6-Bromo-2'-(2-chlorobenzylidene)nicotinohydrazide and 6-Bromo-2'-(3-bromo-5-chloro-2-hydroxybenzylidene) nicotinohydrazide Methanol Solvate: Synthesis, Characterization, Crystal Structures and Antimicrobial Activities.

Two newly synthesized nicotinohydrazones, 6-bromo-2'-(2-chlorobenzylidene)nicotinohydrazide (1) and 6-bromo-2'-(3-bromo-5-chloro-2-hydroxybenzylidene)nicotinohydrazide methanol solvate (2), have been obtained and structurally characterized by spectroscopic method and single crystal X-ray determination. The molecules in both compounds are in E configuration regarding to the azomethine groups. The molecules of compound 1 are linked via hydrogen bonds of N?H∙∙∙O, generating one dimensional chains running along the c-axis direction. The hydrazone molecules of compound 2 are linked by methanol molecules via hydrogen bonds of N?H∙∙∙O and O?H∙∙∙N, generating dimers. The in vitro antimicrobial activities of these compounds indicate that they are interesting antibacterial agents.


Introduction
Hydrazones with the central group -CH=N-NHare of great importance in biological fields, especially for the new drug investigation. 1 These compounds have been reported to show interesting biological activities like antimicrobial, antifungal, anticonvulsant, analgesic, antiplatelet, antitubercular, antiinflammatory, as well as antitumor. 2 Hydrazones are also a kind of interesting ligands in coordination chemistry. 3 The metal complexes with hydrazones are reported to have interesting biological activities. 4 Isoniazide is a front-line antituberculotic drug.

1. Materials and Methods
5-Bromonicotinohydrazide, 2-chlorobenzaldehyde and 3-bromo-5-chloro-2-hydroxybenzaldehyde were purchased from Bide Chemical Reagent Co. Ltd. The other chemicals with AR grade were obtained commercially and used as received. CHN elemental analyses were performed on a Perkin-Elmer 240C elemental analyzer. IR spectra were measured with a FT-IR 170-SX (Nicolet) spectrophotometer. 1 H NMR and 13 C NMR data were measured with a Bruker 500 MHz instrument.

4. X-Ray Structure Analysis
X-Ray diffraction intensities were collected using a Bruker SMART 1000 CCD area detector equipped with graphite-monochromated Mo-Kα radiation (λ = 0.71073 Ǻ) at 298(2) K. Absorption corrections were applied by SADABS. 9 The structures of the compounds were solved by direct methods and refined on F 2 by full-matrix leastsquares methods with SHELXTL. 10 All non-hydrogen atoms were refined anisotropically. The amino and methanol H atoms in both compounds were located in difference Fourier maps and refined isotropically, with N-H and O-H distances restrained to 0.90(1) Å and 0.85(1) Å, respectively, and with U iso (H) values fixed at 1.2U eq (N) and 1.5U eq (O). The other H atoms were placed in idealized positions and constrained to ride on their parent atoms. The Cl atoms in 1 is disordered over two sites, with occupancies of 0.84(2) and 0.16(2). The details of the crystallographic data are summarized in Table 1. Supplementary crystallographic data have been deposited at the Cambridge Crystallographic Data Center (CCDC 850161 and 2022935).

5. Antimicrobial Test
Qualitative determination of antimicrobial activity was done using the disk diffusion method. Suspensions in sterile peptone water from 24 hour cultures of microorganisms were adjusted to 0.5 McFarland. Muller-Hinton Petri dishes of 90 mm were inoculated using these suspensions. Paper disks (6 mm in diameter) containing 10 μL of the substance to be tested (at a concentration of 2048 μg/mL in DMSO) were placed in a circular pattern in each inoculated plate. Incubation of the plates was done at 37 °C for 18-24 h. DMSO impregnated discs were used as negative controls. Toxicity tests of the solvent, DMSO, showed that the concentrations used in antimicrobial activity assays did not interfere with the growth of the microorganisms. Reading of the results was done by measuring the diameters of the inhibition zones generated by the test substance. Penicillin was used as a reference.
Determination of MIC was done using the serial dilutions in liquid broth method. The materials used were 96-well plates, suspensions of microorganism, Muller-Hinton broth and stock solutions of each substance to be tested (2048 μg/mL in DMSO). The following concentrations of the substances to be tested were obtained in the 96-well plates: 1024,512,256,128,64,32,16,8,4, 2, and 1 μg/mL. After incubation at 37 °C for 18-24 h, the MIC for each tested substance was determined by microscopic observation of microbial growth. It corresponds to the well with the lowest concentration of the tested substance where microbial growth was clearly inhibited.

1. Chemistry
The nicotinohydrazones 1 and 2 were facile prepared by the reaction of 1:1 molar ratio of 5-bromonicotinohydrazide with 2-chlorobenzaldehyde and 3-bromo-5chloro-2-hydroxybenzaldehyde, respectively in methanol. The elemental analyses are in good agreement with the formulae proposed for the compounds determined by single crystal X-ray diffraction. The crystals of the compounds are stable in air at room temperature, and easily soluble in DMF, DMSO, methanol, ethanol, chloroform, dichloromethane, and acetonitrile.
Synthesis of the compounds was indicated in their IR spectra by the presence of bands for imine bonds, i.e. 1654 cm -1 for 1 and 1666 cm -1 for 2. In 1 H NMR, the absence of NH 2 signals and the appearance of peaks for NH protons in the region δ 12.24-12.39 ppm and imine CH proton in the region δ 8.92 ppm confirmed the synthesis of the compounds. The aromatic proton signals were found in their respective regions with different multiplicities, confirming their relevant substitution pattern.

Antimicrobial Activity of the Compounds
The antimicrobial activities of the compounds against the organisms Streptococcus pyogenes (S. pyogenes), Streptococcus agalactiae (S. agalactiae), Staphylococcus aureus (S. aureus), Bacillus anthracis (B. anthracis), Klebsiella pneumonia (K. pneumonia) and Pseudomonas aeruginosa (P. aeruginosa) are summarized in Table 5. The results show that both compounds have effective antimicrobial activities against S. pyogenes, S. agalactiae, and B. anthracis, and have relatively poor or negative activities against other bacteria when compared to the Penicillin. Compounds 1 and 2 have similar activities against S. agalactiae and B.  anthracis. Interestingly, compound 2 has stronger activities against S. pyogenes, K. pneumonia and P. aeruginosa than compound 1. This indicates that the Br and Cl substitunts are a good choice in the search for new antimicrobial agents. The activities of the nicotinohydrazone compounds in this work are stronger than the benzohydrazones with Br as substituent. 6a The compounds are more active against S. pyogenes, S. agalactiae, B. anthracis and P. aeruginosa than the benzohydrazone compound with Br, NO 2 and Cl as the substituent. 13 Thus, the present compounds show promising activity against S. pyogenes, S. agalactiae and B. anthracis, which deserves further investigation for developing new antimicrobial drugs.