1 INTRODUCTION

Schiff base ligands have been widely studied recently because of their wide pharmacological activities like antibacterial, antifungal, anticancer activities[1-5]. In this paper, two different hydrazones from 2-fluorobenzhydrazide have been synthesized and their X-ray structure investigations have been carried out to relate the antibacterial activity with the molecular geometry.

2 EXPERIMENTAL

2.1 Reagents and instruments

All chemicals and solvents purchased were of reagent grade and used without further purification.Two bacteria (K. Pneumonia, clinical isolated strains; S. aureus ATCC25923) were presented by pharmacognosy lab of Dalian medical university.FT-IR spectrum (4000–400 cm-1) was recorded on KBr pellet technique using an AVATAR-360 spectrophotometer. The melting point was determined by a XT4 binocular micromelting point apparatus (Beijing). Elemental analyses were performed with a Perkin Elmer 240C apparatus.

2.2 Synthesis of compounds (I) and (II)Compound I

A mixture of 2-fluorobenzhydrazide (0.0463 g,0.3 mmol) and 2-nitrobenzaldehyde (0.0456 g, 0.3 mmol) in 15 mL methanol was stirred for 15 minutes at room temperature to give a clear solution. The brown shiny crystals of compound I suitable for X-ray analysis were obtained by slow evaporation from methanol at room temperature for two weeks.Yield: 91.5% for compound I based on K. Anal.Calcd. (%) for compound I: C, 58.54; H, 3.51; N,14.63. Found (%): C, 58.51; H, 3.53; N, 14.62 and C, 64.63; H, 3.85; N, 10.74. IR (v/cm-1): 3310(νO-H),1228, 1195, 1018, 1613 (νC=N), 1230(νC=O).

Compound II

2-Fluorobenzhydrazide (0.0463 g, 0.3 mmol) was dissolved in 15 mL of methanol and treated with 2-fluorobenzaldehyde (0.0373 g, 0.3 mmol) in a 1:1 molar ratio with stirring for 15 minutes at ambient temperature. The brown solution was left at room temperature for a few days, obtaining brown crystals which were washed with methanol and dried under vacuum. Yield: 92.1% based on K. Anal.Calcd. for compound II (%): C, 64.61; H, 3.87; N,10.76. Found (%): C, 64.63; H, 3.85; N, 10.74. IR(v/cm-1): 3305(vO-H), 1235, 1185, 1020, 1616 (vC=N),1235(νC=O).

2.3 Structure determination of compounds I and II

Intensity data of the two crystal were measured at 298(2) K on a Bruker Smart 1000 CCD diffractometer equipped with a graphite-monochromated MoKα radiation (λ = 0.071073 nm) by using a Ψ-ω scan mode for compounds I and II. And the data were corrected for Lp factors and empirical absorption. The two structures were solved by direct methods and refined by full-matrix least-squares techniques with SHELXL-97 and SHELXS-97[6].The non-hydrogen atoms were refined anisotropically. All hydrogen atoms were located in the calculated positions and refined isotropically. Their crystal data are summarized in Table 1.

Table 1. Crystal Data for the Compounds

2.4 Assessment of the antibacterial activity[7]

Antibacterial activity was evaluated by minimum inhibitory concentration (MIC) using the serial microdilution test. The exponential phase of the growth bacteria was diluted to a density of 105–106CFU/mL, then plated in replicates of five in a 96-well flat bottom microtiter plate and exposed to 5 μL serial dilutions of compounds in sterile medium and DMSO. The addition concentration range of both compounds was 0.1–50 mg/mL. The plates were covered, wrapped in plastic wrap to limit the moisture loss[8], and then incubated at 37 ℃ for 18 h (K. Pneumonia) or 24 h (S. aureus). The optical density at 620 nm (OD620nm) was determined in a Microplate Reader. For the MIC test, the antibacterial activity was defined as the inhibition ratio ≥50%. The time-kill experiments were performed to evaluate the bactericidal activity of the compound which showed good results from MIC data.Time-kill assays were performed at 1/2 ×, 1 ×, 2 ×,4 ×MIC value per CLSI methods[9]. Briefly, growthphase bacterial cultures at 5×105to 5×106CFU/mL were treated with active compounds, and Colony-Forming Units (CFU) were determined at 0, 2, 4, 6,8 and 24 h. For time-kill experiments, bactericidal activity was defined as α≥3log decrease in CFU/mL 24 h after treatment.

3 RESULTS AND DISCUSSION

3.1 Crystal structure of compounds I and II

X-ray single-crystal structural analysis revealed that both compounds are structurally similar Schiff bases (Fig. 1 for compound I, Fig. 2 for compoundII). Single-crystal structural data of the two compounds are shown in Table 1. The bond lengths and bond angles in both compounds are similar to each other and within normal ranges. The bond lengths of 1.260(19) ? in compound I and 1.277(3)? in compound II between atoms C(7) and N(1) are similar to those observed in other Schiff bases[10-15],indicating they are double bonds. The bond lengths of 1.32(2) ? in compound I and 1.350(3) ? in compound II between atoms C(8) and N(2) are intermediate between C–N and C=N bonds due to the conjugation effects in the molecules. The mean planes of the two benzene rings make a dihedral angle of 5.0(13)o for compound I and 10.1(2)o for compound II. As expected, the molecules of the compounds adopt trans configuration about the C=N double bonds. In compound I, the torsion angles C(6)–C(7)–N(1)–N(2), C(7)–N(1)–N(2)–C(8),N(1)–N(2)–C(8)–O(1) and N(1)–N(2)–C(8)–C(9)are 2.0(13), 7.9(13), 2.5(13) and 2.7(13)o, respectively. In compound II, the torsion angles C(1)–C(7)–N(1)–N(2),C(7)–N(1)–N(2)–C(8),N(1)–N(2)–C(8)–O(1) and N(1)–N(2)–C(8)–C(9) are 2.6(2),10.8(2), 1.2(2) and 0.7(2)o, respectively. In the crystal structure of compound I, molecules are linked through intermolecular N–H··O hydrogen bonds,forming chains along the a axis (Table 2, Fig. 3). In the crystal structure of compound II, molecules are linked through intermolecular N–H··O hydrogen bonds, forming chains along the c axis (Table 1, Fig. 4).In addition, weak π··π stacking interactions can be found in both compounds (the π··π stacking interaction parameters can be seen in Table 3). Upon these π··π stacking interactions, two compounds extend into two-dimensional layers along the ab and ac planes, respectively (Figs. 3 and 4).

Table 2. Hydrogen Bonding Details in Compounds I and II (Distances in ? and Angles in o)

Table 3. π··π Stacking Details in Compounds I and II (Distances in ? and Angles in o)

Fig. 1. Molecular structure of I at 30% probability ellipsoids

Fig. 2. Molecular structure of II at 30% probability ellipsoids.Only the major components of the disordered group are shown

Fig. 3. Molecular packing of I viewed along the a axis. Hydrogen bonds are drawn as dashed lines

Fig. 4. Molecular packing of II viewed along the b axis. Hydrogen bonds are drawn as dashed lines

3.2 Effects of compounds I and IIon the antibacterial activity

The both synthesized compounds were screened for antibacterial activity including one Gram(+)bacterial strain (S. aureus) and one Gram(-) bacterial strain (K. Pneumonia). The MICs (minimum inhibitory concentration) of these compounds are listed in Table 4. Compound I shows the highest inhibitory activity against S. aureus (7.8 μg/mL),and compound II exhibits mild antibacterial activity(160 μg/mL). Compound I shows moderate activity against K. Pneumonia (25 μg/mL), and compoundII exhibits mild activity (130 μg/ml). Time-kill assays data of compound I against S. aureus are shown in Fig. 5. Compared with the bacterial growth of tetracycline group, compound I significantly inhibits the S. aureus growth. Concentration(4 ×MIC) was able to reduce the CFU after 2 h, and the reduction was obviously increased at initial 8 h.Concentration (1/2～1×MIC) exerted similar and stable activities all the time. All can achieved a 3 log kill within 8 h for S. aureus.

Table 4. Effect of the Title Compound on Minimum Inhibitory Concentration (MIC) (Unit: μg/mL)

Fig. 5. (S. aureus) Change in CFU/mL over time after the addition of compound I. MIC value was 7.8 μg/mL for S. aureus

3.3 Discussion

K. Pneumonia grows well in the Luria Bertani(LB) medium, and Nutrient Broth medium is the Enrichment Medium of S. aureus[16-17]. Both compounds are dissolved in DMSO and methanol, and insoluble in water. In the antibacterial test, high concentration solution was prepared by DMSO but other low concentration solution was diluted by medium. Thus, the amount of DMSO presented here could not affect the antibacterial results[18].

Tetracycline was used as a positive control in antibacterial test, and the results showed that compound I had significant bacteriostatic effects on Gram-positive bacterial strain (S. aureus) and one Gram-negative bacterial strain (K. Pneumonia),hence it is a broad-spectrum antibacterial agent.Although compound I has lower bacteriostatic effect than the positive control, it may have the ability against MDR (multidrug resistance). Most important of all, compound I has another nitro group in one benzene ring, but compound II has only fluorine atom, which could result in different antibacterial activities.

In the time-kill assay, concentration of the samples was serially diluted 10-fold in the sterile saline in order to control the accuracy of the test.We found that colony counts varied with the incubation time even if the samples were placed at 4℃. This phenomenon suggests that plating should be performed promptly when the samples are removed from the test tubes. The range of quantification using this methodology is 30–100 CFU per plate. In summary, appropriate dilution is considerably important to the time-kill assays.

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