SYNTHESIS, CHARACTERIZATION AND EVALUATION OF BIOLOGICAL ACTIVITY OF SOME SUBSTITUTED( E )-2-BENZYLIDENEHYDRAZINECARBOTHIO AMIDES

. A series of substituted ( E )-2-benzylidenehydrazinecarbothioamide compounds were synthesized by condensation of thiosemicarbazide with substituted benzaldehydes. The synthesized substituted ( E )-2-benzylidenehydrazinecarbothioamide compounds were characterized by their physical constants, UV, IR and NMR spectra. The antimicrobial activities of these synthesized substituted ( E )-2-benzylidenehydrazinecarbothioamide compounds have been screened by Bauer-Kirby method using human pathogenic bacteria and fungal species. The antimicrobial activities of all synthesized ( E )-2-benzylidenehydrazinecarbothioamide compounds have shown significant activity.


INTRODUCTION
The synthesis, structure and biological activity of some new hydrazones prepared from aliphatic hydrazides has been the focus of research. Different methods have been employed to synthesize different types of hydrazones from different starting materials. Hydrazones are characterized by the presence of the triatomic grouping C=N-N-. They can be considered as Schiff bases derived from acid hydrazides. The most important property of hydrazones is their high physiological activity [1][2][3][4][5][6]. Extensive studies have revealed that the lone pair on trigonally hybridized nitrogen atom of the azomethine group is responsible [7][8][9][10][11] for the chemical and biological activity. It has been reported that metal complexes of hydrazones have diverse applications. They find use as plasticizers, polymerization inhibitors and antioxidants. They are used as fungicides and pesticides in biological and biochemical context. Moreover, the hydrazone group plays an important role of the antimicrobial and possesses interesting antibacterial, antifungal [12][13][14] and anti-tubercular activities [15][16][17][18][19][20]. In addition, their varied coordinating behaviour makes them interesting candidates for metal-based drugs. Generally, the ligands act synergistically with metals towards their biological activity. These observations have guided the development of new hydrazones with varied biological activities [21].The biological activity of complexes derived from hydrazones have been studied and contrasted with regard to their antibacterial, antitumoral, antiviral, antimalarial and antitubercular properties [22]. It has also been shown that the azomethine N, which has a lone pair of electrons in a sp 2 hybridised orbital, is biologically important [23].
Hydrazones constitute an important class of biologically active drug molecules [24] which has attracted attention of medicinal chemists due to their wide range of pharmacological properties. These compounds are being synthesized as drugs by many researchers in order to combat diseases with minimal toxicity and maximal effects. These predictions has provided therapeutic pathway to develop new effective biologically active hydrazones. A number of hydrazone derivatives have been reported to exert notably antimicrobial, antihypertensive, anticonvulsant, analgesic, anti- inflammatory, antituberculosis, antitumoral, antiproliferative and antimalarial activities, biological activities of various hydrazones are well reported in literature. This review highlights diverse pharmacological activities shown by hydrazones.
Medicinal chemists have also carried out considerable research for novel antimicrobial and anticancer agents bearing hydrazone moiety. Some studies have confirmed that hydrazone derivatives exhibit antifungal and anticancer activities [25][26][27][28][29]. Some researchers have reported anticancer effects of some antifungal agents and carried out considerable research for deciphering the underlying mechanisms of antitumor activity [30][31][32].In antifungal and anticancer drug design, the lack of selectivity of conventional chemotherapeutic agents and the acquisition of multiple-drug resistance are two major challenging problems. As a consequence of this situation, the search for new effective chemotherapeutic agents has attracted a great deal of interest [33][34][35].
Several hydrazone derivatives have been reported as insecticides, nematocides, herbicides, rodenticides and antituburculosis in addition to that some of the hydrazone were found to be active against leukemia, sarcoma and illnesses [36,37].we now carry out another systematic study of their synthesis and biological activity. Herein, the synthesis of the substituted (E)-2benzylidenehydrazinecarbothioamides are described and their antimicrobial properties are evaluated

General
All the chemicals used in the present investigation, have been procured from Sigma-Aldrich Chemical Company. Melting points of all substituted (E)-2-benzylidenehydrazinecarbothioamides have been determined in open glass capillaries on a Mettler FP51 melting point apparatus and are uncorrected. The UV spectra of all the substituted (E)-2-benzylidenehydrazinecarbothioamides have been recorded with ELICO-BL222 spectrophotometer (λ max nm) in spectral grade methanol solvent. Infrared spectra (KBr, 4000-400 cm -1 ) have been recorded on SHIMADZU Fourier transform spectrophotometer. Bruker AV400 NMR spectrometer operating at 400 MHz has been utilized for recording 1 H NMR spectra and 100 MHz for 13 C NMR spectra in DMSO solvent using TMS as internal standard.

Synthesis of substituted (E)-2-benzylidenehydrazinecarbothio amides
A solution of equimolar quantities of thiosemicarbazide (0.01mol) with substituted benzaldehydes (0.01mol) acetic acid (two drops) and 10 ml of ethanol were shaken occasionally for 1 hour [38]. The completion of the reaction was monitored by TLC continuously. The resultant mixture was cooled at room temperature. Then the precipitate obtained, was filtered at the filter pump and washed several times with cold water then pale yellow solid was obtained as the final product. This crude product was recrystallized from ethanol and glittering colorless solid was obtained.
The general scheme for preparation of substituted (E)-2benzylidenehydrazinecarbothioamides has shown in scheme-1.

CHARACTERIZATION: UV SPECTRUM
In the UV-visible spectra of the compound (fig-1) below 350 nm, a single peak is observed for the of substituted (E)-2-benzylidenehydrazinecarbothioamides of (hydrazone) transition band due to π-π* at 309.00 λ max (nm). The observed peak is assigned to π-π* transition. According to the valence Band theory, as the conjugation increases, the energy difference between the highest occupied and the lowest unoccupied π-orbitals decreases and hence the wave length of the absorption band increases. Fig:1 The UV spectrum of the (E)-2-benzylidenehydrazinecarbothioamide

IR SPECTRUM
The important IR frequencies (fig-2) of substituted (E)-2-benzylidenehydrazinecarbothioamides formed due to the condensation of thiosemaicarbazide with benzaldehyde are present in the table-2. A strong band is observed bending vibration for (E)-2-benzylidenehydrazinecarbothioamides of (CH=N) at around 1620 -1700 cm -1 characteristic of the azomethine. The sharp peak at 3220 cm -1 corresponds to N-H stretching and the broad absorption band at 3385 cm-1 corresponds to the NH 2 stretching. An absorption band was observed around the N-N stretching has been observed at 999 cm -1 . Fig: 2 The IR spectrum of (E)-2-benzylidenehydrazinecarbothioamide

1 H NMR SPECTRUM
The complete assignment of the I H NMR spectra is given here (fig-3). The spectrum was recorded at 400 MHz. The assignment is done on the basis of chemical shifts, multiplicities and coupling constants. The 1H NMR spectrum (E)-2-benzylidenehydrazinecarbothioamide in DMSOd 6

Antimicrobial activities
In an urge to develop new antimicrobial compound, a number of hydrazones were tested for their antimicrobial activities because of the evolution of drug-resistant microbial pathogens. The fast resistance of bacteria against antibiotics has become a widespread medical problem. Treatment options for these infections are often limited, especially in debilitated and immune compromised patients. The dramatically rising incidence of multi-drug resistant microbial infections in the past few decades has become a serious health care problem. The search for new antimicrobial agents will consequently always remain as an important and challenging task for medicinal chemists. The treatment of bacterial and fungal infectious diseases remains a challenging problem because of the increasing number of multi-drug microbial pathogens [39][40][41]. Nowadays, the design of new compounds able to deal with resistant bacteria, having new structures and new targets of action, has become one of the most important areas in the antibacterial research purpose [42].

Antibacterial sensitivity assay
Antibacterial sensitivity assay was performed using Kirby-Bauer [43] disc diffusion technique. In each Petri plate about 0.5 mL of the test bacterial sample was spread uniformly over the solidified Mueller Hinton agar using sterile glass spreader. Then the discs with 5mm diameter made up of Whatmann No.1 filter paper, impregnated with the solution of the compound was placed on the medium using sterile forceps. The plates were incubated for 24 hours at 37 °C by keeping the plates upside down to prevent the collection of water droplets over the medium. After 24 hours, the plates were visually examined and the diameter values of the zone of inhibition were measured. Triplicate results were recorded by repeating the same procedure. The antibacterial screening effect of the synthesized substituted (E)-2-benzylidenehydrazinecarbothioamide compounds is shown in Fig. 4 Plates (1-10).
The antibacterial activities of all the synthesized substituted (E)-2-benzylidenehydrazinecarbothioamide compounds have been studied against three gram positive pathogenic strains Bacillus subtilis, Staphylococcus aureus, Streptococcus and two gram negative strains Escherichia coli and Pseudomonas aeruginosa species. The disc diffusion International Letters of Chemistry, Physics and Astronomy Vol. 59 205 technique was followed using the Kirby-Bauer [43] method, at a concentration of 250 µg/mL with Ciprofloxacin used as the standard drug. The zone of inhibition is compared using Table 3 and the corresponding Clustered column Chart is shown in Fig. 5

Antifungal sensitivity assay
Antifungal sensitivity assay was performed using Kirby-Bauer [43] disc diffusion technique. PDA medium was prepared and sterilized as above. It was poured (ear bearing heating condition) in the Petri-plate which was already filled with 1 ml of the fungal species. The plate was rotated clockwise and counter clock-wise for uniform spreading of the species. The discs were impregnated with the test solution.
The test solution was prepared by dissolving 15 mg of the hydrazone in 1ml of DMSO solvent. The medium was allowed to solidify and kept for 24 hours. Then the plates were visually examined and the diameter values of zone of inhibition were measured. Triplicate results were recorded by repeating the same procedure. Plate-5 Plate-6 Fig. 6 Antifungal activity of substituted (E)-2-benzylidenehydrazinecarbothioamide compounds Antifungal assay has been performed using Kirby-Bauer [43] disc diffusion technique. The antifungal activities of the entire substituted (E)-2-benzylidenehydrazinecarbothioamide have been studied and are shown in Fig. 6 for Plates (1)(2)(3)(4). The zone of inhibition values of the antifungal activities is given in Table 5. The clustered column chart was shown in Fig. 7 and it reveals that the 4-Br and 4-OCH 3 compounds have shown good activity against A. flavus. The 3-Br and 4-Cl substituted compounds have shown excellent activity against A.niger. Also, 3-Br, 4-Br, 4-Cl, 4-F and 4-CH 3 compounds have shown good activity against T.viride. The remaining compounds have shown moderate activity against all the fungi.