Microwave Assisted Synthesis of Thiocarbamoylpyrazoles and Application as an Alternative Latent Fingermark Developers

Fingerprints are unique to each individual, contributing to human identification in forensic cases. The powder technique being widely used is considered one of the most important in latent fingermarks analysis. In this sense, the present work aimed to synthesize 1-thiocarbamoyl-4,5dihydro-1H-pyrazoles and apply them as powder agents to develop latent fingermarks. The compounds were prepared from an effective and green synthesis via the condensation of chalcones with thiosemicarbazide in ethanol under microwave irradiation. Pyrazoles were tested and compared with a standard white powder (Sirchie) on glass surfaces using sebaceous and natural fingermarks so that molecule 2c has been shown to be more promising providing image clarity. The derivatives of pyrazoles were promising as an alternative latent fingermark, presenting high quality images for fingermarks, proving to be a great tool for forensic sciences.


Introduction
Pyrazole and derivatives are interesting heterocyclic compounds since they show a wide range of pharmacological and biological properties including antioxidant, antitumor, antimicrobial, analgesic and anti-inflamatory. [1][2][3][4] In general, the activities are provided because of the presence of the N−N bonds in the pyrazole ring. 5 Microwave assisted organic synthesis highlights key advances in the development of organic chemistry. Its efficiency in accelerating reaction rates has been emulated in several fields of organic synthesis. [6][7][8] This can improve the yield and purity of the final compounds in short reaction times through the precise control of parameters such as power irradiation, pressure and temperature. 9,10 The fingermarks when detected at crime scenes are considered as circumstantial evidence in forensic investigations and criminal justice. 11 Such importance is due to the uniqueness in ridge pattern for each individual and thus are unique and vital for the identification and/or involvement of a suspect. 12 When found in the latent form, for visualization to be possible, it is necessary that some substances react chemically or physically with the compounds present in the fingermark. 13 In this context, heterocyclic compounds have been contributing to papiloscopy for the improvement of new fingermark developers. For example, phenanthro imidazole and benzazoles have been used as new fluorescent powder for the development of latent fingermarks; and 1,2-indanedione and ninhydrins derivatives as novel reagents for detection of amino acids in latent fingermarks. [14][15][16][17][18] Motivated by the importance of forensic chemistry, and in continuation of our research efforts, [19][20][21] we have tried to highlight aspects reported on the chemistry and forensic application of pyrazole nucleous. The aim of this work was to synthesize a series of 1-thiocarbamoyl-3,5-diaryl-4,5-dihydro-1H-pyrazoles under microwave irradiation in order to applied them as novel white powders for the development of latent fingermarks.

Experimental
Apparatus and analysis 1 H and 13 C nuclear magnetic resonance (NMR) spectra were recorded on a Bruker DRX500 spectrometer in CDCl 3 (500 MHz for 1 H and 126 MHz for 13 C) using tetramethylsilane (TMS) as internal standard. Melting points were recorded in open capillary on an electrothermal apparatus (model Fisatom 430). The compounds were analyzed with Fourier transform model Shimadzu Prestige 21 (Shimadzu) scanning from 4000 to 600 cm −1 with a resolution of 4 cm −1 . All solvents and chemicals were research grade and were used as obtained from Sigma-Aldrich (St. Louis, MO, USA).

General procedure for the synthesis of thiocarbamoylpyrazoles
In a 50 mL rounded flask, chalcone 1a-1h (2.0 mmol) and thiosemicarbazide (2.0 mmol) were mixed with ethanol (10 mL) and KOH was added. 22 The reaction mixtures were then inserted in a microwave model Discover System (CEM), with power 100 W, 250 psi at 50 °C for 15 min. For the conventional methodology, the reagents were used at the same quantities and the reaction was refluxed for 4 h. In both cases the solid obtained were filtered and washed with cold ethanol and dried under vacuum to give the pure 1-thiocarbamoyl-4,5-dihydro-1H-pyrazoles 2a-2h. All compounds were macerated with liquid nitrogen for application as a powder.

Latent fingermarks deposition and development
Latent fingermarks were collected from 3 donors on glass surface and natural and sebaceous contents were evaluated. For sebaceous collection, the fingers were rubbed on nose and forehead and homogenized with each other, while for natural collection the donor's hand was washed with soap and water and the latent fingermarks were collected half an hour later. 23 All the revelations were made 24 h after the depositions by physical method. Specific brushes were used for the development and cleaning of excess dust (132LBW and CFB100 from Sirchie ® ). In addition, a Sirchie ® fingerprint developer white powder was used as standard for comparison. The images were taken with a Canon EOS Rebel T6 semi-professional camera with a distance of 9 cm and focus 5.6.
For optimization of the use of the best solvent in the synthesis of thiocarbamoylpyrazoles it was evaluated which of them had the best yield as shown in Figure 1. Optimization was performed using the unsubstituted structure 2a. From this, the best methodology was applied for the series of pyrazoles 2a-2h.
The compounds showed good yields (53-85%) and high conversion (Table 1). These yields are in accordance with other methodologies 24 such as utilization of ultrasound irradiation and more promising than conventional heating methodologies.
Moreover, the most satisfactory yield was obtained using ethanol, which also provided a synthesis process in accordance with green chemistry principles that include low cost and renewable materials. Such efficiency of ethanol to other solvents (Figure 1) may be related to the dielectric constant due to the greater dipole-dipole interaction necessary to cyclocondensation reaction and products obtention. 25 In addition, the column purifications or recrystallization were not required, avoiding the excessive expenditure of solvents.  Moreover, the application of microwave irradiation avoided excessive reaction times and high temperatures.
The compounds 2a-2h were characterized by infrared spectroscopy (IR) and 1 H and 13 C NMR spectroscopy as described previously by our group. 24 Spectroscopic data of the synthesized compounds are described in Supplementary Information section.
The prepared compounds 2a-2h were applied as latent fingerprinting powders and compared with commercial Sirchie ® white powder due to similar staining. In this sense, the evaluation of the image quality of fingermarks developments can be described by the level of detail according to Sears et al., 23 where 0 corresponds to no mark evidence; 1: weak development, evidence of contact but no ridge details; 2: limited development, about 1/3 of ridge details are present but probably cannot be used for identification purposes; 3: strong development, between 1/3 and 2/3 of ridge details, identifiable fingermark; 4: very strong development, full ridge details, identifiable fingermark. Thus, Table 2 shows the efficiency of the development of natural and sebaceous fingermarks using thiocarbamoylpyrazoles 2a-2h and the white standard powder. From this, it was possible to identify that most of the compounds present efficiency similar or superior to the same color standard used by the scientific police. Molecule 2c provided promising results regarding image clarity resulting in visible ridges, strong development and presence of minutiae that assisted in the identification of fingermarks. 26,27 In addition, to investigate the interaction of compounds with the surface, electron microscopy was performed. Figure 2 demonstrates a split assay in which the same fingermark was developed using compound 2c and the white standard powder. Moreover, Figure 2 also shows a scanning electron microcopy analysis of a fingermark enhanced with compound 2c.
In special, from the comparison between compound 2c and the white standard powder it is possible to observe that both are similar, which confirms the efficiency of the new synthesized developer, providing good image quality. According to the SEM images, it is possible to observe the adhesion of the compound 2c to the glass surface. In addition, it was observed that the synthesized powder adhered only to friction ridges avoiding adherence to the glass surface and, thus, provided adequate images of the enhanced fingerprints.

Conclusions
In conclusion, a new synthesis methodology was developed for the 1-thiocarbamoyl-3,5-diaryl-4,5-dihydro-1H-pyrazoles series by microwave irradiation. It has advantages over existing methods such as reaction time and it does not require solvent waste in column purification. This procedure represents a economic and environmentally friendly process for the synthesis of pyrazole derivatives. These compounds represent a new class of latent fingermark developers. They demonstrated high quality images, especially pyrazole 2c, which proved to be the most promising in natural and sebaceous fingerprints. Thus, the prepared compounds are of paramount importance because they provide fingerprints that could be used in human identification, contributing to the forensic sciences.

Supplementary Information
Supplementary data of the synthesized compounds  2a-2h and fingerprint development analysis associated with this work are available free of charge at http://jbcs.sbq.org.br as a PDF file.