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Synthesis, Crystal Structures, and Binding Behaviors with CT-DNA/BSA, Antimicrobial and Cytotoxic Activities of Three New Pyrazine Acylhydrazones

Jie YANG ,  Xiang-Rong LIU ,  Geng-Hui LIAO ,  Ming-Kun YU ,  Zai-Wen YANG ,  Shun-Sheng ZHAO

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Citation:  Jie YANG, Xiang-Rong LIU, Geng-Hui LIAO, Ming-Kun YU, Zai-Wen YANG, Shun-Sheng ZHAO. Synthesis, Crystal Structures, and Binding Behaviors with CT-DNA/BSA, Antimicrobial and Cytotoxic Activities of Three New Pyrazine Acylhydrazones[J]. Chinese Journal of Structural Chemistry, 2020, 39(9): 1639-1654. doi: 10.14102/j.cnki.0254-5861.2011-2770 shu

Synthesis, Crystal Structures, and Binding Behaviors with CT-DNA/BSA, Antimicrobial and Cytotoxic Activities of Three New Pyrazine Acylhydrazones

English

  • 1. INTRODUCTION

    Acylhydrazones have been widely investigated because of their similar structures to natural biological substances[1, 2]. C=N and N–N linkages in acylhydrazone molecule play an essential role in the design and construction of promising bioactive agents[3, 4]. Acylhydrazones as reported in literature display various biological activities such as antimicrobial[5, 6], antitubercular[7], antimalarial[8], anticancer[9] and antioxidant[10].

    Pyrazine is an important skeleton containing two nitrogen atoms in their six-membered ring. The structure of the pyrazine ring resembles some naturally occurring compounds such as nicotinamide and pyrimidine nucleic bases[11]. Thus, pyrazine derivatives exhibit significant pharmacological activities like anticancer[12, 13], antibacterial[14, 15] and anti-hepatitis C virus[16]. Meanwhile, some pyrazines, especially dihydropyrazines, possess the activities to break the DNA strand and induct apoptosis and mutagenesis[17].

    Besides, DNA is significant in gene regulation during the life process. It is the primary target for some kinds of drugs[18-22]. Acylhydrazones could bind to DNA through electrostatic effect, groove binding or intercalation. Serum albumins are key biological macromolecules, which can reversibly bind to drug molecules and transport them to target tissues and organs. Therefore, serum albumins greatly influence the distribution and absorption of drugs[23]. Investigation on the interaction between compound and DNA/serum albumins could ascertain the mechanism of drug effect and be beneficial in the design of new drug[24].

    In this work, we constructed three acylhydrazone derivatives containing the pyrazine rings. All of the compounds were characterized by elemental analyses and single-crystal XRD, FT-IR, 1H NMR and TG-DTG. The binding abilities of three compounds with CT-DNA and BSA were studied by UV-Vis spectroscopy and fluorescence spectroscopy, respectively. The binding behaviors of 1~3 with DNA/BSA were also studied by molecular docking method. Staphylococcus aureus, Escherichia coli and Salmonella typhimurium were used to evaluate the antibacterial activities of 1~3. The cytotoxic activities of 1~3 were studied by MTT assay. Synthetic routes of these three compounds are shown in Scheme 1.

    Scheme 1

    Scheme 1.  Synthetic routes of compounds 1~3
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    2. EXPERIMENTAL

    2.1 Chemicals and methods

    Hydrazine hydrate (80%), acetic acid, ethanol, 2-hydroxy-3-methoxybenzaldehyde, 4-hydroxybenzaldehyde, 4-methylbenzaldehyde, 2-amino-3-pyrazine-carboxylate and etoposide were purchased from commercial sources and were all of analytical grades. CT-DNA, BSA and gentamycin sulfate were biological reagents and purchased from American Sigma Company, J & K Scientific LTD and Jiuding Chemistry, separately. Staphylococcus aureus (CICC No. 10001), Escherichia coli (CICC No. 10003) and Salmonella typhimurium (CICC No. 21484) were obtained from China Center of Industrial Culture Collection and Luria-Bertani (LB) medium used in this experiment and were prepared by 10 g tryptone, 5 g yeast extract, 10 g NaCl and 1 L distilled water. Human lung cancer cells (A549) were purchased from American Type Culture Collection and Dulbecco's Modified Eagle Medium (DMEM) was purchased from Thermo Fisher Scientific.

    The melting points were determined on the WRS-2U melting point apparatus (China). Elemental analyses (C, H, and N) were performed on PE 2400-II elemental analyzer (America). Infrared spectra were measured utilizing a Bruker Tensor-II Fourier infrared spectrometer (Germany). Single-crystal XRD measurements for three compounds were performed on a Bruker Apex-ⅡCCD diffractometer (Germany). Thermogravimetric analysis was carried out by the Mettler Toledo TGA analyzer (Swiss). UV-Vis absorption spectra of interaction between compounds and CT-DNA were obtained on a TU-1900 ultraviolet-visible spectrophotometer (China). Perkin Elmer LS55 fluorescence spectrophotometer (America) was used to record fluorescence spectra of compounds interacting with BSA. BAJIU SAF-680T microplate reader (China) was used to obtain optical density values.

    2.2 Synthesis of three compounds

    2.2.1   Synthesis of 2-amino-3-pyrazine-carbohydrazide (C5H7N5O)

    An ethanolic solution (15 mL) of methyl 4-hydroxy-phenylacetate 2-amino-3-pyrazine-carboxylate (0.3063 g, 2 mmol) was added to 80% hydrazine hydrate (5 mL) under continuous stirring. The mixture was heated at 80 ℃ and refluxed for 4~5 h. Then resultant solutions were filtered and stood for 48 h to obtain the yellow needle crystals.

    2.2.2   Synthesis of three acylhydrazone compounds 1~3

    Synthesis of 4-hydroxybenzaldehyde-2-amino-3-pyrazine acylhydrazone (1): 2-amino-3-pyrazine-carbohydrazide (0.0459 g, 0.3 mmol) and 4-hydroxybenzaldehyde (0.0366 g, 0.3 mmol) were dissolved in ethanol. The mixture was heated at 80 ℃ and refluxed for 3~4 h under continuous stirring. Then resultant solution was filtered and stood for 48 h to obtain the product. The product was recrystallized from ethanol/acetic acid (V(ethanol): V(acetic acid) = 4:1, 20 mL) and bright yellow single crystals were obtained by slow evaporation for 3 d. 4-Methylbenzaldehyde-2-amino-3-pyrazine acylhydrazone (2) and 2-hydroxy-3-methoxy-benzaldehyde-2-amino-3-pyrazine acylhydrazone (3) were synthesized using the same method.

    Compound 1: Yield: 73.60%. m.p. 277.31~278.11 ℃. Anal. Calcd. for C12H11N5O2 (1, %): C, 56.03; H, 4.280; N, 27.24. Found (%): C, 56.23; H, 4.218; N, 27.07. IR (KBr, cm–1) ν: 1664 (C=O), 1599 (C=N)imine, 1537 (C=N)pyrazine. 1H NMR (400 MHz, DMSO-d6) δ: 11.84 (s, 1H, NH), 9.93 (s, 1H, Ar–OH), 8.48 (s, 1H, CH), 8.26 (d, J = 2.3 Hz, 1H, pyrazine-H), 7.88 (d, J = 2.3 Hz, 1H, pyrazine-H), 7.62~7.50 (m, 4H, NH2, 2Ar-H), 6.85 (d, J = 8.6 Hz, 2H, Ar-H).

    Compound 2: Yield: 74.39%. m.p. 213.37~213.87 ℃. Anal. Calcd. for C13H13N5O (2, %): C, 61.18; H, 5.098; N, 27.45. Found (%): C, 61.32; H, 5.008; N, 27.12. IR (KBr, cm–1) ν: 1676 (C=O), 1608 (C=N)imine, 1532 (C=N)pyrazine. 1H NMR (400 MHz, DMSO-d6) δ: 11.99 (s, 1H, NH), 8.56 (s, 1H, CH), 8.28 (d, J = 2.3 Hz, 1H, Pyrazine-H), 7.89 (d, J = 2.3 Hz, 1H, Pyrazine-H), 7.63~7.56 (m, 4H, NH2, 2Ar-H), 7.28 (d, J = 7.9 Hz, 2H, Ar-H), 2.35 (s, 3H, Ar–CH3).

    Compound 3: Yield: 66.51%. m.p. 188.96~189.96 ℃. Anal. Calcd. for C13H13N5O3 (3, %): C, 54.36; H, 4.530; N, 24.39. Found (%): C, 53.24; H, 4.620; N, 24.67. IR (KBr, cm–1) ν: 1671 (C=O), 1593 (C=N)imine, 1551 (C=N)pyrazine. 1H NMR (400 MHz, DMSO-d6) δ: 12.39 (s, 1H, Ar–OH), 11.21 (s, 1H, NH), 8.79 (s, 1H, CH), 8.29 (d, J = 2.3 Hz, 1H, Pyrazine-H), 7.91 (d, J = 2.3 Hz, 1H, Pyrazine-H), 7.09~7.01 (m, 2H, NH2), 6.87 (t, J = 7.9 Hz, 1H, Ar–H), 3.82 (s, 3H, CH), 1.91 (s, 2H, Ar–H).

    2.3 X-ray crystallography

    Three appropriate single crystals with dimensions of 0.35 × 0.24 × 0.13 mm3 (1), 0.37 × 0.31 × 0.22 mm3 (2) and 0.35 × 0.27 × 0.14 mm3 (3) have been selected and mounted on a Bruker ApexⅡCCD diffractometer with a graphite-mono-chromatic MoKα radiation (λ = 0.71073 Å). The diffraction data were collected at normal temperature (25 ℃) and refection data were recorded by using an ω-φ scan mode. The lattice parameters were obtained by the least-squares refinement and crystal structures were solved by direct methods using SHELXS-97 program[25]. All the non-hydrogen atoms positions were refined by full-matrix least-squares using SHELXL-97 and the hydrogen atoms were set in calculated positions.

    2.4 Thermogravimetric experiments

    The TG-DTG curves of 1~3 were obtained in N2 atmosphere within the temperature range of room temperature to 800 ℃ by using thermogravimetry. And the heating rate was set as 5 ℃·min–1.

    2.5 CT-DNA binding experiments

    The CT-DNA solution (2.88 × 10–4 M) was prepared by dissolving CT-DNA in tris-HCl buffer solution (0.01 M, pH = 7.9). Each compound was dissolved in the mixed solution of DMSO and tris-HCl (2:98, V/V) with concentration of 1 × 10–5 M. CT-DNA solution (50 μL for each time) was dropped into the solutions of test compound (3 mL) and reference tris-HCl buffer solution. After each addition of CT-DNA, the UV-Vis spectra were recorded in the range of 250~500 nm.

    2.6 BSA binding experiments

    BSA solution (1 × 10–7 M) was prepared by dissolving BSA in tris-HCl-NaCl buffer solution (0.01 M, pH = 7.2). The solution of each compound (1 × 10–5 M) was prepared by dissolving the compound into a mixture of DMSO and tris-HCl-NaCl buffer (2:98, V/V). Then the solution of test compound (30 μL for each time) was dropped into the BSA solution (3 mL) and reference tris-HCl-NaCl buffer solution. The mixed solution was monitored by fluorescence spectrometer in the wavelength range of 300~540 nm. The excitation wavelength was 280 nm and the excitation/emission slit widths were all 5 nm.

    2.7 Molecular docking studies

    The crystal structures of DNA (PDB ID: 425D) and BSA (PDB ID: 4F5S) were chosen from RCSB Protein Data Bank (http://www.rcsb.org/pdb). The crystal structures of 1~3 were obtained from single-crystal XRD data, and CIF files were converted into PDB format using Mercury software. Polar hydrogen atoms and Gasteiger charges were added and water molecules were removed by Autodock tools. Molecular docking studies of 1~3 and DNA/BSA interactions were done on the Autodock 4.0 software. For each docking case, the obtained conformations were ranked based on the lowest free binding energy and the lowest energy conformation was selected as the binding mode. The visualization of the docked pose was performed using Discovery Studio 4.5[26].

    2.8 Antibacterial activity

    Microplate reader method[27] was used to evaluate the antibacterial activities of 1~3 and gentamycin sulfate against Staphylococcus aureus (S. aureus), Escherichia coli (E. coli) and Salmonella typhimurium (S. typhimurium). All the strains were incubated in Luria-Bertani (LB) medium at 30 ℃ for 15 h. Then bacteria were diluted to obtain a density of 105 colony forming units per mL (CFU/mL). The sterile 96-well plate filled with 5 μL bacterial suspensions and a series of 250 μL compound solutions with concentrations ranging from 10 to 50 μM. The plates were incubated at 30 ℃ for 6 h. Optical densities (OD) values were measured by a microplate reader at 630 nm and inhibition rates were determined by following equation[28]:

    $ \text { Inhibition rate }=\left(1-\mathrm{OD}_{\text {sample }} / \mathrm{OD}_{\text {control }}\right) \times 100 \% $

    		(1)

    where ODsample is the optical density of strains incubated in medium alone, and ODcontrol the optical density of strains treated with the test compound. Half inhibitory concentrations (IC50) were calculated by PASW Statistics software.

    2.9 Cytotoxic activity

    MTT (3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyl-tetrazolium bromide) assay was used to investigate the cytotoxic activities of three compounds and etoposide against human lung cancer cells (A549). The A549 cells were placed in 96-well plate incubated in Dulbecco's Modified Eagle Medium (DMEM) at 37 ℃ for 24 h before the addition of compounds solution with various concentrations (0~100 μM). After adding the compound solution, the plate was incubated under 5% CO2 at 37 ℃ for 24 h. Then each well was added 10 uL of MTT solution (5 mg/mL in PBS) and 90 uL of DMEM and incubated for 24 h at 37 ℃. The DMEM was removed and purple formazan crystals were dissolved by 100 uL DMSO solution. OD values were measured at 570 nm by a microplate reader and inhibition rates were calculated by the formula (1).

    3. RESULTS AND DISCUSSION

    3.1 Crystal structures of 1~3

    Crystal structures of 1~3 with atomic numbering scheme were depicted in Figs. 1~3. Views of the hydrogen bonds are shown in Figs. 4~6. Selected bond distances and bond angles for 1~3 are given in Table 1. 1 crystallized in the monoclinic system, P21/c space group and possessed a condensing unit of 2-amino-3-pyrazine-carbohydrazide and 4-hydroxybenzaldehyde with a lattice acetic acid molecule as an asymmetric unit with Z = 4. In 1, the C(6)–N(5) bond distance (1.277(2) Å) is close to the typical C=N double bond which is found in other acylhydrazone compounds[29, 30]. The C(5)–O(1) bond length is 1.236(2) Å, which is comparable to the C=O bond length (1.21 Å)[31]. These data show that 1 includes a keto group. Pyrazine and benzene rings form a dihedral angle of 4.77°, as shown in Table 1, indicating that these two rings are not in exact parallel planes. Moreover, the torsion angles of C(8)–C(7)–C(6)–N(5) (1.8(3)°) and C(12)–C(7)–C(6)–N(5) (–178.50(16)°) indicate a small twist between C(6)–N(5) and the benzene ring. The O(1) atom is involved in an intramolecular hydrogen bond with H(3)B. And the O(1) atom also forms an intermolecular hydrogen bond with atom H(2) of another molecule. There exist hydrogen bonds between the compound and lattice acetic acid molecule. Thus, the molecules of 1 are interlinked by hydrogen bonds N(3)–H(3)A···O(3), O(2)– H(2)···O(1) and O(4)–H(4)A···N(2) into a 2D structure. Hydrogen bond distances (Å) and angles (°) of the compounds are set out in Table 2.

    Figure 1

    Figure 1.  Crystal structure of 1 with 30% probability
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    Figure 2

    Figure 2.  Crystal structure of 2 with 30% probability
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    Figure 3

    Figure 3.  Crystal structure of 3 with 30% probability
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    Figure 4

    Figure 4.  View of the hydrogen bonds of 1
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    Figure 5

    Figure 5.  View of the hydrogen bonds of 2
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    Figure 6

    Figure 6.  View of the hydrogen bonds of 3
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    Table 1

    Table 1.  Selected Bond Distances (Å) and Bond Angles (º) for 1~3
    DownLoad: CSV
    Compound 1
    Bond Dist. Bond Dist. Bond Dist.
    O(1)–C(5) 1.236(2) N(5)–N(4) 1.379(19) N(3)–C(1) 1.330(2)
    O(2)–C(10) 1.359(2) N(4)–C(5) 1.338(2) N(2)–C(1) 1.349(2)
    N(5)–C(6) 1.277(2) C(5)–C(4) 1.482(2) C(4)–C(1) 1.421(2)
    Angle (°) Angle (°) Angle (°)
    N(5)–C(6)–C(7) 122.39(17) O(1)–C(5)–C(4) 122.73(17) C(4)–N(1)–C(3)–C(2) 1.0(3)
    C(6)–N(5)–N(4) 115.15(16) C(6)–N(5)–N(4)–C(5) 179.23(16) C(8)–C(7)–C(6)–N(5) 1.8(3)
    C(5)–N(4)–N(5) 120.43(15) C(10)–C(9)–C(8)–C(7) 0.7(3) C(12)–C(7)–C(6)–N(5) –178.50(16)
    N(4)–C(5)–C(4) 114.31(16) C(9)–C(10)–C(11)–C(12) 0.6(3) N(4)–C(5)–C(4)–C(1) –177.05(15)
    O(1)–C(5)–N(4) 122.96(17) C(1)–N(2)–C(2)–C(3) 0.3(3) N(4)–C(5)–C(4)–N(1) 2.1(2)
    Compound 2
    Bond Dist. Bond Dist. Bond Dist.
    O(1)–C(5) 1.227(4) N(4)–C(5) 1.343(5) N(3)–C(1) 1.316(4)
    N(5)–C(6) 1.283(5) C(10)–C(13) 1.515(6) N(2)–C(1) 1.346(5)
    N(5)–N(4) 1.384(4) C(5)–C(4) 1.488(6) C(4)–C(1) 1.321(4)
    Angle (°) Angle (°) Angle (°)
    N(5)–C(6)–C(7) 120.6(4) O(1)–C(5)–C(4) 122.5(5) C(4)–N(1)–C(3)–C(2) 0.6(8)
    C(6)–N(5)–N(4) 113.5(4) C(6)–N(5)–N(4)–C(5) 178.3(4) C(8)–C(7)–C(6)–N(5) –6.7(7)
    C(5)–N(4)–N(5) 118.8(3) C(10)–C(9)–C(8)–C(7) 0.6(8) C(12)–C(7)–C(6)–N(5) 174.4(4)
    N(4)–C(5)–C(4) 114.6(4) C(9)–C(10)–C(11)–C(12) 0.3(7) N(4)–C(5)–C(4)–C(1) –177.2(4)
    O(1)–C(5)–N(4) 122.8(4) C(1)–N(2)–C(2)–C(3) –0.6(7) N(4)–C(5)–C(4)–N(1) 2.5(5)
    Compound 3
    Bond Dist. Bond Dist. Bond Dist.
    O(1)–C(5) 1.218(4) N(5)–N(4) 1.367(4) N(3)–C(1) 1.346(5)
    O(2)–C(8) 1.358(5) N(4)–C(5) 1.351(5) N(2)–C(1) 1.345(5)
    N(5)–C(6) 1.263(5) C(5)–C(4) 1.497(5) C(4)–C(1) 1.409(6)
    Angle (°) Angle (°) Angle (°)
    N(5)–C(6)–C(7) 120.8(3) O(1)–C(5)–C(4) 123.3(4) C(4)–N(1)–C(3)–C(2) –0.4(6)
    C(6)–N(5)–N(4) 119.5(3) C(6)–N(5)–N(4)–C(5) 177.8(4) C(8)–C(7)–C(6)–N(5) –2.3(6)
    C(5)–N(4)–N(5) 118.4(3) C(10)–C(9)–C(8)–C(7) –0.1(6) C(12)–C(7)–C(6)–N(5) 176.4(4)
    N(4)–C(5)–C(4) 113.9(3) C(9)–C(10)–C(11)–C(12) 1.2(7) N(4)–C(5)–C(4)–C(1) 178.3(3)
    O(1)–C(5)–N(4) 122.7(4) C(1)–N(2)–C(2)–C(3) 0.8(7) N(4)–C(5)–C(4)–N(1) –1.5(5)

    Table 2

    Table 2.  Hydrogen Bond Distances (Å) and Angles (°) of 1~3
    DownLoad: CSV
    Compound D–H···A d(D–H)/Å d(H···A)/Å d(D···A)/Å ∠DHA (º)
    1 N(3)–H(3)A···O(3)a 0.860 2.191 3.044 170.99
    N(3)–H(3)B···O(1) 0.860 2.075 2.713 130.51
    O(2)–H(2)···O(1)b 0.820 2.080 2.799 146.20
    O(4)–H(4)A···N(2)c 0.820 1.916 2.721 166.73
    2 N(3)–H(3)A···O(4)a 0.860 2.123 2.970 168.24
    N(3)–H(3)B···O(1) 0.860 2.104 2.732 129.43
    N(4)–H(4)···O(2)b 0.860 2.312 3.092 150.79
    O(3)–H(3)···O(1)c 0.820 1.899 2.719 177.42
    O(5)–H(5)···N(2)d 0.820 1.948 2.738 161.24
    3 N(4)–H(4)···N(7)a 0.860 2.448 3.279 162.42
    O(2)–H(2)···N(5) 0.820 1.877 2.595 145.59
    O(5)–H(5)···N(10) 0.820 1.959 2.674 145.31
    N(8)–H(8)A···O(2)b 0.860 2.332 3.152 159.69
    N(8)–H(8)B···O(4) 0.860 2.091 2.738 131.49
    N(3)–H(3)A···O(5)c 0.860 2.431 3.069 131.55
    N(3)–H(3)A···O(6)d 0.860 2.474 3.156 136.82
    N(3)–H(3)B···O(1) 0.860 2.112 2.746 130.08
    Symmetry codes: for 1 (a) –x + 1/2, y + 1/2, –z + 1/2; (b) –x + 1/2, y – 1/2, –z + 3/2; (c) –x + 1/2, y – 1/2, –z + 1/2; for 2 (a) x + 1, y – 1, z; (b) –x + 1, y – 1/2, –z + 1/2; (c) x, –y + 1/2, z – 1/2; (d) x – 1, y + 1, z; for 3 (a) x – 1, y, z – 1; (b) –x + 3/2, –y + 3/2, –z + 1; (c) –x + 1, –y + 2, –z + 1; (d) –x + 1, –y + 2, –z + 1

    Similarly, compound 2 crystallized in P21/c monoclinic space with one molecule in a unit cell, whereas 3 crystallized in C2/c monoclinic space and two molecules exist in a unit cell. Typical C=N and C=O double bonds are both found in 2 and 3. Dihedral angles between pyrazine and benzene rings are 3.73° and 6.23° in 2 and 3, respectively. Therefore, 2 is more coplanar among 1~3. C(6)–N(5) and benzene ring in 2 and 3 are also not planar but torsion angles are quite small. Moreover, there exist a series of π-π stacking interactions between the adjacent pyrazine and benzene rings in 2 and 3. The parameters of π-π stacking interactions of compounds 2 and 3 are listed in Table 3. Both the units of 2 and 3 are linked by intermolecular hydrogen bond and π-π stacking interactions to produce a 2D supramolecular network.

    Table 3

    Table 3.  Selected π-π Stacking Interactions of 2 and 3
    DownLoad: CSV
    Compound Parameters of π-π stacking
    Dentroid-centroid separation (Å) Dihedral angle (°) Vertical displacements between ring centroids (Å)
    2 3.753(11) 3.54 3.545, 3.509
    3 3.811(4) 6.23 3.307, 3.370

    3.2 Thermal stabilities

    The thermal stabilities of 1~3 were studied at a heating rate of 5 ℃·min–1 in the temperature range from room temperature to 800 ℃ by using thermogravimetry. The TG curves of three compounds at the heating rate of 5 ℃·min–1 are plotted in Fig. 7 and DTG curves of 1~3 are shown in Fig. 8. It can be seen that the thermal decomposition processes of 1 and 3 are characterized by two stages, whereas 2 only undergoes one stage of decomposition. As seen from Figs. 7 and 8, the first decomposition stage of 1 at the temperature range of 72~129 ℃ with the mass loss of 7.3% might be the loss of -OH group on benzene ring (calcd. 6.6%). In the second stage, the weight loss is 43.47% at 250~399 ℃ resulting from the bond breaking of C(5)–N(4) (calcd. 42.7%). The first step for 3 loses 16.60% of its mass at 60~123 ℃, which is assigned to the loss of -OH and -OCH3 groups on benzene ring (calcd. 16.7%). The second stage from 194 to 348 ℃ occurs with the mass loss of 67.38% corresponding to the bond breaking of C(5)–N(4) and C(6)–C(7) (calcd. 68.6%). According to Figs. 7 and 8, 2 decomposes in one step from 194 to 313 ℃ with weight loss of 99.27%, indicating that compound 2 undergoes a complete decomposition. The temperature of the maximum thermal decomposition peaks for 1~3 is 284, 289 and 276 ℃, showing that three compounds all possess better thermal stabilities and 2 displays the most thermal stable one among them.

    Figure 7

    Figure 7.  TG curves of 1~3 (β = 5 ℃·min–1)
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    Figure 8

    Figure 8.  DTG curves of 1~3 (β = 5 ℃·min–1)
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    3.3 Interactions of 1~3 with CT-DNA studied by UV-Vis spectra

    The binding behaviors of small molecules with CT-DNA were often investigated by UV-Vis absorption spectra. Generally, the changes in absorption spectra are closely related to DNA-binding mode. The absorption spectra of 1~3 interacting with CT-DNA are given in Figs. 9~11, respectively. As an increasing amount of CT-DNA, the absorption spectra band of 1~3 at 369, 364 and 368 nm showed hypochromism about 9.0, 8.7 and 9.5% without wavelength shift. It indicated that the interaction modes of all the three compounds with CT-DNA are groove binding[32, 33].

    Figure 9

    Figure 9.  UV-Vis absorption of CT-DNA interacting with 1 (Inset: plot of [CT-DNA]/(εa–εf) against [CT-DNA])
    DownLoad: Full-Size Img PowerPoint

    Figure 10

    Figure 10.  UV-Vis absorption of CT-DNA interacting with 2 (Inset: plot of [CT-DNA]/(εa–εf) against [CT-DNA])
    DownLoad: Full-Size Img PowerPoint

    Figure 11

    Figure 11.  UV-Vis absorption of CT-DNA interacting with 3 (Inset: plot of [CT-DNA]/(εa – εf) against [CT-DNA])
    DownLoad: Full-Size Img PowerPoint

    To further investigate the binding affinities of compounds with the CT-DNA, the binding constants (Kb) of 1~3 with CT-DNA were determined according to the following equation (2)[34, 35]:

    $ \frac{[\mathrm{CT}-\mathrm{DNA}]}{\varepsilon_{\mathrm{a}}-\varepsilon_{\mathrm{f}}}=\frac{[\mathrm{CT}-\mathrm{DNA}]}{\varepsilon_{\mathrm{b}}-\varepsilon_{\mathrm{f}}}+\frac{1}{K_{\mathrm{b}}\left(\varepsilon_{\mathrm{b}}-\varepsilon_{\mathrm{f}}\right)} $

    		(2)

    where [CT-DNA] is the concentration of CT-DNA solution, εa the apparent extinction coefficient of the compound in the presence of CT-DNA, εb the molar extinction coefficient for the free compound, and εf the molar extinction coefficient for compound fully bound with CT-DNA. In plots of [CT-DNA]/(εa–εf) versus [CT-DNA] (as insets in Figs. 9~11),

    the binding constants (Kb) for 1~3 were calculated by the ratio of the slope to intercept and listed in Table 4. The calculated Kb values for 1~3 are 3.63 × 105, 8.32 × 106 and 7.95 × 108 M–1 following the order 3 > 2 > 1. And the value of Kb for 3 illustrated the interaction of 3 with CT-DNA is stronger than that of some pyrazine derivatives, such as 2, 6-bis-[4-(4, 5-dihydro-1H-imidazol-2-yl-methoxy)-phenyl]-pyrazine (Kb = 4.30 × 105 M–1)[36], 4-chlorobenzaldehyde-2-amino-3-pyrazine hydrazone (Kb = 9.02 × 107 M–1)[37] and pyrazine complexes, [Ni(C17H15N4O2)2]·2CH3OH (Kb = 1.01 × 107 M–1)[38] and [Cu(H2O(Gly)(PZTA)]·ClO4 (Kb = 3.91 × 103 M–1)[39].

    Table 4

    Table 4.  Binding Constants Kb, Fitting Equations and R2 of 1~3 Bound to CT-DNA
    DownLoad: CSV
    Compound Kb (M–1) Linear fitting equation R2
    1 3.63 × 105 CDNA/(εa– εf) = 8.51 × 10–5CDNA + 2.34 × 10–10 0.9985
    2 8.32 × 106 CDNA/(εa– εf) = 3.35 × 10–5CDNA + 4.03 × 10–12 0.9999
    3 7.95 × 108 CDNA/(εa– εf) = 6.41 × 10–5CDNA + 8.06 × 10–14 0.9999

    3.4 Interactions of 1~3 with BSA studied by fluorescence spectra

    Bovine serum albumins (BSA) were used in this experiment due to its low cost, wide availability and structural similarity to human serum albumins. Figs. 12~14 show the effects of 1~3 on the fluorescence intensities of BSA. It can be seen that BSA emits a strong fluorescence peak at 341.5 nm when the exciting wavelength is 280 nm. With continuous addition of compounds into BSA solution, the endogenous fluorescence of BSA was quenched. It indicated that 1~3 could bind to the BSA and result in microenvironment change around the tryptophan of BSA[40, 41].

    Figure 12

    Figure 12.  Fluorescence quenching curves of BSA in the absence and presence of 1 (Inset: Stern-Volmer plot: F0/F vs Q)
    DownLoad: Full-Size Img PowerPoint

    Figure 13

    Figure 13.  Fluorescence quenching curves of BSA in the absence and presence of 2 (Inset: Stern-Volmer plot: F0/F vs Q)
    DownLoad: Full-Size Img PowerPoint

    Figure 14

    Figure 14.  Fluorescence quenching curves of BSA in the absence and presence of 3 (Inset: Stern-Volmer plot: F0/F vs Q)
    DownLoad: Full-Size Img PowerPoint

    There are two kinds of fluorescence quenching: static quenching and dynamic quenching. To further clarify the quenching process, fluorescence quenching data were studied by the Stern-Volmer equation (3)[42]:

    $ F / F_0=1+K_{\mathrm{q}} \tau_0[Q]=1+K_{\mathrm{sv}}[Q]$

    		(3)

    where [Q] is the concentration of 1~3, F and F0 the fluorescence intensities of BSA with and without compounds, Kq the quenching rate constant, τ0 the average lifetime of BSA without the quencher, about 10–8 s–1 [43], Ksv the Stern-Volmer dynamic quenching constant which was determined by the linear fitting of the plots of F/F0 against [Q] (as insets in Figs. 12~14) and presented in Table 5. In Table 5, the values of the quenching rate constants Kq go beyond the limit of dynamic quenching rate constant (2.0 × 1010 M–1·s–1)[44]. Thus, the quenching processes of three compounds interacting with BSA are static quenching. The binding constants (KA) and the number of binding sites (n) in static quenching can be given in the following equation[45]:

    $ \lg \left[\left(F_{0}-F\right) / F\right]=\ln K_{\mathrm{A}}+n \operatorname{lr}[Q] $

    		(4)

    Table 5

    Table 5.  Stern-volume Quenching Constants Ksv, Quenching Rate Constants Kq, Linear Fitting Equations and R2 of 1~3 Bound to BSA
    DownLoad: CSV
    Compound Ksv (M–1) Kq (M–1·s–1) Linear fitting equation R2
    1 4.19 × 105 4.19 × 1013 F0/F = 4.19 × 105[Q] + 1.01 0.9948
    2 3.57 × 105 3.37 × 1013 F0/F = 3.37 × 105[Q] + 1.01 0.9926
    3 5.43 × 105 5.43 × 1013 F0/F = 5.43 × 105[Q] + 1.01 0.9965

    KA and n were determined from the intercepts and slope of lg[(F0 – F)/F] against lg[Q] (Fig. 15) and the data are listed in Table 6.

    Figure 15

    Figure 15.  Plots of lg[(F0–F)/F] vs lg[Q] of 1~3
    DownLoad: Full-Size Img PowerPoint

    Table 6

    Table 6.  Binding Constants KA, Number of Binding Sites n, Linear Fitting Equations and R2 of 1~3 Bound to BSA
    DownLoad: CSV
    Compound KA (M–1) n Linear fitting equation R2
    1 2.96 × 105 0.98 lg[(F0 – F)/F] = 0.98lg[Q] + 5.47 0.9972
    2 5.89 × 104 0.87 lg[(F0 – F)/F] = 0.87lg[Q] + 4.77 0.9984
    3 1.63 × 106 1.07 lg[(F0 – F)/F] = 1.07lg[Q] + 6.21 0.9862

    In Table 6, the values of KA follow the sequence 3 > 1 > 2, suggesting that 3 has the strongest protein-binding ability. Moreover, the binding sites' values are approximately equal to 1, so there exists a single binding site between BSA and each compound. Thus, the tryptophan residues involved in the interactions might be Trp213 or Trp134[46]. Overall, BSA can be considered as a good carrier to transport the entire three compounds in vivo.

    3.5 Molecular docking studies

    3.5.1   Molecular docking results of 1~3 with DNA

    The molecular docking technique was used to identify binding locations and mechanisms of 1~3 interacting with DNA. Docking studies have been performed on DNA with the sequence of d(ACCGGTACCGGT)2 in the presence of compounds 1~3. The best docking perspectives of three compounds and DNA are shown in Figs. 16~18, indicating the groove binding mode of DNA with 1~3. And 1 and 2 could bind within the major groove of DNA, but 3 binds in the minor groove of DNA. Four hydrogen bonds were formed between N(4)–H(4), O(2)–H(2), N(3)–H(3)A and N(3)–H(3)B groups of compound 1 with DC8, DC21, DT18 and DT19 nucleotides, respectively. And the N–H group in DA19 also interacted with O(1) to form a hydrogen bond. In compound 2, two N–H···O hydrogen bonds formed between 2 and DC21, DG22. N–H group of DG5 displayed hydrogen bonding interaction with N(1) in 2. There also exist hydrophobic interactions between 2 and DG4, DG5. In the case of 3, N–H group in DG10 and DG16 exhibited hydrogen bonding with N(5), O(1) separately. Additionally, N(3)–H(3)B group interacted with oxygen atom of DG17 to form a hydrogen bond. The lowest free energies of docking were obtained as –6.93, –6.98 and –7.29 kcal·mol–1 for 1~3, respectively, which indicated that compound 3 possesses the strongest binding affinity among 1~3. All the molecular docking results are consistent with the spectroscopy experimental results.

    Figure 16

    Figure 16.  (a) Molecular docking pose of 1 bound to the groove of DNA, (b) Detailed view of the bonding mode
    DownLoad: Full-Size Img PowerPoint

    Figure 17

    Figure 17.  (a) Molecular docking pose of 2 bound to the groove of DNA, (b) Detailed view of the bonding mode
    DownLoad: Full-Size Img PowerPoint

    Figure 18

    Figure 18.  (a) Molecular docking pose of 3 bound to the groove of DNA, (b) Detailed view of the bonding mode
    DownLoad: Full-Size Img PowerPoint
    3.5.2   Molecular docking results of 1~3 with BSA

    The best docking poses of 1~3 with BSA are shown in Figs. 19~21. As can be seen, 1 and 3 docked around Trp213 in subdomain IIA, whereas 2 docked around Trp134 in subdomain IB[47]. It also supports the fluorescence quenching of tryptophan in BSA in the presence of 1~3 which is obtained from the fluorescence spectroscopy experimental results (Section 3.4). In the case of compound 1, N(4)–H(4) and N(3)–H(3)B groups interacted oxygen atoms of ALA209, LEU346 to form two hydrogen bonds. In addition, 1 had hydrophobic contacts with TRP213, LEU197, ARG198, LEU210, ALA209, ALA212 and LEU346. N–H groups of ARG185 formed two hydrogen bonds with O(1) and N(5) atoms in 2. Also, hydrogen bond was observed between N(3)–H(3)A group of 2 and oxygen atom in PRO113. There are several hydrophobic contacts between 2 and TYR137, MET184, ARG185, VAL188, ILE141, ARG185, VAL186, LYS144, ARG144, TYR137 and TYR160. For compound 3, N–H group of ARG217 displayed three hydrogen bonds with O(3) and N(5). N–H group of VAL342 formed a hydrogen bond with O(1). N(3)–H(3)A group in 3 exhibited hydrogen bond with oxygen atom of PRO338. Further, three hydrophobic interactions were detected between 3 and PRO446, VAL342 and ALA341. The lowest free energies of docking are –6.25, –7.09 and –7.15 kcal·mol–1 for 1~3, respectively. Compound 3 showed the strongest ability to interact with BSA which is in good agreement with the experimental results.

    Figure 19

    Figure 19.  (a) Molecular docking pose of 1 bound to BSA, (b) Detailed view of the bonding mode
    DownLoad: Full-Size Img PowerPoint

    Figure 20

    Figure 20.  (a) Molecular docking pose of 2 bound to BSA, (b) Detailed view of the bonding mode
    DownLoad: Full-Size Img PowerPoint

    Figure 21

    Figure 21.  (a) Molecular docking pose of 3 bound to BSA, (b) Detailed view of the bonding mode
    DownLoad: Full-Size Img PowerPoint

    3.6 Antibacterial activity

    The antibacterial activities of 1~3 were screened against S. aureus, E. coli and S. typhimurium, and gentamycin sulfate was used as a control drug. The antibacterial activities of three compounds and gentamycin sulfate are listed in Table 7. As seen from Table 7, all three compounds show higher antibacterial properties against S. aureus than E. coli and S. typhimurium. The IC50 value of compound 3 against S. aureus (IC50 = 9.58 μM) is smaller than that of gentamycin sulfate (IC50 = 40.20 μM), exhibiting a noticeable antibacterial activity of 3 on S. aureus. The antibacterial activity of 2 on S. aureus (IC50 = 40.31 μM) is comparable to that of gentamycin sulfate (IC50 = 40.20 μM). And compound 3 has generally stronger antibacterial activity than 1 and 2 because 3 possesses excellent binding ability with CT-DNA/BSA. Furthermore, E. coli and S. typhimurium are more sensitive to gentamycin sulfate than 1~3.

    Table 7

    Table 7.  IC50 of 1~3 Treated with the Tested Bacteria
    DownLoad: CSV
    Compound IC50 (μM)
    S. aureus E. coli S. typhimurium
    1 83.78 216.45 161.12
    2 40.31 111.62 139.91
    3 9.58 80.97 105.57
    Gentamycin sulfate 56.96 15.96 2.85

    3.7 In vitro cytotoxicity

    The positive results received from the above DNA/BSA binding experiments of three compounds encourage us to investigate their cytotoxicities against human lung cancer cell line (A549) by MTT method. Etoposide was used as a positive control to evaluate the cytotoxicities of 1~3 under the same experimental conditions. With increasing the concentration of compounds, the inhibition rates are shown in Fig. 22. The IC50 values of 1~3 are 115.78, 191.04 and 84.52 μM, respectively. Compounds 1~3 showed anticancer activities against A549 in different degrees. Among three compounds, 3 exhibited best cytotoxic property against A549. But compared to etoposide (IC50 = 25.03 μM), 3 showed low anticancer effect.

    Figure 22

    Figure 22.  Inhibition effects of 1~3 on cell A549
    DownLoad: Full-Size Img PowerPoint

    4. CONCLUSION

    The single crystal structures of C12H11N5O2·CH3COOH (1), C13H13N5O·2CH3COOH (2) and C13H13N5O3·C13H13N5O3 (3) presented that 1 and 2 belong to monoclinic, space group P21/c and 3 belongs to monoclinic, space group C2/c. 1~3 have good thermal stability, and their temperature of maximum thermal decomposition peaks is all not less than 276 ℃. 1~3 can interact with CT-DNA by groove binding and bind with BSA in static mechanism. The binding mode between compounds and DNA/BSA was confirmed by molecular docking. 1~3 can inhibit the growth of S. aureus, E. coli and S. typhimurium. In particular, compound 3 showed higher antibacterial activity against S. aureus than the familiar antibiotic gentamycin sulfate. Cytotoxicity experiments suggested that 1~3 had lower cytotoxicity activities than etoposide and 3 exhibited best cytotoxicity property against A549 among three compounds with an IC50 value of 84.52 μM.


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      Mandal, G.; Bardhan, M.; Ganguly, T. Interaction of bovine serum albumin and albumin-gold nanoconjugates with l-aspartic acid. A spectroscopic approach. Colloids Surf. B 2010, 81, 178−184. doi: 10.1016/j.colsurfb.2010.07.002

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      Protas, A. V.; Popova, E. A.; Mikolaichuk, O. V.; Porozov, Y. B.; Mehtiev, A. R.; Ott, I.; Alekseev, G. V.; Kasyanenko, N. A.; Trifonov, R. E. Synthesis, DNA and BSA binding of Pd(II) and Pt(II) complexes featuring tetrazolylacetic acids and their esters. Inorg. Chim. Acta 2018, 473, 133−144. doi: 10.1016/j.ica.2017.12.040

  • Scheme 1  Synthetic routes of compounds 1~3

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    Figure 1  Crystal structure of 1 with 30% probability

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    Figure 2  Crystal structure of 2 with 30% probability

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    Figure 3  Crystal structure of 3 with 30% probability

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    Figure 4  View of the hydrogen bonds of 1

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    Figure 5  View of the hydrogen bonds of 2

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    Figure 6  View of the hydrogen bonds of 3

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    Figure 7  TG curves of 1~3 (β = 5 ℃·min–1)

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    Figure 8  DTG curves of 1~3 (β = 5 ℃·min–1)

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    Figure 9  UV-Vis absorption of CT-DNA interacting with 1 (Inset: plot of [CT-DNA]/(εa–εf) against [CT-DNA])

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    Figure 10  UV-Vis absorption of CT-DNA interacting with 2 (Inset: plot of [CT-DNA]/(εa–εf) against [CT-DNA])

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    Figure 11  UV-Vis absorption of CT-DNA interacting with 3 (Inset: plot of [CT-DNA]/(εa – εf) against [CT-DNA])

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    Figure 12  Fluorescence quenching curves of BSA in the absence and presence of 1 (Inset: Stern-Volmer plot: F0/F vs Q)

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    Figure 13  Fluorescence quenching curves of BSA in the absence and presence of 2 (Inset: Stern-Volmer plot: F0/F vs Q)

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    Figure 14  Fluorescence quenching curves of BSA in the absence and presence of 3 (Inset: Stern-Volmer plot: F0/F vs Q)

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    Figure 15  Plots of lg[(F0–F)/F] vs lg[Q] of 1~3

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    Figure 16  (a) Molecular docking pose of 1 bound to the groove of DNA, (b) Detailed view of the bonding mode

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    Figure 17  (a) Molecular docking pose of 2 bound to the groove of DNA, (b) Detailed view of the bonding mode

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    Figure 18  (a) Molecular docking pose of 3 bound to the groove of DNA, (b) Detailed view of the bonding mode

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    Figure 19  (a) Molecular docking pose of 1 bound to BSA, (b) Detailed view of the bonding mode

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    Figure 20  (a) Molecular docking pose of 2 bound to BSA, (b) Detailed view of the bonding mode

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    Figure 21  (a) Molecular docking pose of 3 bound to BSA, (b) Detailed view of the bonding mode

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    Figure 22  Inhibition effects of 1~3 on cell A549

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    Table 1.  Selected Bond Distances (Å) and Bond Angles (º) for 1~3

    Compound 1
    Bond Dist. Bond Dist. Bond Dist.
    O(1)–C(5) 1.236(2) N(5)–N(4) 1.379(19) N(3)–C(1) 1.330(2)
    O(2)–C(10) 1.359(2) N(4)–C(5) 1.338(2) N(2)–C(1) 1.349(2)
    N(5)–C(6) 1.277(2) C(5)–C(4) 1.482(2) C(4)–C(1) 1.421(2)
    Angle (°) Angle (°) Angle (°)
    N(5)–C(6)–C(7) 122.39(17) O(1)–C(5)–C(4) 122.73(17) C(4)–N(1)–C(3)–C(2) 1.0(3)
    C(6)–N(5)–N(4) 115.15(16) C(6)–N(5)–N(4)–C(5) 179.23(16) C(8)–C(7)–C(6)–N(5) 1.8(3)
    C(5)–N(4)–N(5) 120.43(15) C(10)–C(9)–C(8)–C(7) 0.7(3) C(12)–C(7)–C(6)–N(5) –178.50(16)
    N(4)–C(5)–C(4) 114.31(16) C(9)–C(10)–C(11)–C(12) 0.6(3) N(4)–C(5)–C(4)–C(1) –177.05(15)
    O(1)–C(5)–N(4) 122.96(17) C(1)–N(2)–C(2)–C(3) 0.3(3) N(4)–C(5)–C(4)–N(1) 2.1(2)
    Compound 2
    Bond Dist. Bond Dist. Bond Dist.
    O(1)–C(5) 1.227(4) N(4)–C(5) 1.343(5) N(3)–C(1) 1.316(4)
    N(5)–C(6) 1.283(5) C(10)–C(13) 1.515(6) N(2)–C(1) 1.346(5)
    N(5)–N(4) 1.384(4) C(5)–C(4) 1.488(6) C(4)–C(1) 1.321(4)
    Angle (°) Angle (°) Angle (°)
    N(5)–C(6)–C(7) 120.6(4) O(1)–C(5)–C(4) 122.5(5) C(4)–N(1)–C(3)–C(2) 0.6(8)
    C(6)–N(5)–N(4) 113.5(4) C(6)–N(5)–N(4)–C(5) 178.3(4) C(8)–C(7)–C(6)–N(5) –6.7(7)
    C(5)–N(4)–N(5) 118.8(3) C(10)–C(9)–C(8)–C(7) 0.6(8) C(12)–C(7)–C(6)–N(5) 174.4(4)
    N(4)–C(5)–C(4) 114.6(4) C(9)–C(10)–C(11)–C(12) 0.3(7) N(4)–C(5)–C(4)–C(1) –177.2(4)
    O(1)–C(5)–N(4) 122.8(4) C(1)–N(2)–C(2)–C(3) –0.6(7) N(4)–C(5)–C(4)–N(1) 2.5(5)
    Compound 3
    Bond Dist. Bond Dist. Bond Dist.
    O(1)–C(5) 1.218(4) N(5)–N(4) 1.367(4) N(3)–C(1) 1.346(5)
    O(2)–C(8) 1.358(5) N(4)–C(5) 1.351(5) N(2)–C(1) 1.345(5)
    N(5)–C(6) 1.263(5) C(5)–C(4) 1.497(5) C(4)–C(1) 1.409(6)
    Angle (°) Angle (°) Angle (°)
    N(5)–C(6)–C(7) 120.8(3) O(1)–C(5)–C(4) 123.3(4) C(4)–N(1)–C(3)–C(2) –0.4(6)
    C(6)–N(5)–N(4) 119.5(3) C(6)–N(5)–N(4)–C(5) 177.8(4) C(8)–C(7)–C(6)–N(5) –2.3(6)
    C(5)–N(4)–N(5) 118.4(3) C(10)–C(9)–C(8)–C(7) –0.1(6) C(12)–C(7)–C(6)–N(5) 176.4(4)
    N(4)–C(5)–C(4) 113.9(3) C(9)–C(10)–C(11)–C(12) 1.2(7) N(4)–C(5)–C(4)–C(1) 178.3(3)
    O(1)–C(5)–N(4) 122.7(4) C(1)–N(2)–C(2)–C(3) 0.8(7) N(4)–C(5)–C(4)–N(1) –1.5(5)
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    Table 2.  Hydrogen Bond Distances (Å) and Angles (°) of 1~3

    Compound D–H···A d(D–H)/Å d(H···A)/Å d(D···A)/Å ∠DHA (º)
    1 N(3)–H(3)A···O(3)a 0.860 2.191 3.044 170.99
    N(3)–H(3)B···O(1) 0.860 2.075 2.713 130.51
    O(2)–H(2)···O(1)b 0.820 2.080 2.799 146.20
    O(4)–H(4)A···N(2)c 0.820 1.916 2.721 166.73
    2 N(3)–H(3)A···O(4)a 0.860 2.123 2.970 168.24
    N(3)–H(3)B···O(1) 0.860 2.104 2.732 129.43
    N(4)–H(4)···O(2)b 0.860 2.312 3.092 150.79
    O(3)–H(3)···O(1)c 0.820 1.899 2.719 177.42
    O(5)–H(5)···N(2)d 0.820 1.948 2.738 161.24
    3 N(4)–H(4)···N(7)a 0.860 2.448 3.279 162.42
    O(2)–H(2)···N(5) 0.820 1.877 2.595 145.59
    O(5)–H(5)···N(10) 0.820 1.959 2.674 145.31
    N(8)–H(8)A···O(2)b 0.860 2.332 3.152 159.69
    N(8)–H(8)B···O(4) 0.860 2.091 2.738 131.49
    N(3)–H(3)A···O(5)c 0.860 2.431 3.069 131.55
    N(3)–H(3)A···O(6)d 0.860 2.474 3.156 136.82
    N(3)–H(3)B···O(1) 0.860 2.112 2.746 130.08
    Symmetry codes: for 1 (a) –x + 1/2, y + 1/2, –z + 1/2; (b) –x + 1/2, y – 1/2, –z + 3/2; (c) –x + 1/2, y – 1/2, –z + 1/2; for 2 (a) x + 1, y – 1, z; (b) –x + 1, y – 1/2, –z + 1/2; (c) x, –y + 1/2, z – 1/2; (d) x – 1, y + 1, z; for 3 (a) x – 1, y, z – 1; (b) –x + 3/2, –y + 3/2, –z + 1; (c) –x + 1, –y + 2, –z + 1; (d) –x + 1, –y + 2, –z + 1
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    Table 3.  Selected π-π Stacking Interactions of 2 and 3

    Compound Parameters of π-π stacking
    Dentroid-centroid separation (Å) Dihedral angle (°) Vertical displacements between ring centroids (Å)
    2 3.753(11) 3.54 3.545, 3.509
    3 3.811(4) 6.23 3.307, 3.370
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    Table 4.  Binding Constants Kb, Fitting Equations and R2 of 1~3 Bound to CT-DNA

    Compound Kb (M–1) Linear fitting equation R2
    1 3.63 × 105 CDNA/(εa– εf) = 8.51 × 10–5CDNA + 2.34 × 10–10 0.9985
    2 8.32 × 106 CDNA/(εa– εf) = 3.35 × 10–5CDNA + 4.03 × 10–12 0.9999
    3 7.95 × 108 CDNA/(εa– εf) = 6.41 × 10–5CDNA + 8.06 × 10–14 0.9999
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    Table 5.  Stern-volume Quenching Constants Ksv, Quenching Rate Constants Kq, Linear Fitting Equations and R2 of 1~3 Bound to BSA

    Compound Ksv (M–1) Kq (M–1·s–1) Linear fitting equation R2
    1 4.19 × 105 4.19 × 1013 F0/F = 4.19 × 105[Q] + 1.01 0.9948
    2 3.57 × 105 3.37 × 1013 F0/F = 3.37 × 105[Q] + 1.01 0.9926
    3 5.43 × 105 5.43 × 1013 F0/F = 5.43 × 105[Q] + 1.01 0.9965
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    Table 6.  Binding Constants KA, Number of Binding Sites n, Linear Fitting Equations and R2 of 1~3 Bound to BSA

    Compound KA (M–1) n Linear fitting equation R2
    1 2.96 × 105 0.98 lg[(F0 – F)/F] = 0.98lg[Q] + 5.47 0.9972
    2 5.89 × 104 0.87 lg[(F0 – F)/F] = 0.87lg[Q] + 4.77 0.9984
    3 1.63 × 106 1.07 lg[(F0 – F)/F] = 1.07lg[Q] + 6.21 0.9862
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    Table 7.  IC50 of 1~3 Treated with the Tested Bacteria

    Compound IC50 (μM)
    S. aureus E. coli S. typhimurium
    1 83.78 216.45 161.12
    2 40.31 111.62 139.91
    3 9.58 80.97 105.57
    Gentamycin sulfate 56.96 15.96 2.85
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  • 发布日期:  2020-09-01
  • 收稿日期:  2020-02-19
  • 接受日期:  2020-06-19
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