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 Chimica Techno Acta ARTICLE published by Ural Federal University 2021, vol. 8(4), № 20218404 eISSN 2411-1414; chimicatechnoacta.ru DOI: 10.15826/chimtech.2021.8.4.04 1 of 5 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 Alena I. Siutkina ab , Ramiz R. Makhmudov ac, Daria A. Shipilovskikh d a: Perm State University, 614068 Bukireva st., 15, Perm, Russia b: Perm State Pharmaceutical Academy, 614990 Ekaterininskaya st., 101, Perm, Russia c: Federal Scientific Center for Medical and Preventive Health Risk Management Technologies, 614045 Monastyrskaya st., 82, Perm, Russia d: Perm National Research Polytechnic University, 614077 Komsomolsky Prospekt, 29, Perm, Russia * Corresponding author: syutkina.alyona@yandex.ru This article belongs to the MOSM2021 Special Issue. © 2021, The Authors. This article is published in open access form under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). Abstract The synthesis of new 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 is described. Starting 2-{[5-aryl-2-oxofuran-3(2H)- ylidene]amino}thiophene-3-carboxylic acids were obtained by in- tramolecular cyclisation of substituted 4-aryl-4-oxo-2- thienylaminobut-2-enoic acids in acetic anhydride. New derivatives of 2-[(1,4-dioxo-1-amino-4-arylbutyl-2-en-2-yl)amino]-4,5,6,7- tetrahydrobenzo[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 1H and 13C NMR spectros- copy, IR spectrometry and elemental analysis methods. Analgesic ac- tivity 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-2- en-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 analgesic activity Gewald reaction 2,4-dioxobutanoic acids 3-(thiophen-2-yl)iminofuran- 2(3H)-one Received: 01.11.2021 Revised: 19.11.2021 Accepted: 19.11.2021 Available online: 22.11.2021 1. Introduction The Gewald aminothiophene fragment is a promising pharmacophore group, since it was found in both natural and synthetic physiologically active compounds [1–6]. The synthesis of substituted Gewald aminothiophenes can be carried out using the Gewald reaction [7–9]. In turn, 3- imino(hydrazono)-3H-furan-2-ones have high reactivity, leading to the production of compounds of various struc- tures [10–17]. Decyclization reactions of 3-imino-3H- furan-2-ones lead to the production of 2,4-dioxobutanoic acid derivatives, for which antiviral [18–22], analgesic [23], anti-inflammatory [24], antimicrobial [25] activity was determined. It was previously shown that 3-imino(hydrazono)-3H- furan-2-ones can be decyclized under the action of aliphat- ic, aromatic, and heterocyclic amines to form amides of 4- aryl(tert-butyl)-4-oxo-2-amino(hydrazono)-2-eno acids [26, 27]. In this paper, synthesis and analgesic activity of new 2-[(1,4-dioxo-1-amino-4-arylbutyl-2-en-2-yl)amino]- 4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylic acid derivatives is discussed. 2. Experimental IR spectra were recorded on an FSM-1202 instrument from liquid paraffin. 1H and 13C NMR spectra were record- ed 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. http://chimicatechnoacta.ru/ https://doi.org/10.15826/chimtech.2021.8.4.04 http://creativecommons.org/licenses/by/4.0/ https://orcid.org/0000-0001-6702-7716 Chimica Techno Acta 2021, vol. 8(4), № 20218404 ARTICLE 2 of 5 Starting substituted 4-aryl-4-oxo-2-thienylaminobut-2- enoic acids 1a,b and substituted 3-thienylimino-3H-furan- 2-ones 2a,b were obtained according to the procedure de- scribed in [28–30]. All data correspond to the previously obtained ones. 2.1. General procedure for the synthesis of N- substituted amides of 4-aryl-4-oxo-2-[(3-thiophen-2- yl)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. 2.2. Ethyl 2-((1-((4-methylpyrimidin-2-yl)amino)-1,4- dioxo-4-phenylbut-2-en-2-yl)amino)-4,5,6,7- tetrahydrobenzo[b]thiophene-3-carboxylate (3a) Yield 0.36 g (74%), orange crystals, mp 172–173 °С (iso- propanol). IR spectrum, ν, cm–1: 1671 (CONH), 1738, (COOEt), 3186, 3351 (NH). 1Н NMR spectrum (CDCl3), , ppm: 1.31 t (3H, CH3CH2O, Jнн = 7.2 Hz), 1.76 m (4H, 2CH2), 2.40 s (3H, CH3), 2.57 m (2H, CH2), 2.80 m (2H, CH2), 4.37 q (2H, CH3CH2O, Jнн = 7.1 Hz), 6.19 s (1Н, С=CH), 6.83 m (2H, Harom), 7.22 m (3H, Harom), 7.45 m (2H, Harom), 8.43 s (1Н, NН), 10.21 s (1Н, NH). Found, %: С 63.60; Н 5.37; N 11.43; S 6.52. C26H26N4O4S. Calculated, %: С 63.66; Н 5.34; N 11.42; S 6.54. 2.3. Ethyl 2-((1-((5-bromopyridin-2-yl)amino)-1,4- dioxo-4-phenylbut-2-en-2-yl)amino)-4,5,6,7- tetrahydrobenzo[b]thiophene-3-carboxylate (3b) Yield 0.40 g (73%), orange crystals, mp 188–189 °С (iso- propanol). IR spectrum, ν, cm–1: 1667 (CONH), 1708, (COOEt), 3323, 3416 (NH). 1Н NMR spectrum (CDCl3), , ppm: 1.40 t (3H, CH3CH2O, Jнн = 6.9 Hz), 1.76 m (4H, 2CH2), 2.58 m (2H, CH2), 2.77 m (2H, CH2), 4.36 q (2H, CH3CH2O, Jнн = 6.9 Hz), 6.17 s (1Н, С=CH), 6.71 s (1Н, NН), 7.27 m (3H, Harom), 7.38 m (2H, Harom), 7.83 m (1H, Harom), 8.13 m (1H, Harom), 8.37 m (1H, Harom), 10.49 s (1Н, NH). Found, %: С 56.37; Н 4.33; N 7.56; S 5.74. C26H24BrN3O4S. Calculated, %: С 56.32; Н 4.36; N 7.58; S 5.78. 2.4. Ethyl 2-((1,4-dioxo-4-phenyl-1-(thiazol-2- ylamino)but-2-en-2-yl)amino)-4,5,6,7- tetrahydrobenzo[b]thiophene-3-carboxylate (3c) Yield 0.41 g (85%), yellow crystals, mp 200–201 °С (iso- propanol). IR spectrum, ν, cm–1: 1663 (CONH), 1718, (COOEt), 3234, 3439 (NH). 1Н NMR spectrum (DMSO-d6), , ppm: 1.38 t (3H, CH3CH2O, Jнн = 7.2 Hz), 1.74 m (4H, 2CH2), 2.63 m (2H, CH2), 2.75 m (2H, CH2), 4.35 q (2H, CH3CH2O, Jнн = J 7.2 Hz), 6.25 s (1Н, С=CH), 7.38 m (5H, Harom), 7.62 m (2H, Harom), 8.12 s (1Н, NH), 10.44 s (1Н, NH). Found, %: С 59.83; Н 4.85; N 8.72; S 13.36. C24H23N3O4S2. Calculated, %: С 59.86; Н 4.81; N 8.73; S 13.31. 2.5. 2-((1-((4-Bromophenyl)amino)-4-(4- methoxyphenyl)-1,4-dioxobut-2-en-2-yl)amino)- 4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (3d) Yield 0.47 g (84%), orange crystals, mp 193–195 °С (iso- propanol). IR spectrum, ν, cm–1: 1666, 1686 (CONH, CONH2), 3294 (NH, NH2). 1Н NMR spectrum (DMSO-d6), , ppm: 1.69 m (4Н, СН2), m 2.50 (2H, CH2), 2.58 m (2H, CH2), 3.84 s (3H, OCH3), 6.37 s (1Н, С=CH), 7.04 m (2H, Harom), 7.47 m (2H, NH2; 4H, Harom), 8.02 m (2H, Harom), 11.17 s (1Н, NH), 12.67 s (1Н, NH). Found, %: С 56.30, Н 4.35, N 7.53, S 5.77. C26H24BrN3O4S. Calculated, %: С 56.32, Н 4.36, N 7.58, S 5.78. 2.6. 2-((4-(4-methoxyphenyl)-1-morpholino-1,4- dioxobut-2-en-2-yl)amino)-4,5,6,7- tetrahydrobenzo[b]thiophene-3-carboxamide (3e) Yield 0.41 g (87%), yellow crystals, mp 146–148 °С (iso- propanol). IR spectrum, ν, cm–1: 1658 (CON, CONH2), 3169, 3344 (NH, NH2). 1Н NMR spectrum (DMSO-d6), , ppm: 1.73 m (4Н, 2СН2), 2.62 m (4H, 2CH2), 3.48 m (8H, 4CH2), 3.83 s (3H, OCH3), 6.14 s (1Н, С=CH), 7.00 m (2H, Harom), 7.40 br s (1H, NH2), 7.49 br s (1H, NH2), 7.97 m (2H, Harom), 12.94 s (1Н, NH). Found, %: С 61.35, Н 5.82, N 8.96, S 6.80. C24H27N3O5S. Calculated, %: С 61.39, Н 5.80, N 8.95, S 6.83. 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 intraperitone- ally 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 per- formed 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 try- ing 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 de- fensive reflex of no more than 15 s. Each compound was tested on 6 animals. The results were evaluated by in- creasing the time of the onset of the defensive reflex com- pared 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 Chimica Techno Acta 2021, vol. 8(4), № 20218404 ARTICLE 3 of 5 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. 3. Results and discussion Starting furanones 2a,b were obtained by known literature method via intramolecular cyclization of 4-aryl-4-oxo-2- tienylaminobut-2-enoic acids 1a,b in acetic anhydride. The reaction of 3-thienylimino-3H-furan-2-ones 2a,b with al- kyl-, aryl-, hetarylamines in inert aprotic solvent proceed- ed with the formation of N-substituted amides of but-2- enic 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 nucleo- philic reagents was published based on the detailed large- scale study of kinetic data [34–36] 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 mecha- nism for the transformations 3-thienylimino-3H-furan-2- ones 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 elec- tron pair (aprotic nonpolar or weakly polar solvents), a non-catalytic reaction occurs. The use of a nonpolar sol- vent 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 for- mation 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 1H 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 hydro- gen 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 stud- ied compounds have a pronounced analgesic effect, sur- passing the effect of the comparison drug metamizole. Table 1 Analgesic activity of amides 3a-c Compound Dosage, mg/kg The latent period of the defensive reflex (120 min), s 3a 50 21.20±1.24 3b 50 22.40±1.83 3c 50 21.00±1.46 Metamizole 93 (ED50) 16.60±1.00 Control – 10.30±0.60 4. Conclusions New 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 were obtained with 73-87% yields by the decyclization reaction of 2-{[5-aryl-2-oxofuran-3(2H)- ylidene]amino}thiophene-3-carboxylic acids under the action of aliphatic, aromatic and heterocyclic amines. It was found that the obtained compounds exhibited signifi- cant analgesic activity, reliably exceeding the effect of a referral drug. Scheme 1 The reaction of 3-thienylimino-3H-furan-2-ones 2a,b with alkyl-, aryl-, hetarylamines in inert aprotic solvent Chimica Techno Acta 2021, vol. 8(4), № 20218404 ARTICLE 4 of 5 Scheme 2 The transformations 3-thienylimino-3H-furan-2-ones 2a,b under the action of amines as nucleophiles (NuH) Acknowledgements The research was supported by the Perm Research and Education Centre for Rational Use of Subsoil, 2021. References 1. 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