnS (530.64): C, 47.53; H, 4.18; N, 13.20. Found: C, 47.50; H, 4.12; N, 13.17. IR (KBr, cm−1): 1506 (C=N), 1256 (N–N), 734 (C=S). UV-vis [λ(nm), CH3OH ]: 296, 448. 4.3 Evaluation of antibacterial activity Antibacterial activity was assessed by agar-well diffusion to evaluate minimal inhibitory concentration (MIC). Antibacterial activities of ligands and complexes were investigated by the method reported in the literature [46]. A series of concentrations were obtained on the basis of 1 mg/mL using a double step-by-step dilution method, respectively, from which 100 microliters were added to the agar well to obtain the respective MIC according to the size of the bacteriostatic ring. In addition, results were explicated by the diameter of inhibition zone (mm). Ampicillin and Kanamycin were used as positive control. Solvent DMSO was used as blank control. Each experiment was carried out in triplicate under sterile conditions and the reported results were obtained from three independent measurements. 4.4 Antibacterial mechanism The excellent performance can always prompt us to further explore its mechanism of action. In view of the documents reported, the way of antibacterial action is the synergistic effect of multiple influencing factors. Up to date, a large number of research results have explained the mechanism of bacterial death including injuries of cell membrane, cell structure change, protein leakage and oxidative stress and so on. These aspects will be tested in this paper and the conclusions will be drawn. Taking sample 1 as an example, the mechanism was investigated. E. coli was used as a test strain. Detection of cell membrane integrity of bacteria, protein leakage test, determination of respiratory chain dehydrogenase activity and thioalcohol oxidation and quantitative determination were carried out according to the literature [39]. A colony was selected to be cultured in a 37oC with 150 rpm for 16 hours. The filtrate obtained by centrifugation at 4000 rpm was washed three times with phosphate buffer (PBS), and then suspended in sodium chloride, and a bacterial solution of optical density at 600 nm (OD600 = 0.5) was obtained by dilution, this concentration of bacteria was applied to the following procedure. 4.4.1 Bacterial cell membrane integrity detection The rupture of cell membrane will lead to the leakage of intracellular components of bacteria. The integrity of bacterial cell membrane was judged by the leakage of intracellular components, which was intuitively proved by measuring the absorbance at 260 nm. The group without sample was used as the control group. The bacterial suspension mixed with 4 mg complex 1 was cultured at 37 oC for 15 minutes, then filtered with a filter membrane of 0.22 microns and measured by UV-vis spectrophotometer. 4.4.2 Protein leakage test The damage of bacterial cell membrane leads to the leakage of intracellular components such as RNA, protein, potassium ion and so on, so the leakage of protein can also be used as an index to evaluate the integrity of cell membrane [47]. 1 mL bacterial suspension and 4 mg 1 were added into EP tube, then 5 mL Coomaslan
solution also was added. The mixture was shaken well and reacted for two minutes, and the absorbance at 595 nm was measured [48]. 4.4.3 Detection of respiratory chain dehydrogenase activity The activity of respiratory chain dehydrogenase has a great effect on the survival of bacteria [39]. The untreated bacteria were used as positive control. Bacteria boiled for 20 min were regarded as negative control. 0.1 mL (0.5%) INT solution and 4 mg complex 1 were added to 3 mL bacterial suspension. The mixture was incubated for 15 minutes at a dark condition of 37 oC. Then 50 µL formaldehyde was added to stop the reaction and the product in the tube was collected by centrifugation. The INF solution was extracted twice in a mixture of acetone and ethanol with a volume ratio of 1 : 1. The supernatant was mixed together, and the absorbance at 490 nm was measured. 4.4.4 Oxidation and quantitative detection of Glutathion Glutathion plays an important role in biological system, the thiol group in GSH is responsible for its biological activity [49] and its detection is completed according to literature [39]. The concentration of thiols in GSH was quantified by the Ellman's assay [50]. Bicarbonate buffer (pH 8.6) was used as reaction solution and all samples were prepared in triplicate. GSH solution without 1 was used as a negative control. GSH (0.8 mM) oxidization by H2O2 (1 mM) was used as a positive control. 1 (4 mg) was added into GSH (0.8 mM, 1mL) solution and the mixture was incubated with a speed of 150 rpm at 37 oC for 2 hours. After incubation, 3.5 mL Tris-HCl and 70 µL DNTB (Ellman's reagent, 5,50-dithio-bis-(2-nitrobenzoic acid) were added and the mixture became yellow solution. The yellow product was filtrated with 0.22 µm microporous filtering film. Finally, their absorbance at 412 nm was measured. The loss of GSH was calculated by the following formula: loss of GSH % = (absorbance of negative control - absorbance of sample)/absorbance of negative control͢ × 100%. Conflict of interest The authors declare no competing interests. Acknowledgements This work was supported by the National Natural Science Foundation of China (21671055) and the Program for Innovation Teams in Science and Technology in Universities of Henan Province [20IRTSTHN004]. Appendix A. Supplementary material CCDC numbers 1820173–1820174 contain the supplementary crystallogphic datas of 1–2. These data can be obtained free of charge from the Cambridge Crystallographic Centre via www.ccdc.cam.ac.uk/data_ request/cif. References [1] K. E. Jones, N. G. Patel, M. A. Levy, A. Storeygard, D. Balk, J. L. Gittleman, P. Daszak, Nature. 451 (2008) 990. [2] C. Huedo, F. Zani, A. Mendiola, S. Pradhan, C. Sinha, E. López‐Torres. Appl. Organomet.
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Highlights • 1 and 2 were successfully synthesized and characterized. • The compounds 1 and 2 have good biological activity, and 1 shows a more efficient antibacterial action than standard antibacterial substances. • The antibacterial performances of two compounds are different, which can be attributed to the difference of the ligand N-terminal substituents. • The possible antibacterial mechanism of 1 was explored systematically.
The authors declare no competing interests.