English

• 1. INTRODUCTION

  N-Acylhydrazone scaffold (-CONHN=) was brought into sharp focus for decades in many research fields owing to their particular physical, chemical, and biological properties. Recently, many compounds containing this moiety have been reported to possess a highly potential activity, such as antiviral^[1], antimicrobial^[2], antitumor^[3] and antioxidant^[4] activities, etc. Some of them have already been approved as pharmaceuticals (e.g., nifuroxazide, iproniazide, isocarboxazide, and desferrioxamine), and some others are under clinical trials^[5]. Thus, acylhydrazone and its derivatives are promising in disease treatment and prevention.

  Pyridine, as one of the active sites of some anti-diabetic drugs (e.g., pioglitazone, rosiglitazone), plays an essential role in pharmacological action^[6]. A survey of the literatures reveals that pyridine deriva-tives also exhibit an extensive range of biological activities like antithyroid^[7], antiviral, anti-HⅣ^[8], anti-inflammatory, analgesic^[9] and antimicrobial^[10] activities, etc.

  Moreover, halogenation of organic compound has been widely used in the processes of hit-to-lead or lead-to-drug conversions, and many drugs and drug candidates in clinical development contain halo-gen^[11]. The biological activity of a compound can be effectively enhanced by halogenation, e. g., Byeon et al. evaluated the Aβ fibrillation inhibition activities of 34 bisstyryl derivatives, and found that the IC₅₀ values increased in the order of halogen < Me < OMe < NMe₂^[12]. According to the research of Fais et al., simultaneous introduction of halogen at position 7 of the benzofuran scaffold could bring an enhancement of inhibitory activity against the butyrylcholinesterase enzyme^[13]. Zhang et al. reported a series of new thiothiazole benzoyl-hydrazone derivatives, and the structure-activity relationship analysis showed that the presence of halogen at the para position on phenyl effectively improved the antibacterial activity^[14].

  It is generally accepted that the aggregation of different active groups in a molecule would result in an enhancement of its biological activity. Thus, a combination of suitable active groups was considered as a practical approach to construct biological active compounds. Along this line, the expectation of obtaining potential bioactive compounds prompted us to introduce pyridine ring and halogen atoms into the design and synthesis of acylhydrazones.

  To gain insight into biological activity, the interac-tions between bioactive compounds and biomacro-molecules (e.g., DNA, protein) have attracted growing attention^{[15, 16]}. Study of the interaction of compound with DNA is of great importance since many drugs target DNA in vivo. The binding of drug to DNA can affect its transcription, replication, expression of genetic information in cells, and thereby influence its physiological function^[17]. Serum albumin (SA) is the most abundant protein in plasma as well as the key point in the disposition and transportation of various exogenous and endogenous compounds. The interac-tion between SA and bioactive compound plays a critical role in its absorption, distribution, and metabolism^[18].

  Herein, we have recently designed and synthesized three halogenated pyridyl hydrazones 3a, 3b, and 3c, which share an identical scaffold but have different substituents, Cl, Br and I, at the 4-position of the phenyl ring (Scheme 1). They were characterized by elemental analysis, IR, ¹H NMR, and single-crystal X-ray diffraction. Thermogravimetric analysis was used to evaluate the thermal stabilities of them. In addition, the binding affinities with ct-DNA/BSA of 3a~3c were studied and compared by experimental and theoretical methods including UV-vis, fluore-scence spectroscopy and molecule docking. Spectral experiments were used to prove that 3a~3c could effectively bind to ct-DNA/BSA and obtain the structure-activity relationship. Molecular docking was carried out to support the experimental results, observe the binding sites, and understand the binding properties as well as the role of halogens. The antibacterial activities of 3a~3c were studied by cylinder plate method in vitro.

  Scheme 1

  

  Scheme 1.  Synthesis route of 3a~3c

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  2. EXPERIMENTAL

  2.1 Reagents and apparatus

  Unless otherwise noted, all solvents and starting chemicals were of commercially analytical grade and used without further purification. The ct-DNA was purchased from Sigma company, and BSA from J&K Chemicals.

  The melting points were determined on an XT4-100B microscopic melting point apparatus. Elemental analyses for carbon, hydrogen, and nitrogen were carried out on a Perkin-Elmer 2400-Ⅱ analyzer. ¹H NMR spectra were recorded with Bruker Avance Ⅱ 400 MHz spectrometer. The single-crystal X-ray diffraction measurements were performed on a Bruker Smart Apex Ⅱ CCD diffrac-tometer. Thermal data were collected on a Mettler Toledo TG-DSC1 HT instrument. The UV-vis spectra were recorded on a TU-1900 spectrophoto-meter. Fluorescence spectra were determined on a Perkin Elmer LS55 spectrophotometer.

  2.2 Syntheses

  2.2.1   Synthesis of 4-chloropicolinohydrazide (2)

  Hydrazine hydrate (8.0 mL, 80%) was dropwise added to a 0 ℃ cooled solution of methyl 4-chloropicolinate (1, 30.0 mmol) in ethanol (30.0 mL). The resulting mixture was stirred for 2 h at room temperature before filtration. Then, the separated solid was washed with water and ethanol, dried and recrystallized with ethanol to get pure white crystalline solid. Yield: 87%. m.p.: 170.11~170.81 ℃. Anal. Calcd. (%) for C₆H₆ClN₃O: C, 42.00; N, 24.49; H, 3.52. Found (%): C, 42.32; N, 23.03; H, 3.82.

  2.2.2   Synthesis of 3a~3c

  Synthesis of 4-chlorobenzaldehyde-4-chloropy-ridine-2-formyl acylhydrazone (3a): 4-chloropico-lino-hydrazide (2, 0.4 mmol) and methanol (10.0 mL) were stirred together in a flask for 30 min at 65 ℃ before a solution of p-chlorobenzaldehyde (0.4 mmol) in glacial acetic acid (5.0 mL) was added slowly. The resulting mixture was stirred for 4 h at 65 ℃ to react completely before filtration, and concentrated under reduced pressure. The residue was purified by recrystallization with ethanol to get white rod crystals of 3a suitable for single-crystal X-ray analysis. Yield: 92%. m.p.: 227.84~228.74 ℃. Anal. Calcd. (%) for C₁₃H₉Cl₂N₃O: C, 53.17; N, 13.40; H, 3.31. Found (%): C, 53.09; N, 14.29; H, 3.08. IR (KBr, cm^-1) ν: 3301 (N–H), 1686 (C=O), 1609 (C=N), 753 (C–Cl). ¹H NMR (400 MHz, DMSO-d₆, 298 K): δ (ppm) :12.31 (s, 1H, N–H), 8.70 (s, 1H, 6-pyridine-H), 8.65 (s, 1H, 3-pyridine-H), 8.13 (s, 1H, 5-pyridine-H), 7.84 (s, 1H, N=C-H), 7.74 (s, 2H, p-Ar),7.53 (s, 2H, o-Ar).

  Synthesis of 4-bromobenzaldehyde-4-chloropyri-dine-2-formyl acylhydrazone (3b): The 3b as white rod crystals suitable for single-crystal X-ray analysis was obtained by the same procedure as 3a except using p-bromobenzaldehyde instead of p-chloroben-zaldehyde. Yield: 88%. m.p.: 247.23~247.78 ℃. Anal. Calcd. (%) for C₁₃H₉BrClN₃O: C, 46.12; N, 12.41; H, 2.68. Found (%): C, 46.38; N, 11.71; H, 2.94. IR (KBr, cm^-1) ν: 3301 (N–H), 1693 (C=O), 1609 (C=N), 597 (C–Br). ¹H NMR (400 MHz, DMSO-d₆, 298 K): δ (ppm): 12.32 (s, 1H, N–H), 8.70 (s, 1H, 6-pyridine-H), 8.63 (s, 1H, 3-pyridine-H), 8.13 (s, 1H, 5-pyridine-H), 7.84 (s, 1H, N=C-H), 7.67 (s, 4H, Ar).

  Synthesis of 4-iodobenzaldehyde-4-chloropyri-dine-2-formyl acylhydrazone (3c): The 3c as white rod crystals suitable for single-crystal X-ray analysis was obtained by the same procedure as 3a except using p-iodobenzaldehyde instead of p-chloro-benzaldehyde. Yield: 85%. m.p.: 259.10~259.90 ℃. Anal. Calcd. (%) for C₁₃H₉ClIN₃O: C, 40.49; N, 10.90; H, 2.35. Found (%): C, 40.60; N, 11.43; H, 2.45. IR (KBr, cm^-1) ν: 3302 (N–H), 1690 (C=O), 1606 (C=N), 519 (C–I). ¹H NMR (400 MHz, DMSO-d₆, 298 K): δ (ppm): 12.31 (s, 1H, N–H), 8.70 (s, 1H, 6-pyridine-H), 8.60 (s, 1H, 3-pyridine-H), 8.13 (s, 1H, 5-pyridine-H), 7.84 (s, 1H, N=C-H), 7.84 (s, 2H, p-Ar), 7.51 (s, 2H, o-Ar).

  2.3 X-ray structure determinations

  The single-crystal X-ray diffraction was performed on a Bruker Smart Apex Ⅱ CCD diffractometer. Intensities of reflections were measured using MoKα monochromatized radiation (λ = 0.71073 Å) at 293(2) K. Data reduction and cell refinement were performed by SMART and SAINT programs^[19], and semiempirical absorption correction was applied to the intensity data using the SADABS program^[20]. The structures were solved by direct methods and refined by full-matrix least-squares methods on F² using the SHELXS-97 and SHELXL-97 programs^{[21, 22]}. The non-H atoms in the structure were treated as anisotropic. Hydrogen atoms were located geome-trically and refined in a riding mode.

  The crystal data and refinement parameters of 3a~3c are summarized in Table 1.

  Table 1

  

  Table 1.  Crystallographic Data for 3a~3c

  DownLoad: CSV

  Compound	3a	3b	3c
  CCDC No.	1548090	1545302	1584146
  T/K	296(2)	296(2)	296(2)
  Crystal size (mm)	0.36 × 0.30 × 0.21	0.34 × 0.27 × 0.14	0.31 × 0.25 × 0.13
  Molecular formula	C13H9Cl2N3O	C13H9BrClN3O	C13H9ClIN3O
  Formula weight	294.13	338.59	385.58
  Crystal system	Monoclinic	Monoclinic	Monoclinic
  Space group	Cc	Cc	Cc
  a (Å)	12.316(4)	12.345(3)	12.323(18)
  b (Å)	13.253(4)	13.362(4)	13.567(18)
  c (Å)	8.130(2)	8.147(2)	8.293(12)
  α (°)	90.00	90.00	90.00
  β (°)	93.842(5)	93.814(5)	93.956(3)
  γ (°)	90.00	90.00	90.00
  V (Å3)	1324.0(7)	1341.0(6)	1383.1(3)
  Z	4	4	4
  μ (mm–1)	0.484	3.259	2.503
  h/k/l (max, min)	–14, 14/–15, 10/–9, 9	–14, 13/–15, 13/–8, 9	–13, 14/–16, 12/–9, 9
  Dc (mg·m–3)	1.476	1.677	1.852
  F(000)	600	672	744
  θ range	2.26~25.09	2.25~25.10	2.24~25.09
  Reflections collected/independent/Rint	3181/2163/0.0217	3280/1839/0.0325	3374/2213/0.0207
  Data/restraints/parameters	2163/2/172	1839/2/172	2213/2/173
  Completeness (%)	99.9	99.8	100
  Final R, wR indices (I > 2σ(I))	0.0378/0.0817	0.0366/0.0727	0.0282/0.0727
  R, wR indices (all data)	0.0444/0.0857	0.0490/0.0777	0.0293/0.0736
  Goodness of fit on F2	1.046	1.026	1.062
  Largest diff. peak/hole (e·Å–3)	0.183/–0.201	0.532/–0.358	0.759/–0.590

  2.4 Thermal stability experiments

  Thermal gravimetric analyses were investigated under nitrogen at heating rates of 5.00, 10.00, and 15.00 ℃·min⁻¹ from room temperature to 800 ℃.

  2.5 ct-DNA binding experiments

  2.5.1   UV-vis absorption titration

  DNA stock solution was prepared by dilution of ct-DNA to buffer (0.01 mol·L^-1 tris-HCl, pH = 7.9, tris = tris(hydroxymethyl)aminomethane), followed by exhaustive stirring at 4 ℃ for 2 h and kept at 4 ℃ for no longer than a week. The stock solution of ct-DNA gave a ratio of UV absorbance at 260 and 280 nm (A260/A280) of 1.87, indicating that ct-DNA was sufficiently free of protein contamination^[23]. UV-vis absorption titrations were carried out by the stepwise addition of ct-DNA solutions following a certain ratio to a cuvette containing a fixed con-centration of the acylhydrazones (1×10^-5 mol·L^-1), and spectra were recorded in the 200~600 nm range at room temperature.

  2.5.2   Ethidium bromide (EB) experiment

  EB displacement experiments were performed by monitoring changes in the fluorescence intensities after aliquot addition of the acylhydrazones to an aqueous solution of the EB-DNA system ([DNA] = [EB] = 2×10^-5 mol·L^-1). Before measurements, the mixture solutions of EB-DNA were shaken up and incubated at room temperature for 2 h. The fluore-scence spectra were recorded with excitation at 500 nm and emission at 450~750 nm, and the EB concentration was kept constant while the acylhydrazone concentration was changed from 0 to 2×10^-5 mol·L^-1.

  2.5.3   Molecular docking calculation

  The docking experiments were performed using the Lamarckian genetic algorithm (LGA) of AutoDock 4.2^[24]. The DNA structure (PDB ID: 425D) was retrieved from RCSB Protein Data Bank as the docking model. Hydrogen atoms and Gasteiger charges were added to DNA by AutoDock Tools. The single bonds of acylhydrazones were set to be rotatable. Grid maps of 100×70×70 Å grid points and 0.375 Å grid spacing were generated using AutoGrid. AutoDock was run with the following LGA parameters: GA population size, 150; maximum number of energy evaluations, 2.5×10⁶; numbers of generations, 2.7×10³. A total of 100 runs were performed. The minimum energy conformers were picked out based on their rank and score. Discovery Studio 4.5 Client^[25] was used to visualize docking poses and ligand-receptor interactions.

  2.6 BSA binding experiments

  2.6.1   UV-vis absorption assay

  Tris-NaCl-HCl buffer solution (0.01 mol·L^-1, pH = 7.2, tris = tris-(hydroxymethyl)aminomethane) was used in all BSA-binding experiments, BSA stock solution was prepared by dilution of BSA to buffer, and stored at 4 ℃ for no longer than a week. The concentrations of BSA and the acylhydrazones were all 5×10^-6 mol·L^-1 and their mixture solutions were shaken up and incubated at room temperature for 2 h before measurements. Absorption spectra were recorded in the 200~600 nm range at room temperature.

  2.6.2   Tryptophan quenching experiment

  Tryptophan quenching experiments were done by keeping the concentration of BSA constant (1×10^-6 mol·L^-1) while varying the acylhydrazones concen-tration from 0 to 1×10^-6 mol·L^-1 at 25 ℃. The fluorescence spectra were recorded with excitation at 295 nm and emission at 300~540 nm.

  2.6.3   Molecular docking calculation

  The available crystal structure of BSA (PDB ID: 4F5S) was retrieved from RCSB Protein Data Bank as the docking model. By setting the x, y and z dimensions of the grid box to 126, 102 and 126 with a grid spacing of 0.375 Å to ensure the binding sites cover the main cavities of BSA after assigning the ligands and receptors with Gasteiger charges. The procedure and other parameters were the same as the above DNA docking process.

  2.7 Determination of antibacterial activities

  The antibacterial activities of the 3a~3c against four pathogens, namely E. coli, S. aureus, B. subtilis, and P. aeruginosa, were determined by the cylinder plate method. The initial concentrations of 3a~3c were 500 μg·mL^-1, and the minimum inhibitory concentrations were determined by the two-fold dilution method. Carrier plates with bacteria were prepared from agar medium as the lower layer, and the nutrient agar medium mixed with the bacteria as the upper layer. The Oxford cups (7.8 mm) were placed vertically on the surface of the medium, 200 μL of the sample was added to each cup before culturing at 37 ℃ for 16~18 h and the average diameters of the zones of inhibition were measured. The positive control in the experiment was gentami-cin sulfate, and the negative control was 20% DMF (V_DMF:V_water = 1:4).

  3. RESULTS AND DISCUSSION

  3.1 Description of molecular structures

  3a~3c crystallize in the monoclinic Cc space group. Molecular plots with atom-labeling schemes and crystal packing diagrams of 3a~3c are presented in Figs. 1 and 2, respectively. Selected intramolecu-lar bond lengths and bond angles for 3a~3c are listed in Table 2. The parameters of the hydrogen bonds and π-π stacking interactions are summarized in Tables 3 and 4, respectively.

  Figure 1

  

  Figure 1.  ORTEP views of 3a~3c. Non-H atoms, represented as displacement ellipsoids, are plotted at the 30% probability level, while H atoms are shown as small spheres of arbitrary radius

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  Figure 2

  

  Figure 2.  Crystal packing diagrams of 3a~3c. Dotted lines represent intermolecular hydrogen bonds and π-π stacking

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  Table 2

  

  Table 2.  Selected Bond Lengths (Å) and Bond Angles (°) for 3a~3c

  DownLoad: CSV

  3a
  Bond	Dist.		Bond	Dist.		Bond	Dist.
  O(1)–C(6)	1.219(3)		N(2)–N(3)	1.383(3)		Cl(1)–C(3)	1.738(3)
  N(2)–C(6)	1.351(4)		N(3)–C(7)	1.278(4)		Cl(2)–C(11)	1.752(3)
  Angle	(°)		Angle	(°)		Angle	(°)
  O(1)–C(6)–N(2)	124.0(3)		C(7)–N(3)–N(2)	114.1(3)		C(6)–N(2)–N(3)–C(7)	178.4(3)
  N(2)–C(6)–C(5)	113.2(2)		N(1)–C(5)–C(6)	117.5(3)		N(3)–N(2)–C(6)–C(5)	179.8(3)
  C(6)–N(2)–N(3)	121.4(3)		C(13)–C(8)–C(7)	119.3(3)		N(2)–N(3)–C(7)–C(8)	178.8(3)
  3b
  Bond	Dist.		Bond	Dist.		Bond	Dist.
  O(1)–C(6)	1.225(6)		N(2)–N(3)	1.382(6)		Cl(1)–C(3)	1.748(6)
  N(2)–C(6)	1.334(6)		N(3)–C(7)	1.270(7)		Br(1)–C(11)	1.898(5)
  Angle	(°)		Angle	(°)		Angle	(°)
  O(1)–C(6)–N(2)	125.3(5)		C(7)–N(3)–N(2)	114.4(5)		C(6)–N(2)–N(3)–C(7)	–179.7(5)
  N(2)–C(6)–C(5)	113.8(4)		N(1)–C(5)–C(6)	117.1(4)		N(3)–N(2)–C(6)–C(5)	–178.4(4)
  C(6)–N(2)–N(3)	122.1(4)		C(13)–C(8)–C(7)	119.0(4)		N(2)–N(3)–C(7)–C(8)	–179.0(4)
  3c
  Bond	Dist.		Bond	Dist.		Bond	Dist'
  O(1)–C(6)	1.213(6)		N(2)–N(3)	1.380(6)		Cl(1)–C(3)	1.745(6)
  N(2)–C(6)	1.352(7)		N(3)–C(7)	1.276(7)		I(1)–C(11)	2.088(5)
  Angle	(°)		Angle	(°)		Angle	(°)
  O(1)–C(6)–N(2)	124.8(5)		C(7)–N(3)–N(2)	114.8(4)		C(6)–N(2)–N(3)–C(7)	–177.9(5)
  N(2)–C(6)–C(5)	113.1(4)		N(1)–C(5)–C(6)	117.2(4)		N(3)–N(2)–C(6)–C(5)	176.4(4)
  C(6)–N(2)–N(3)	121.8(4)		C(13)–C(8)–C(7)	118.9(4		N(2)–N(3)–C(7)–C(8)	178.7(4)

  Table 3

  

  Table 3.  Hydrogen Bond Lengths (Å) and Bond Angles (º) for 3a~3c

  DownLoad: CSV

  Compound	D–H···A	d(D–H)/Å		d(H···A)/Å	d(D···A)/Å	∠DHA/º
  3a	N(2)–H(2)···Cl(2)a	0.86		2.97	3.759(3)	154
  3b	N(2)–H(2)···Br(1)a	0.86		3.01	3.786(5)	151
  3c	N(2)–H(2)···I(1)a	0.86		3.13	3.866(5)	146
  Symmetry codes: for 3a (a) x–1/2, –y+1/2, z+1/2; for 3b (a) x–1/2, –y+3/2, z+1/2; for 3c (a) x+1/2, –y+3/2, z–1/2

  Table 4

  

  Table 4.  Parameters of π-π Stacking Interactions of 3a~3c

  DownLoad: CSV

  Compound	π-π interaction	Centroid-centroid separation (Å)	Dihedral angle (°)	Horizontal displacements between ring centroids (Å)	Vertical displacements between ring centroids (Å)
  3a	i	3.702(10)	4.788	1.543, 1.282	3.365, 3.473
  	ii	3.613(8)	5.664	0.612, 0.356	3.561, 3.595
  3b	i	3.702(8)	4.519	1.282, 1.542	3.473, 3.365
  	ii	3.614(6)	4.666	0.932, 0.674	3.492, 3.550
  3c	i	3.702(4)	3.469	1.249, 1.453	3.485, 3.405
  	ii	3.619(4)	3.452	0.876, 0.727	3.511, 3.545

  As shown by X-ray data, acylhydrazone molecules present in the keto tautomeric form, in which C(7)=N(3) double bonds are trans-configuration and C(6)(=O(1))–N(2)–N(3) moiety is cis-configura-tion^[5]. In the case of 3a, the bond lengths of O(1)–C(6) and N(3)–C(7) are 1.219(3) and 1.278(4) Å respectively, which are both the typical double bonds^[26]. The N(2)–N(3) bond distance is 1.383(3) Å, which is slightly shorter than the single bond distance (1.411(7) Å), indicating some double bond characters^[27]. Besides, the bond length of N(2)–C(6) is 1.351(4) Å, which is between carbon-nitrogen single bond and carbon-nitrogen double bond, suggesting a conjugation among O(1)–C(6)–N(2). The C(5)–C(6)–N(2)–N(3)–C(7)–C(8) unit between the two rings is almost completely planar with all-trans extended chain conformations, as evident by the relevant torsion angles (C(6)–N(2)–N(3)–C(7) 178.4(3)°, N(3)–N(2)–C(6)–C(5) 179.8(3)°, N(2)–N(3)–C(7)–C(8) 178.8(3)°) close to 180°. Besides, the dihedral angle between the benzene and pyridine rings in the molecule is 5.72°, which indicates that 3a has great molecular coplanarity. The molecular structures of 3b and 3c are analogous to that of 3a, and the numbering marks of the same atom are identical. Intermolecular interactions of 3a~3c, as shown in Fig. 2, include intermolecular hydrogen bonds N–H···X (X=Cl, Br, I) and π-π stacking. The units of 3a~3c were linked by intermolecular hydrogen bonds into a 2D layered structure, which was further extended into a 3D network by a series of π-π stacking interactions.

  3.2 Thermal analysis

  3a~3c were analyzed by thermogravimetric technology (TG-DTG) to explore their thermal stabilities. The thermal decomposition curves for 3a~3c at the heating rate of 5.00 ℃·min^-1 are given in Fig. 3. The thermal decomposition processes of 3a~3c involve only one step, and the weight loss percentages are all close to 100%, indicating that 3a~3c are almost completely decomposed. Moreover, the decomposition processes did not occur below 200 ℃, suggesting that they have high thermal stabilities. To compare the relative thermal stabilities of 3a~3c quantitatively, the apparent activation energies E_a were calculated by the Kissinger and Ozawa equation^[28] :

  $ \ln \left(\frac{\beta}{T_{\mathrm{p}}^2}\right)=\ln \left(\frac{A R}{E_{\mathrm{a}}}\right)-\frac{E_{\mathrm{a}}}{R T_{\mathrm{p}}}\ \ \ \ \ \ {\text{ (Kisinger)}} $

  (1)

  $ \lg \beta=\lg \left[\frac{A E_{\mathrm{a}}}{R G(\alpha)}\right]-2.315-0.4567 \frac{E_{\mathrm{a}}}{R T_{\mathrm{p}}}\ \ \ \ \ \ {\text{ (Ozawa) }} $

  (2)

  Figure 3

  

  Figure 3.  TG-DTG curves of 3a~3c at the heating rate of 5 ℃·min^-1

  DownLoad: Full-Size Img PowerPoint

  where E_a is the apparent activation energy, G(α) the integral mechanism constant, T_p, A, R and β correspond to the temperature of the decomposition peak, the pre-exponential factor, the gas molar constant and the heating rate, respectively. For Kissinger equation, a plot of ln(β/T_p²) versus 1/T_p gives a slope and the intercept equals to E_a/R and ln(AR/E_a), respectively. For Ozawa equation, a plot of lgβ versus 1/T_p gives a slope and the intercept equals to 0.4567E_a/R and lg(AE_a/RG(α)), respectively. The apparent activation energies E_a of thermal decomposition processes of 3a~3c were calculated from the slope and the intercept and listed in Table 5. The greater the apparent activation energy, the higher the thermal stability. The order of thermal stability is 3c >3b > 3a.

  Table 5

  

  Table 5.  Kinetic Parameters of Thermal Decomposition for 3a~3c at Three Heating Rates

  DownLoad: CSV

  Compound	β (℃·min-1)	Tp/℃	Kissinger			Ozawa
  			Ea (kJ·mol-1)	lgA		Ea (kJ·mol-1)
  3a	5	280.74	74.09	4.29		79.59
  	10	300.19
  	15	315.49
  3b	5	287.23	76.69	4.639		81.95
  	10	307.23
  	15	323.95
  3c	5	294.44	85.95	5.32		90.94
  	10	317.93
  	15	325.46

  3.3 ct-DNA binding studies

  3.3.1   UV-vis absorption titration analysis

  There are two modes between active compound interaction with DNA: non-covalent and covalent. The great majority of active compounds binds to DNA in a weak non-covalent interaction, including electrostatic binding, minor/major groove binding, and intercalative binding^[29]. Absorption titration was performed to find out the bind modes of 3a~3c with ct-DNA via monitoring the changes in absorbance of 3a~3c with increasing the concentration of ct-DNA. In general, intercalation/groove binding between active compounds and DNA causes hypochromism with or without red/blue shift while electrostatic binding results in hyperchromism^[30]. The spectral variations of 3a~3c in the presence of ct-DNA with increasing concentrations are shown in Fig. 4. With the increase of ct-DNA concentration, the absorption intensities of 3a~3c decreased significantly (hypochromism), but there were no noticeable shifts, implying 3a~3c can bind to ct-DNA effectively. After the interaction with ct-DNA, the π* orbital of the small molecule could couple with a π orbital of base pairs, resulting in the decrease of π-π* transition energy levels^[31]. Thus, the above observations preliminarily indicate that 3a~3c preferentially bind to ct-DNA via non-covalent groove binding mode. Since it is well established that the unremarkable (or no) shift in the absorption spectrum is most likely observed in the groove binding, instead, the remarkable shift is in intercalation^[32].

  Figure 4

  

  Figure 4.  Normalized absorption spectra of (i) 3a, (ii) 3b and (iii) 3c in the absence and presence of DNA. Inset: plot of [DNA] against [DNA]/(ε_a – ε_f)

  DownLoad: Full-Size Img PowerPoint

  To quantitatively evaluate the affinities of the 3a~3c binding to ct-DNA, the equilibrium binding parameters K_b were determined using the Benesi-Hildebrand equation^[33]:

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

  (3)

  where [DNA] is the concentration of ct-DNA in base pairs, ε_a the apparent extinction coefficient for the acylhydrazone in the presence of ct-DNA, ε_f the extinction coefficient of the free acylhydrazone, and ε_b the extinction coefficient of the acylhydrazone that fully binds to ct-DNA. The ratios of slopes to intercepts in the plots of [DNA]/(ε_a – ε_f) versus [DNA] give the values of K_b. The binding constants were found to be 1.45×10⁴, 2.41×10⁴ and 4.12×10⁴ L·mol^-1 for 3a~3c, respectively, which are collected in Table 6. The K_b of 3c is sharp higher than that of 3b and 3c, indicating 3c has the highest binding affinity with ct-DNA.

  Table 6

  

  Table 6.  Spectrophysical Data of Acylhydrazones Binding with ct-DNA

  DownLoad: CSV

  Compound	UV titration				EB displacement
  	Δλ (nm)	ΔAbs (%)	Kb (L·mol-1)		Δλ (nm)	ΔF (%)
  3a	0	14.67	1.45×104		2.5	25.62
  3b	1.0	22.52	2.41×104		2.0	26.68
  3c	1.0	40.67	4.12×104		4.5	33.90

  Table 7

  

  Table 7.  DNA Docking Results of the Acylhydrazones 3a~3c (Unit: kcal/mol)

  DownLoad: CSV

  Compound	Binding free energy (ΔGbinding)	Vdw_hb_desolv energy (ΔGvdW+hb+desolv)	Electrostatic energy (ΔGelec)	Total internal energy (ΔGtotal)	Torsional free Energy (ΔGtor)	Unbound system's energy (ΔGunb)
  3a	–8.14	–8.95	–0.09	–0.69	+0.89	–0.69
  3b	–8.37	–9.13	–0.14	–0.66	+0.89	–0.66
  3c	–8.66	–9.50	–0.06	–0.89	+0.89	–0.89
  ΔGbinding = ΔGvdW+hb+desolv + ΔGelec + ΔGtotal + ΔGtor – ΔGunb.

  3.3.2   Ethidium bromide (EB) displacement analysis

  The EB displacement experiments were employed to further understand the binding behavior^[34]. It is well known that the replacement of EB from DNA sequence by quencher results in a decrease of fluorescence emission of DNA-EB. Emission spectra of the DNA-EB with increasing concentrations of 3a~3c are depicted in Fig. 5. Regular decreases in the fluorescence intensities with insignificant shifts of emission peaks of DNA-EB are observed by increasing concentrations of 3a~3c. The results indicate interactions between 3a~3c and ct-DNA, but the microenvironment of DNA-EB does not change significantly^[35]. Combined with the above electric absorption titration results, the low fluorescence quenching suggests that the primary binding modes of acylhydrazones with ct-DNA are groove binding^{[32, 35]}.

  Figure 5

  

  Figure 5.  Emission spectra of EB and EB bound with ct-DNA, and in the presence of (i) 3a, (ii) 3b and (iii) 3c with increasing concentrations

  DownLoad: Full-Size Img PowerPoint

  To better understand the docking events, the minimum docking energy conformers of 3a~3c are illustrated in Fig. 6(i). It can be observed that 3a~3c enter into the minor groove of DNA, and they have the same docking site and quite similar docking poses, which result in the same types of interactions between 3a~3c and DNA and the same base pairs involved in the interactions. The interactions between 3c and DNA are presented in Fig. 6(ii). Carbonyl oxygen and nitrogen of azomethine in the acylhydrazone scaffold act as donors to form hydrogen bonds with DG16 base. Moreover, there are some hydro-phobic interactions, mainly T-shaped π-π stacking interactions.

  $ \lg \left[\left(F_0-F\right) / F\right]=\lg K_{\mathrm{q}}+n \lg [Q] $

  (4)

  Figure 6

  

  Figure 6.  Molecular docking results of 3a~3c with DNA. (i) Energy-minimized docking sites and poses of 3a~3c with DNA. (ii) Interactions of 3c binding to DNA with minimum energy

  DownLoad: Full-Size Img PowerPoint

  where F₀ and F are the fluorescence intensities of BSA in the absence and presence of quencher, and K_q and n the binding constant and binding sites, respectively. The dependence of lg[(F₀ – F)/F] on the value of lg[Q] is linear with slope equal to the value of n and the value of lgK_q is fixed on the ordinate. The values of K_q and n are presented in Table 8. Obviously, the values of K_q increase in the order of 3a, 3b, 3c, and the numbers of binding sites n are all close to 1, indicating that 3c has the strongest binding affinity to BSA as well as 3a~3c bind to BSA in the ratio of 1:1, which is consistent with the above analysis.

  Table 8

  

  Table 8.  Spectrophysical Data of Acylhydrazones Binding with BSA

  DownLoad: CSV

  Compound	UV absorption		Tryptophan quenching
  	ΔAbs		Δλ (nm)	ΔF (%)	Kq (L·mol–1)	n
  3a	0.013		1.0	27.00	1.85×104	0.78
  3b	0.015		1.0	27.85	8.95×104	0.89
  3c	0.019		1.0	28.27	2.09×105	0.95

  Recently, halogen bond, noncovalent intermolecu-lar interaction occurring between Lewis bases (O, N, and S) and Lewis acids (Cl, Br, and I), has been recognized as prevalent interaction between halogenated ligand and target protein^[45]. Because the differences among 3a~3c are only the difference of halogen atoms involved in halogen bonds, it is considered that the difference of binding strength of halogen bonds is the key to determine the difference of binding affinities between 3a~3c and BSA. The distances (d_X···O) of C–Cl···O, C‒Br···O and C‒I···O are 3.33, 3.33 and 3.21, respectively. The values of d_X···O suggest that the binding strength of iodine to oxygen might be the strongest among the three halogen bonds between ARG194 and receptors 3a, 3b, and 3c^[11]. The minimum binding free energies of 3a~3c with BSA are –8.59, –8.69 and –9.19, respectively, which are listed in Table 9. The values of 3a and 3b are very close, while that of 3c is much higher, which is consistent with the strength of halogen bonds. The above results show that iodinated acylhydrazone has the strongest binding affinity, probably because iodine-oxygen halogen bond plays a stronger role in the binding of acylhydrazone with BSA than chlorine-oxygen halogen bond and bromi-ne-oxygen halogen bond.

  Table 9

  

  Table 9.  BSA Docking Results of the Acylhydrazones 3a~3c (Unit: kcal/mol)

  DownLoad: CSV

  Compound	Binding free energy (ΔGbinding)	Vdw_hb_desolv energy (ΔGvdW+hb+desolv)	Electrostatic energy (ΔGelec)	Total internal energy (ΔGtotal)	Torsional free energy (ΔGtor)	Unbound system's energy (ΔGunb)
  3a	–8.59	–9.39	–0.09	–0.46	+0.89	–0.46
  3b	–8.69	–9.49	–0.09	–0.42	+0.89	–0.42
  3c	–9.19	–9.99	–0.09	–0.47	+0.89	–0.47
  ΔGbinding = ΔGvdW+hb+desolv + ΔGelec + ΔGtotal + ΔGtor – ΔGunb

  3.3.3   Molecular docking analysis

  Molecular docking analysis is an attractive approach for predicting the preferred orientation of ligand bind to a receptor^[36]. Generally, the large molecule could effectively bind to the major groove of DNA, while the small molecule is more likely to interact with the minor groove of DNA. The docking results are presented as minimum binding free energies, as summarized in Table 7, from which it can be noticed that the binding free energy values of 3a~3c are negative, indicating that the binding processes of 3a~3c to DNA are spontaneous^[37]. Besides, the minimum binding free energy of 3c is lower than that of the others, suggesting that 3c has stronger DNA-binding affinity than the others, which is identical with the results of the above absorption titration.

  3.4 BSA binding studies

  3.4.1   UV-vis absorption analysis

  BSA was selected as the model SA owing to an 88% homology similarity with HSA (human serum albumin)^[38]. The binding of acylhydrazone to BSA may cause the quenching of endogenous fluore-scence of BSA, including static quenching and dynamic quenching. UV analysis is an effective method to discriminate the static or dynamic quenching. It is well accepted that static quenching refers to the formation of a new complex between BSA and quencher in the ground state as well as a change of the absorption spectrum of BSA. By contrary, dynamic quenching is mainly caused by collision and energy transfer between BSA and quencher, which would not cause a change in the absorption spectrum^[39]. The absorption spectra of BSA in the absence and presence of 3a~3c are shown in Fig. 7, from which it can be observed that the absorption spectra of BSA change after the adding of 3a~3c. The spectral change values are presented in Table 8. The results suggest that the binding of 3a~3c to BSA cause changes in the microenvironment of aromatic amino acids, so quenching mechanisms of 3a~3c to BSA are identified as static quenching.

  Figure 7

  

  Figure 7.  UV-vis absorption spectra of (i) 3a, (ii) 3b, (iii) 3c and BSA in the absence and presence of 3a, 3b, 3c. Lines D (C-B, calculated data) are derived from lines C (experimental data) minu lines B (experimental data)

  DownLoad: Full-Size Img PowerPoint

  3.4.2   Tryptophan quenching analysis

  The fluorescence experiments were devoted to further evaluating protein-binding affinities and predicting the binding sites of 3a~3c with BSA. The endogenous fluorescence of BSA originates from three kinds of amino acid residues, i.e., tyrosine (Tyr), tryptophan (TRP), and phenylalanine (Phe)^[40]. Generally, tryptophan fluorescence is employed to monitor the changes in the structural conformation of BSA and predict the local environment in which the probe binds to BSA^[41]. To ensure that the endogenous fluorescence of BSA is only derived from TRP134 and TRP213 residues, the excitation wavelength was thus set to 295 nm. TRP134 is well-exposed to the hydrophilic region, while TRP 213 is located in a hydrophobic cavity of the BSA.

  Fig. 8 shows that the emission intensities of BSA are decreased regularly with a small blue shift by increasing the concentration of 3a~3c, which in turn imply they effectively bind with BSA. The con-comitant blue shifts mean that the hydrophobicity of the local environment around the tryptophan residues becomes stronger, probably due to the proximity of acylhydrazone molecules to the tryptophan resi-dues^[42]. In addition, the fluorescence intensities do not decrease significantly when the concentrations of 3a~3c are equal to that of BSA, indicating that the additions of 3a~3c could not completely influence the microenvironments of two tryptophan residues, that is to say, 3a~3c molecules prefer to bind with BSA near one of TRP residues. To substantiate the above statements, the binding constants and the number of binding sites of the acylhydrazones with BSA were calculated by Scatchard equation^[43]:

  Figure 8

  

  Figure 8.  Emission spectra of BSA and in the presence of (i) 3a, (ii) 3b and (iii) 3c with increasing concentrations. Inset: Scatchard plot of lg[(F₀ – F)/F] against lg[Q]

  DownLoad: Full-Size Img PowerPoint

  3.4.3   Molecular docking analysis

  Molecular docking was performed to study the selection of binding sites and display the probable interactions of 3a~3c bind to BSA. Because the number of binding sites of 3a~3c in BSA is all 1, the minimum docking energy conformer was selected for analysis. The minimum docking energy conformers and binding sites of 3a~3c are visualized in Fig. 9(i). Since the molecular structures of 3a~3c differ only in the halogen atom at the 4 position on the benzene ring, their docking sites are the same and the docking poses are almost the same in BSA. This results in the identical interactions and residues of interactions between 3a~3c and BSA. Fig. 9(ii) shows that 3c enters the hydrophobic pocket of BSA and is surrounded by various residues. The interactions between 3c and BSA include hydrogen bond, halogen bond, and hydrophobic interactions. Hydrogen bond and hydrophobic interactions play a role in stabilizing the 3c-BSA complex^[44]. Fig. 9(iii) displays the hydrophobic interactions on TRP213 and the halogen bonds of 3a~3c bind with BSA. The involvement of TRP213 in turn implies the quenching of emission of TRP213, which is consistent with the previous experimental results and shows the reliability of docking.

  Figure 9

  

  Figure 9.  Molecular docking results of 3a~3c with BSA. (i) Energy-minimized docking site and poses of 3a~3c binding to BSA. (ii) Interactions of 3c binding to BSA with minimum energy (iii) Interactions on TRP213 and the halogen bonds of 3a~3c binding with BSA. Distances (in Å) are labeled

  DownLoad: Full-Size Img PowerPoint

  3.5 In vitro antibacterial analysis

  Based on the premise that 3a~3c can bind effectively to biological macromolecules DNA and BSA, it was pertinent to examine their in vitro antibacterial activities which were studied together with the conventional antibacterial drug Gentamycin. The microorganisms used in this work include B. subtilis and S. aureus (as Gram-positive bacteria), E. coli and P. aeruginosa (as Gram-negative bacteria). The sizes of the inhibition zones of 3a~3c against the organisms at five concentrations are listed in Table 10. The antibacterial potential of 3a~3c is evaluated by comparing the sizes of inhibitory zones. 3a~3c exhibit moderate antibacterial activities compared with the conventional drug Gentamycin. As shown in Fig. 10, 3c exhibits larger inhibitory zones than 3a and 3b, whereas inhibitory zone of 3c against B. subtilis was the largest one. Besides, the minimal inhibitory concentrations (MIC) of 3c against B. subtilis and S. aureus are both 62.5 μg·mL^-1, while the other MICs are more than or equal to 125 μg·mL^-1, suggesting that 3c has better activity than 3a and 3b under identical experimental conditions. Noteworthily, the order of the in vitro antibacterial activities of 3a~3c to the selected bacteria is in consistent with their DNA/BSA binding affinities, indicating that the antibacterial activities of 3a~3c may be related to or originate from their abilities of interaction with DNA and protein.

  Table 10

  

  Table 10.  in Vitro Antibacterial Activities of 3a~3c against Bacteria

  DownLoad: CSV

  Compound	Conc.	Inhibition zone (mm)
  	(μg/mL)	E. coli	S. aureus	B. subtilis	P. aeruginosa
  3a	500	11.1	13.1	14.0	11.2
  	250	10.1	11.2	12.6	9.7
  	125	8.8	9.6	9.9	–
  	62.5	–	–	–	–
  	31.2	–	–	–	–
  	15.6	–	–	–	–
  3b	500	11.5	13.5	14.3	10.7
  	250	10.3	11.9	12.2	9.3
  	125	8.3	9.4	9.3	–
  	62.5	–	–	–	–
  	31.2	–	–	–	–
  	15.6	–	–	–	–
  3c	500	13.8	15.6	16.9	12.1
  	250	11.6	13.2	14.5	10.9
  	125	9.3	10.6	11.8	8.8
  	62.5	–	8.7	9.2	–
  	31.2	–	–	–	–
  	15.6	–	–	–	–
  Gentamicin	500	24.6	23.8	24.7	20.9
  "–" = no activity

  Figure 10

  

  Figure 10.  Inhibition zones of 3a~3c against bacteria (i) E.coli, (ii) S.aureus, (iii) B.subtilis, (iv) P.aeruginosa. d and e represent negative and positive controls, respectively

  DownLoad: Full-Size Img PowerPoint

  4. CONCLUSION

  The X-ray diffraction analysis revealed that C₁₃H₉Cl₂N₃O (3a), C₁₃H₉BrClN₃O (3b), and C₁₃H₉ClIN₃O (3c) all crystallize in monoclinic space group Cc. 3a~3c maintained good thermal stabilities because their thermal decomposition temperatures are all above 200 ℃. Interactions of 3a~3c with ct-DNA through minor groove as well as BSA through the hydrophobic pocket near TRP 213 have been proved by UV-vis, fluorescence spectroscopy, and molecular docking studies. The binding affinity of iodinated acylhydrazone (3c) with BSA was the strongest, presumably because of the strongest binding strength of iodine-oxygen halogen bond. In vitro antibacterial assay showed 3a~3c exhibited moderate antibacterial activities relative to the conventional drug Gentamycin.

  
  
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• 

  Scheme 1  Synthesis route of 3a~3c

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  Figure 1  ORTEP views of 3a~3c. Non-H atoms, represented as displacement ellipsoids, are plotted at the 30% probability level, while H atoms are shown as small spheres of arbitrary radius

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  Figure 2  Crystal packing diagrams of 3a~3c. Dotted lines represent intermolecular hydrogen bonds and π-π stacking

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  Figure 3  TG-DTG curves of 3a~3c at the heating rate of 5 ℃·min^-1

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  Figure 4  Normalized absorption spectra of (i) 3a, (ii) 3b and (iii) 3c in the absence and presence of DNA. Inset: plot of [DNA] against [DNA]/(ε_a – ε_f)

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  Figure 5  Emission spectra of EB and EB bound with ct-DNA, and in the presence of (i) 3a, (ii) 3b and (iii) 3c with increasing concentrations

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  Figure 6  Molecular docking results of 3a~3c with DNA. (i) Energy-minimized docking sites and poses of 3a~3c with DNA. (ii) Interactions of 3c binding to DNA with minimum energy

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  Figure 7  UV-vis absorption spectra of (i) 3a, (ii) 3b, (iii) 3c and BSA in the absence and presence of 3a, 3b, 3c. Lines D (C-B, calculated data) are derived from lines C (experimental data) minu lines B (experimental data)

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  Figure 8  Emission spectra of BSA and in the presence of (i) 3a, (ii) 3b and (iii) 3c with increasing concentrations. Inset: Scatchard plot of lg[(F₀ – F)/F] against lg[Q]

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  Figure 9  Molecular docking results of 3a~3c with BSA. (i) Energy-minimized docking site and poses of 3a~3c binding to BSA. (ii) Interactions of 3c binding to BSA with minimum energy (iii) Interactions on TRP213 and the halogen bonds of 3a~3c binding with BSA. Distances (in Å) are labeled

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  Figure 10  Inhibition zones of 3a~3c against bacteria (i) E.coli, (ii) S.aureus, (iii) B.subtilis, (iv) P.aeruginosa. d and e represent negative and positive controls, respectively

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  Table 1.  Crystallographic Data for 3a~3c

  Compound	3a	3b	3c
  CCDC No.	1548090	1545302	1584146
  T/K	296(2)	296(2)	296(2)
  Crystal size (mm)	0.36 × 0.30 × 0.21	0.34 × 0.27 × 0.14	0.31 × 0.25 × 0.13
  Molecular formula	C13H9Cl2N3O	C13H9BrClN3O	C13H9ClIN3O
  Formula weight	294.13	338.59	385.58
  Crystal system	Monoclinic	Monoclinic	Monoclinic
  Space group	Cc	Cc	Cc
  a (Å)	12.316(4)	12.345(3)	12.323(18)
  b (Å)	13.253(4)	13.362(4)	13.567(18)
  c (Å)	8.130(2)	8.147(2)	8.293(12)
  α (°)	90.00	90.00	90.00
  β (°)	93.842(5)	93.814(5)	93.956(3)
  γ (°)	90.00	90.00	90.00
  V (Å3)	1324.0(7)	1341.0(6)	1383.1(3)
  Z	4	4	4
  μ (mm–1)	0.484	3.259	2.503
  h/k/l (max, min)	–14, 14/–15, 10/–9, 9	–14, 13/–15, 13/–8, 9	–13, 14/–16, 12/–9, 9
  Dc (mg·m–3)	1.476	1.677	1.852
  F(000)	600	672	744
  θ range	2.26~25.09	2.25~25.10	2.24~25.09
  Reflections collected/independent/Rint	3181/2163/0.0217	3280/1839/0.0325	3374/2213/0.0207
  Data/restraints/parameters	2163/2/172	1839/2/172	2213/2/173
  Completeness (%)	99.9	99.8	100
  Final R, wR indices (I > 2σ(I))	0.0378/0.0817	0.0366/0.0727	0.0282/0.0727
  R, wR indices (all data)	0.0444/0.0857	0.0490/0.0777	0.0293/0.0736
  Goodness of fit on F2	1.046	1.026	1.062
  Largest diff. peak/hole (e·Å–3)	0.183/–0.201	0.532/–0.358	0.759/–0.590

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  Table 2.  Selected Bond Lengths (Å) and Bond Angles (°) for 3a~3c

  3a
  Bond	Dist.		Bond	Dist.		Bond	Dist.
  O(1)–C(6)	1.219(3)		N(2)–N(3)	1.383(3)		Cl(1)–C(3)	1.738(3)
  N(2)–C(6)	1.351(4)		N(3)–C(7)	1.278(4)		Cl(2)–C(11)	1.752(3)
  Angle	(°)		Angle	(°)		Angle	(°)
  O(1)–C(6)–N(2)	124.0(3)		C(7)–N(3)–N(2)	114.1(3)		C(6)–N(2)–N(3)–C(7)	178.4(3)
  N(2)–C(6)–C(5)	113.2(2)		N(1)–C(5)–C(6)	117.5(3)		N(3)–N(2)–C(6)–C(5)	179.8(3)
  C(6)–N(2)–N(3)	121.4(3)		C(13)–C(8)–C(7)	119.3(3)		N(2)–N(3)–C(7)–C(8)	178.8(3)
  3b
  Bond	Dist.		Bond	Dist.		Bond	Dist.
  O(1)–C(6)	1.225(6)		N(2)–N(3)	1.382(6)		Cl(1)–C(3)	1.748(6)
  N(2)–C(6)	1.334(6)		N(3)–C(7)	1.270(7)		Br(1)–C(11)	1.898(5)
  Angle	(°)		Angle	(°)		Angle	(°)
  O(1)–C(6)–N(2)	125.3(5)		C(7)–N(3)–N(2)	114.4(5)		C(6)–N(2)–N(3)–C(7)	–179.7(5)
  N(2)–C(6)–C(5)	113.8(4)		N(1)–C(5)–C(6)	117.1(4)		N(3)–N(2)–C(6)–C(5)	–178.4(4)
  C(6)–N(2)–N(3)	122.1(4)		C(13)–C(8)–C(7)	119.0(4)		N(2)–N(3)–C(7)–C(8)	–179.0(4)
  3c
  Bond	Dist.		Bond	Dist.		Bond	Dist'
  O(1)–C(6)	1.213(6)		N(2)–N(3)	1.380(6)		Cl(1)–C(3)	1.745(6)
  N(2)–C(6)	1.352(7)		N(3)–C(7)	1.276(7)		I(1)–C(11)	2.088(5)
  Angle	(°)		Angle	(°)		Angle	(°)
  O(1)–C(6)–N(2)	124.8(5)		C(7)–N(3)–N(2)	114.8(4)		C(6)–N(2)–N(3)–C(7)	–177.9(5)
  N(2)–C(6)–C(5)	113.1(4)		N(1)–C(5)–C(6)	117.2(4)		N(3)–N(2)–C(6)–C(5)	176.4(4)
  C(6)–N(2)–N(3)	121.8(4)		C(13)–C(8)–C(7)	118.9(4		N(2)–N(3)–C(7)–C(8)	178.7(4)

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  Table 3.  Hydrogen Bond Lengths (Å) and Bond Angles (º) for 3a~3c

  Compound	D–H···A	d(D–H)/Å		d(H···A)/Å	d(D···A)/Å	∠DHA/º
  3a	N(2)–H(2)···Cl(2)a	0.86		2.97	3.759(3)	154
  3b	N(2)–H(2)···Br(1)a	0.86		3.01	3.786(5)	151
  3c	N(2)–H(2)···I(1)a	0.86		3.13	3.866(5)	146
  Symmetry codes: for 3a (a) x–1/2, –y+1/2, z+1/2; for 3b (a) x–1/2, –y+3/2, z+1/2; for 3c (a) x+1/2, –y+3/2, z–1/2

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  Table 4.  Parameters of π-π Stacking Interactions of 3a~3c

  Compound	π-π interaction	Centroid-centroid separation (Å)	Dihedral angle (°)	Horizontal displacements between ring centroids (Å)	Vertical displacements between ring centroids (Å)
  3a	i	3.702(10)	4.788	1.543, 1.282	3.365, 3.473
  	ii	3.613(8)	5.664	0.612, 0.356	3.561, 3.595
  3b	i	3.702(8)	4.519	1.282, 1.542	3.473, 3.365
  	ii	3.614(6)	4.666	0.932, 0.674	3.492, 3.550
  3c	i	3.702(4)	3.469	1.249, 1.453	3.485, 3.405
  	ii	3.619(4)	3.452	0.876, 0.727	3.511, 3.545

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  Table 5.  Kinetic Parameters of Thermal Decomposition for 3a~3c at Three Heating Rates

  Compound	β (℃·min-1)	Tp/℃	Kissinger			Ozawa
  			Ea (kJ·mol-1)	lgA		Ea (kJ·mol-1)
  3a	5	280.74	74.09	4.29		79.59
  	10	300.19
  	15	315.49
  3b	5	287.23	76.69	4.639		81.95
  	10	307.23
  	15	323.95
  3c	5	294.44	85.95	5.32		90.94
  	10	317.93
  	15	325.46

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  Table 6.  Spectrophysical Data of Acylhydrazones Binding with ct-DNA

  Compound	UV titration				EB displacement
  	Δλ (nm)	ΔAbs (%)	Kb (L·mol-1)		Δλ (nm)	ΔF (%)
  3a	0	14.67	1.45×104		2.5	25.62
  3b	1.0	22.52	2.41×104		2.0	26.68
  3c	1.0	40.67	4.12×104		4.5	33.90

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  Table 7.  DNA Docking Results of the Acylhydrazones 3a~3c (Unit: kcal/mol)

  Compound	Binding free energy (ΔGbinding)	Vdw_hb_desolv energy (ΔGvdW+hb+desolv)	Electrostatic energy (ΔGelec)	Total internal energy (ΔGtotal)	Torsional free Energy (ΔGtor)	Unbound system's energy (ΔGunb)
  3a	–8.14	–8.95	–0.09	–0.69	+0.89	–0.69
  3b	–8.37	–9.13	–0.14	–0.66	+0.89	–0.66
  3c	–8.66	–9.50	–0.06	–0.89	+0.89	–0.89
  ΔGbinding = ΔGvdW+hb+desolv + ΔGelec + ΔGtotal + ΔGtor – ΔGunb.

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  Table 8.  Spectrophysical Data of Acylhydrazones Binding with BSA

  Compound	UV absorption		Tryptophan quenching
  	ΔAbs		Δλ (nm)	ΔF (%)	Kq (L·mol–1)	n
  3a	0.013		1.0	27.00	1.85×104	0.78
  3b	0.015		1.0	27.85	8.95×104	0.89
  3c	0.019		1.0	28.27	2.09×105	0.95

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  Table 9.  BSA Docking Results of the Acylhydrazones 3a~3c (Unit: kcal/mol)

  Compound	Binding free energy (ΔGbinding)	Vdw_hb_desolv energy (ΔGvdW+hb+desolv)	Electrostatic energy (ΔGelec)	Total internal energy (ΔGtotal)	Torsional free energy (ΔGtor)	Unbound system's energy (ΔGunb)
  3a	–8.59	–9.39	–0.09	–0.46	+0.89	–0.46
  3b	–8.69	–9.49	–0.09	–0.42	+0.89	–0.42
  3c	–9.19	–9.99	–0.09	–0.47	+0.89	–0.47
  ΔGbinding = ΔGvdW+hb+desolv + ΔGelec + ΔGtotal + ΔGtor – ΔGunb

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  Table 10.  in Vitro Antibacterial Activities of 3a~3c against Bacteria

  Compound	Conc.	Inhibition zone (mm)
  	(μg/mL)	E. coli	S. aureus	B. subtilis	P. aeruginosa
  3a	500	11.1	13.1	14.0	11.2
  	250	10.1	11.2	12.6	9.7
  	125	8.8	9.6	9.9	–
  	62.5	–	–	–	–
  	31.2	–	–	–	–
  	15.6	–	–	–	–
  3b	500	11.5	13.5	14.3	10.7
  	250	10.3	11.9	12.2	9.3
  	125	8.3	9.4	9.3	–
  	62.5	–	–	–	–
  	31.2	–	–	–	–
  	15.6	–	–	–	–
  3c	500	13.8	15.6	16.9	12.1
  	250	11.6	13.2	14.5	10.9
  	125	9.3	10.6	11.8	8.8
  	62.5	–	8.7	9.2	–
  	31.2	–	–	–	–
  	15.6	–	–	–	–
  Gentamicin	500	24.6	23.8	24.7	20.9
  "–" = no activity

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•