Synthesis and evaluation of biological activities of 4-cyclopropyl-5-(2-fluorophenyl) arylhydrazono-2,3-dihydrothiazoles as potent antioxidant agents

A new series of 4-cyclopropyl-5-(2-fluorophenyl)arylhydrazono-2,3-dihydrothiazole derivatives was synthesized via the reaction of prepared thiosemicarbazones with 2-bromo-1-cyclopropyl-2-(2-ﬂuorophenyl)ethanone in the presence of Et3N as a catalyst through a semi Hantzsch cyclization. The optimized reaction conditions for this one-pot reaction were achieved. The products were obtained in short reaction times, high yields and high purities. Antioxidant activity of products was evaluated using DPPH (2,2-diphenyl-2-picrylhydrazyl) and ABTS 2,2-azinobis(3-ethylbenzothiazoline-sulfonate) assays. Products showed higher antioxidant activity using the ABTS method. Compounds 5c and 5g showed lower IC50 values compared with ascorbic acid as a standard. Compounds 5a–5h possessed moderate to high antioxidant activity by both methods. Also, antibacterial activity of 5a–5h was evaluated against gram-positive and gram-negative bacterial strains. None of the compounds inhibited A. hydrophila, while they had moderate to low inhibitory activity against other tested bacterial strains. GRAPHICAL ABSTRACT

With the aim to obtain the optimal reaction conditions, preparation of 5a was selected as a model reaction. Results are summarized in Table 1. The effects of different basic catalysts, amounts of catalyst, and solvents were studied. According to the results, the reaction proceeded slowly in low yield in the absence of catalyst (entry 1), while product 5a was obtained in a higher yield and shorter reaction time in the presence of Et 3 N (entry 2). Increasing the amount of catalyst showed no substantial improvement in the yield (entry 5), while the yield was decreased by reducing the amount of catalyst to 10% (entry 4). Moreover, the effects of other polar solvents such as DMF, methanol (MeOH) and CH 3 CN on the yield and time of the reaction were studied (entry 6-11). Thus, compound 5a was obtained from the reaction of thiosemicarbazone 3a and 4 in the presence of Et 3 N (20 mol%) in high yield (95%) and short reaction time (30 min) in EtOH as the solvent under reflux conditions. The formation of product 5a was indicated by a reaction color change from yellow to dark orange within 5 min, however completion of the reaction took place within 30 min. The scope of this method was explored for the synthesis of new 4-cyclopropyl-5-(2-fluorophenyl)arylhydrazono-2,3-dihydrothiazoles 5a-5h with different carbonyl compounds 1a-1h under the optimized reaction conditions. The results showed that this procedure is reliable, simple setup, reproducible, high yield, economic and products purified without chromatographic methods. All products were fully characterized by IR, 1 H NMR and 13 C NMR spectra. The physicochemical properties of synthesized compounds are presented and summarized in Table 2.
IR spectra of products 5a-5h showed characteristic absorption bands at 3320-3200, 1620-1600 and 1070-1050 cm −1 due to N-H, C=N and C-F bonds, respectively. 1 H NMR spectra of products were in accordance with expected number, chemical shifts and coupling constants. Aromatic protons appeared at 8.28-6.66 ppm. Also, aliphatic proton of cyclopropyl moiety (H c ) and methylene protons appeared as multiples at 1.99-1.86 and 1.20-0.88 ppm, respectively. In addition, N-H can be endo-or exo-cyclic. However, according to the literature [33] a signal at 14.50-11.45 ppm is related to exocyclic N-H. So, we can propose an exo-cyclic N-H for compound 5g according to a down field signal at 12 ppm and an endo-cyclic N-H for other compounds. 13 C NMR spectra of compounds 5a-5h corresponded to the expected number and types of carbons. Aromatic and olefinic carbons appeared at 168.6-108.4 ppm and aliphatic carbons appeared at their expected chemical shifts at 55.9-7.8 ppm. 13 C NMR spectra of products represented C-F couplings clearly. Fluorine atom has split all carbons of the phenyl ring. All chemical shifts and coupling constants of important carbon atoms are summarized in Table 3.

Antioxidant activity
All products were screened for their in vitro antioxidant activity. DPPH (2,2-diphenyl-2picrylhydrazyl) radical and ABTS 2,2-azinobis(3-ethylbenzothiazoline-sulfonate) radical cation are widely used, rapid, simple, and inexpensive methods to evaluate antioxidant ability of compounds. In this research, the antioxidant activity of compounds was evaluated using colorimetric DPPH and ABTS methods. When a compound acts as an antioxidant it can scavenge free radicals and lead to a decrease in absorption band at 517 and 734 nm for DPPH and ABTS solutions, respectively. Moreover, potential antioxidant activity leads to a rapid decrease in absorbance. ABTS is also frequently used by the food industry and agricultural researchers to measure the antioxidant capacities of foods. ABTS is converted to its radical cation (ABTS •+ ) by the addition of sodium persulfate or potassium persulfate. ABTS •+ scavenging is considered as an electron transfer reaction. [34] ABTS radical cation is blue in color, when it reacts with an antioxidant compound the blue color changes to yellow or colorless. Also, the DPPH radical changes from purple to colorless in reaction with an antioxidant compound. DPPH · scavenging may be through donation of a radical hydrogen atom (H • ) to form a stable DPPH-H molecule. [35] The antioxidant activities of compounds were screened at concentrations of 125-4000 μg/mL at 517 and 734 nm for DPPH and ABTS assays, respectively. Also, IC 50 Table 3. 13 C NMR spectroscopy data of significant carbon atoms of 5a-5h.     values (the concentration of compounds to scavenge 50% of DPPH or ABTS) were calculated by plotting radical scavenging activity against concentration and obtaining a line equation. Ascorbic acid was used as a standard. The investigation of antioxidant activity revealed that all the newly synthesized compounds showed potent to moderate radical scavenging activity when compared with ascorbic acid as a standard. As it is depicted in Figures 2 and 3 radical scavenging activity of products 5a-5h was dose dependent. In the DPPH assay, compounds 5a, 5b, 5e and 5g showed higher antioxidant activity at lower concentration (125-500 μg/mL) in comparison with others. While, at higher concentrations the antioxidant activity of all products was approximately equal. Moreover, compounds 5a and 5e showed a chigher antioxidant activity at low concentration (120 μg/mL) in the ABTS assay, while higher concentrations represented potent antioxidant activity.
In addition, IC 50 values of products 5a-5h were calculated ( Figure 4). The IC 50 values were in the range of 1.92-0.17 and 0.96-0.08 μM for DPPH and ABTS assays, respectively.  As it is evident, the ABTS assay showed more potent antioxidant activity. It shows that compounds 5a-5h can donate radical electron better than the hydrogen radical. Product 5g was more active than ascorbic acid according to the DPPH assay, while compound 5c represented better antioxidant activity when compared with ascorbic acid in the ABTS assay. However, compounds 5a, 5c, 5e and 5g showed potent antioxidant activity. Other compounds showed moderate activity.
Thiazole moiety has an important role in antioxidant activity. [25,12] As it is depicted in Scheme 2 the endo-or exo-N-H readily can donate a hydrogen radical to the DPPH radical and generate a new radical species which can resonate through the thiazole ring and =C-N-N=C moieties. So, the new radical can be stable by resonance through this structure. Although the thiazole and =C-N-N=C moieties have an important role in antioxidant activity, however other parts of products can affect this activity as well. Probably, the high antioxidant activity of 5c can be due to the fused aromatic rings which can stabilize the free radical by resonance through a longer system. Low antioxidant activity of 5h is due to the presence of an electron-withdrawing group NO 2 , which resulted in destabilization of radicals (Scheme 2). [36] Scheme 2. Proposed mechanism for antioxidant activity of compounds 5a-5h.

Antibacterial activity
The new synthesized compounds 5a-5h were screened for their in vitro antibacterial activity against Gram-positive and Gram-negative bacterial strains including: Staphylococcus aureus (S. aureus), Micrococcus luteus (M. luteus), Escherichia coli (E. coli), Pseudomonas aeruginosa (Ps. aeruginosa), Bacillus subtilis (B. subtilis), and Aeromonas hydrophila (A. hydrophila) using the well-diffusion method. Penicillin G and Gentamicin were used as positive controls. DMSO was used as a negative control and showed no activity against mentioned bacterial strains. The antibacterial activity of products 5a-5h was screened at a concentration of 1000 μg/mL in DMSO. The experiments were performed in triplicate. The results are presented as mean ± standard deviation in millimeter.
According to Table 4 all of the compounds were inactive against A. hydrophila, while all of the compounds showed antibacterial activity against Ps. aeruginosa. Moreover, 5a and 5f showed low antibacterial activity against S. aureus; however, most of the compounds possessed antibacterial activity against E. coli and B. subtilis. Furthermore, 5e showed the highest antibacterial activity against B. subtilis (14.3 ± 0.57) among all of the compounds. Also, 5f and 5b possessed higher antibacterial activity against M. luteus (10.6 ± 0.57) and E. coli (11.6 ± 1.15), respectively. Moreover, compounds 5a and 5c showed similar activity (10.3 ± 0.57) against Ps. aeruginosa. In addition, the antibacterial activity of compounds 5a-5h was not comparable to standard drugs.

Conclusion
In conclusion we have reported an efficient, convenient and easy setup procedure for synthesis of new 4-cyclopropyl-5-(2-fluorophenyl)arylhydrazono-2,3-dihydrothiazole derivatives. The optimized procedure led to high yields and high purities products in short reaction time. The obtained data revealed that products namely 5a, 5c, 5e and 5g exhibited promising antioxidant activity. The important role of hydrazinyl-thiazole moiety of synthesized compounds in antioxidant activity was discussed. The antioxidant activity by the ABTS method was higher than the DPPH method; this presents potential electron donation capacity of products besides their hydrogen atom transfer capacity. Products showed moderate to low antibacterial activity.

Materials and instruments
Starting materials containing ketones, thiosemicarbazide, and 2-bromo-1-cyclopropyl-2-(2-fluorophenyl)ethanone were obtained from Fluka company (Switzerland) and Merck company (Germany), antioxidant reagents were supplied from Sigma company (Germany), and biological cultures were obtained from Merck company (Germany) and Quelab company (Canada). All chemicals were used without further purification. All reactions were monitored by TLC performed on silica gel plates (60 F 254 Merck). IR spectra were recorded on a Shimadzu IR-470 spectrophotometer in anhydrous potassium bromide (KBr). 1 H NMR and 13 C NMR spectra were recorded on a 400 MHz Bruker spectrometers using CDCl 3 as the solvent and chemical shifts are expressed relative to TMS. Coupling constants were expressed in hertz (Hz). Melting points were determined using a Mettler Fp5 apparatus and are uncorrected. Absorbance of antioxidant assays was recorded on the Unico 2100 spectrophotometer. Elemental analyses were made by a Carlo-Erba EA1110 CNNO-S analyzer.

General procedure for synthesis of 5a-5h
Thiosemicarbazide 2 (2 mmol, 0.18 g) was added to a solution of carbonyl compound 1a-1h (2 mmol) in 10 mL EtOH as the solvent in the presence of a few drops of AcOH and refluxed for 2 h. The mixture was cooled down and the resulting precipitate was filtered off and dried at room temperature. Resulted thiosemicarbazone 3a-h (1 mmol) was dissolved in EtOH (5 mL) and 2-bromo-1-cyclopropyl-2-(2-fluorophenyl)ethanone 4 (1 mmol) was added to the mixture. The solution was refluxed in the presence of Et 3 N (20% mole) for appropriate time. The progress of the reaction was monitored by TLC (n-hexane:EtOAc 7:2). After completion of the reaction, pH was controlled and the mixture was neutralized with saturated Na 2 CO 3 solution. The solids was filtered off and dried at room temperature. The products obtained from EtOH:H 2 O recrystallization.    13

Biology
DPPH radical-scavenging activity assay DPPH radical-scavenging activity of compounds was evaluated according to the literature. [29] Appropriate amount of DPPH was dissolved in MeOH to give a concentration of 6.25 × 10 −5 M. A series of sample solution at concentrations of 4000, 2000, 1000, 500, 250, 125 μg/mL in MeOH was prepared by two-fold serial dilution. To 0.1 mL of each sample solution was added 3.9 mL of fresh DPPH solution and was shaken vigorously. Samples were kept in darkness for 30 min then their absorbance was measured at 517 nm. MeOH was used as a blank. Radical-scavenging activity was calculated as follows: where A control is the absorbance of negative control (3.9 mL DPPH + 0.1 mL MeOH) and A sample the absorbance of the test compounds.