Synthesis, Spectral Characteristics, and Molecular Docking Studies of 2,4-Dichloro-N -(2,2,2-trichloro-1-((5-(phenylamino)- 1,3,4-thiadiazol-2-yl)amino)ethyl)benzamide †

: Derivatives of 1,3,4-thiadiazole are of great interest for scientiﬁc and practical human activities as biologically active substances, dyes, components for creating semiconductors, energy ac-cumulators, liquid crystals, polymers, nanomaterials, etc. Here we report the synthesis of 2,4-dichloro-N -(2,2,2-trichloro-1-((5-(phenylamino)-1,3,4-thiadiazol-2-yl)amino)ethyl)benzamide based on N , N’ - disubstituted hydrazinecarbothioamide—2,4-dichloro-N -(2,2,2-trichloro-1-(2-(phenylcarbamothioyl)- hydrazine-1-carbothioamido)ethyl)benzamide. The method for obtaining the target product is based on the dehydrosulfurization reaction of the starting hydrazinecarbothioamide under the action of a mixture of iodine and triethylamine in a DMF medium. A new derivative of 1,3,4-thiadiazole was obtained in 84% yield, and its structure was conﬁrmed by 1 H and 13 C NMR spectroscopy data. Molecular docking studies were carried out with the structure of the resulting compound and dihydrofolate reductase (DHFR) in the AutoDock Vina program. The resulting compound is a potential inhibitor of DHFR and surpasses several known analogues in terms of the strength of the complex formed with the active site of this enzyme.

Derivatives of 1,3,4-thiadiazoles are widely used in coordination chemistry as ligands [42][43][44], in the synthesis of polymers [45], and the creation of polymer films [46,47]. The prospects of using 1,3,4-thiadiazoles in optoelectronics [48], in the purification of water from heavy metal ions [49], for the separation of minerals by the flotation method [50], and the creation of corrosion-resistant coatings [51] are discussed in the literature. A large number of works are devoted to the development of dyes and fluorescent markers based on 1,3,4-thiadiazoles [46,52,53].

Chemistry
1 H NMR (400 MHz) and 13 C NMR (100 MHz) spectra were measured for solutions in DMSO-d 6 on a Varian VXR-400 spectrometer. Elemental analysis was performed on a LECO CHNS-900 instrument (see Supplementary Materials). The reaction and purity of the compounds were monitored by TLC on Silufol UV-254 plates using a chloroform/acetone mixture (3:1) as an eluent.

Molecular Docking Studies
The dihydrofolate reductase enzyme, whose structure was downloaded from the Protein Data Bank (PDB ID: 1DLS) [57], was used as a potential biological target for molecular docking. The preparation of the enzyme structure for docking was carried out using the Chimera 1.14 program [58], while water and Methotrexate molecules were removed. The ligand structures were constructed and optimized by the PM3 method [59] in the ArgusLab 4.0.1 program [60][61][62][63][64]. Molecular docking was performed using the AutoDock Vina program [65] implemented in PyRx 0.8. During the docking procedure, the center of the grid, whose coordinates were: X = 23.4 Å, Y = 16.7 Å, and Z = 1.

Chemistry
The starting 2,4-dichloro-N-(2,2,2-trichloro-1-(2-(phenylcarbamothioyl)hydrazine-1-carbothioamido)e thyl)benzamide (3) was obtained by the addition reaction of N-phenylhydrazinecarbothioamide (2) [54] to 2,4-dichloro-N-(2,2,2-trichloro-1-isothiocyanatoethyl)benzamide (1) [55,56] (Scheme 1). The reaction was carried out in an acetonitrile medium, bringing the reaction mass to a boil, and then leaving it for 24 h. Hydrazinecarbothioamide (3) precipitated quantitatively from the reaction mass. The yield of the product purified by recrystallization from acetonitrile was 87%. Under the action of iodine on hydrazinecarbothioamide (3) in a DMF medium, sulfur was eliminated, HI was formed, and the target 1,3,4-thiadiazole cycle was closed. The resulting sulfur precipitated, and HI bound to the corresponding salt with triethylamine. After removing the precipitated sulfur by filtration, the target product, 2,4-dichloro-N-(2,2,2-trichloro-1-((5-(phenylamino)-1,3,4-thiadiazol-2-yl)amino)ethyl)ben zamide (4), was precipitated from the filtrate with 1% aqueous sodium thiosulfate solution. The target derivative of 1,3,4-thiadiazole was quantitatively precipitated with water, and after recrystallization from acetonitrile, the yield was 84%. The 1 H NMR spectrum of the starting hydrazinecarbothioamide (3) showed five broadened singlet NH proton signals at 10.28-7.97 ppm ( Figure S1), while the spectrum of compound 4 showed three NH proton signals, a singlet at 9.73 ppm, a broadened singlet at 9.59 ppm, and a doublet at 8.12 ppm ( Figure S2). The 13 C NMR spectrum of compound 3 was characterized by two closely located C=S carbon signals at 182.8 and 182.3 ppm ( Figure S3). In turn, in the spectrum of compound 4, there were no C=S carbon signals, but two C=N carbon signals could be observed at 157.9 and 156.6 ppm ( Figure  S4). All of the above points to the formal elimination of H2S and the closure of the 1,3,4-thiadiazole ring. The 1 H NMR spectrum of the starting hydrazinecarbothioamide (3) showed five broadened singlet NH proton signals at 10.28-7.97 ppm ( Figure S1), while the spectrum of compound 4 showed three NH proton signals, a singlet at 9.73 ppm, a broadened singlet at 9.59 ppm, and a doublet at 8.12 ppm ( Figure S2). The 13 C NMR spectrum of compound 3 was characterized by two closely located C=S carbon signals at 182.8 and 182.3 ppm ( Figure S3). In turn, in the spectrum of compound 4, there were no C=S carbon signals, but two C=N carbon signals could be observed at 157.9 and 156.6 ppm ( Figure S4). All of the above points to the formal elimination of H 2 S and the closure of the 1,3,4-thiadiazole ring.
According to the results of molecular docking, the resulting 1,3,4-thiadiazole derivative is a potential inhibitor of DHFR and can be recommended for further in vitro investigations. Further work in the development of DHFR inhibitors based on 1,3,4-thiadiazole derivatives containing an alkylamide fragment seems to be very promising.

Conflicts of Interest:
The author declares no conflict of interest.