Introduction

N-Substituted amino acids as well as their hydrazides, amides, and hydrazones have continuously attracted attention of researchers owing to their properties that are useful in technological applications as well as biological activity. They are also suitable compounds as synthons in the synthesis of various heterocyclic compounds. Cyclization of hydrazine derivatives is a well-known method for azole synthesis [1]. The five-membered heterocyclic nucleus plays a vital role in many biological activities; thus, azole derivatives have been demonstrated to possess antibacterial [2, 3], anti-inflammatory [4], antitumor [5], antifungal [3], and antimicrobial [6–9] activities. 5-Mercapto-1,2,4-triazoles containing the furyl radical are potential anti-HIV-1 agents [10], whereas substituted triazole-3-thiones have demonstrated antidepressant activity [11].

Previously, we described the cyclization of N-phenyl-β-alanine hydrazide, N-phenyl- and N-(p-tolyl)-N-carboxyphenyl-β-alanine dihydrazides, N,N′-bis(hydrazinocarbonylethyl)-1,4-phenylenediamine, and their semicarbazides and thiosemicarbazides into compounds containing one or several azole fragments [2, 12–14]. As a continuation of our search for compounds possessing antibacterial and antifungal activities, we report herein the synthesis of heterocyclic compounds from 3-(4-methylphenylamino)propanehydrazide (1) and its carbamoyl derivatives. Cyclization of N′-phenylcarbamoyl derivatives to oxazole and thiazole, oxadiazole and thiadiazole, as well as triazole derivatives was carried out. Some of the synthesized compounds were tested for antibacterial activity.

Results and discussion

N-(4-Methylphenyl)-β-alanine hydrazide (1) was obtained from N-phenyl-β-alanine by heating at reflux with hydrazine in toluene as described elsewhere [15]. Reaction of 1 in a solution of ethyl xanthogenate in ethanol, without isolation of the intermediate potassium salt of propanedithionate, and subsequent acidification of the reaction mixture with hydrochloric acid provided 5-[2-[(4-methylphenyl)amino]ethyl]-1,3,4-oxadiazol-2(3H)-thione (2) (Scheme 1). Heating of oxadiazole-2-thione 2 at reflux with hydrazine and subsequent acidification of the reaction mixture with acetic acid resulted in formation of 4-amino-2,4-dihydro-5-[2-[(4-methylphenyl)amino]ethyl]-3H-1,2,4-triazole-3-thione (3). In our previous work [16], 3 was obtained by melting N-(2-carboxyethyl)-N-(4-methylphenyl)-β-alanine with thiocarbohydrazide.

Scheme 1
scheme 1

 

Full size image

Oxadiazol-2-one 4 was synthesized by the reaction of 1 with 1,1′-carbonyldiimidazole (CDI) in dioxane. In the 1H NMR spectrum of 4, a singlet attributed to the NH proton in the 1,3,4-oxadiazole moiety is observed at 10.30 ppm, whereas the proton of the same group in its thio analogue 2 resonated at 13.99 ppm owing to the stronger deshielding effect of the thioxo group.

Reaction of 1 with 2,5-hexanedione in propan-2-ol in the presence of a catalytic amount of acetic acid provided N-(2,5-dimethyl-1H-pyrrol-1-yl)-3-[(4-methylphenyl)amino]propanamide (5). Carbon resonances ascribed to the CH3 groups in the pyrrole moiety are observed at 10.86 ppm in the 13C NMR spectrum of 5, whereas resonances at 102.77 ppm and 126.65 ppm confirm the existence of a pyrrole ring in this compound.

Acid semicarbazides and thiosemicarbazides are easily synthesized from the respective hydrazides and organic isocyanates. On cyclization, they produce oxadiazoles and triazolones and their thio analogues [2, 13]. Depending on the ratio of the reacting substances, reaction of 1 with phenyl isocyanates resulted in formation of propanehydrazide phenylcarbamoyl derivatives 6 and 9 and di(phenylcarbamoyl) derivative 10 (Scheme 2). In a similar way, phenylthiocarbamoyl propanamides 7 and 8 and di(phenylthiocarbamoyl) propanamides 11 and 12 were synthesized when phenyl isothiocyanates were used.

Scheme 2
scheme 2

 

Full size image

The rates of reaction of propanehydrazide 1 with phenyl isocyanates may be different. Its reaction with p-tolyl isothiocyanate was very facile, e.g., after 15 min the crystals of 9 started precipitating from the reaction mixture. In the 1H NMR spectra of semicarbazides 6–9, additional aromatic proton resonances are present in comparison with the spectrum of 1, and two triplets attributed to two methylene CH2 group protons are shifted downfield by 0.13–0.41 and 0.05–0.67 ppm. The introduction of the second phenylcarbamoyl group into molecules of semicarbazides 6–9 markedly increased the solubility of the compounds obtained. Semicarbazides 6–9 are soluble in DMSO and DMF only, whereas their phenylcarbamoyl derivatives 10–12 are soluble in such organic solvents as aromatic hydrocarbons, alcohols, dioxane, and tetrahydrofuran.

The direction of semicarbazide cyclization depends on the reaction conditions. At room temperature on treatment with phosphorus oxychloride or concentrated sulfuric acid, semicarbazides undergo dehydration and cyclize to oxadiazole or thiadiazole derivatives [2], whereas triazolones or triazolethiones are obtained under the action of alkali [2, 6].

Reaction of 6–9 in aqueous solutions of KOH or NH2NH2 at reflux temperature and subsequent acidification of the reaction mixtures provided 2,4-dihydro-5-[2-[(4-methylphenyl)amino]ethyl]-4-phenyl-3H-1,2,4-triazol-3-one (13), dihydrotriazolone 14, 2,4-dihydro-5-[2-[(4-methylphenyl)amino]ethyl]-4-phenyl-3H-1,2,4-triazole-3-thione (15), and dihydrotriazolethione 16, accordingly (Scheme 3). Carbamoyl groups attached to the secondary nitrogen atom in dicarbamoyl derivatives 10–12 underwent hydrolysis on treatment with alkali or hydrazine and, thus, triazole derivatives 13–16 were obtained.

Scheme 3
scheme 3

 

Full size image

In the 1H NMR spectra of 13–16, a deshielding effect of the triazole ring on the adjacent methylene group is observed. Its triplets are shifted downfield to the region of 2.57–2.64 ppm when compared with the chemical shifts of methylene group protons in the spectra of 6–9.

At room temperature when dissolved in concentrated sulfuric acid, semicarbazides 6, 7 and thiosemicarbazides 8, 9 underwent cyclization to 5-[2-[(4-methylphenyl)amino]ethyl]-N-phenyl-1,3,4-oxadiazol-2-amine (20), its dimethyl homologue 21, and their thio analogues 5-[2-[(4-methylphenyl)amino]ethyl]-N-phenyl-1,3,4-thiadiazol-2-amine (22) and dihydrothiadiazol-2-amine 23 (Scheme 4).

Scheme 4
scheme 4

 

Full size image

In the 1H NMR spectrum of 17, proton resonances of NHAr and NHAr′ are shifted downfield in comparison with the spectrum of 10 owing to the influence of the oxadiazole moiety and are observed at 8.68 and 9.75 ppm. Singlets of the NHNH group protons, which are present in the spectrum of phenylcarbamoyl semicarbazide 10 at 7.89 and 8.03 ppm, are absent. In a similar manner, in the 1H NMR spectrum of 18, proton resonances of NHAr and NHAr′ are shifted downfield in comparison with the spectrum of 11 owing to the influence of the thiadiazole moiety and are observed at 8.71 and 10.29 ppm. Singlets of the NHNH group protons, characteristic of phenylcarbamoyl semicarbazide 11 and observed at 9.56 and 9.59 ppm, are absent in the spectrum of 18. The proton in the NH group adjacent to Ar′ resonates in the region of 9.79–10.35 ppm in the 1H NMR spectra of 20–23.

N-(3,4-Diphenyl-2(3H)-thiazolylidene)-3-[(4-methylphenyl)amino]propanehydrazide (24) was synthesized by heating 8 at reflux with phenacyl bromide. The formation of 24 was supported by the presence of one resonance of an NH group at 10.90 ppm, absence of two NH group spectral signals, a new singlet at 7.99 ppm ascribed to the SCH group, and new proton resonances attributed to the aromatic moiety in the 1H NMR spectrum.

Some of the synthesized compounds were evaluated for their antibacterial activity against a strain of Rhizobium radiobacter (Agrobacterium tumefaciens) by the diffusion technique [16, 17]. R. radiobacter is a soil-borne, Gram-negative bacterium, which is a causative agent of “crown gall” disease, an economically important disease of many plants [18]. Activities of the tested compounds were compared with the known antibacterial agent ampicillin.

As seen from the data presented in Table 1, R. radiobacter is sensitive to compounds 5, 18, 22, and 23; furthermore, 1,3,4-thiadiazol-2-amines 22 and 23 are active in a broader range of concentrations, whereas 2, 3, and 21 did not exhibit antibacterial activity against the tested microorganism strain.

Table 1 Antibacterial activity of 2, 3, 5, 18, and 21–23 against Rhizobium radiobacter determined by the diffusion method
Full size table

Experimental

1H and 13C NMR spectra were recorded on a Varian Unity Inova 300 spectrometer (300 and 75 MHz, respectively) using TMS as an internal standard and DMSO-d 6 as a solvent. IR spectra were taken on a Perkin-Elmer Spectrum Bx FT-IR spectrometer. Mass spectra were recorded on a Waters Micromass ZQ 2000 instrument. Elemental analyses (C, H, N) were performed on an Elemental Analyzer CE-440, and their results were found to be in good agreement (±0.2 %) with the calculated values. Melting points were determined on an Auto probe analyzer APA 1. Monitoring of the reaction course and purity of the compounds prepared was carried out using TLC on silica gel plates (Merck, F254). Silica gel (Acros Organics, New Jersey, USA, 0.035–0.070 mm) was used for column chromatography.

3-[(4-Methylphenyl)amino]propanehydrazide (1, C10H15N3O)

A mixture of 35.84 g N-(4-methylphenyl)-β-alanine (0.2 mol) and 10 g hydrazine (0.2 mol) in 100 cm3 toluene was heated at reflux with complete azeotropic separation of the formed water. The liquid fractions were concentrated in vacuo. Recrystallization of the residue from propan-2-ol afforded 26.63 g (69 %) 1. m.p.: 135–136 °C (Ref. [15] m.p. 135–136 °C).

5-[2-[(4-Methylphenyl)amino]ethyl]-1,3,4-oxadiazol-2(3H)-thione (2, C11H13N3OS)

To 1.68 g KOH (30 mmol) dissolved in 50 cm3 ethanol, 3 cm3 CS2 (3.8 g, 50 mmol) was added dropwise and the mixture was stirred at room temperature for 15 min. Afterwards 5.79 g 1 (30 mmol) dissolved in 50 cm3 ethanol was added and the reaction mixture was heated at reflux for 24 h. The liquid fractions were concentrated in vacuo and the residue was dissolved in 150 cm3 H2O and acidified with HCl to pH 3–4. The precipitate was isolated by filtration and washed with methanol. Recrystallization from methanol afforded 5.01 g (71 %) 2. m.p.: 66–67 °C; 1H NMR (300 MHz, DMSO-d 6): δ = 2.14 (s, 3H, CH3), 2.93 (t, J = 6.60 Hz, 2H, CH2CN), 3.37 (t, J = 6.60 Hz, 2H, CH2N), 5.48 (br s, 1H, NHCH2), 6.49 (d, J = 8.40 Hz, 2H, H(2,6)Ar), 6.90 (d, J = 8.40 Hz, 2H, H(3,5)Ar), 13.99 (s, 1H, NNH) ppm; 13C NMR (75 MHz, DMSO-d 6): δ = 19.99 (CH3), 25.25 (C-8), 40.21 (C-7), 112.35 (C-2, 6), 124.58 (C-4), 129.36 (C-3, 5), 145.61 (C-1), 162.64 (C-9), 177.68 (C-10) ppm; IR (KBr): \( \bar{\nu } \) = 3,364, 3,276 (NH), 1,618 (C=N), 1,318 (C=S) cm−1; MS (ESI, 20 eV): m/z (%) = 236 ([M+1]+, 100).

4-Amino-2,4-dihydro-5-[2-[(4-methylphenyl)amino]ethyl]-3H-1,2,4-triazole-3-thione (3, C11H15N5S)

A mixture of 2.35 g 2 (10 mmol), 0.32 g NH2NH2 (10 mmol), and 30 cm3 dioxane was heated at reflux for 8 h, and then acidified with acetic acid to pH 3. The precipitate was isolated by filtration and washed with H2O until pH 7. Recrystallization from propan-2-ol afforded 1.39 g (56 %) 3. m.p.: 105–106 °C (Ref. [19], oily residue); 1H NMR, IR, and MS spectra were found to be identical with the literature ones [19].

5-[2-[(4-Methylphenyl)amino]ethyl]-1,3,4-oxadiazol-2(3H)-one (4, C11H13N3O2)

A mixture of 1.93 g 1 (10 mmol) and 1.94 g 1,1′-carbonyldiimidazole (12 mmol) in 40 cm3 dry dioxane was heated at reflux for 12 h. H2O (30 cm3) was added and the crystalline precipitate was isolated by filtration. Purification by column chromatography (acetone/hexane 1:1, R f = 0.8) afforded 1.34 g (61 %) 4. m.p.: 144–145 °C; 1H NMR (300 MHz, DMSO-d 6): δ = 2.14 (s, 3H, CH3), 2.93–2.99 (m, 2H, CH2CN), 3.18–3.27 (m, 2H, CH 2NH), 5.25 (t, J = 6.10 Hz, 1H, NHCH2), 6.49 (d, J = 8.10 Hz, 2H, H(2,6)Ar), 6.90 (d, J = 8.10 Hz, 2H, H(3,5)Ar), 10.29 (s, 1H, NNH) ppm; 13C NMR (75 MHz, DMSO-d 6): δ = 19.99 (CH3), 32.97 (C-8), 43.53 (C-7), 112.33 (C-2, 6), 124.36 (C-4), 129.32 (C-3, 5), 146.07 (C-1), 150.98 (C-9), 169.63 (C-10) ppm; IR (KBr): \( \bar{\nu } \) = 3,361, 2,975 (NH), 1,691 (C=O), 1,522 (C=N) cm−1; MS (ESI, 25 eV): m/z (%) = 220 ([M+1]+, 50).

N-(2,5-Dimethyl-1H-pyrrol-1-yl)-3-[(4-methylphenyl)amino]propanamide (5, C16H21N3O)

A mixture of 0.97 g 1 (5 mmol), 40 cm3 propan-2-ol, 0.46 g 2,5-hexanedione (10 mmol), and 1 cm3 acetic acid was heated at reflux for 12 h. Afterwards 20 cm3 cold H2O was added. Recrystallization of the precipitate from ethanol afforded 0.85 g (63 %) 5. m.p.: 131–132 °C; 1H NMR (300 MHz, DMSO-d 6): δ = 1.97 (s, 6H, 2 CH3), 2.14 (s, 3H, CH3), 2.54 (t, J = 6.90 Hz, 2H, COCH2), 3.32 (q, J = 6.6 Hz, J = 12.90 Hz, 2H, NHCH 2), 5.38 (t, J = 8.40 Hz, 1H, NH), 5.62 (s, 2H, 2 CH), 6.53 (d, J = 8.40 Hz, 2H, H(2,6)Ar), 6.91 (d, J = 8.40 Hz, 2H, H(3,5)Ar), 10.59 (s, 1H, NH) ppm; 13C NMR (75 MHz, DMSO-d 6): δ = 10.86 (2 CH3), 19.99 (CH3), 33.18 (C-8), 39.70 (C-7), 102.77 (C-11, 12), 112.23 (C-2, 6), 124.18 (C-4), 126.65 (C-10, 13), 129.31 (C-3, 5), 146.06 (C-1), 170.30 (C=O) ppm; IR (KBr): \( \bar{\nu } \) = 3,360, 3,277 (NH), 1,663 (C=O), 1,524 (CN) cm−1; MS (ESI, 25 eV): m/z (%) = 272 ([M+1]+, 70).

3-[(4-Methylphenyl)amino]-N-[(phenylcarbamoyl)amino]propanamide (6, C17H20N4O2)

To 1.93 g 1 (10 mmol) dissolved in 20 cm3 methanol, 1.09 cm3 phenyl isocyanate (1.19 g, 10 mmol) was added dropwise and heated at reflux for 1 h. The crystalline precipitate was isolated by filtration and washed with methanol. Recrystallization from DMF/H2O afforded 2.54 g (81 %) 6. m.p.: 132–133 °C; 1H NMR (300 MHz, DMSO-d 6): δ = 2.14 (s, 3H, CH3), 2.27 (t, J = 7.20 Hz, 2H, CH2CO), 3.18 (q, J = 6.90 Hz, J = 13.20 Hz, 2H, CH2NH), 4.18 (s, 2H, NHAr′), 5.28 (t, J = 5.55 Hz, 1H, NHCH2), 6.47 (d, J = 8.40 Hz, 2H, H(2,6)Ar), 6.88 (d, J = 8.40 Hz, 2H, H(3,5)Ar), 6.95 (t, J = 8.10 Hz, 1H, H(4)Ar′), 7.25 (t, J = 8.10 Hz, 2H, H(3,5)Ar′), 7.49 (d, J = 8.10 Hz, 2H, H (2,6) Ar′), 8.78, 9.02 (2 s, 2H, NHNHCO) ppm; 13C NMR (75 MHz, DMSO-d 6): δ = 19.99 (C-17), 33.38 (C-8), 39.71 (C-7), 112.16 (C-2, 6), 118.37 (C-12, 16), 121.74 (C-14), 124.03 (C-4), 128.53 (C-13, 15), 129.27 (C-3, 5), 139.61 (C-11), 146.23 (C-1), 155.95 (C-10), 170.13 (C-9) ppm; IR (KBr): \( \bar{\nu } \) = 2,829–3,311 (NH), 1,669, 1,643 (CO), 1,525 (O=C–N) cm−1; MS (APCI+, 25 eV): m/z (%) = 335 ([M+Na]+, 100).

N-[[(3,5-Dimethylphenyl)carbamoyl]amino]-3-[(4-methylphenyl)amino]propanamide (7, C19H24N4O2)

Prepared from 1.93 g 1 (10 mmol) and 1.045 cm3 3,5-dimethylphenyl isocyanate (1.41 g, 10 mmol) by following the same procedure as for the synthesis of 6 except that the duration of heating at reflux was 3 h. Yield 1.33 g (39 %); m.p.: 145–146 °C (from DMF/H2O); 1H NMR (300 MHz, DMSO-d 6): δ = 2.13 (s, 3H, CH3), 2.18 (s, 6H, 2 CH3), 2.40 (t, J = 7.05 Hz, 2H, CH2CO), 3.84 (t, J = 7.05 Hz, 2H, CH 2NH), 6.57 (d, J = 9.30 Hz, 2H, H(2,6)Ar), 6.91–7.10 (m, 5H, H(3,5)Ar + HAr′), 7.63 (s, 1H, NH), 7.95 (s, 1H, NH), 8.55 (s, 1H, NH), 9.72 (s, 1H, NH) ppm; 13C NMR (75 MHz, DMSO-d 6): δ = 20.04 (CH3Ar), 21.05 (2 CH3), 32.24 (C-8), 46.08 (C-7), 112.36 (C-2, 6), 124.33 (C-4), 127.95 (C-12, 16), 129.34 (C-3, 5), 137.25 (C-14), 137.57 (C-11), 139.40 (C-13, 15), 146.31 (C-1), 155.38 (C-9), 170.72 (C-10) ppm; IR (KBr): \( \bar{\nu } \) = 2,861–3,275 (NH), 1,668, 1,616 (CO), 1,527 (O=C–N) cm−1; MS (APCI+, 25 eV): m/z (%) = 341 ([M+1]+, 100).

3-[(4-Methylphenyl)amino]-N-[(phenylcarbamothioyl)amino]propanamide (8, C17H20N4OS)

Prepared from 1.93 g 1 (10 mmol) and 1.90 cm3 phenyl isothiocyanate (1.35 g, 10 mmol) by following the same procedure as for the synthesis of 6 except that the duration of heating at reflux was 1.5 h. Yield 2.20 g (67 %); m.p.: 162–163 °C (from DMF/H2O); 1H NMR (300 MHz, DMSO-d 6): δ = 2.33 (s, 3H, CH3), 2.63 (t, J = 7.80 Hz, 2H, CH2CO), 4.35 (t, J = 7.80 Hz, 2H, CH 2NH), 7.21–7.32 (m, 9H, HAr + HAr′), 8.73 (s, 1H, NH), 9.52 (s, 1H, NH), 9.58 (s, 1H, NH), 9.96 (s, 1H, NH) ppm; 13C NMR (75 MHz, DMSO-d 6): δ = 20.65 (CH3), 30.85 (C-8), 50.45 (C-7), 112.22 (C-2, 6), 124.65 (C-4), 125.78 (C-14), 127.57 (C-12, 16), 127.79 (C-13, 15), 129.41 (C-3, 5), 139.70 (C-11), 140.47 (C-1), 170.10 (C-9), 181.56 (C-10) ppm; IR (KBr): \( \bar{\nu } \) = 2,966–3,363 (NH), 1,702 (CO), 1,504 (O=C–N), 1,330 (C=S) cm−1; MS (ESI, 20 eV): m/z (%) = 351 ([M+Na]+, 60).

3-[(4-Methylphenyl)amino]-N-[[(4-methylphenyl)carbamothioyl]amino]propanamide (9, C18H22N4OS)

Prepared from 1.93 g 1 (10 mmol) and 2.99 cm3 p-tolyl isothiocyanate (1.49 g, 25 mmol) by following the same procedure as for the synthesis of 6 except that the duration of heating at reflux was 20 min. Yield 2.02 g (59 %); m.p.: 184–185 °C (from DMF/H2O); 1H NMR (300 MHz, DMSO-d 6): δ = 2.15 (s, 3H, CH3), 2.29 (s, 3H, CH3), 2.46 (t, J = 6.90 Hz, 2H, CH2CO), 3.26 (t, J = 6.90 Hz, 2H, CH 2NH), 5.34 (s, 1H, NH), 6.50 (d, J = 8.40 Hz, 2H, H(2,6)Ar), 6.90 (d, J = 8.40 Hz, 2H, H(3,5)Ar), 7.09–7.17 (m, 2H, H(3,5) Ar′), 7.25–7.33 (m, 2H, H(2,6) Ar′), 9.51 (s, 2H, 2 NH), 9.96 (s, 1H, NHAr′) ppm; 13C NMR (75 MHz, DMSO-d 6): δ = 20.05 (CH3-Ar′), 20.52 (CH3-Ar), 33.29 (C-8), 39.28 (C-7), 112.29 (C-2, 6), 124.25 (C-4), 125.80 (C-11), 128.50 (C-12, 16), 129.34 (C-3, 5), 134.22 (C-13, 15), 136.42 (C-14), 146.27 (C-1), 170.74 (C-9), 180.95 (C-10) ppm; IR (KBr): \( \bar{\nu } \) = 3,360, 3,270, 3,138, 2,969 (NH), 1,684 (CO), 1,512 (O=C–N), 1,378 (C=S) cm−1; MS (ESI, 25 eV): m/z (%) = 365 ([M+Na]+, 100).

3-[(4-Methylphenyl)(phenylcarbamoyl)amino]-N-[(phenylcarbamoyl)amino]propanamide (10, C24H25N5O3)

Method A. Prepared from 1.93 g 1 (10 mmol) and 2.7 cm3 phenyl isocyanate (2.97 g, 25 mmol) by following the same procedure as for the synthesis of 6 except that the duration of heating at reflux was 4 h. Yield 3.23 g (75 %) (from propan-2-ol).

Method B. A mixture of 0.78 g 6 (2.5 mmol), 10 cm3 DMF, and 0.27 cm3 phenyl isocyanate (0.30 g, 2.6 mmol) was heated at reflux for 6 h. Afterwards 30 cm3 H2O was added. The resin-like product formed was washed with 3 × 10 cm3 H2O. Twice repeated recrystallization from propan-2-ol afforded 0.79 g (73 %) 10. m.p.: 145–146 °C; 1H NMR (300 MHz, DMSO-d 6): δ = 2.34 (s, 3H, CH3), 2.44 (t, J = 7.20 Hz, 2H, CH2CO), 3.88 (t, J = 7.20 Hz, 2H, CH2N), 6.89–7.01 (m, 4H, HAr), 7.16–7.29 (m, 5H, HAr′), 7.37–7.51 (m, 5H, HAr″), 7.89 (s, 1H, NH), 8.03 (s, 1H, NH), 8.72 (s, 1H, NH), 9.78 (s, 1H, NH) ppm; 13C NMR (75 MHz, DMSO-d 6): δ = 20.63 (CH3), 32.19 (C-8), 46.04 (C-7), 112.25, 118.54, 119.72, 121.84, 121.92, 127.81, 128.17, 128.54, 128.69, 139.05, 139.52, 139.88 (CAr), 154.47 (CO), 155.28 (CO), 170.51 (CS) ppm; IR (KBr): \( \bar{\nu } \) = 2,924–3,422 (NH), 1,705, 1,656 (CO), 1,529 (OCN) cm−1; MS (ESI, 25 eV): m/z (%) = 431 ([M+1]+, 100).

3-[(4-Methylphenyl)(phenylcarbamothioyl)amino]-N-[(phenylcarbamothioyl)amino]propanamide (11, C24H25N5OS2)

Method A. Prepared from 1.93 g 1 (10 mmol) and 4.52 cm3 phenyl isothiocyanate (3.38 g, 25 mmol) by following the same procedure as for the synthesis of 6. Yield 3.24 g (70 %).

Method B. Prepared from 1.93 g 1 (10 mmol) and 0.47 cm3 phenyl isothiocyanate (0.34 g, 2.6 mmol) by following the same procedure as for the synthesis of 10 (method B). Yield 0.79 g (68 %); m.p.: 157–158 °C (from propan-2-ol); 1H NMR (300 MHz, DMSO-d 6): δ = 2.33 (s, 3H, CH3), 2.64 (t, J = 7.80 Hz, 2H, CH2CO), 4.35 (t, J = 7.80 Hz, 2H, CH2N), 7.06–7.48 (m, 14H, HAr, HAr′, HAr″), 8.73 (s, 1H, NH), 9.56 (s, 1H, NH), 9.59 (s, 1H, NH), 9.96 (s, 1H, NH) ppm; 13C NMR (75 MHz, DMSO-d 6): δ = 20.63 (CH3), 30.85 (C-8), 50.42 (C-7), 112.81, 119.78, 124.63, 125.75, 127.55, 127.76, 127.96, 130.46, 137.00, 139.03, 139.69, 140.44 (CAr), 170.15 (CO), 180.79 (CS), 181.55 (CS) ppm; IR (KBr): \( \bar{\nu } \) = 2,960–3,360 (NH), 1,709 (CO), 1,369 (CS) cm−1; MS (ESI, 25 eV): m/z (%) = 464 ([M+1]+, 40).

3-[(4-Methylphenyl)[(4-methylphenyl)carbamothioyl]amino]-N-[[(4-methylphenyl)carbamothioyl]amino]propanamide (12, C26H29N5OS2)

Method A. A mixture of 1.93 g 1 (10 mmol) and 2.29 cm3 p-tolyl isothiocyanate (3.73 g, 25 mmol) was heated at reflux for 1 h. The crystalline precipitate was isolated by filtration. Recrystallization from propan-2-ol afforded 3.83 g (78 %) 12.

Method B. Prepared from 0.86 g 9 (2.5 mmol) and 0.24 cm3 p-tolyl isothiocyanate (0.39 g, 2.6 mmol) by following the same procedure as for the synthesis of 10 (method B). Yield 0.81 g (66 %); m.p.: 172–173 °C (propan-2-ol); 1H NMR (300 MHz, DMSO-d 6): δ = 2.16 (s, 3H, CH3), 2.28 (s, 3H, CH3), 2.33 (s, 3H, CH3), 2.46 (t, J = 6.90 Hz, 2H, CH2CO), 3.25 (t, J = 6.90 Hz, 2H, CH2N), 6.49 (d, J = 8.40 Hz, 2H, H(2,6)Ar), 6.90 (d, J = 8.40 Hz, 2H, H(3,5)Ar), 7.02–7.08 (m, 4H, HAr′), 7.19–7.25 (m, 4H, HAr″), 9.50 (s, 2H, NH), 9.95 (s, 2H, NH) ppm; 13C NMR (75 MHz, DMSO-d 6): δ = 20.03, 20.47, 20.63 (CH3), 33.31 (C-8), 50.42 (C-7), 112.25 (C-2, 6), 123.82 (C-4), 125.87 (C-14, 21), 128.25, 128.80 (C-13, 15, 20, 22), 129.31 (C-3, 5), 133.53, 130.48 (C-12, 16, 19, 23), 136.42 (C-11), 136.78 (C-18), 146.26 (C-1), 169.43 (CO), 171.23 (CS), 179.52 (CS) ppm; IR (KBr): \( \bar{\nu } \) = 3,360, 3,271, 3,138, 2,969 (NH), 1,684 (CO), 1,513 (O=C–N), 1,378, 1,356 (C=S) cm−1; MS (ESI, 25 eV): m/z (%) = 492 ([M+1]+, 90).

2,4-Dihydro-5-[2-[(4-methylphenyl)amino]ethyl]-4-phenyl-3H-1,2,4-triazol-3-one (13, C17H18N4O)

A mixture of 0.78 g 6 (2.5 mmol) and 25 cm3 20 % aqueous KOH solution was heated at reflux for 4 h and then cooled to room temperature. Afterwards conc. HCl was added to pH 4. The crystalline precipitate was isolated by filtration and washed with H2O. Recrystallization from DMF/H2O afforded 0.35 g (48 %) 13. m.p.: 153–154 °C; 1H NMR (300 MHz, DMSO-d 6): δ = 2.12 (s, 3H, CH3-Ar), 2.61 (t, J = 7.20 Hz, 2H, CH2C), 3.09–3.19 (m, 2H, NHCH 2), 5.37 (s, 1H, NHAr), 6.27 (d, J = 8.25 Hz, 2H, H(2,6)Ar), 6.82 (d, J = 8.25 Hz, 2H, H(3,5)Ar), 7.38–7.45 (m, 2H, HAr′), 7.48–7.55 (m, 3H, HAr′), 11.70 (s, 1H, NH) ppm; 13C NMR (75 MHz, DMSO-d 6): δ = 19.96 (CH3), 25.75 (C-8), 39.60 (C-7), 111.98 (C-2, 6), 124.14 (C-4), 127.54 (C-12, 16), 128.60 (C-14), 129.28 (C-13, 15), 129.38 (C-3, 5), 132.87 (C-11), 145.21 (C-1), 145.71 (C-9), 154.33 (C-10) ppm; IR (KBr): \( \bar{\nu } \) = 3,326, 2,890 (NH), 1,670 (CO), 1,528 (C=N), 1,499 (C–N) cm−1; MS (APCI+, 25 eV): m/z (%) = 295 ([M+1]+, 20), 317 ([M+Na]+, 70).

4-(3,5-Dimethylphenyl)-2,4-dihydro-5-[2-[(4-methylphenyl)amino]ethyl]-3H-1,2,4-triazol-3-one (14, C19H22N4O)

Prepared from 0.68 g 7 (2 mmol) by following the same procedure as for the synthesis of 13 to afford 0.42 g (65 %) 14. m.p.: 202–203 °C; 1H NMR (300 MHz, DMSO-d 6): δ = 2.11 (s, 3H, CH3), 2.28 (s, 6H, 2 CH3), 2.57 (t, J = 7.20 Hz, 2H, CH2C), 3.15 (t, J = 7.20 Hz, 2H, NHCH 2), 5.41 (s, 1H, NHAr), 6.26 (d, J = 8.40 Hz, 2H, H(2,6)Ar), 6.81 (d, J = 8.40 Hz, 2H, H(3,5)Ar), 6.96 (d, J = 0.60 Hz, 2H, H(2,6)Ar′), 7.10 (d, J = 0.60 Hz, 1H, H(4)Ar′), 11.75 (br s, 1H, NH) ppm; 13C NMR (75 MHz, DMSO-d 6): δ = 20.09 (CH3), 20.71 (2 CH3), 25.77 (C-8), 39.90 (C-7), 112.11 (C-2, 6), 124.33 (C-4), 125.17 (C-12, 16), 129.43 (C-3, 5), 130.14 (C-14), 132.76 (C-11), 138.93 (C-13, 15), 145.46 (C-1), 145.81 (C-9), 154.58 (C-10) ppm; IR (KBr): \( \bar{\nu } \) = 3,355, 2,917 (NH), 1,698 (CO), 1,527 (C=N), 1,455 (C–N) cm−1; MS (APCI+, 25 eV): m/z (%) = 323 ([M+1]+, 90).

2,4-Dihydro-5-[2-[(4-methylphenyl)amino]ethyl]-4-phenyl-3H-1,2,4-triazole-3-thione (15, C17H18N4S)

Method A. A mixture of 1.57 g 8 (5 mmol) and 50 cm3 20 % aqueous KOH solution was heated at reflux for 3 h and then cooled to room temperature. Afterwards conc. HCl was added to pH 4. The crystals formed were isolated by filtration and washed with H2O. Recrystallization from DMF/H2O afforded 0.64 g (83 %) 15.

Method B. A mixture of 0.66 g 7 (2 mmol), 0.064 g NH2NH2 (2 mmol), and 10 cm3 methanol was heated at 50–60 °C for 4 h. Afterwards 40 cm3 cold H2O was added to the reaction mixture. The precipitate was isolated by filtration and purified by column chromatography (acetone/hexane 1:1, R f = 0.87). Recrystallization from DMF/H2O afforded 0.31 g (50 %) 15.

Method C. A mixture of 0.93 g 11 (2 mmol) and 20 cm3 10 % aqueous KOH solution was heated at reflux for 4 h. The crystalline precipitate was isolated by filtration. Recrystallization from DMF/H2O afforded 0.61 g (98 %) 15. m.p.: 173–174 °C; 1H NMR (300 MHz, DMSO-d 6): δ = 2.11 (s, 3H, CH3), 2.63 (t, J = 7.20 Hz, 2H, CH2C), 3.16 (q, J = 7.20 Hz, J = 13.80 Hz, 2H, NHCH 2), 5.43 (t, J = 6.60 Hz, 1H, NHAr), 6.24 (d, J = 8.25 Hz, 2H, H(2,6)Ar), 6.79 (d, J = 8.25 Hz, 2H, H(3,5)Ar), 7.34–7.49 (m, 5H, HAr′), 13.76 (s, 1H, NH) ppm; 13C NMR (75 MHz, DMSO-d 6): δ = 19.99 (CH3), 25.30 (C-8), 39.71 (C-7), 111.98 (C-2, 6), 124.22 (C-4), 127.24 (C-12, 16), 128.38 (C-14), 128.55 (C-13, 15), 129.42 (C-3, 5), 133.66 (C-11), 145.58 (C-1), 150.52 (C-9), 167.54 (C-10) ppm; IR (KBr): \( \bar{\nu } \) = 3,310, 2,920 (NH), 1,525 (C=N), 1,497 (C–N), 1,298 (C=S) cm−1; MS (ESI, 20 eV): m/z (%) = 331 ([M+Na]+, 100).

2,4-Dihydro-4-(4-methylphenyl)-5-[2-[(4-methylphenyl)amino]ethyl]-3H-1,2,4-triazole-3-thione (16, C18H20N4S)

Method A. Prepared from 0.51 g 9 (45 mmol) by following the same procedure as for the synthesis of 15 (method A) to afford 0.64 g (83 %) 16.

Method B. Prepared from 0.34 g 9 (1 mmol) and 0.064 g NH2NH2 (2 mmol) by following the same procedure as for the synthesis of 15 (method B) to afford 0.15 g (50 %) 16. m.p.: 192–193 °C; 1H NMR (300 MHz, DMSO-d 6): δ = 2.12 (s, 3H, CH3), 2.40 (s, 3H, CH3), 2.62 (t, J = 7.20 Hz, 2H, CH2C), 3.16 (q, J = 7.05 Hz, J = 14.10 Hz, 2H, CH 2NH), 5.42 (t, J = 6.00 Hz, 1H, NHAr), 6.25 (d, J = 8.10 Hz, 2H, H(2,6)Ar), 6.81 (d, J = 8.10 Hz, 2H, H(3,5)Ar), 7.26–7.38 (m, 4H, HAr′), 13.72 (s, 1H, NHAr′) ppm; 13C NMR (75 MHz, DMSO-d 6): δ = 19.99 (CH3-Ar), 20.73 (CH3-Ar′), 25.26 (C-8), 39.71 (C-7), 112.00 (C-2, 6), 124.19 (C-4), 128.09 (C-14), 129.27 (C-12, 16), 129.87 (C-13, 15), 131.05 (C-3, 5), 139.11 (C-11), 145.58 (C-1), 150.61 (C-9), 167.59 (C-10) ppm; IR (KBr): \( \bar{\nu } \) = 3,392, 2,935 (NH), 1,524 (C = N), 1,494 (C–N), 1,338 (C=S) cm−1; MS (APCI+, 25 eV): m/z (%) = 348 ([M+Na+1]+, 50).

3-(4-Methylphenyl)-1-phenyl-3-[2-[5-(phenylamino)-1,3,4-oxadiazol-2-yl]ethyl]urea (17, C24H23N5O2)

To 5 cm3 conc. H2SO4 was added 0.52 g 10 (1.2 mmol) in portions and the reaction mixture was stirred at room temperature for 30 min. Afterwards, it was added dropwise into ice-water mixture. The crystalline precipitate was isolated by filtration. Recrystallization from ethanol afforded 0.43 g (87 %) 17. m.p.: 98–99 °C; 1H NMR (300 MHz, DMSO-d 6): δ = 2.34 (s, 3H, CH3), 2.43 (t, J = 7.20 Hz, 2H, CH2CO), 3.87 (t, J = 7.20 Hz, 2H, CH2N), 8.68, 9.75 (2 s, 2H, ArNH, Ar′NH) ppm; 13C NMR (75 MHz, DMSO-d 6): δ = 20.58 (CH3), 32.18 (C-8), 39.71 (C-7), 110.22, 118.50, 119.68, 121.78, 121.88, 127.76, 128.12, 128.49, 130.08, 136.07, 139.47, 139.83 (CAr), 154.42 (C-9), 155.21 (C-10), 170.44 (CS) ppm; IR (KBr): \( \bar{\nu } \) = 3,360, 2,929 (NH), 1,512 (CN) cm−1; MS (ESI, 25 eV): m/z (%) = 414 ([M+1]+, 100).

3-(4-Methylphenyl)-1-phenyl-3-[2-[5-(phenylamino)-1,3,4-thiadiazol-2-yl]ethyl]thiourea (18, C24H23N5S2)

Prepared from 0.56 g 11 (1.2 mmol) by following the same procedure as for the synthesis of 17 except that POCl3 was used instead of conc. H2SO4 to afford 0.45 g (84 %) 18. m.p.: 93–94 °C (ethanol); 1H NMR (300 MHz, DMSO-d 6): δ = 2.32 (s, 3H, CH3), 3.28 (t, J = 7.35 Hz, 2H, CH2CN), 4.47 (t, J = 7.35 Hz, 2H, CH2NH), 6.96–7.58 (m, 14H, HAr, HAr′, HAr″), 8.71 (s, 1H, NHAr′), 10.29 (s, 1H, NHAr″) ppm; 13C NMR (75 MHz, DMSO-d 6): δ = 20.60 (CH3), 27.66 (C-8), 53.57 (C-7), 117.22, 121.70, 124.76, 125.97, 127.43, 127.74, 128.98, 130.44, 137.07, 139.55, 140.39, 140.60 (CAr), 156.28 (C-9), 164.35 (C-10), 181.75 (C=S) ppm; IR (KBr): \( \bar{\nu } \) = 3,367, 2,924 (NH), 1,510 (CN) cm−1; MS (ESI, 25 eV): m/z (%) = 446 ([M+1]+, 100).

1,3-Bis(4-methylphenyl)-3-(2-[5-[(4-methylphenyl)amino]-1,3,4-thiadiazol-2-yl]ethyl)thiourea (19, C26H27N5S2)

Prepared from 0.49 g 12 (1 mmol) by following the same procedure as for the synthesis of 17 to afford 0.21 g (44 %) 19. m.p.: 97–98 °C (propan-2-ol); 1H NMR (300 MHz, DMSO-d 6): δ = 2.16 (s, 3H, CH3), 2.24 (s, 3H, CH3), 2.32 (s, 3H, CH3), 3.13 (t, J = 6.60 Hz, 2H, CH2CN), 3.36 (t, J = 6.60 Hz, 2H, CH2N), 6.57 (d, J = 8.40 Hz, 2H, H(2,6)Ar), 6.93 (d, J = 8.40 Hz, 2H, H(3,5)Ar), 7.08–7.17 (m, 4H, HAr′), 7.42–7.54 (m, 4H, HAr″), 10.19 (s, 2H, NH) ppm; 13C NMR (75 MHz, DMSO-d 6): δ = 20.03, 20.26, 20.46 (CH3), 29.21 (C-8), 42.72 (C-7), 112.84, 117.20, 124.99, 126.07, 127.50, 128.24, 129.31, 129.39, 130.34, 130.48, 138.39, 145.33 (CAr), 157.02 (C-9), 164.42 (C-10), 181.75 (CS) ppm; IR (KBr): \( \bar{\nu } \) = 3,358, 2,910 (NH), 1,515 (CN) cm−1; MS (ESI, 25 eV): m/z (%) = 474 ([M+1]+, 70).

5-[2-[(4-Methylphenyl)amino]ethyl]-N-phenyl-1,3,4-oxadiazol-2-amine (20, C17H18N4O)

A mixture of 0.62 g 6 (2 mmol) and 5 cm3 conc. H2SO4 was stirred at room temperature until fully dissolved (approx. 20 min). The reaction mixture was added dropwise to an ice/water mixture. The crystalline precipitate was isolated by filtration and washed with H2O. Recrystallization from methanol afforded 0.57 g (98 %) 20. m.p.: 114–115 °C; 1H NMR (300 MHz, DMSO-d 6): δ = 2.23 (s, 3H, CH3), 2.29 (t, J = 7.20 Hz, 2H, CH2CO), 3.47 (t, J = 7.20 Hz, 2H, CH 2NH), 6.89–6.99 (m, 2H, HAr), 7.15–7.21 (m, 2H, HAr), 7.36–7.48 (m, 5H, HAr′), 8.76 (s, 1H, NHAr), 9.78 (s, 1H, NHAr′) ppm; 13C NMR (75 MHz, DMSO-d 6): δ = 20.70 (CH3), 32.32 (C-8), 46.12 (C-7), 112.63 (C-2, 6), 119.85 (C-12, 16), 124.27 (C-4), 128.29 (C-14), 128.69 (C-13, 15), 130.24 (C-3, 5), 136.29 (C-11), 139.90 (C-1), 155.39 (C-9), 169.61 (C-10) ppm; IR (KBr): \( \bar{\nu } \) = 3,310, 2,919 (NH), 1,520 (CN) cm−1; MS (APCI+, 25 eV): m/z (%) = 295 ([M+1]+, 90).

N-(3,5-Dimethylphenyl)-5-[2-[(4-methylphenyl)amino]ethyl]-1,3,4-oxadiazol-2-amine (21, C19H22N4O)

Prepared from 0.68 g 7 (2 mmol) by following the same procedure as for the synthesis of 20 to afford 0.67 g (69 %) 21. m.p.: 191–192 °C (methanol); 1H NMR (300 MHz, DMSO-d 6): δ = 2.19 (s, 3H, CH3), 2.29 (s, 6H, 2 CH3), 2.59 (t, J = 7.20 Hz, 2H, CH2CO), 3.51 (t, J = 7.20 Hz, 2H, CH 2NH), 7.05–7.39 (m, 7H, HAr + HAr′), 8.79 (s, 1H, NHAr), 9.87 (s, 1H, NHAr′) ppm; 13C NMR (75 MHz, DMSO-d 6): δ = 20.65 (CH3), 23.16 (2 CH3), 29.37 (C-8), 46.54 (C-7), 111.70 (C-2, 6), 119.07 (C-12, 16), 122.25 (C-4), 130.54 (C-3, 5), 136.97 (C-14), 138.42 (C-11), 138.92 (C-13,15), 144.20 (C-1), 155.26 (C-9), 169.40 (C-10) ppm; IR (KBr): \( \bar{\nu } \) = 3,309, 2,920 (NH), 1,599 (CN) cm−1; MS (APCI+, 25 eV): m/z (%) = 323 ([M+1]+, 70).

5-[2-[(4-Methylphenyl)amino]ethyl]-N-phenyl-1,3,4-thiadiazol-2-amine (22, C17H18N4S)

Prepared from 0.66 g 8 (2 mmol) by following the same procedure as for the synthesis of 20 to afford 0.58 g (94 %) 22. m.p.: 152–153 °C (methanol); 1H NMR (300 MHz, DMSO-d 6): δ = 2.15 (s, 3H, CH3), 3.13 (t, J = 7.50 Hz, 2H, CH2CO), 3.35 (t, J = 7.50 Hz, 2H, CH 2NH), 5.56 (s, 1H, NH), 6.53 (d, J = 8.40 Hz, 2H, HAr(2,6)), 6.91 (d, J = 8.40 Hz, 2H, HAr(3,5)), 6.96 (t, J = 7.80 Hz, 1H, HAr′(4)), 7.32 (t, J = 7.80 Hz, 2H, HAr′(3,5)), 7.59 (d, J = 7.80 Hz, 2H, HAr′(2,6)), 10.21 (s, 1H, NHAr′) ppm; 13C NMR (75 MHz, DMSO-d 6): δ = 20.00 (CH3), 29.38 (C-8), 42.39 (C-7), 112.36 (C-2, 6), 117.07 (C-12, 16), 121.45 (C-14), 124.41 (C-4), 128.94 (C-13, 15), 129.35 (C-3, 5), 140.76 (C-11), 145.90 (C-1), 157.53 (C-9), 164.25 (C-10) ppm; IR (KBr): \( \bar{\nu } \) = 3,360, 2,923 (NH), 1,510 (CN) cm−1; MS (APCI+, 25 eV): m/z (%) = 311 ([M+1]+, 70).

5-[2-(4-Methylphenylamino)ethyl]-N-phenyl-1,3,4-thiadiazol-2-amine (23, C18H20N4S)

Prepared from 0.86 g 9 (2.5 mmol) by following the same procedure as for the synthesis of 20 to afford 0.56 g (69 %) 23. m.p.: 105–106 °C (ethanol); 1H NMR (300 MHz, DMSO-d 6): δ = 2.23 (s, 3H, CH3Ar′), 2.30 (s, 3H, CH3Ar), 3.29 (t, J = 6.60 Hz, 2H, CH 2CN), 3.55 (t, J = 6.60 Hz, 2H, NHCH 2), 5.61 (s, 1H, NHCH2), 7.05–7.18 (m, 4H, HAr), 7.44–7.56 (m, 4H, HAr′), 10.35 (s, 1H, NHAr′) ppm; 13C NMR (75 MHz, DMSO-d 6): δ = 20.34 (CH3Ar′), 20.51 (CH3Ar), 27.30 (C-8), 46.51 (C-7), 112.29 (C-2, 6), 117.38 (C-12, 16), 121.96 (C-11), 123.09 (C-4), 129.38 (C-3, 5), 129.94 (C-13, 15), 138.37 (C-14), 155.56 (C-1), 157.42 (C-9), 164.67 (C-10) ppm; IR (KBr): \( \bar{\nu } \) = 3,232, 2,919 (NH), 1,514 (CN) cm−1; MS (ESI, 25 eV): m/z (%) = 325 ([M+1]+, 40).

N-(3,4-Diphenyl-2(3H)-thiazolylidene)-3-[(4-methylphenyl)amino]propanehydrazide (24, C25H24N4OS)

To 3.28 g 8 (10 mmol) dissolved in 40 cm3 ethanol, 1.98 g phenacyl bromide (10 mmol) and 16.4 g sodium acetate (200 mmol) were added and the reaction mixture was heated at reflux for 10 h. Afterwards it was cooled to room temperature, poured into ice-cold water while stirring and left overnight in the cold. The precipitate formed was isolated by filtration and washed with H2O three times. Recrystallization from H2O afforded 1.19 g (55 %) 24. m.p.: 126–127 °C; 1H NMR (300 MHz, DMSO-d 6): δ = 2.13 (s, 3H, CH3), 2.32–2.45 (m, 2H, CH2CO), 3.14 (t, J = 7.05 Hz, 2H, CH 2NH), 5.23 (s, 1H, NHAr), 6.36–6.53 (m, 4H, HAr), 6.84–7.08 (m, 5H, HAr′), 7.31–7.48 (m, 5H, HAr″), 7.99 (s, 1H, SCH), 10.90 (s, 1H, NH) ppm; 13C NMR (75 MHz, DMSO-d 6): δ = 19.97, 33.31, 47.69, 112.34, 120.87, 123.07, 124.39, 127.57, 128.33, 128.70, 128.88, 129.26, 129.43, 129.69, 139.09, 145.82, 150.22, 156.77, 170.55 ppm; IR (KBr): \( \bar{\nu } \) = 3,354, 2,916 (NH), 1,707 (CO), 1,580 (O=C–N) cm−1; MS (ESI, 25 eV): m/z (%) = 468 ([M+K]+, 40).

Biology

Antibacterial activity was tested using the disk diffusion technique. The microbial agent R. radiobacter was commercially available from the German Collection of Microorganisms and Cell Cultures (DSMZ). The zone of inhibition of bacterial growth was investigated. The main solution (1 mg/cm3) of synthesized compounds was prepared in DMSO and was diluted at various concentrations (50, 150, 250, 450, 550 μg/cm3) in DMSO. Cultures of R. radiobacter were cultivated in petri dishes for 24 h at 37 °C on Luria–Bertani (LB) agar medium. A bacterial suspension was prepared from cultivated bacterial cultures and 100 mm3 inoculum containing bacterial cells (108 CFU/cm3) was spread over LB agar medium. Filter paper disks were prepared by adding 25 mm3 of each compound solution and then disks were put on LB agar medium. Ampicillin was used as the positive control, and DMSO was used as a negative control. The petri dishes were incubated for 24 h at 37 °C and zones of inhibition were then ascertained for each sample.