Synthesis and analgesic activity evaluation of derivatives of 2-[(1,4-dioxo-1-amino-4-arylbutyl-2-en-2-yl)amino]-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylic acid

The synthesis of new derivatives of 2-[(1,4-dioxo-1-amino-4arylbutyl-2-en-2-yl)amino]-4,5,6,7-tetrahydrobenzo[b]thiophene-3carboxylic acid is described. Starting 2-{[5-aryl-2-oxofuran-3(2H)ylidene]amino}thiophene-3-carboxylic acids were obtained by intramolecular cyclisation of substituted 4-aryl-4-oxo-2thienylaminobut-2-enoic acids in acetic anhydride. New derivatives of 2-[(1,4-dioxo-1-amino-4-arylbutyl-2-en-2-yl)amino]-4,5,6,7tetrahydrobenzo[b]thiophene-3-carboxylic acids were obtained via decyclization reaction of 2-{[5-aryl-2-oxofuran-3(2H)ylidene]amino}thiophene-3-carboxylic acids. The structure of the compounds obtained was confirmed by the H and C NMR spectroscopy, IR spectrometry and elemental analysis methods. Analgesic activity of new compounds has been studied by the “hot plate” method on outbred white mice of both sexes with intraperitoneal injection. It was found that derivatives of 2-[(1,4-dioxo-1-amino-4-arylbutyl-2en-2-yl)amino]-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylic acid possess analgesic effect exceeding the effect of the comparison drug metamizole. Keywords


Experimental
IR spectra were recorded on an FSM-1202 instrument from liquid paraffin. 1 H and 13 C NMR spectra were recorded on a Bruker Avance III instrument (400 and 100 MHz) from CDCl3 and DMSO-d6 solutions relative to residual signals of the non-deuterated solvent. Elemental analysis was performed on a Leco CHNS-932 instrument. Reaction progress and individuality of obtained compounds was monitored by TLC on Sorbfil plates, eluting with a diethyl ether-benzene-acetone system (10:9:1); detecting in UV light and iodine vapor. Melting points were determined on an SMP40 instrument.
2.1. General procedure for the synthesis of Nsubstituted amides of 4-aryl-4-oxo-2-[(3-thiophen-2yl)amino]-but-2-enoic acids 3a-e A mixture of 0.001 mol of compound 2a-e and 0.001 mol of the corresponding amine in anhydrous toluene (20 mL) was stirred at 50 °С for 2 h. After cooling, the precipitate was filtered off and recrystallized. Evaluation of analgesic activity was carried out in the Perm State National Research University, the Research Laboratory of Biologically Active Substances. Analgesic activity was determined on outbred white mice of both sexes weighing 18-22 g using the "hot plate" method [31]. The studied compounds were administered intraperitoneally in the form of a suspension in a 2% starch solution at a dose of 50 mg/kg 30 min before the animals were placed on a metal plate heated to 53.5 °C [32]. Studies were performed 30, 60, 90, 120 min after administration of the compound.
The indicator of the change in pain sensitivity was the length of time the animals stay on the hot plate until a defensive pain reflex occurs -licking the hind legs or trying to tear off all four paws from the surface of the plate. The time of onset of this reflex from the beginning of the placement of the animal on the plate was measured in seconds (latent period). The maximum duration of the latent period is the interval of 40 s. In the experiment we used animals with the initial time of the onset of the defensive reflex of no more than 15 s. Each compound was tested on 6 animals. The results were evaluated by increasing the time of the onset of the defensive reflex compared with the initial data. The control group of animals was injected with 2% starch mucus. Metamizole sodium (Farmkhimkomplekt LLC) at a dose of 93 mg/kg (ED50) was used as a comparison compound.
Statistical processing of experimental data was carried out using Student's confidence criteria. The effect was considered significant at p<0.05 [33]. The studies were carried out in accordance with all applicable international, national and institutional guidelines for the care and use of animals.

Results and discussion
Starting furanones 2a,b were obtained by known literature method via intramolecular cyclization of 4-aryl-4-oxo-2tienylaminobut-2-enoic acids 1a,b in acetic anhydride. The reaction of 3-thienylimino-3H-furan-2-ones 2a,b with alkyl-, aryl-, hetarylamines in inert aprotic solvent proceeded with the formation of N-substituted amides of but-2enic acids 3a-e (Scheme 1). As a result, it was found that the attack of the amino group was directed at the carbon atom of the lactone carbonyl moiety of compounds 2a,b and led to the products of the furanone cycle decyclization. The ester and amide groups under the conditions of the reaction did not participate in interaction with amines, which does not contradict the literature data.
The mechanism of the decyclization reaction of 5-aryl-2,3-dihydro-2,3-furandiones under the action of nucleophilic reagents was published based on the detailed largescale study of kinetic data [34][35][36] as well as quantum chemical calculations [37]. Assuming the similarity of these structures with the iminofuranones discussed in the current paper, we can assume the validity of this mechanism for the transformations 3-thienylimino-3H-furan-2ones 2a,b under the action of amines as nucleophiles (NuH) described here (Scheme 2).
If the solvent cannot be a donor or acceptor of an electron pair (aprotic nonpolar or weakly polar solvents), a non-catalytic reaction occurs. The use of a nonpolar solvent contributes to the displacement of the equilibrium from the transition state TS1 to the intermediate I that leads to the limiting stage of the process with the formation of the transition state TS2.
Compounds 3a-e are crystalline substances of orange or yellow color, obtained with yields up to 87%.
We have studied the 1 H NMR spectra of compounds 3a,b in DMSO-d6 and 3c-e CDCl3. It was established that compounds 3a-e are characterized by a proton singlet of the NH group involved in a strong intramolecular hydrogen bond at 10.44-12.94 ppm, proton signals of the NHCO group at 6.71, 8.12-11.17 ppm and a proton singlet of the CH group at 6.14-6.37 ppm.
Some of the compounds obtained were examined for analgesic activity. It is shown in Table 1 that all the studied compounds have a pronounced analgesic effect, surpassing the effect of the comparison drug metamizole.