Microwave-Assisted Synthesis of New N1,N4-Substituted Thiosemicarbazones

We present an efficient procedure for the synthesis of thirty-six N1,N4-substituted thiosemicarbazones, including twenty-five ones that are reported for the first time, using a microwave-assisted methodology for the reaction of thiosemicarbazide intermediates with aldehydes in the presence of glacial acetic acid in ethanol and under solvent free conditions. Overall reaction times (20–40 min when ethanol as solvent, and 3 min under solvent free conditions) were much shorter than with the traditional procedure (480 min); satisfactory yields and high-purity compounds were obtained. The thiosemicarbazide intermediates were obtained from alkyl or aryl isothiocyanates and hydrazine hydrate or phenyl hydrazine by stirring at room temperature for 60 min or by microwave irradiation for 30 min, with lower yields for the latter. The preliminary in vitro antifungal activity of thiosemicarbazones was evaluated against Aspergillus parasiticus and Candida albicans.


Introduction
Thiosemicarbazones and thiosemicarbazides are now well established as an important class of sulfur/nitrogen donor ligands, particularly for transition metal ions [1,2], because of the remarkably diverse biological activities observed for these compounds. These activities include antiviral [3][4][5], antitumor [6,7], and antimicrobial properties [8], as well as other industrially important activities, including anticorrosion [9] and antifouling [10] effects. Considering all of these properties, it is important to be able to synthesize new series of thiosemicarbazones.
The increasing demand for clean and efficient chemical procedures has been a target for the synthesis of organic compounds. The combined use of microwave irradiation and solvent-free conditions has shown advantages from economic and environmental standpoints [11,12]. Furthermore, microwave-assisted organic reactions generally provide high yields of pure products, minimize the use of organic solvents, and allow for a simplified work-up and shorter reaction times [13].
Our research group has been working on more efficient and cleaner synthetic methods, focusing on microwave irradiation and solvent-free conditions [14][15][16][17]. To extend our investigation and considering the special importance of thiosemicarbazone class, in this paper we report the synthesis of thirty six thiosemicarbazones N 1 ,N 4 -substituted from thiosemicarbazides with low reaction times and good yields by microwave-assisted reactions. Furthermore, the evaluation of antifungal activity against the Aspergillus parasiticus and Candida albicans was realized.

Results and Discussion
Thirty-six thiosemicarbazones were synthesized using a microwave-assisted methodology; twentyfive are new compounds. The synthetic procedure was performed in two steps starting with aryl thiosemicarbazides 5a-d or alkyl thiosemicarbazides 5e-i, also prepared in this work, and cinnamaldehyde (6a), 3-methyl-indolcarboxaldehyde (6b), 2,6-dimethoxy-pyridincarboxaldehyde (6c) or 4-quinolinecarboxaldehyde (6d) in ethanol as a solvent, and under solvent free conditions, with a few drops of added glacial acetic acid, as outlined in Scheme 1. The reaction mixtures were irradiated in a scientific microwave reactor for 20-40 min at 100 W when ethanol used as solvent, and for 3 min at 800 W in the absence of solvent. The products, a cinnamaldehyde series (compounds 7-15), a 3-methyl-indolcarboxaldehyde series (compounds 16-24), a 2,6-dimethoxypyridincarboxaldehyde series (compounds 25-33) and a 4-quinolinecarboxaldehyde series (compounds 34-42), were obtained with both procedures as fine crystals in high-purity and satisfactory yields after a short time, when compared with the reaction time for the traditional procedure (480 min). The best yields (88-98%) and the short reaction times (3 min) were obtained when the solvent free conditions were used. The thiosemicarbazones 7-10 and 16-19 were also prepared using a reflux method; after 8 h under reflux in ethanol, lower yields were obtained for all the compounds than in the microwave-assisted synthesis. Table 1 shows the reaction times and yields for the target compounds, 7-42. The thiosemicarbazone structures 7-42 were fully characterized by 1 H-and 13 C-NMR and IR spectroscopy. The 1 H-and 13 C-NMR shifts () were assigned based on literature data [18][19][20][21][22] and were consistent with the structures proposed. The 1 H chemical shifts of H-C=N for the thiosemicarbazone cinnamaldehyde series products 7-15 showed lower values than for the other series. In contrast, the 13 C chemical shifts of C=N were assigned with lower and similar values for the 2,6-dimethoxy-3pyridinecarboxaldehyde and 4-quinolinecarboxaldehyde series products 25-33 and 34-42. The values ranged from 137.02 to 138.62 for the cinnamaldehyde ones 7-15 and from 140.10 to 146.93 for the 3-indolecarboxaldehyde series 16-24. The electronic substituent effects were as expected. The Figures 1-4 show the numbered structures.
The aryl thiosemicarbazides 5a-d or alkyl thiosemicarbazides 5e-i used as intermediates in thiosemicarbazone preparation were prepared by both traditional and microwave-assisted procedures. Scheme 2 shows the reactions and the conditions for both procedures.   Different from the thiosemicarbazones, thiosemicarbazide preparation via the traditional methodology with toluene as the solvent under stirring at room temperature was more efficient than the microwave-assisted procedure, affording very good yields in comparison, especially for the alkyl isothiosemicarbazides. This performance may be due to the higher volatility of the alkyl isothiocyanates (1e-i). However, the reaction times using the microwave irradiation method were 30 minutes, whereas stirring at room temperature required 60 minutes. Table 2 lists the yields in the preparation of 5a-i. The thiosemicarbazides were identified by comparison with analytical data found in the literature [23].
The preliminary in vitro antimicrobial activity of all thiosemicarbazones synthesized on Aspergillus parasiticus and Candida albicans was evaluated. The thiosemicarbazones showed weak or moderate activity as compared to the standard fungicide itroconazole. However, the observed antimicrobial effects do not exclude from further investigations of these compounds against other fungal strains. Table 3 lists the MIC (g/mL) values obtained for active thiosemicarbazones.

General
Melting points were determined with a Meltemp II apparatus and were uncorrected. Infrared spectra (KBr pellets) were recorded on a Bruker Vertex 70 spectrophotometer. The 1 H-and 13 C-NMR spectra were obtained on a Bruker Avance II 400 spectrometer ( 1 H, 400 MHz; 13 C, 100 MHz) using tetramethylsilane TMS as the internal standard and acetone-d 6 and pyridine-d 5 as the solvent. Elemental analyses were performed on a Perkin-Elmer Model 2400 instrument. The microwave-assisted organic reactions were performed in a CEM Discovery System reactor.

General Procedure for the Preparation of Thiosemicarbazides 5a-i
The thiosemicarbazides were prepared according to methods described elsewhere [23]. Briefly, the alkyl or aryl isothiocyanate (25 mmol) and hydrazine hydrate or phenyl hydrazine (25 mmol) were mixed in the presence of toluene (20 mL). The reaction mixture was kept under stirring for 1 hour at room temperature. The solid obtained was filtered and washed with ice-cold toluene. All thiosemicarbazides were identified by the comparison of analytical data (melting points and NMR) with literature reports.

General Procedure for the Preparation of Thiosemicarbazides 5a-i Using Microwave Irradiation
The alkyl or aryl isothiocyanate (0.74 mmol) and hydrazine hydrate or phenyl hydrazine (0.74 mmol) were mixed in the presence of toluene (2 mL) and submitted to microwave irradiation for 30 min at 100 W. The solid obtained was filtered and washed with ice-cold toluene. All thiosemicarbazides were identified by comparing their melting points with those previously obtained.

General Procedure for the Preparation of Thiosemicarbazones 7-42 Using Microwave Irradiation and Ethanol as Solvent
The aldehyde (0.84 mmol) and alkyl or aryl thiosemicarbazide (0.84 mmol) were mixed in the presence of ethanol (5 mL) and few drops of glacial acetic acid and submitted to microwave irradiation for 20-40 min at 100 W. The solid obtained was filtered and washed with ice-cold ethanol several times.

General Procedure for the Preparation of Thiosemicarbazones 7-42 Using Microwave Irradiation in Solvent-Free Conditions
The aldehyde (0.84 mmols) and alkyl or aryl thiosemicarbazide (0.84 mmols) were mixed in an agate mortar with few drops of glacial acetic acid and submitted to microwave irradiation for 3 min at 800 W. The solid obtained was extracted and recrystallized from ethanol to furnish the pure products.

General Procedure for the Preparation of Thiosemicarbazones 7-10 and 16-19 Using Traditional Reflux
The aldehyde (1.19 mmol) and aryl thiosemicarbazide (1.19 mmol) were mixed in the presence of ethanol (10 mL) and few drops of glacial acetic acid. The reaction mixture was kept under reflux for 8 hours. The solid obtained was filtered and washed with ice-cold ethanol several times.  Cinnamaldehyde-4-phenyl-thiosemicarbazone (7  Cinnamaldehyde-4-(p-methyl-phenyl)-thiosemicarbazone (8

Conclusions
In summary, four series of thiosemicarbazones derived from cinnamaldehyde, 3-indolecarboxaldehyde, 2,6-dimethoxypyridinecarboxaldehyde and 4-quinolinecarboxaldehyde and the corresponding thiosemicarbazides were prepared using microwave-assisted reactions in the presence of ethanol as solvent and under solvent free conditions, resulting in good yields, high purity and lower reaction times in comparison with the traditional reflux method, especially when solvent free conditions were utilized.