Synthesis and antiradical activity of 4-aryl (hetaryl)-substituted 3 ...

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such as amrinone.3 Certain derivatives of 3-aminopyridin-. 2-(1Н)-ones possess antiviral, including antiHIV,4,5 activity or act as thrombin inhibitors.6 The ...
DOI 10.1007/s10593-016-1809-7 Chemistry of Heterocyclic Compounds 2015, 51(11/12), 991–996

Synthesis and antiradical activity of 4-aryl(hetaryl)substituted 3-aminopyridin-2(1Н)-ones Ivan V. Kulakov1*, Mariya V. Matsukevich1, Zarina T. Shulgau3, Shyngys Sergazy3, Tulegen M. Seilkhanov4, Amrit Puzari5, Alexander S. Fisyuk1,2 1

Department of Organic Chemistry, F. M. Dostoevskii Omsk State University, 55a Mira Ave., Omsk 644077, Russia; [email protected] 2 Omsk State Technical University, 11 Mira Ave., Omsk 644050, Russia; [email protected] 3 RSE " National Center for Biotechnology" of the Ministry of Education and Science, 13/5 Kurgalzhynskoe road, Astana 010000, Kazakhstan; [email protected] 4 Sh. Ualikhanov Kokshetau State University, 76 Abaya St., Kokshetau 020000, Kazakhstan; [email protected] 5 Department of Science & Humanities, National Institute of Technology Nagaland, Pin: 797 103, Nagaland, India; [email protected] Submitted November 4, 2015 Accepted November 25, 2015

Translated from Khimiya Geterotsiklicheskikh Soedinenii, 2015, 51(11/12), 991–996

The reaction of aryl(hetaryl)-substituted 1,3-diketones with chloroacetamide gave the respective N-(3-oxoalkenyl)chloroacetamides, which were converted to 3-pyridinium-substituted pyridin-2(1Н)-ones upon heating with excess of pyridine in n-butanol. The decomposition of pyridinium salts with hydrazine hydrate resulted in new 4-aryl(hetaryl)-substituted 3-aminopyridin-2(1Н)-ones, which showed strong antiradical activity with respect to ABTS and DPPH radicals. Keywords: cyclization.

3-aminopyridin-2(1Н)-ones, 1,3-diketones, N-(3-oxoalkenyl)chloroacetamides, antiradical activity, intramolecular

3-Aminopyridin-2(1Н)-ones are known to be of interest as potential biologically active compounds.1,2 Some medicinal compounds of this class have been accepted in clinical practice, such as amrinone.3 Certain derivatives of 3-aminopyridin2-(1Н)-ones possess antiviral, including antiHIV,4,5 activity or act as thrombin inhibitors.6 The presence of an amino acid structure offers opportunities for using these compounds as building blocks in the synthesis of peptide mimics.7–9 The preparation of 3-aminopyridin-2(1Н)-ones 3 has been frequently based on 3-functionalized pyridin-2(1Н)ones 2,4,10–14 which in turn have been prepared by the condensation of 1,3-diketones 1 or enamino ketones with amides of α-functionalized acetic acids. Such condensation occurs as a Knoevenagel reaction between the amide as СНacidic component and the more active carbonyl group of the unsymmetrical 1,3-diketone, followed by heterocyclization of the obtained intermediate. In the case of 1-alkyl3-aryl-1,3-diketones, the reaction leads to 4-alkyl-6-arylsubstituted pyridin-2(1Н)-ones15–17 (Scheme 1), which can not be converted to 4-aryl-3-aminopyridin-2(1Н)-ones. 0009-3122/15/51(11/12)-0991©2015 Springer Science+Business Media New York

991

Apparently, for this reason only a few examples for the synthesis of such compounds are known.18–21 At the same time, there is another approach for the synthesis of substituted pyridin-2(1Н)-ones, based on the cyclization of N-(3-oxoalkenyl)amides.22–25 In this case, the first step of synthesis involved an interaction of 1,3-diketones 1a–c with ammonia, which occurred at the more reactive carbonyl group (Scheme 2). The subsequent acylation of enamino ketones 4a–c with chloroacetyl chloride led to N-(3-oxoalkenyl)chloroacetamides 5a–c, followed by nucleophilic substitution of chlorine atom with pyridine giving the open form of pyridinium salts 6a–c, which were transformed by intramolecular Knoevenagel cyclization to the pyridin-2(1Н)-ones 7a–c (Scheme 2). These two complementary approaches were used to convert 1,3-diketones 1 to pyridin-2(1Н)-ones with various sets of substituents at the ring positions 4 and 6. We previously demonstrated such a possibility in the synthesis of alkyl derivatives of pyridin-2(1Н)-one 8a,c, as well as 3-amino4-phenylpyridin-2(1Н)-one 8b.24

Chemistry of Heterocyclic Compounds 2015, 51(11/12), 991–996 Scheme 1

Scheme 2

The biological studies with 3-amino-4,6-dimethylpyridin-2(1Н)-one (8а) revealed a strong antiradical activity.26 The modification of compound 8а at the 3-amino group did not improve its antiradical activity.26 Therefore, we set out to search for new antioxidants among 3-aminopyridin-2(1Н)-ones with various substituents at positions 4–6. During this project, we synthesized a series of new 3-amino-4-aryl(hetaryl)pyridin-2(1Н)-ones 8d–h for the purpose of testing these compounds for possible antiradical activity. Previously we used a simplified one-step method for the synthesis of N-(3-oxoalkenyl)chloroacetamides 5a,b,24 based on the condensation of 1,3-diketones 1a,b with α-chloroacetamide, avoiding the intermediate step of obtaining the enamino ketones 4a,b. The same method was utilized by us for the preparation of compounds 5d–h. The starting compounds were 1,3-diketones 1d–h, obtained by condensation of aryl(hetaryl) methyl ketones27 with ethyl acetate.28–30 Heating of the 1,3-diketones 1d–h with α-chloroacetamide in benzene for 24 h led to the formation of N-(3-oxoalkenyl)chloroacetamides 5d–h in satisfactory yields (Scheme 2). Nucleophilic substitution of the halogen atom in compounds 5d–h and the subsequent cyclization of the intermediate pyridinium salts 6d–h occurred as a one-pot synthesis with the formation of 3-pyridinium-substituted pyridin-2(1Н)-ones 7d–h in 30– 60% yields. The 3-aminopyridin-2(1H)-ones 8d–h were obtained in 65–86% yields by heating compounds 7d–h in an 80% hydrazine hydrate solution. The structure of all

compounds was confirmed unequivocally by IR spectroscopy, elemental analysis, as well as 1Н and 13С NMR spectroscopy data. The antiradical (antioxidative) properties of antioxidants are largely based on the ability of these compounds to "quench" active radicals, thus the estimation of this ability is a common method for quantitative characterization of antiradical properties. Antiradical activity evaluation may be performed with respect to specific radicals (ABTS+• or DPPH•), as well as by nonspecific reduction of certain substrates. In order to identify new antioxidants among the previously synthesized compounds 8a–с24 and the newly obtained 3-amino-4-aryl(hetaryl)pyridin-2(1H)-ones 8d–h, we performed antiradical activity tests with respect to two radicals: 1) 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS+•);31 2) 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•).32 The method of antiradical activity determination for compounds with respect to the ABTS+• radical cation is based on the fact that ABTS+• is a stable species, which can exist in aqueous solutions for a sufficiently long time, but interacts with various antiradical agents added to the solution and is rapidly consumed by such reactions, resulting in the quenching of ABTS+•. The consumption of ABTS+• is accompanied by characteristic spectral changes, which enable recording of the reaction rate. The possibilities of adjusting the initial concentration of 992

Chemistry of Heterocyclic Compounds 2015, 51(11/12), 991–996 Table 2. The IC50(DPPH) values of compounds 8a–h

radicals in the system and control of the quenching rate have led to a widespread use of ABTS+• for the standardization of antiradical activity of various compounds. In this work, we have compared the quenching (inhibition) dynamics of ABTS+• in the presence of study compounds and a reference compound. In our case a semisynthetic water-soluble analog of vitamin Е was used which is also known by the brand name of Trolox. The use of Trolox allowed us to evaluate the antiradical effectiveness in accordance with the Trolox Equivalent Antioxidant Capacity (TEAC). The TEAC values correspond to the concentration of Trolox in mmol/l (mM), which quenches ABTS+• with the same effectiveness as the analyzed compounds at 1 mM concentration. All the study compounds were tested at the concentration of 0.1 mM. The statistical processing of results was performed with the Statistica 6.0 software. The obtained results are presented in Table 1 as mean values ± standard deviation of the mean value.33 Apparently, compound 8f showed practically no antiradical activity with respect to the ABTS+• radical cation under the conditions of this test, while compounds 8a and 8e showed moderate activity, and in the case of compounds 8b–d,g,h the activity was slightly above that of Trolox.

Compound 8a 8b

TEAC, mM

Compound

25.6 30.4

8h

23.0

Ascorbic acid

19.4

8c

15.1

8d

13.1

8e

15.6

–* 20.8

* The IC50(DPPH) value could not be measured.

According to literature data,34 the half maximal inhibitory concentration of 2,2-diphenyl-1-picrylhydrazyl radical, IC50 (DPPH), for a series of standard antioxidants ranges from 8 (for quercetin) to 49 µМ (for glutathione). This means that the study compounds, IC50(DPPH) values of which is from 13.1 to 30.4 µМ, have a great potential for studying their antioxidant properties. Thus, we have developed a simple and convenient method for the preparation of 3-amino-4-aryl(hetaryl)substituted pyridin-2(1Н)-ones, which were otherwise difficult to obtain, from the respective aryl(hetaryl)-substituted 1,3-diketones. The antiradical activity of some 4-aryl(hetaryl)-substituted 3-aminopyridin-2(1Н)-ones with respect to the DPPH radical and ABTS+• radical cation was shown to be as strong as the antiradical activity of such commonly known antioxidants such as Trolox and ascorbic acid.

Table 1. The TEAC values of compounds 8a–h Compound

IC50(DPPH), M

Compound 8f 8g

IC50(DPPH), M

TEAC, mM

8a

0.68±0.03 (n = 6) *

8e

0.88 ± 0.03 (n = 4)

8b

1.28±0.06 (n = 4)

8f

0.28±0.02 (n = 4)

8c

1.33±0.08 (n = 4)

8g

1.24 ± 0.06 (n = 4)

8d

1.20 ± 0.05 (n = 4)

8h

1.50 ± 0.15 (n = 3)

Experimental IR spectra were recorded on an FT-801 FT-IR spectrometer in KBr pellets. 1H and 13С NMR spectra were acquired on a JEOL JNM-ECA 400 instrument (400 and 100 MHz, respectively) in CDCl3 (compounds 5d–h, 8d–h) and DMSO-d6 (compounds 7d–h) with TMS as internal standard. 13С NMR signals were assigned by using the APT (Attached Proton Test). Melting points were determined on a Kofler bench. Elemental analysis was performed with a Carlo Erba 1106 CHN analyzer. The reaction progress and purity of the isolated compounds were controlled by TLC on Sorbfil plates. 2-Chloro-N-[4-(4-methoxyphenyl)-4-oxobut-2-en-2-yl]acetamide (5d). A mixture of diketone 1d (3.85 g, 20 mmol), α-chloroacetamide (2.40 g, 26 mmol), and p-toluenesulfonic acid monohydrate (0.95 g, 5 mmol) in benzene (30 ml) was refluxed with a Dean–Stark trap for 24 h. After the reaction was complete, the reaction mixture was filtered to remove the excess of chloroacetamide, the precipitate was washed with benzene. The organic fractions were combined, washed with water until neutral pH, and dried over anhydrous sodium sulfate. Benzene was removed by distillation at reduced pressure. The oily product was purified by recrystallization from hexane (or its mixture with a suitable solvent). Yield 3.63 g (68%), white crystals, mp 48-50°C. 1H NMR spectrum, δ, ppm (J, Hz): 2.55 (3H, d, 4J = 0.9, СН3); 3.86 (3H, s, ОСН3); 4.20 (2H, s, СH2Cl); 6.11 (1H, br. s, =СH); 7.04 (2H, d, 3 J = 8.7, Н-3,5 Ar); 7.96 (2Н, d, J = 8.7, H-2,6 Ar), 13.20 (1Н, br. s, NH). 13C NMR spectrum, δ, ppm: 22.2 (CH3);

* n – number of repetitions.

The antiradical activity of the study samples was tested with respect to DPPH radical by using a methanol solution of DPPH (100 µМ), to which 20 μl of the study compound solution in methanol was added. The final concentrations of the study compounds thus were in the range from 5 to 50 µМ. A compound with known antiradical properties, ascorbic acid, was used as a reference compound. The antiradical activity of the same compounds with respect to the DPPH radical was expressed by the determined values of IC50(DPPH) (Table 2) – the concentrations of study compounds resulted in 50% optical density decrease of 100 µМ DPPH radical solution (the half maximal inhibitory concentration). The activity of compound 8f did not reach a sufficient level for the calculation of IC50, since the optical density of 100 µМ solution of DPPH radical after 10 min of incubation with the study compound at the final 50 µМ concentration was not different from the optical density of reference solution without the study compound. Compounds 8c–e showed a pronounced antiradical activity during the test with DPPH radical, with IC50(DPPH) values even lower than those of the reference antioxidant, ascorbic acid. 993

Chemistry of Heterocyclic Compounds 2015, 51(11/12), 991–996

reaction mixture was refluxed for 5 h. The solution was diluted with hexane and cooled, the salt that precipitated was filtered off, washed several times with a cold 1:1 mixture of 2-propanol–hexane, and dried. Yield 1.08 g (33%), grayish beige crystals, mp >300°C. 1H NMR spectrum, δ, ppm: 2.35 (3H, s, 6-CH3); 3.82 (3H, s, ОCH3); 6.37 (1H, s, H-5); 7.09 (2H, d, J = 8.6, Н-3,5 Ar); 7.52 (2Н, d, J = 8.6, H-2,6 Ar); 8.20–8.22 (2H, m, H-3,5 Py+); 8.74 (1H, t, 3J = 7.7, H-4 Py+); 9.04 (2H, d, 3J = 5.4, H-2,6 Py+); 12.98 (1H, br. s, NH). 13C NMR spectrum, δ, ppm: 18.8 (CH3); 55.5 (ОСН3); 108.6 (C-5); 114.8 (C-3,5 Ar); 128.4 (C-3,5 Py+); 128.6 (C-1 Ar); 129.1 (C-2,6 Ar); 129.8 (C-3); 130.2 (C-4); 134.2 (C-6); 148.1 (C-4 Py+); 148.4 (C-2,6 Py+); 157.1 (C-4 Ar); 158.8 (C-2). Found, %: C 66.22; H 5.09; N 8.26. C18H17ClN2O2. Calculated, %: C 65.75; H 5.21; N 8.52. Compounds 7е–h were obtained analogously. 1-[4-(3,4-Dimethoxyphenyl)-6-methyl-2-oxo-1,2-dihydropyridin-3-yl]pyridinium chloride (7e). Yield 1.82 g (51%), beige crystals, mp >300°C. 1H NMR spectrum, δ, ppm: 2.38 (3H, s, 6-CH3); 3.54 (3H, s, ОCH3); 3.70 (3H, s, ОCH3); 6.46 (1H, s, H-5); 6.69–6.72 (2H, m, H-2,5 Ar); 6.90 (1H, d, 3J = 8.3, H-6 Ar); 8.20 (2H, dd, 3J = 7.8, 3 J = 5.5, H-3,5 Py+); 8.70 (1H, t, 3J = 7.8, H-4 Py+); 9.05 (2H, d, 3J = 5.6, H-2,6 Py+); 12.94 (1H, br. s, NH). 13 C NMR spectrum, δ, ppm: 18.8 (CH 3 ); 55.5 (2OCH 3 ); 106.5 (C-5); 111.1 (C-5 Ar); 111.8 (C-2 Ar); 120.7 (C-6 Ar); 125.7 (C-1,4 Ar); 126.6 (С-3); 128.0 (C-3,5 Py+); 147.4 (C-4 Py+); 147.7 (C-2,6 Py+); 148.6 (C-4 Ar); 149.0 (C-3 Ar); 149.7 (C-6); 157.9 (C-2). Found, %: C 63.91; H 5.33; N 8.25. C19H19ClN2O3. Calculated, %: C 63.60; H 5.34; N 7.81. 1-[4-(Biphenyl-4-yl)-6-methyl-2-oxo-1,2-dihydropyridin3-yl)pyridinium chloride (7f). Yield 2.31 g (62%), beige crystals, mp >300°C. 1H NMR spectrum, δ, ppm: 2.19 (3H, s, 6-СН3); 6.60 (1H, s, Н-5); 7.37–7.51 (3H, m, Н-3,4,5 Ph); 7.74 (2Н, d, 3J = 7.3, H-2,6 Ph); 7.81 (2Н, d, 3J = 8.3, H-2,6 Ar); 8.03 (2Н, d, 3J = 8.3, H-3,5 Ar); 8.20 (2H, m, H-3,5 Py+); 8.69 (1H, t, 3J = 7.9, H-4 Py+); 9.07 (2H, d, 3 J = 5.5, H-2,6 Py+); 12.86 (1H, br. s, NH). 13C NMR spectrum, δ, ppm: 18.8 (CH3); 106.2 (C-5); 126.6 (C-3); 126.8 (C-2,6 Ar); 127.0 (C-3,5 Ar); 127.6 (C-2,6 Ph); 128.0 (C-4 Ph); 128.3 (C-3,5 Py+); 129.0 (C-3,5 Ph), 129.1 (C-1 Ar); 132.6 (C-4); 135.1 (C-6); 138.8 (C-4 Ar); 141.0 (C-1' Ph); 144.9 (C-4 Py+); 147.5 (C-2,6 Py+); 157.9 (C-2). Found, %: C 74.19; H 4.85; N 7.75. C23H19ClN2O. Calculated, %: C 73.69; H 5.11; N 7.47. 1-[6-Methyl-2-oxo-4-(2-thienyl)-1,2-dihydropyridin3-yl]pyridinium chloride (7g). Yield 1.30 g (43%), white crystals, mp 298–300°C. 1H NMR spectrum, δ, ppm: 2.39 (3H, s, 6-CH3); 6.71 (1H, s, H-5); 7.11 (1H, dd, 3J = 5.0, 3 J = 3.7, H-4 thiophene); 7.24 (1H, dd, 3J = 3.7, 4J = 1.4, H-5 thiophene); 7.79 (1H, dd, 3J = 5.0, 4J = 1.4, H-3 thiophene); 8.35–8.39 (2H, dd, 3J = 7.8, 3J = 6.5, H-3,5 Py+); 8.87 (1H, t, 3J = 7.8, H-4 Py+); 9.20 (2H, d, 3J = 6.4, H-2,6 Py+); 12.90 (1H, br. s, NH). 13C NMR spectrum, δ, ppm: 18.7 (CH3); 104.4 (C-5); 125.1 (C-3); 128.4 (C-3 thiophene); 129.0 (C-3,5 Py+); 130.7 (C-4 thiophene); 131.7 (C-5 thiophene); 133.8 (C-2 thiophene); 140.8 (C-4);

43.1 (CH2); 55.1 (OCH3); 103.0 (=CH); 113.8 (C-3,5 Ar); 127.5 (C-1 Ar); 128.9 (C-2,6 Ar); 149.0 (=C–N); 158.5 (C-4 Ar); 167.4 (NHCO); 189.6 (CO). Found, %: C 58.62; H 5.02; N 5.49. C13H14ClNO3. Calculated, %: C 58.33; H 5.27; N 5.23. Compounds 5е–h were obtained analogously. 2-Chloro-N-[4-(3,4-dimethoxyphenyl)-4-oxobut-2-en2-yl]acetamide (5e). Yield 3.32 g (56%), white crystals, mp 115–117°C (hexane). 1H NMR spectrum, δ, ppm (J, Hz): 2.53 (3H, d, J = 0.9, СН3); 3.95 (3H, s, ОСН3); 3.96 (3H, s, ОСН3); 4.15 (2H, s, СH2Cl); 6.15 (1H, br. s, =СH); 6.89 (1H, d, J = 9.2, Н-5 Ar); 7.55–7.57 (2Н, m, H-2,6 Ar), 13.37 (1Н, br. s, NH). 13C NMR spectrum, δ, ppm: 22.4 (CH3); 43.2 (CH2); 56.0 (2OCH3); 103.2 (=CH–); 109.9 (C-2 Ar), 110.2 (C-5 Ar), 122.0 (C-6 Ar), 131.2 (C-1 Ar), 149.0 (=C–N); 153.0 (C-4 Ar); 154.6 (C-3 Ar); 166.2 (NHCO); 190.0 (CO). Found, %: C 56.71; H 5.11; N 4.36. C14H16ClNO4. Calculated, %: C 56.48; H 5.42; N 4.70. 2-Chloro-N-[4-(biphenyl-4-yl)-4-oxobut-2-en-2-yl]acetamide (5f). Yield 2.94 g (47%), white crystals, mp 152– 155°C (1:1 hexane–chloroform). 1H NMR spectrum, δ, ppm (J, Hz): 2.21 (3H, s, СН3); 4.15 (2H, s, СH2Cl); 6.21 (1H, br. s, =СH); 7.46 (3H, t, J = 7.3, Н-3,4,5 Ph); 7.62 (2Н, d, J = 7.4, H-2,6 Ph); 7.67 (2Н, d, J = 8.7, H-3,5 Ar); 7.95 (2Н, d, J = 8.2, H-2,6 Ar), 12.95 (1Н, br. s, NH). 13 C NMR spectrum, δ, ppm: 25.5 (CH3); 43.1 (CH2); 96.9 (=CH); 127.4 (C-2,6 Ar); 127.8 (C-4 Ph); 128.1 (C-3,5 Ar); 129.0 (C-2,6 Ph); 129.8 (C-3,5 Ph); 132.7 (C-1 Ar); 138.4 (C-4 Ar); 140.5 (C-1 Ph); 149.2 (=C–N); 168.2 (NHCO); 191.0 (CO). Found, %: C 69.11; H 4.95; N 4.21. C18H16ClNO2. Calculated, %: C 68.90; H 5.14; N 4.46. 2-Chloro-N-[4-oxo-4-(2-thienyl)but-2-en-2-yl]acetamide (5g). Yield 3.65 g (75%), white crystals, mp 81–83°C (hexane). 1H NMR spectrum, δ, ppm: 2.52 (3H, d, 4J = 0.9, =ССH3); 4.14 (2H, s, CH2Cl); 6.02 (1Н, br. s, =СH); 7.14 (1H, dd, 3J = 5.0, 3J = 3.7, Н-4 thiophene); 7.64 (1H, dd, 3 J = 5.0, 4J = 0.9, Н-3 thiophene); 7.73 (1H, dd, 3J = 3.7, 4 J = 0.9, H-5 thiophene), 13.10 (1Н, br. s, NH). 13C NMR spectrum, δ, ppm: 22.3 (CH3); 43.2 (CH2); 103.6 (=CH); 128.3 (C-4 thiophene); 130.9 (C-3 thiophene); 133.7 (C-5 thiophene); 145.1 (C-2 thiophene); 155.1 (=C–N); 166.1 (NHCO); 183.8 (CO). Found, %: C 49.53; H 3.88; N 5.31. C10H10ClNO2S. Calculated, %: C 49.28; H 4.14; N 5.75. 2-Chloro-N-[4-(5-methylfuran-2-yl)-4-oxobut-2-en2-yl]acetamide (5h). Yield 3.24 g (67%), white crystals, mp 73–75°C (hexane). 1H NMR spectrum, δ, ppm: 2.37 (3H, s, СH3 furan); 2.47 (3H, d, 4J = 0.9, =ССH3); 4.09 (2H, s, CH2Cl); 5.96 (1Н, br. s, =СH); 6.15 (1H, d, 3 J = 3.2, Н-4 furan); 7.11 (1H, d, 3J = 3.2, Н-3 furan), 13.05 (1Н, br. s, NH). 13C NMR spectrum, δ, ppm: 14.0 (СH3 furan), 22.1 (CH3); 43.1 (CH2); 103.2 (=CH); 109.4 (C-4 furan); 118.6 (C-3 furan); 151.7 (=C–N); 154.7 (C-2 furan); 157.9 (C-5 furan); 166.1 (NHCO); 179.0 (CO). Found, %: C 54.78; H 5.22; N 5.44. C11H12ClNO3. Calculated, %: C 54.67; H 5.01; N 5.80. 1-[4-(4-Methoxyphenyl)-6-methyl-2-oxo-1,2-dihydropyridin-3-yl]pyridinium chloride 7d. Pyridine (2.4 ml, 30.0 mmol) was added to a solution of chloroacetamide 5d (2.67 g, 10.0 mmol) in anhydrous n-butanol (20 ml). The 994

Chemistry of Heterocyclic Compounds 2015, 51(11/12), 991–996

148.1 (C-2,6 Py+); 148.3 (C-4 Py+); 149.0 (C-6); 158.2 (C-2). Found, %: C 59.49; H 4.58; N 8.85. C15H13ClN2OS. Calculated, %: C 59.11; H 4.30; N 9.19. 1-[6-Methyl-4-(5-methylfuran-2-yl)-2-oxo-1,2-dihydropyridin-3-yl]pyridinium chloride (7h). Yield 1.63 g (54%), white crystals, mp >300°C. 1H NMR spectrum, δ, ppm: 1.93 (3H, s, 5-CH3 furan); 2.34 (3H, s, 6-CH3); 6.28 (1H, d, 3J = 3.7, H-4 furan); 6.74 (1H, s, H-5); 7.04 (1H, d, 3 J = 3.7, H-3 furan); 8.33–8.36 (2H, m, H-3,5 Py+); 8.87 (1H, t, 3J = 7.8, H-4 Py+); 9.17 (2H, d, 3J = 5.5, H-2,6 Py+); 12.75 (1H, br. s, NH). 13C NMR spectrum, δ, ppm: 12.9 (СH3 furan), 18.7 (CH3); 100.6 (C-5); 109.4 (C-4 furan); 117.6 (C-3 furan); 121.9 (C-3); 128.3 (C-3,5 Py+); 135.3 (C-4); 144.8 (C-6); 147.6 (C-4 Py+); 147.8 (C-2,6 Py+); 148.4 (C-2 furan); 156.4 (C-5 furan); 158.5 (C-2). Found, %: C 63.82; H 4.86; N 8.88. C16H15ClN2O2. Calculated, %: C 63.48; H 4.99; N 9.25. 3-Amino-4-(4-methoxyphenyl)-6-methylpyridin-2(1H)one (8d). The pyridinium salt 7d (2.93 g, 10 mmol) was dissolved in 80% hydrazine hydrate (4.0 ml, 50.0 mmol). The mixture was refluxed for 3 h, cooled, and diluted with water (5 ml). The precipitate that formed was filtered off and recrystallized from aqueous ethanol or 2-propanol. Yield 1.97 g (86%), beige crystals, mp 218–219°C (2-propanol). 1H NMR spectrum, δ, ppm: 2.30 (3H, s, 6-СН3); 3.86 (3H, s, ОСН3); 4.16 (2H, br. s, NH2); 5.96 (1H, s, H-5); 6.99 (2H, d, J = 8.7, Н-3',5' Ar); 7.44 (2Н, d, J = 8.7, H-2',6' Ar); 12.33 (1H, br. s, NH). 13C NMR spectrum, δ, ppm: 18.4 (CH3); 55.3 (ОСН3); 108.1 (C-5); 114.3 (C-3,5 Ar); 127.8 (C-1 Ar); 129.3 (C-2,6 Ar); 130.1 (C-4, 3); 130.8 (C-6); 159.1 (C-4 Ar); 159.7 (C-2). Found, %: C 68.08; H 6.47; N 12.56. C13H14N2O2. Calculated, %: C 67.81; H 6.13; N 12.17. Compounds 8е–h were obtained analogously. 3-Amino-4-(3,4-dimethoxyphenyl)-6-methylpyridin2(1H)-one (8e). Yield 1.69 g (65%), beige crystals, mp 210– 212°C (1:1 EtOH–H2O). 1H NMR spectrum, δ, ppm: 2.32 (3H, s, 6-СН3); 3.91 (3H, s, ОСН3); 3.93 (3H, s, ОСН3); 4.21 (2H, br. s, NH2); 5.97 (1H, s, H-5); 6.96 (1H, d, J = 8.2, Н-5' Ar); 7.04 (1Н, s, H-2' Ar); 7.07 (1Н, d, J = 8.2, H-6' Ar); 12.62 (1H, br. s, NH). 13C NMR spectrum, δ, ppm: 18.3 (CH3); 55.9 (2 OCH3); 108.1 (C-5); 111.1 (C-2 Ar), 111.3 (C-5', Ar), 120.5 (C-6 Ar), 128.1 (C-1 Ar), 130.4 (C-3); 130.8 (C-4); 131.2 (C-6); 148.5 (C-4 Ar); 149.1 (C-3 Ar); 159.8 (C-2). Found, %: C 64.86; H 5.76; N 10.30. C14H16N2O3. Calculated, %: C 64.60; H 6.20; N 10.76. 3-Amino-4-(biphenyl-4-yl)-6-methylpyridin-2(1H)one (8f). Yield 2.09 g (76%), beige crystals, mp 254–255°C (EtOH). 1H NMR spectrum, δ, ppm: 2.32 (3H, s, 6-СН3); 4.27 (2H, br. s, NH2); 6.01 (1H, s, Н-5); 7.36–7.49 (3H, m, Н-3,4,5 Ph); 7.58 (2Н, d, J = 8.3, H-2,6 Ar); 7.63–7.65 (2Н, m, H-2,6 Ph); 7.69 (2Н, d, J = 8.3, H-3,5 Ar); 11.69 (1Н, br. s, N-H). 13C NMR spectrum, δ, ppm: 18.5 (CH3); 108.0 (C-5); 125.9 (C-3); 127.0 (C-2,6 Ar); 129.4 (C-4 Ph); 127.6 (C-3,5 Ar); 128.5 (C-2,6 Ph); 128.9 (C-3,5 Ph), 130.7 (C-1 Ar); 131.2 (C-4); 136.8 (C-6); 140.5 (C-4' Ar); 140.7 (C-1' Ph); 159.5 (C-2). Found, %: C 78.47; H 5.54; N 10.33. C18H16N2O. Calculated, %: C 78.24; H 5.84; N 10.14.

3-Amino-6-methyl-4-(2-thienyl)pyridin-2(1Н)-one (8g). Yield 1.50 g (73%), beige crystals, mp 180-181°C (2-PrOH). 1H NMR spectrum, δ, ppm: 2.31 (3H, s, 6-CH3); 4.52 (2H, br. s, 3-NH2); 6.13 (1H, s, H-5); 7.15 (1H, dd, 3 J = 4.9, 3J = 3.8, Н-4 thiophene); 7.38 (1H, d, 3J = 3.8, H-5 thiophene); 7.40 (1H, d, 3J = 4.9, H-3 thiophene); 12.47 (1H, br. s, NH). 13C NMR spectrum, δ, ppm: 18.3 (CH3); 107.10 (C-5); 120.7 (C-4); 125.7 (C-3 thiophene); 126.2 (C-4 thiophene); 127.7 (C-5 thiophene), 130.9 (C-3); 131.1 (C-2 thiophene); 139.8 (C-6); 160 (C-2). Found, %: C 58.52; H 5.02; N 13.24. C10H10N2OS. Calculated, %: C 58.23; H 4.89; N 13.58. 3-Amino-6-methyl-4-(5-methylfuran-2-yl)pyridin-2 (1Н)-one (8h). Yield 1.67 g (82%), light-yellow crystals, mp 194–196°C (2-PrOH). 1H NMR spectrum, δ, ppm: 2.29 (3H, s, 6-CH3); 2.39 (3H, s, 5-CH3 furan); 4.88 (2H, br. s, 3 -NH2); 6.13 (1H, d, 3J = 3.3, H-4 furan); 6.20 (1H, s, H-5); 6.57 (1H, d, 3J = 3.3, H-3 furan); 12.53 (1H, br. s, NH). 13C NMR spectrum, δ, ppm: 13.8 (СH3 furan), 18.4 (6-CH3); 103.0 (C-5); 107.8 (C-4 furan); 109.6 (C-3 furan); 116.0 (C-6); 128.9 (C-3); 130.7 (C-6); 150.2 (C-2 furan); 152.2 (C-5 furan); 160.3 (C-2). Found, %: C 64.88; H 5.56; N 13.46. C11H12N2O2. Calculated, %: C 64.69; H 5.92; N 13.72. Determination of antioxidant activity. The antiradical activity of the obtained samples was studied with respect to the radical cation ABTS+•, using the Antioxidant Assay Kit (Sigma) according to the instruction manual. The method was based on using ferrylmyoglobin radicals, obtained from metmyoglobin and hydrogen peroxide, to oxidize ABTS with the formation of radical cation – ABTS+•. The addition of various antiradical agents to the solution resulted in their interaction with ABTS+• and rapid consumption (quenching) of the latter. The consumption of ABTS+• was accompanied by characteristic spectral changes, which enabled the study of inhibition dynamics.31 The optical density changes were recorded at 405 nm wavelength on a automatic microplate reader Elx800 (Bio Tek Instruments, Inc.). Addition to the solution of various antiradical agents leads to their interaction with the radical ABTS+• and rapid consumption (quenching) of the latter in a concentration-dependent manner, and the color intensity (or optical density) decreases proportionally. The numerical value of the concentration of the test samples expressed in equivalent concentrations of Trolox (TEAC) in mmol/l, using a calibration curve.31 Proceedings: a working solution of ABTS substrate was prepared by adding 25 l of 3% hydrogen peroxide solution to 10 ml of ABTS substrate. Calibration solution (10 l) and 0.1 mM solution of the test compounds (10 l) were added to the wells of a 96-well plate. Trolox tested concentrations were 0; 0.015; 0.045; 0.105; 0.210; and 0.420 mmol/l. Working solution of myoglobin (20 l) was added into each well. ABTS substrate working solution (150 l) was added to each well. Incubated for exactly for 5 min at room temperature, after which stop solution (100 l) was pipetted to each well. Photometry was performed at 405 nm, and a calibration curve for Trolox was prepared. Calculated the TEAC values of the study objects in the program Excel, using a calibration curve.31 995

Chemistry of Heterocyclic Compounds 2015, 51(11/12), 991–996 Morrow, D. M.; Fries, H. E.; Wu, C. W.; Edwards, R. M.; Jin, J. Bioorg. Med. Chem. Lett. 2010, 20 (22), 6744. 11. Zhang, X.; Schmitt, A. C.; Decicco, C. P. Tetrahedron Letters. 2002, 43 (52), 9663. 12. Seger, H.; Geyer, A. Synthesis. 2006, 19, 3224. 13. Singh, B.; Lesher, G. Y. J. Heterocyclic Chem. 1990, 27 (7), 2085. 14. Damewood, J. R.; Edwards, P.D.; Feeney, S.; Gomes, B. C.; Steelman, G. B.; Tuthill, P. A.; Williams, J. C.; Warner, P.; Woolson, S. A.; Wolanin, D. J; Veale, C. A. J. Med. Chem. 1994, 37 (20), 3303. 15. Elzanate, A. M. Heterocycl. Comm. 2002, 8 (2), 145. 16. Elgemeie, G. E. H.; Hussain, B. A. W.; Elgemeie, G. H.; ElEzbawy, S. R.; Ramiz, M. M.; Mansour, O. A. Org. Prep. and Proc. Int. 1991, 23 (5), 645. 17. Elgemeie, G. E. H.; El-Ezbawy, S. R.; Ali, H. A.; Mansour, A.-K. Org. Prep. and Proc. Int. 1994, 26 (4), 465. 18. Biediger, R. J.; Chen, Q.; Decker, E. R.; Holland, G. W.; Kassir, J. M.; Li, W.; Market, R. V.; Scott, I. L.; Wu, C.; Li, J. US Patent 2004/63955. 19. Muraoka, M.; Morishita, K.; Aida, N.; Tanaka, M.; Yuri, M.; Ohashi, N. US Patent 6300500. 20. Dominguez, C.; Harvey, T. S.; Liu, L.; Siegmund, A. US Patent 2005020592. 21. Hanfeld, V.; Leistner, S.; Wagner, G.; Lohmann, D.; Poppe, H.; Heer, S. Pharmazie. 1988, 43 (10), 677. 22. Fisyuk, A. S.; Bogza, Y. P.; Poendaev, N. V.; Goncharov, D. S. Chem. Heterocycl. Compd. 2010, 46, 844. [Khim. Geterotsikl. Soedin. 2010, 1044.] 23. Goncharov, D. S.; Kostuchenko, A. S.; Fisyuk, A. S. Chem. Heterocycl. Compd. 2009, 45, 793. [Khim. Geterotsikl. Soedin. 2009, 1005.] 24. Fisyuk, A. S.; Kulakov, I. V.; Goncharov, D. S.; Nikitina, O. S.; Bogza, Y. P.; Shatsauskas, A. L. Chem. Heterocycl. Compd., 2014, 50, 217. [Khim. Geterotsikl. Soedin. 2014, 241.] 25. Goncharov, D. S.; Garkushenko,·A. K.; Savelieva, A. P.; Fisyuk, A. S. ARKIVOC 2015, (v), 176. 26. Kulakov, I. V.; Nikitina, O. S.; Fisyuk, A. S.; Goncharov, D. S.; Shul'gau, Z. T., Gulyaev, A. E. Chem. Heterocycl. Compd., 2014, 50, 670. [Khim. Geterotsikl. Soedin. 2014, 729.] 27. Practicum on Organic Chemistry [in Russian]; V. I. Terenin; Ed.; BINOM. Laboratoriya Znanii: Moscow, 2010, p. 323. 28. Sneed, J. K.; Levine, R. J. Am. Chem. Soc. 1950, 72 (11), 5219. 29. Davis, B. R.; Hinds, M.G.; Ting, P. P. C. Austr. J. Chem., 1992, 45, 865. 30. Berti, F.; Bincoletto, S.; Donati, I.; Fontanive, G.; Fregonese, M.; Benedetti, F. Org. and Biomol. Chem., 2011, 9 (6), 1987. 31. Miller, N. J.; Rice-Evans, C.; Davies, M. J.; Gopinathan, V.; Milner, A. Clin. Sci. 1993, 84, 407. 32. Brand-Williams, W.; Cuvelier, M. E.; Berset, C. Lebensm.Wiss. Technol. 1995, 28, 25. 33. Biometriya [in Russian]; Lakin, G. F., Ed.; Vysshaya shkola: Moscow, 1980, p. 293. 34. Plattner, S.; Erb, R.; Chervet, J.-P. and Oberacher, H. Anal. Bioanal. Chem. 2014, 406 (1), 213. 35. Kamal, Z.; Ullah, F.; Ayaz, M.; Sadiq, A; Ahmad, S.; Zeb, A.; Hussain, A.; Imran, M. Biol. Res. 2015; 48, 21. DOI: 10.1186/ s40659-015-0011-1.

The antiradical activity of the samples was studied with respect to the DPPH radical by using a 100 µМ methanol solution of DPPH.32 Compounds with more pronounced antiradical activity were identified by mixing a 100 µМ methanol solution of DPPH (2 ml) with 20 μl of the study sample dissolved in methanol at 5 mM concentration. Thus, the final concentration of the studied compound in the reaction mixture was 50 µМ. The optical density decrease at 515 nm was measured 10 min after the addition of study compound to the solution of DPPH radical. The final concentrations of compounds capable of reducing the optical density by more than 50% were 50, 25, 20, 15, 10, and 5 µМ during the test with DPPH radical. After that, the concentration of study compounds producing a 50% decrease of the optical density – the IC50(DPPH) value was determined. Ascorbic acid was used as a reference compound. This study was performed with financial support from the Russian Foundation for Basic Research (project 15-5345084 IND_a). References 1. Kusakabe, K.; Tada, Y.; Iso, Y.; Sakagami, M.; Morioka, Y.; Chomei, N.; Shinonome, S.; Kawamoto, K.; Takenaka, H.; Yasui, K.; Hamana, H.; Hanasaki, K. Bioorg. Med. Chem. 2013, 21, 2045. 2. Zhang, Y.-M.; Fan, X.; Chakaravarty, D.; Xiang, B.; Scannevin, R.H.; Huang, Z.; Ma, J.; Burke, S.L.; Karnachi, P.; Rhodesa, K.J.; Jackson, P.F. Bioorg. Med. Chem. Lett. 2008, 18, 409. 3. Ward, A.; Brogden, R.N.; Heel, R. C.; Speight, T.M.; Avery, G. S. Drugs, 1983, 26(6), 468. 4. Hoffman, J. M.; Wai, J. S.; Thomas, C. M.; Levin, R. B.; O'Brien, J. A.; Goldman, M. E. J. Med. Chem. 1992, 35 (21), 3784. 5. Saari, W. S.; Wai, J. S.; Fisher, T. E.; Thomas, C. M.; Hoffman, J. M.; Rooney, C. S.; Smith, A. M.; Jones, J. H.; Bamberger, D. L. J. Med. Chem. 1992, 35 (21), 3792. 6. Sanderson, P. E.J.; Cutrona, K. J.; Savage, K. L.; NaylorOlsen, A. M.; Bickel, D. J.; Bohn, D. L.; Clayton, F. C.; Krueger, J. A.; Lewis, S.D.; Lucas, B. J.; Lyle, E. A.; Wallace, A. A.; Welsh, D. C.; Yan, Y. Bioorg. Med. Chem. Lett. 2003, 13, 1441. 7. Dragovich, P. S.; Prins, T. J.; Zhou, R.; Brown, E. L.; Maldonado, F. C.; Fuhrman, S. A.; Zalman, L. S.; Tuntland, T.; Lee, C. A.; Patick, A. K.; Matthews, D. A.; Hendrickson, T. F.; Kosa, M. B.; Liu, B.; Batugo, M. R.; Gleeson, J.-P. R.; Sakata, S. K.; Chen, L.; Guzman, M. C.; Meador, J. W.; Ferre, R. A.; Worland, S. T. J. Med. Chem. 2002, 45, 1607. 8. Verissimo, E.; Berry, N.; Gibbons, P.; Lurdes, M.; Cristiano, S.; Rosenthal, P. J.; Gut, J.; Ward, S. A., O'Neill, P. M. Bioorg. Med. Chem. Lett. 2008, 18, 4210. 9. Zhu, S.; Hudson, T. H.; Kyle, D. E.; Lin, A. J. J. Med. Chem., 2002, 45, 3491. 10. Li, Y. H.; Tseng, P.-S.; Evans, K. A.; Jaworski, J.-P.;

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