Synthesis and characterization of substituted acetophenone-4, 4-dimethyl-3-thiosemicarbazone derivatives with an evaluation of antimicrobial and antioxidant activities

Thiosemicarbazones are organic compounds; these are widely used in the medical ield. It is primarily in the manufacture of pharmaceutical drugs and bio molecules. These compounds have the azomethine functional group (-N=CH-) used to derive organic open chain and heterocyclic compounds. Thiosemicarbazones are similar to semicarbazones, and carbonyl group oxygen is replaced by sulphur. Thiosemicarbazone contains nitrogen and sulfur atoms, so they are used as ligands in coordination chemistry and form metal complexes. Thiosemicarbazone and its metal complexes are used in a variety of ways. It is especially widely used in the preparation of polymers and biochemicals products. Thiosemicarbazone and its metal complexes are closely correlated with biological properties and act as antioxidants, antifungals, antimicrobials and anticancer. Subsequently, in the current study, substituted thiosemicarbazoneswere synthesized and examined for their antimicrobial and antioxidant activity. Newly substituted thiosemicarbazones were obtained from the respective acetophenones and thiosemicarbazides. The structure of the newly prepared compounds was con irmed by spectroscopic studies such as IR, H, C NMR, Mass and elemental analysis. The antimicrobial activity of all the new compounds showed signi icant activity against the selected bacteria and fungi in the studies. The compound TZ04 exhibited good antioxidant activity comparedwith standard ascorbic acid and butylated hydroxytoluene.


INTRODUCTION
Thiosemicarbazones is a thiourea derivative, and the general formula is R 1 R 2 C=N-NH-CS-NR'R". R 1 , R 2 , R' and R" may be alkyl/aryl/hydragen or heterocyclic part (Reddy et al., 2016). Thiosemicarbazones are generally prepared from condensation of carbonyl compounds and thiosemicarbazide with alcohol and dehydrating agents. Chemically, Thiosemicarbazones are generally known as Schiff base compounds and contain azomethine (-C=N-) group (Mohan et al., 2018). In the presences, azomethein group thiosemicarbazones are intermediate and form heterocyclic compounds (Salman et al., 2017) with suitable agents and also forms, metal complexes (Suvarapu et al., 2012) with metal ions due to in the presence of N and S donor atoms. The derivatives of thiosemicarbazones in organic and metal bonding compounds are of high biological importance.
The biological activity of metal complexes varies from metal ions or ligands, and this is indicated to increase or decrease the biological activity of many metal complexes. Based on the above information, we have studied the synthesis, characterization, antimicrobial and antioxidant activities of the substituted acetophenone-4,4-dimethyl-3-thiosemi carbazones. A thin layer chromatography was used to determine the purity of the compounds. The structures of the synthesized compounds were determined by spectral and elemental analysis. The physical properties of the synthesized compounds are given in the experimental section, and all newly synthesized compounds were screened to antimicrobial and antioxidant activity.

MATERIALS AND METHODS
The chemicals used in this study are brought from Sigma-Aldrich Chemicals and was used directly in the experimental part without re inement. The compounds were synthesized by the reporting method , and the method is described in Scheme 1 and Table 1. Reaction completion was checked and con irmed by thin-layer chromatography. The melting points were determined and are uncorrected using the open capillary tube method. The structures of the products were con irmed by elemental and spectral analysis.

Antimicrobial activity
The Kirby-Bauer disc diffusion method (Hussain et al., 2016) of in vitro antibacterial activity was used  to evaluate all the synthesized compounds. Bacteria such as B.subtilis, S.aureus, S.typhi and E.coli were used to test the activity of the compounds. Cipro loxacin was used as the reference antibacterial drug. For the anti fungal assay, Candida albicans was used to test the activity of the compounds. Fluconazole was kept as the standard drug. The inhibition zone of synthesized compounds was compared with standard drugs. The results of the zone of inhibition for the antimicrobial activity of the synthesized compounds are given in Table 2.

Antioxidant activity
All synthesized compounds for antioxidant activity were screened using the DPPH evaluation method (Meeran and Hussain, 2017). The antioxidant data of all the samples are given in Table 3. DPPH method is a reduction principle of the purple DPPH (free radical) reduced and changes to yellow colored diphenylpicrylhydrazine. The remaining purple colored DPPH exhibited maximum absorption of 517 nm. The 2 ml different concentration of the synthesized compounds or standards were added with 2ml of DPPH solution (0.1 mM), and these are kept in the dark. After 20 min of incuba-tion at 37 • C, the solution absorbance was measured at 517 nm. AA and BHA were used as positive controls. The following formula was used to calculate the percentage of inhibition.

RESULTS AND DISCUSSION
The inal compounds were puri ied by re crystallization with ethanol. The structure of the compounds was con irmed based on spectral and elemental analysis. The spectral characterization of 2-[1-(4-chlorophenyl) ethylidene]-N, N-dimethylhydrazinecarbothioamide (TZ03) is described as an example. The IR spectrum revealed 3471, 3062 & 2985 and 1589 cm −1 values respectively. It is obtained due to the compound which contains the characteristics of groups of amide NH, aromatic & aliphatic CH and imine C = N respectively. 1 H NMR spectrum reported a singlet at δ 11.39 ppm assignable to NH proton. Two doublets at δ 7.82 (J=10.4) ppm and δ 7.44 (J=10.8Hz) ppm each for two protons are assignable to H-7, H-11 and H-8, H-10 respectively.
A singlet at δ 3.11 ppm for six protons is due to -N(CH 3 ) 2 and a singlet at δ 2.29 ppm is related to C 5 -CH 3 protons. The 13 C NMR spectral results are described below. The signal exhibited at δ177.5 ppm due to the thiocarbonyl carbon (C=S) and the carbon of C=N showed at δ147.4 ppm. The aromatic carbons C 6 , [C 7 and C 11 ], [C 8 and C 10 ] and C 9 appeared at δ 135.6, 128.2, 128.9 and 136.6ppm respectively. The methyl carbon, C 12 is observed at 23.3ppm and the peak at 42.6 ppm due to Dimethyl carbons of C 13 and C 14 (NMe 2 ) respectively. The mass spectrum of the molecular ion peak is reported at 255 [M + ]. This value refers to the molecular weight of the compound. Hence, the above spectral data are compatible with the structure of the desired product, 2-[1-(4-chlorophenyl) ethylidene]-N, N-dimethylhydrazinecarbothioamide.
All synthesized compounds were screened for in vitro antimicrobial activity by the Kirby-Bauer disc diffusion method. The inhibition zone was compared with standards. The results of the antibacterial and anti fungal activity are given in Table 2 and Graph 1. The newly synthesized compounds showed signi icant activity against selected bacteria. High anti fungal activity was observed in the TZ04 compared to other compounds due to the Bromo substitution of TZ04. The results of the antioxidant activity of synthesized compounds at different concentrations are shown in Table 3. The calculated IC 50 values are given in Table 3 and Graph 2. The most active compound among the synthesized compounds is TZ04, which gave an IC 50 value of 14.8 µg/ml, while AA and BHA gave 8.09 and 11.52 µg/ml respectively.

CONCLUSIONS
Serious substituted acetophenone-4,4-dimethyl-3thiosemicarbazones derivatives have been synthesized, and the elemental and spectral analysis conirmed the structures of the compounds. Newly synthesized compounds exhibited signi icant antimicrobial activity against selected bacteria and fungi. The compound TZ04 showed high antioxidant activity. Finally, 2-[1-(4-bromophenyl)ethylidene]-N, Ndimethylhydrazinecarbothio amide is observed to have good anti fungal and high antioxidant activity.