A facile microwave assisted green chemical synthesis of novel piperidino 2-thioxoimidazolidin-4-ones and their in vitro microbiological evaluation

Abstract

A series of novel hybrid heterocyclic compounds, 3-(3-alkyl-2,6-diarylpiperin-4-ylidene)-2-thioxoimidazolidin-4-ones were synthesised and a comparative study was also carried out under microwave irradiation. The synthesised compounds were characterised by their melting points, elemental analysis, MS, FT-IR, one-dimensional NMR (1H, D₂O exchanged 1H and ¹³C), two dimensional HOMOCOSY and NOESY spectroscopic data. All the synthesised title compounds were screened for their in vitro antibacterial and antifungal activity against clinically isolated strains namely B. subtilis, M. luteus, S. typhii, S. paratyphii B, S. felxneri, P. vulgaris, A. niger, Mucor, Rhizopus and M. gypsuem and the results were discussed.

Keywords::

• 3-(3-alkyl-2,6-diarylpiperin-4-ylidene)-2-thioxoimidazolidin-4-ones
• NaHSO₄.SiO₂
• microwave irradiation
• antibacterial activity
• antifungal activity

Introduction

Sulphur analogs of hydantoins with one or both carbonyl groups replaced by thiocarbonyl groups are called as thiohydantoins. There has been much interest in the synthesis and properties of 2-thiohydantoin derivatives used as synthetic intermediates with a wide range of applications [1,2] as hypolipidaemic, anticarcinogenic, antimutagenic, antithyroidal, antiviral (e.g. against herpes simplex virus, HSV, human immunodeficiency virus (HIV), tuberculosis, antimicrobial (antifungal and antibacterial), anti-ulcer and anti-inflammatory agents, as well as pesticides. Additionally, 2-thiohydantoins have been used as reference standards for the development of C-terminal protein sequencing, as reagents for the development of dyes and in textile printing, metal cation complexation and polymerisation catalysis. For these reasons, an alternative synthetic methodology is of paramount importance for the synthesis of 2-thiohydantoins.

Microwave irradiation is well known to promote the synthesis of a variety of compounds, where chemical reactions can be accelerated due to selective absorption of microwaves by polar molecules. Microwave-induced rate acceleration technology [3–5] has become a powerful tool in organic synthesis in view of the mild, clean, and convenient methodology and the enhanced selectivity of the reaction process in comparison to conventional solution reactions, and the associated ease of manipulation. The coupling of microwave irradiation together with solid supports under solvent free conditions have received considerable attention recently as it is one of the novel approaches to eco-friendly chemistry. Heterogeneous reactions [6–11] facilitated by supported reagents on various mineral oxides have received special attention in recent years.

Silica gel supported sodium hydrogen sulphate (NaHSO₄.SiO₂), is a non-toxic and inexpensive catalyst, which has been used for a variety of organic transformations [7, 9–11]. Baliah et al. have reviewed the importance of piperidin-4-ones as intermediates in the synthesis of several physiologically active compounds [12,13]. In corollary of the interesting biological and pharmaceutical properties and synthetic utility, there is substantial interest in piperidones; this substructure containing compounds are widely present in numerous alkaloids and synthetically derived molecules of biological importance [14].

In view of our continued interest in the development of simpler and more convenient synthetic routes for achieving biologically challenging hybrid heterocyclic systems [3–11], we have reported a simple method to synthesise some piperidones based on 2-thiohydantoins namely 3-(3-alkyl-2,6-diarylpiperin-4-ylidene)-2-thioxoimidazolidin-4-ones with good yields by the reaction of 3-alkyl-2,6-diarylpiperidin-4-one thiosemicarbazones with chloroethyl acetate catalysed by NaHSO₄.SiO₂ under microwave irradiation in dry media and their in vitro antibacterial and antifungal activities against clinically isolated bacterial and fungal strains were evaluated.

Experimental

General remarks

TLC was used to assess the reactions and the purity of the products. All the reported melting points were taken in open capillaries and were uncorrected. A Biotage Initiator microwave synthesiser, Sweden a scientific microwave oven was used for the irradiation. IR spectra were recorded in KBr (pellet forms) on a Thermo Nicolet-Avatar–330 (Thermo Fisher Scientific Inc., Waltham, MA) FT-IR spectrophotometer (Bruker Biospin International, Ag, Aegeristrasse, Switzerland) and note worthy absorption values (cm⁻¹) were listed. One dimensional ¹H and ¹³C NMR spectra were recorded at 400 MHz and 100 MHz respectively on Bruker AMX 400 NMR spectrometer (Bruker Biospin International, Ag, Aegeristrasse, Switzerland) using DMSO-d as solvent. Two dimensional HOMOCOSY and NOESY spectra were recorded at 500 MHz on Bruker DRX 500 NMR spectrometer using DMSO-d as solvent. The electron spray impact (ESI) positive (+ve) mass (MS) spectra were recorded on a Bruker Daltonics LC-MS spectrometer. Satisfactory microanalysis was obtained on Carlo Erba 1106 CHN analyser (Thermo Fisher Scientific Inc., Waltham, MA).

General procedure for the synthesis of 3-alkyl-2,6-diarylpiperidin-4-ones 1–15

A mixture of ammonium acetate (0.1 mol), the respective substituted benzaldehyde (0.2 mol) and appropriate ketone (0.1 mol) (s.d. Fine chemicals, Mumbai, India) were dissolved in 95% alcohol (80 mL) and the solution was heated on a hot plate with gentle swirling until the colour of the mixture changed to orange. The mixture was cooled and poured into diethyl ether (100 mL) and concentrated hydrochloric acid (14 mL) was added. The precipitated hydrochloride was collected by filtration and recrystallised from the ethanol-ether mixture. The hydrochloride was dispersed in acetone and concentrated aqueous ammonia added dropwise until a clear solution was obtained. The clear solution was poured into cold water and the solid precipitation was collected and recrystallised from ethanol.

General procedure for the synthesis of 3-alkyl-2,6-diarylpiperidin-4-one thiosemicarbazones 16–30

A mixture of the respective 3-substituted-2,6-diarylpiperidin-4-ones 1–15 (0.01 mol) and thiosemicarbazide (0.01 mol) in ethanol (60 mL) was refluxed for 2 h on a steam bath. It was cooled and then the separated solid was filtered and washed with water. The solid was subjected to column chromatography using benzene:chloroform (1:1) as eluent to afford compounds 16–30.

General procedure for the synthesis of 3-(3-alkyl-2,6-diarylpiperin-4-ylidene)-2-thioxoimidazolidin-4-ones 31–45

To a well stirred solution of 3-alkyl-2,6-diphenylpiperidin-4-one thiosemicarbazones 31–45 (0.01 mmol) and anhydrous sodium acetate (0.01 mol) in 30 mL of ethanol, chloroethyl acetate (0.01 mmol) in 15 mL of ethanol was added drop wise through a funnel for about 10 min and the reaction mixture was refluxed for 4 h. After completion of the reaction, the reaction mixture was poured into ice cold water and the solid mass was collected and recrystallised twice from ethanol.

General procedure for the microwave assisted synthesis of 3-(3-alkyl-2,6-diarylpiperin-4-ylidene)-2-thioxoimidazolidin-4-ones catalysed by NaHSO₄.SiO₂ 31–45

A mixture containing 3-alkyl-2,6-diphenylpiperidin-4-ones 1–15 (0.01 mmol), thiosemicarbazide (0.01 mmol), and chloroethyl acetate (0.01 mmol) and NaHSO₄.SiO₂ (25 mg) was added in an alumina bath, mixed thoroughly with the aid of a glass rod (10 s) and then irradiated in a microwave oven for 2–4 min. at 320 W (monitored by TLC). After completion of the reaction, the reaction mixture was extracted with dichloromethane (3 × 5 mL). The catalyst and other solid wastes were removed by filtration. The combined organic layer was washed with water three times and then dried over anhydrous MgSO₄. The organic layer was concentrated in vacuo to furnish the crude products and recrystallised twice from ethanol.

Spectroscopic data

3-(2,6-diphenylpiperidin-4-ylideneamino)-2-thioxoimidazolidin-4-one 31

Irradiation reaction time = 3 min; IR (KBr) (cm⁻¹): 3400, 3306, 3060, 3029, 2980, 2896, 2797, 1728, 1635, 1598, 1215, 701, 758, 1041; ¹H NMR (δ ppm): 1.97–2.05 (m, 1H, H_3a), 2.37–2.41 (dd, 1H, H_3e, J_3e,3a = 13.64 Hz, J_3e,2a = 2.96 Hz); 2.43–2.52 (m, 1H, H_5a), 2.83 (s, 1H, NH of piperidine), 3.62–3.66 (dd, H_5e, J_5e,5a = 2.96 Hz, J_5e,6a = 13.52 Hz), 3.8 (s, 2H, CH₂ of imidazolidine), 3.88–3.92 (dd, 1H, H_2a, J_2a,3e = 3.08 Hz, J_2a,3a = 11.76 Hz), 4.15–4.19 (dd, 1H, H_6a, J_6a,5e = 3.2 Hz, J_6a,5a = 11.88 Hz), 7.23–7.5 (m, 10H, Ar-H’s), 11.78 (s, NH of imidazolidine); In the D₂O exchanged ¹H NMR spectrum, two peaks at 2.83 ppm and 11.78 ppm which resonances due to NH of piperidine and imidazolidine respectively disappeared; ¹³C NMR (δ ppm): 29.6 C-3, 37.3 C-5, 43.6 CH₂ of imidazolidine, 60.2 C-2, 61.1 C-6, 126.5–128.1 Ar-C’s, 144, 144.1 ipso-C, 163.1 C=N, 167.6 C=O, 173.8 C=S.

3-(2,6-bis(4-fluorophenyl)piperidin-4-ylideneamino)-2-thioxoimidazolidin-4-one 32

Irradiation reaction time = 4 min; IR (KBr) (cm⁻¹): 3430, 3317, 3076, 3030, 2956, 2924, 2854, 1717, 1641, 1604, 1092, 835, 733, 519; ¹H NMR (δ ppm): 1.98–2.05 (m, 1H, H_3a), 2.39–2.44 (dd, 1H, H_3e, J_3e,3a = 13.52 Hz, J_3e,2a = 2.96 Hz); 2.44–2.49 (m, 1H, H_5a), 2.86 (s, 1H, NH of piperidine), 3.51–3.55 (dd, 1H, H_5e, J_5e,5a = 2.96 Hz, J_5e,6a = 13.4 Hz), 3.77 (s, 2H, CH₂ of imidazolidine), 3.85–3.89 (dd, 1H, H_2a, J_2a,3e = 2.96 Hz, J_2a,3a = 11.76 Hz), 4.16–4.2 (dd, 1H, H_6a, J_6a,5e = 3.32 Hz, J_6a,5a = 11.96 Hz), 7.18–7.37 (m, 8H, Ar-H’s), 11.81 (s, 1H, NH of imidazolidine); ¹³C NMR (δ ppm): 30.1 C-3, 38.2 C-5, 43.4 CH₂ of imidazolidine, 61.8 C-2, 62.9 C-6, 114.3–142.6 Ar-Cs, 142.9, 159.6 ipso-C, 162 C=N, 166.7 C=O, 173.7 C=S.

3-(2,6-bis(4-chlorophenyl)piperidin-4-ylideneamino)-2-thioxoimidazolidin-4-one 33

Irradiation reaction time = 4 min; IR (KBr) (cm⁻¹): 3400, 3309, 3065, 3032, 2978, 2923, 2852, 1724, 1628, 1595, 1195, 1014, 826, 722, 676, 634; ¹H NMR (δ ppm): 1.98–2.05 (m, 1H, H_3a), 2.38–2.42 (dd, 1H, H_3e, J_3e,3a = 13.52 Hz, J_3e,2a = 2.98 Hz); 2.43–2.48 (m, 1H, H_5a), 2.79 (s, 1H, NH of piperidine), 3.52–3.56 (dd, 1H, H_5e, J_5e,5a = 3.08 Hz, J_5e,6a = 13.42 Hz), 3.78 (s, 2H, CH₂ of imidazolidine), 3.87–3.91 (dd, 1H, H_2a, J_2a,3e = 3.08 Hz, J_2a,3a = 11.76 Hz), 4.15–4.19 (dd, 1H, H_6a, J_6a,5e = 3.14 Hz, J_6a,5a = 11.70 Hz), 7.3–7.58 (m, 8H, Ar-H’s), 11.79 (s, 1H, NH of imidazolidine); ¹³C NMR (δ ppm): 30 C-3, 38.8 C-5, 43.5 CH₂ of imidazolidine, 60.9 C-2, 61.2 C-6, 126.7–140.4 Ar-C’s, 144.7, 145.3 ipso-C, 163.1 C=N, 166.8 C=O, 173.5 C=S.

3-(2,6-bis(4-methoxyphenyl)piperidin-4-ylideneamino)-2-thioxoimidazolidin-4-one 34

Irradiation reaction time = 3 min; IR (KBr) (cm⁻¹): 3400, 3306, 3065, 3020, 2962, 2924, 2853, 1728, 1630, 1601, 1251, 1031, 830, 749, 527; ¹H NMR (δ ppm): 1.97–2.04 (m, 1H, H_3a), 2.35–2.39 (dd, 1H, H_3e, J_3e,3a = 13.52 Hz, J_3e,2a = 2.96 Hz); 2.43–2.5 (m, 1H, H_5a), 2.89 (s, 1H, NH of piperidine), 3.48–3.53 (dd, 1H, H_5e, J_5e,5a = 3.16 Hz, J_5e,6a = 13.4 Hz), 3.59 (s, 6H, OCH₃ at the phenyl rings), 3.72 (s, 2H, CH₂ of imidazolidine), 3.87–3.91 (dd, 1H, H_2a, J_2a,3e = 3.16 Hz, J_2a,3a = 11.68 Hz), 4.13–4.17 (dd, 1H, H_6a, J_6a,5e = 3.08 Hz, J_6a,5a = 11.68 Hz), 7.19–7.42 (m, 8H, Ar-H’s), 11.78 (s, 1H, NH of imidazolidine); ¹³C NMR (δ ppm): 30.1 C-3, 37 C-5, 43.1 CH₂ of imidazolidine, 54.2 -OCH₃ at the phenyl rings, 60.2 C-2, 61.1 C-6, 127.3–144.0 Ar-C’s, 144.1, 159.9, ipso-C, 162.3 C=N, 167.7 C=O, 173.8 C=S.

3-(2,6-dip-tolylpiperidin-4-ylideneamino)-2-thioxoimidazolidin-4-one 35

Irradiation reaction time = 3 min; IR (KBr) (cm⁻¹): 3403, 3308, 3063, 3018, 2960, 2921, 2850, 1727, 1631, 1600, 1249, 1023, 828, 742, 524; ¹H NMR (δ ppm): 1.97–2.05 (m, 1H, H_3a), 2.18 (s, 6H, CH₃ at the phenyl rings), 2.34–2.38 (dd, 1H, H_3e, J_3e,3a = 13.5 Hz, J_3e,2a = 2.97 Hz); 2.45–2.52 (m, 1H, H_5a), 2.84 (s, 1H, NH of piperidine), 3.49–3.52 (dd, 1H, H_5e, J_5e,5a = 3.14 Hz, J_5e,6a = 13.42 Hz), 3.76 (s, 2H, CH₂ of imidazolidine), 3.88–3.9 (dd, 1H, H_2a, J_2a,3e = 3.14 Hz, J_2a,3a = 11.66 Hz), 4.14–4.18 (dd, 1H, H_6a, J_6a,5e = 3.07 Hz, J_6a,5a = 11.66 Hz), 7.17–7.39 (m, 8H, Ar-H’s), 11.76 (s, 1H, NH of imidazolidine); ¹³C NMR (δ ppm): 22.2 -CH₃ at the phenyl rings, 30.3 C-3, 37.2 C-5, 43.3 CH₂ of imidazolidine, 60.1 C-2, 61 C-6, 127.1–143.9 Ar-C’s, 144.1, 147.2, ipso-C, 162.8 C=N, 167.5 C=O, 173.5 C=S.

3-(3-methyl-2,6-diphenylpiperidin-4-ylideneamino)-2-thioxoimidazolidin-4-one 36

Irradiation reaction time = 2 min; IR (KBr) (cm⁻¹): 3420, 3311, 3063, 3031, 2978, 2933, 1713, 1634, 1600, 1232, 755, 702; ¹H NMR (δ ppm): 0.77–0.79 (d, 3H, CH₃ at C-3, J_CH3,3a = 6.45 Hz), 2.05–2.11 (m, 1H, H_5a), 2.55–2.59 (m, 1H, H_3a), the signal for 1H, NH of piperidine merged with water peak, 2.69–2.79 (m, 1H, H_5e), 3.45–3.48 (d, 1H, H_2a, J_2a,3a = 9.98 Hz), 3.54–3.58 (dd, 1H, H_6a, J_6a,5e = 2.5 Hz, J_6a,5a = 12.95 Hz), 3.76 (s, 2H, CH₂ of imidazolidine), 7.24–7.47 (m, 10H, Ar-H’s), 11.71 (s, NH of imidazolidine); ¹³C NMR (δ ppm): 11.9 -CH₃ at C-3, 32.5 C-5, 37.3 C-3, 44.4 CH₂ of imidazolidine, 60.4 C-6, 68.7 C-2, 126.5–128.1 Ar-C’s, 144.9, 144, ipso-C, 163.1 C=N, 167.5 C=O, 173.8 C=S.

3-(2,6-bis(4-fluorophenyl)-3-methylpiperidin-4-ylideneamino)-2-thioxoimidazolidin-4-one 37

Irradiation reaction time = 3 min; IR (KBr) (cm⁻¹): 3412, 3304, 3071, 3030, 2985, 2931, 2869, 1717, 1635, 1597, 1221, 1030, 535, 836, 766; ¹H NMR (δ ppm): 0.78–0.8 (d, 3H, CH₃ at C-3, J_CH3,3a = 6.58 Hz), 2.07–2.09 (m, 1H, H_5a), 2.54–2.59 (m, 1H, H_3a), the signal for 1H, NH of piperidine merged with water peak, 2.67–2.79 (m, 1H, H_5e), 3.46–3.48 (d, 1H, H_2a, J_2a,3a = 10.61 Hz), 3.54–3.58 (dd, 1H, H_6a, J_6a,5e = 2.46 Hz, J_6a,5a = 12.46 Hz), 3.78 (s, 2H, CH₂ of imidazolidine), 7.12–7.51 (m, 8H, Ar-H’s), 11.72 (s, NH of imidazolidine); ¹³C NMR (δ ppm): 11.8 -CH₃ at C-3, 32.5 C-5, 37.2 C-3, 44.5 CH₂ of imidazolidine, 61 C-6, 67.7 C-2, 114.6–160.7 Ar-C’s, 162.4, 162.5, ipso-C, 163.4 C=N, 169.2 C=O, 173.9 C=S.

3-(2,6-bis(4-chlorophenyl)-3-methylpiperidin-4-ylideneamino)-2-thioxoimidazolidin-4-one 38

Irradiation reaction time = 4 min; IR (KBr) (cm⁻¹): 3429, 3282, 3070, 3032, 2985, 2931, 1718, 1625, 1592, 1210, 1017, 897, 823; ¹H NMR (δ ppm): 0.77–0.79 (d, 3H, CH₃ at C-3, J_CH3,3a = 6.57 Hz), 2.01–2.07 (m, 1H, H_5a), 2.53–2.57 (m, 1H, H_3a), the signal for 1H, NH of piperidine merged with water peak, 2.68–2.8 (m, 1H, H_5e), 3.46–3.48 (d, 1H, H_2a, J_2a,3a = 10.82 Hz), 3.54–3.58 (dd, 1H, H_6a, J_6a,5e = 2.44 Hz, J_6a,5a = 12.45 Hz), 3.9 (s, 2H, CH₂ of imidazolidine), 7.37–7.49 (m, 8H, Ar-H’s), 11.74 (s, NH of imidazolidine); ¹³C NMR (δ ppm): 11.8 -CH₃ at C-3, 31.8 C-5, 36.8 C-3, 44.3 CH₂ of imidazolidine, 61 C-6, 67.7 C-2, 128-131.7 Ar-C’s, 141.8, 142.8 ipso-C, 160.8 C=N, 166.9 C=O, 171.5 C=S.

3-(2,6-bis(4-methoxyphenyl)-3-methylpiperidin-4-ylideneamino)-2-thioxoimidazolidin-4-one 39

Irradiation reaction time = 2 min; IR (KBr) (cm⁻¹): 3426, 3286, 3080, 2981, 2927, 1722, 1627, 1594, 1200, 1025, 897, 812, 744, 527; ¹H NMR (δ ppm): 0.78–0.8 (d, 3H, CH₃ at C-3, J_CH3,3a = 6.45 Hz), 2.04–2.08 (m, 1H, H_5a), 2.55–2.59 (m, 1H, H_3a), the signal for 1H, NH of piperidine merged with water peak, 2.69–2.79 (m, 1H, H_5e), 3.46–3.48 (d, 1H, H_2a, J_2a,3a = 10.72 Hz), 3.54–3.58 (dd, 1H, H_6a, J_6a,5e = 2.56 Hz, J_6a,5a = 12.87 Hz), 3.28 (s, 6H, OCH₃ at the phenyl rings), 3.78 (s, 2H, CH₂ of imidazolidine), 6.86–7.36 (m, 8H, Ar-H’s), 11.82 (s, NH of imidazolidine); ¹³C NMR (δ ppm): 11.9 -CH₃ at C-3, 32.5 C-5, 37.3 C-3, 44.6 CH₂ of imidazolidine, 54.9 -OCH₃ at the phenyl rings, 60.5 C-6, 68.1 C-2, 127.6–158.4 Ar-C’s, 160.5, 163.1 ipso-C, 166.9 C=N, 169.7 C=O, 173.9 C=S.

3-(3-methyl-2,6-dip-tolylpiperidin-4-ylideneamino)-2-thioxoimidazolidin-4-one 40

Irradiation reaction time = 2 min; IR (KBr) (cm⁻¹): 3441, 3312, 3229, 3060, 2971, 2931, 1727, 1634, 1607, 1247, 1031, 542, 833, 753; ¹H NMR (δ ppm): 0.78–0.8 (d, 3H, CH₃ at C-3, J_CH3,3a = 6.38 Hz), 2.01–2.07 (m, 1H, H_5a), 2.19 (s, 6H, CH₃ at the phenyl rings), 2.54–2.58 (m, 1H, H_3a), the signal for 1H, NH of piperidine merged with water peak, 2.69–2.79 (m, 1H, H_5e), 3.44–3.47 (d, 1H, H_2a, J_2a,3a = 10.85 Hz), 3.54–3.58 (dd, 1H, H_6a, J_6a,5e = 2.55 Hz, J_6a,5a = 12.89 Hz), 3.89 (s, 2H, CH₂ of imidazolidine), 7.1–7.33 (m, 8H, Ar-H’s), 11.75 (s, NH of imidazolidine); ¹³C NMR (δ ppm): 11.9 -CH₃ at C-3, 20.6 -CH₃ at the phenyl rings, 32.5 C-5, 37 C-3, 44.5 CH₂ of imidazolidine, 60.2 C-6, 68.4 C-2, 126.3–136.2 Ar-C’s, 139.9, 140.9 ipso-C, 166.8 C=N, 169.6 C=O, 171.5 C=S.

3-(3,3-dimethyl-2,6-diphenylpiperidin-4-ylideneamino)-2-thioxoimidazolidin-4-one 41

Irradiation reaction time = 2 min; IR (KBr) (cm⁻¹): 3496, 3307, 3060, 3027, 2973, 2926, 2852, 1719, 1625, 1206, 1028, 758, 703; ¹H NMR (δ ppm): 0.97 (s, 3H, CH₃ at C-4), 1.19 (s, 3H, CH₃ at C-4), 2.52–2.56 (dd, 1H, H_5e, J_5e,5a = 13.96 Hz, J_5e,6a = 3.64 Hz), 2.75 (s, 1H, H₁); 3.18–3.25 (m, 1H, H₅a), 3.72 (s, 1H, H_2a), 3.91 (s, 2H, CH₂ of imidazolidine), 4.13–4.18 (dd, 1H, H_6a, J_6a,5e = 3.64 Hz, J_6a,5a = 14.44 Hz), 7.21–7.53 (m, 10H, H_arom), 11.8 (s, 1H, NH of imidazolidine); ¹³C NMR (δ ppm): 20.4, 20.8 two CH₃ at C-4, 43.5 CH₂ of imidazolidine, 46.4 C-5, 48.8 C-3, 61 C-6, 69.8 C-2, 126.5–140.3 Arom-C’s, 143.5, 144.2 ipso Cs, 163.1 C=N, 167.3 C=O, 173.8 C=S.

3-(2,6-bis(4-fluorophenyl)-3,3-dimethylpiperidin-4-ylideneamino)-2-thioxoimidazolidin-4-one 42

Irradiation reaction time = 3 min; IR (KBr) (cm⁻¹): 3480, 3309, 3147, 3072, 2975, 2923, 1719, 1628, 1222, 836, 702, 656, 525; ¹H NMR (δ ppm): 0.96 (s, 3H, CH₃ at C-4), 1.18 (s, 3H, CH₃ at C-4), 2.53–2.58 (dd, 1H, H_5e, J_5e,5a = 13.8 Hz, J_5e,6a = 3.6 Hz), 2.8 (s, 1H, H₁); 3.19–3.27 (m, 1H, H₅a), 3.78 (s, 1H, H_2a), 3.92 (s, 2H, CH₂ of imidazolidine), 4.15–4.19 (dd, 1H, H_6a, J_6a,5e = 3.6 Hz, J_6a,5a = 14.44 Hz), 7.3–7.59 (m, 8H, H_arom), 11.79 (s, 1H, NH of imidazolidine); ¹³C NMR (δ ppm): 20.3, 20.7 two CH₃ at C-4, 43.5 CH₂ of imidazolidine, 46.4 C-5, 48.8 C-3, 61 C-6, 69.9 C-2, 113.9–140.4 Arom-C’s, 160.4, 160.6 ipso Cs, 163.3 C=N, 166.9 C=O, 173.8 C=S.

3-(2,6-bis(4-chlorophenyl)-3,3-dimethylpiperidin-4-ylideneamino)-2-thioxoimidazolidin-4-one 43

Irradiation reaction time = 4 min; IR (KBr) (cm⁻¹): 3400, 3306, 3093, 3030, 2974, 2928, 2852, 1722, 1630, 1203, 1015, 827, 766, 523; ¹H NMR (δ ppm): 0.98 (s, 3H, CH₃ at C-4), 1.28 (s, 3H, CH₃ at C-4), 2.54–2.58 (dd, 1H, H_5e, J_5e,5a = 13.92 Hz, J_5e,6a = 3.64 Hz), 2.81 (s, 1H, H₁); 3.18–3.25 (m, 1H, H₅a), 3.77 (s, 1H, H_2a), 3.94 (s, 2H, CH₂ of imidazolidine), 4.16–4.21 (dd, 1H, H_6a, J_6a,5e = 3.64 Hz, J_6a,5a = 14.48 Hz), 7.37–7.89 (m, 8H, H_arom), 11.85 (s, 1H, NH of imidazolidine); ¹³C NMR (δ ppm): 20.2, 20.8 two CH₃ at C-4, 43.4 CH₂ of imidazolidine, 46 C-5, 48.5 C-3, 61.4 C-6, 68.6 C-2, 113.4–139.2 Arom-C’s, 158.8, 159.2 ipso Cs, 163.2 C=N, 165.9 C=O, 173.5 C=S.

3-(2,6-bis(4-methoxyphenyl)-3,3-dimethylpiperidin-4-ylideneamino)-2-thioxoimidazolidin-4-one 44

Irradiation reaction time = 3 min; IR (KBr) (cm⁻¹): 3448, 3317, 3098, 3027, 2976, 2924, 2861, 1702, 1626, 1211, 1028, 818, 747, 519; ¹H NMR (δ ppm): 0.98 (s, 3H, CH₃ at C-4), 1.16 (s, 3H, CH₃ at C-4), 2.54–2.58 (dd, 1H, H_5e, J_5e,5a = 13.94 Hz, J_5e,6a = 3.66 Hz), 2.76 (s, 1H, H₁); 3.19–3.25 (m, 1H, H₅a), 3.56 (s, 6H, OCH₃ at the phenyl rings),3.79 (s, 1H, H_2a), 3.91 (s, 2H, CH₂ of imidazolidine), 4.17–4.22 (dd, 1H, H_6a, J_6a,5e = 3.66 Hz, J_6a,5a = 14.5 Hz), 7.19–7.76 (m, 8H, H_arom), 11.79 (s, 1H, NH of imidazolidine); ¹³C NMR (δ ppm): 20.3, 20.7 two CH₃ at C-4, 43.5 CH₂ of imidazolidine, 46.5 C-5, 48.9 C-3, 54 -OCH₃ at the phenyl rings, 61 C-6, 68.2 C-2, 126.4–141.1 Arom-C’s, 141.3, 142.2 ipso C’s, 163.1 C=N, 166.8 C=O, 173.8 C=S.

3-(3,3-dimethyl-2,6-dip-tolylpiperidin-4-ylideneamino)-2-thioxoimidazolidin-4-one 45

Irradiation reaction time = 2 min; IR (KBr) (cm⁻¹): 3450, 3318, 3093, 3022, 2974, 2925, 2864, 1705, 1628, 1209, 1023, 817, 749, 516; ¹H NMR (δ ppm): 0.99 (s, 3H, CH₃ at C-4), 1.18 (s, 3H, CH₃ at C-4), 2.24 (s, 6H, CH₃ of phenyl rings), 2.55–2.59 (dd, 1H, H_5e, J_5e,5a = 13.96 Hz, J_5e,6a = 3.68 Hz), 2.77 (s, 1H, H₁); 3.19–3.26 (m, 1H, H₅a), 3.8 (s, 1H, H_2a), 3.92 (s, 2H, CH₂ of imidazolidine), 4.18–4.23 (dd, 1H, H_6a, J_6a,5e = 3.68 Hz, J_6a,5a = 14.52 Hz), 7.21–7.8 (m, 8H, H_arom), 11.8 (s, 1H, NH of imidazolidine); ¹³C NMR (δ ppm): 20.3, 20.7 two CH₃ at C-4, 22.7 (-CH₃ of phenyl rings), 43.5 CH₂ of imidazolidine, 46.5 C-5, 48.9 C-3, 61 C-6, 68.2 C-2, 126.4–141.1 Arom-C’s, 141.3, 142.2 ipso Cs, 163.1 C=N, 166.8 C=O, 173.8 C=S.

Microbiology

Materials

All the clinically isolated bacterial strains namely Bacillus subtilis, Micrococcus luteus, Salmonella typhii, Salmonella paratyphii-B, Shigella felxneri, Proteus vulgaris and fungal strains namely Aspergillus niger, Mucor, Rhizopus and Microsporum gypsuem, were obtained from the Faculty of Medicine, Annamalai University, Annamalainagar-608 002, Tamil Nadu, India.

In vitro antibacterial and antifungal activity

The MIC as μg/mL values was determined by a two-fold serial dilution method [15]. The respective test compounds 31–45 were dissolved in dimethyl sulphoxide (DMSO) to obtain a 1 mg mL⁻¹ stock solution. Seeded broth (broth containing microbial spores) was prepared in nutrient broth (NB) from 24 h old bacterial cultures on nutrient agar (Hi-media, Mumbai) at 37 ± 1°C while fungal spores from 1 to 7 days old Sabouraud’s agar (Hi-media, Mumbai) slant cultures were suspended in Sabouraud’s dextrose broth (SDB). The colony forming units (CFU) of the seeded broth were determined by a plating technique and adjusted within the range of 10⁴–10⁵ CFU/mL. The final inoculum size was 10⁵ CFU/mL for the antibacterial assay and 1.1–1.5 × 10² CFU/mL for the antifungal assay. The testing was performed at pH 7.4 ± 0.2 for bacteria (NB) and at pH 5.6 for fungi (SDB). Exactly 0.4 mL of the test compound solution was added to 1.6 mL of the seeded broth to form the first dilution. One milliliter of this was diluted with a further 1 mL of seeded broth to give the second dilution and so on till six such dilutions were obtained. A set of assay tubes containing only the seeded broth was kept as a control. The tubes were incubated in biochemical oxygen demand (BOD) (Sigma Instruments, Chennai, India) incubators at 37 ± 1°C for bacteria and 28 ± 1°C for fungi. MICs were recorded by visual observations after 24 h (for bacteria) and 72–96 h (for fungi) of incubation. Penicillin was used as the standard for bacterial studies and amphotericin B was used as the standard for fungal studies.

Results and discussion

In the current work, the classical synthetic strategy adopted to obtain the a new series of hybrid heterocycles comprising both piperidine and thiohydantoin nuclei namely 3-(3-alkyl-2,6-diarylpiperin-4-ylidene)-2-thioxoimidazolidin-4-ones 31–45 was synthesised by the treatment of the respective thiosemicarbazones 16–30 with chloroethyl acetate and anhydrous sodium acetate in refluxing ethanol for 4 h. The synthetic route for the formation of compounds 31–45 is given in Scheme 1. The physical and analytical data is in Table 1. In order to avoid the use of solvent, to minimise the reaction time and to improve the yield of the product, a ‘one-pot’ reaction procedure was developed by the treatment of 3-alkyl-2,6-diarylpiperidin-4-ones 1–15, thiosemicarbazide and chloroethyl acetate in the ratio of 1:1:1 the same reaction was also performed under microwave irradiation in a scientific microwave oven using a catalytic amount of NaHSO₄.SiO₂ (25 mg) to afford the title compounds in high yields in dry media. The NaHSO₄.SiO₂ catalyst was shown to be one of the most efficient MW absorbers with a very high specificity to MW heating. It was able to reach a temperature of 110°C after 3 minutes of irradiation in a domestic oven (320 W). A mere 25 mg of NaHSO₄.SiO₂ catalyst to 0.01 moles of substrates was the most acceptable ratio in terms of efficiency and safety; a power level of 320 watts was the most suitable. Moreover, the catalyst assists in the removal of water molecules and facilitates the condensation reaction to form the title compounds.

Table 1.  Physical and analytical data of compounds 31–45.

Compound	R^1	R^2	X	Yield Δ (MW) (%)	mp (°C)	Elemental analysis (%)			m/z (M+1)^+. Molecular formula
						C Found (calculated)	H Found (calculated)	N Found (calculated)
31	H	H	H	74 (90)	108	65.85 (65.91)	5.45 (5.53)	15.3 (15.37)	365.2 C20H20N4OS
32	H	H	F	78 (92)	103	59.91 (59.99)	4.44 (4.53)	13.9 (13.99)	400.4 C20H18F2N4OS
33	H	H	Cl	67 (82)	167	55.39 (55.43)	4.15 (4.19)	12.81 (12.93)	432.3 C20H18Cl2N4OS
34	H	H	OCH3	72 (90)	158	62.2 (62.24)	5.66 (5.7)	13.17 (13.20)	424.0 C22H24N4O3S
35	H	H	CH3	70 (88)	142	67.3 (67.32)	6.1 (6.16)	14.23 (14.27)	392.3 C22H24N4OS
36	CH3	H	H	75 (95)	171	66.6 (66.64)	5.82 (5.86)	14.76 (14.8)	378.2 C21H22N4OS
37	CH3	H	F	78 (92)	182	60.81 (60.85)	4.82 (4.86)	13.48 (13.52)	414.1 C21H20F2N4OS
38	CH3	H	Cl	75 (85)	187	56.35 (56.38)	4.4 (4.51)	12.43 (12.52)	446.3 C21H20Cl2N4OS
39	CH3	H	OCH3	72 (90)	182	62.95 (62.99)	5.95 (5.98)	12.68 (12.78)	438.5 C23H26N4O3S
40	CH3	H	CH3	70 (94)	136	67.9 (67.95)	6.41 (6.45)	13.75 (13.78)	406.4 C23H26N4OS
41	CH3	CH3	H	76 (96)	148	67.28 (67.32)	6.1 (6.16)	14.21 (14.27)	392 C22H24N4OS
42	CH3	CH3	F	77 (95)	134	61.64 (61.67)	5.11 (5.17)	13.03 (13.08)	428.2 C22H22F2N4OS
43	CH3	CH3	Cl	75 (90)	176	57.21 (57.27)	4.78 (4.81)	12.11 (12.14)	460 C22H22Cl2N4OS
44	CH3	CH3	OCH3	72 (85)	132	63.6 (63.69)	6.1 (6.24)	12.31 (12.38)	452.4 C24H28N4O3S
45	CH3	CH3	CH3	71 (88)	124	68.44 (68.54)	6.65 (6.71)	13.2 (13.32)	420.1 C24H28N4OS

Scheme 1.  A facile synthetic route for the synthesis of 3-(3-substituted-2,6-diarylpiperidin-4-ylideneamino)-2-thioxoimidazolidin-4-ones.

The conversion of 3-alkyl-2,6-diarylpiperidin-4-ones 1–15 into 3-(3-alkyl-2,6-diarylpiperin-4-ylidene)-2-thioxoimidazolidin-4-ones 31–45 using this method was followed via the 3-alkyl-2,6-diarylpiperidin-4-one thiosemicarbazone derivatives 16–31. In the first step, 3-alkyl-2,6-diarylpiperidin-4-ones were converted to their respective thiosemicarbazones and rapidly rearrange to give 3-(3-alkyl-2,6-diarylpiperin-4-ylidene)-2-thioxoimidazolidin-4-ones 31–45 in the second step. The attempt to isolate the respective thiosemicarbazones from the reaction mixture was unsuccessful. The formation of 3-(3-alkyl-2,6-diarylpiperin-4-ylidene)-2-thioxoimidazolidin-4-ones 31–45 via the thiosemicarbazones were confirmed by the same kind of reactions carried out using the NaHSO₄.SiO₂ catalyst and 3-alkyl-2,6-diarylpiperidin-4-ones thiosemicarbazones 16–30 and under microwave irradiation for 2–4 min. The products formed from the above two methods were found to be the same. The structures of all the synthesised compounds 3-(3-alkyl-2,6-diarylpiperin-4-ylidene)-2-thioxoimidazolidin-4-ones 31-45 were characterised on the basis of their mps, elemental analysis, FT-IR, MS, one-dimensional NMR (¹H, ¹³C), two dimensional HOMOCOSY and NOESY spectra which were in good agreement with the proposed structures.

Spectral assignments for the newly synthesised compound 31

In order to investigate the spectral assignments, compound 31 was chosen as a representative compound. The IR spectrum of 3-(2,6-diphenylpiperidin-4-ylideneamino)-2-thioxoimidazolidin-4-one 31 shows characteristic frequencies at 1728, 1215 cm⁻¹ were due to the presence of carbonyl and thiocarbonyl groups. The absorption frequencies shown in the region of 3300-3426 cm-1 suggested the presence of -NH groups. In addition, the absorption frequency at 1635 cm⁻¹ was due to C=N stretching vibration. The mass spectrum of compound 31 shows a molecular ion peak at an m/z of 365.25 (M+•+1) which was consistent with the proposed molecular formula of 31. The elemental analysis (Ccal 65.87, Cobs 65.91; Hcal 5.53, Hobs 5.5; Ncal 15.37, Nobs 15.33) were consistent with the proposed molecular formula (C₂₀H₂₀N₄₀S) of 31.

The assignment of signals in the ¹H NMR spectrum of compound 31 were based on total widths and spin multiplicities. There were two double doublet centered at 3.9 ppm and 4.17 ppm. Each signal corresponded to one proton. These two signals were due to the benzylic protons H_2a and H_6a (3.9/H_2a, 4.17/H_6a). Two coupling constants were extracted from the double doublet at 3.88–3.92 and the values were found to be 11.76 Hz and 3.08 Hz. The lower value was due to the vicinal coupling of J_2a,3e and the higher value corresponds to the trans coupling of J_2a,3a. The two coupling constants were calculated from the double doublet at 4.15–4.19 ppm and the values were 11.88 Hz and 3.20 Hz. The lower value was due to the vicinal coupling of J_6a,5e and the higher value is due to the trans coupling of J_6a,5a. The double doublets observed at 2.41–2.37 ppm and 3.62–3.66 ppm were due to the equatorial methylenic protons H_3e and H_5e respectively. A double doublet was expected for the axial methylenic proton H_5a, but a multiplet was obtained at 2.43–2.52 ppm corresponding to the H_5a proton. A double doublet at 2.37–2.41 ppm had two coupling constants and the coupling values were 2.96 Hz and 13.64 Hz. The lower value corresponds to vicinal coupling of J_3e,2a and the higher value corresponds to geminal coupling of J_3e,3a. The two coupling constant values were extracted from the double doublet at 3.62–3.66 ppm. The lower value 2.96 Hz was due to vicinal coupling of J_{5e, 6a} and the higher value 13.52 Hz is due to geminal coupling of J_{5e, 5a}. A sharp singlet observed at 3.80 ppm was due to the methylene protons of the imidazolidine moiety. A double doublet was expected for the axial methylenic proton H_3a. but a multiplet was obtained at 1.97–2.05 ppm corresponding to the H_3a proton. The NH proton of the piperidone moiety was observed as a broad singlet at 2.83 ppm. The broad signal at 11.78 ppm was due to the NH proton of the imidazolidine moiety. A multiplet appeared in the range of 7.23 −7.5 ppm due to the aromatic ring protons at the C-2 and C-6 positions.

In the ¹³C NMR spectrum of 31, resonances in the aliphatic range 29.62, 37.3, 60.23, 61.12, 43.64 ppm were observed. The signals appeaing at 29.62 and 37.3 ppm were due to the C-3 and C-5 carbons respectively. Among the signals at 60.23 ppm and 61.12 ppm for the benzylic carbons, the one at 60.23 ppm was due to C-2 and the signal at 61.12 ppm was due to C-6 carbon. The ¹³C resonance at 43.64 ppm must be due to the methylene carbon of the imidazolidine moiety (at C-5). The signal at 163.19 ppm was conveniently assigned to the C=N carbon of the piperidone moiety. The ¹³C resonance of the carbonyl carbon and the thiocarbonyl carbon at the C-4 and C-2 of the midazolidine moiety appeared at 167.63 and 173.87 ppm (C=S/173.87; C=O/167.63) respectively. The ipso carbons appeared at 144.01 and 144.16 ppm. The signals appearing in the region of 126.51–128.15 were due to the aromatic carbons in the two phenyl rings at the C-2 and C-6 positions. All the above mentioned assignments were further confirmed by HOMOCOSY and NOESY spectra.

In the HOMOCOSY spectrum of 31, the signal at 3.9 ppm showed cross peaks with the signals at 2.01 ppm and 2.39 ppm. The signal at 4.17 ppm showed cross peaks with the signals at 2.48 and 3.64 ppm. Consequently, the signal at 3.9 ppm must be due to the benzylic proton H_2a. Since this can have coupling only with the H_3e and H_3a protons, the two signals at 2.01 ppm and 2.39 ppm were assigned to the H_3a and H_3e protons respectively. The signal at 4.17 ppm must be due to the benzylic proton H_6a, since this can have coupling with the H_5a and H_3e protons. The two signals at 2.48 and 3.64 ppm were assigned to the H_5a and H_5e protons respectively. The individual assignments can be made by using its NOESY spectrum.

In the NOESY spectrum of 31, the signals of the benzylic proton (H_2a,H_6a) have a strong nOe with the signals of the methylene protons (H_3e and H_5a proton). Hence the signal at 3.9 ppm has a strong nuclear Overhauser effect (nOe) with the signal at 2.39 ppm. The signal at 4.17 ppm has a strong nOe with the signal 2.48 ppm. From this it was concluded that the signal at 3.9 ppm must be due to the benzylic proton H_2a. The signal at 4.17 ppm must be due to the benzylic proton H_6a. The signals at 2.39 ppm and 2.48 ppm must be due to the H_3e and the H_5a proton respectively. Moreover, an interesting observation from the NOESY spectrum of compound 31 was that the signal at 11.78 ppm had a strong nOe with the signal at 2.01 ppm. Hence, the signal at 11.78 must be due to the NH proton of the imidazolidine moiety and the signal at 2.01 ppm must be due to the H_3a proton. The signal at 3.80 ppm has strong nOe with the signals at 2.01 ppm and 2.39 ppm. The signal at 3.8 ppm must be due to the methylene proton of the imidazolidine moiety. The signal at 2.39 ppm was due to the H_3e proton. From the NOESY spectrum, we concluded that the imidazolidine moiety of 46 presents toward the H_3a and H_3e protons of the piperidone moiety of 31. From the view of nOes of -NH and CH₂ protons of the imidazolidine moiety, the -NH signal (11.78 ppm) and CH₂ signal (3.8 ppm) must be very close to the methylene protons of the piperidone moiety at the C-3 position.

Therefore, taking into account the HOMOCOSY and NOESY correlations for compound 31, the tentative assignments made for its ring and substituent protons were confirmed. It has been shown that the 2,6-diarylpiperidin-4-one ring mostly and favourably adopts the chair conformation with all its substituents at an equatorial disposition [12]. Moreover the equatorial disposition of the phenyl group at the C-2 and C-6 makes the chair conformation more rigid thereby preventing interconvertion of one chair form into another. Hence, based on the obtained chemical shifts and coupling constant values, the normal chair conformation (Figure 1) was proposed for compound 31.

Figure 1.  Chair conformation for compound 31.

Two dimensional HOMOCOSY and NOESY NMR spectral assignments for the newly synthesised 3-(3-methyl-2,6-diphenylpiperidin-4-ylideneamino)-2-thioxoimidazolidin-4-one 36

In the HOMOCOSY spectrum of 36, the double doublet at 3.56 ppm had cross peaks with the signals at 2.08 ppm and 2.74 ppm and vice versa. This mutual correlation clearly showed that these two signals must be due to the H_5a and H_5e proton. The signal at 3.56 ppm must be due to the benzylic proton H_6a. Besides, the doublet at 3.46 ppm had a cross peak with the signal at 2.59 ppm and 0.78 ppm. Therefore, the signal at 3.46 ppm must be due to the benzylic proton H_2a and the signal at 2.59 ppm should be due to the H_3a proton. Likewise, the methyl proton doublet at 0.78 ppm had a strong cross peak with the signal at 2.59 ppm. Hence, the signal at 2.59 ppm must be due to the H_3a proton. The individual assignments can be made by using the NOESY spectrum.

From the NOESY spectrum of compound 36, it was interesting to note that the signal at 0.78 ppm of C-3 methyl protons had strong nOe with the benzylic proton signal at 3.46 ppm and the methine proton signal at 2.59 ppm. It further confirmed the assignment of the doublet at 3.46 ppm to the benzylic proton H_2a and the signal centred at 2.59 ppm to the methine proton using the HOMOCOSY spectrum of the same compound. The benzylic proton signal at 3.46 ppm had a nOe with the methine proton signal at 2.59 ppm and the C-3 methyl proton signal at 0.78 ppm. Hence, the signals at 2.59 ppm and 3.46 ppm must be due to the H_3a and H_2a protons respectively. The double doublet at 3.56 ppm had a cross peak with the methine proton signals at 2.08 ppm and 2.74 ppm. This suggested that the signal at 2.08 ppm was due to the H_5a proton and the signal at 2.74 ppm must be due to the H_5e proton. This showed that the signal at 3.56 ppm should be due to the benzylic proton H_6a. The signal at 3.76ppm had a strong nOe with the signal at 2.08 ppm. Hence the signal at 3.76 ppm must be due to the methylene proton of the imidazolidine moiety. The NH proton signal at 11.71 ppm of the imidazolidine moiety had a strong nOe with the methine proton signal at 2.74 ppm. Hence, the NH and CH₂ protons of the imidazolidine moiety must be very close to the methylene protons at C-5 position of piperidone moiety. Therefore, with reference to HOMOCOSY and NOESY correlations in compound 36, the assignments made for its ring and substituent protons were confirmed. Hence based on the obtained chemical shifts and coupling constant values, a normal chair conformation (Figure 2) was proposed for compound 36.

Figure 2.  Chair conformation for compound 36.

Two dimensional HOMOCOSY and NOESY NMR spectral assignments for the newly synthesised 3-(3,3-dimethyl-2,6-diphenylpiperidin-4-ylideneamino)-2-thioxoimidazolidin-4-one 41

Two singlets of the two methyl proton at 0.97 and 1.19 ppm had a cross peak with the signal at 3.72 ppm. Hence, the signal at 3.72 ppm must be due to the H_2a proton. The double doublet at 4.15ppm had cross peaks with the signal at 2.54ppm and vice versa. This mutual correlation clearly showed that the signal at 4.15ppm was due to the H_6a proton and the signal at 2.54 ppm must be due to the H_5e proton. The individual assignments can be made by using its NOESY spectrum.

From the NOESY spectrum of compound 41, it was interesting to note that the two methyl protons at 0.97 ppm and 1.19 ppm had a nOe with protons at 3.72 ppm. This shows that the signal at 3.72 ppm should be due to the H_2a proton. The double doublet at 4.15 ppm had a strong nOe with the proton at 2.54 ppm. This revealed that the signal at 2.54 ppm must be due to the H_5e proton. In addition, the signal at 3.91 ppm had a strong nOe with the proton signal at 2.54 ppm and a weak nOe with the proton signal at 3.21 ppm. Hence, the signal at 3.91 ppm must be due to the methylene proton of the imidazolidine moiety. The signal at 3.21 ppm must be due to the H_5a proton. The NH proton signal (11.8 ppm) of the imidazolidine moiety had a strong nOe with the methine proton signals at 2.54 and 3.21 ppm. This showed clearly that the NH and CH₂ protons of the imidazolidine moiety must be very close to the methylene protons at the C-5 position of piperidone moiety. Therefore, with reference to HOMOCOSY and NOESY correlations in compound 41, the assignments made for its ring and substitutent protons were confirmed. Hence based on the obtained chemical shifts and coupling constant values, a normal chair conformation (Figure 3) was proposed for compound 41.

Figure 3.  Chair conformation for compound 41.

Antibacterial activity

New 3-(3-alkyl-2,6-diarylpiperin-4-ylidene)-2-thioxoimidazolidin-4-ones 31–45 were tested for their antibacterial activity in vitro against clinically isolated bacterial strains namely B. subtilis, M. luteus, S. typhii, S. paratyphii B, S. felxneri and P. vulgaris. Penicillin was used as the standard drug. The minimum inhibitory concentration (MIC) in μg/ mL values was reproduced in Table 2. Compound 31 which doesn’t have a substituent at the para position of the phenyl ring at positions 2 and 6 of the piperidone ring exhibited antibacterial activity against B. subtilis at a MIC value of 100 μg/mL. Introduction of a methyl substituent at position 3 of the piperidone ring in compound 36 enhanced its activity with a MIC value of 12.5  μg/mL. Further introduction of a dimethyl substituent at position 3 of the piperidone ring in compound 41 enhanced the antibacterial activity against B. subtilis with a MIC value of 6.25 μg/mL. Compounds 32 and 33 which have electron withdrawing fluoro and chloro functional group at the para position of the phenyl rings at positions 2 and 6 of the piperidone ring respectively were inactive against B. subtilis and exhibited a MIC of 200 μg/ mL. Similar results were observed for compounds 37, 38, 42 and 43 against B. subtilis. But introduction of electron donating methoxy and methyl functional group at the para position of the phenyl rings at positions 2 and 4 of the piperidone ring in compounds 34 and 35 showed moderate activity against B. subtilis. In addition to an electron donating methoxy and methyl functional group at the para position of the phenyl rings at positions 2 and 4 of the piperidone ring, a methyl substituent at position 3 of piperidone ring in compounds 39 and 40 were inactive against B. subtilis. Against B. subtilis, analogous results were observed in the dimethyl substituted compounds, 44 and 45. Compound 31, the methyl substituted compound 36 and the dimethyl substituted compound 41 none of which had a substituent at the para position of the phenyl rings at positions 2 and 6 of the piperidone ring exhibited antibacterial activity against M. luteus at a MIC value of 200 μg/mL. The introduction of an electron withdrawing fluoro and chloro functional group at the para position of the phenyl rings at positions 2 and 6 of the piperidone ring respectively in compounds 32 and 33 were active against M. luteus and exhibited a MIC of 12.5 μg/ mL. Methyl substitution at position 3 of the piperidone ring for compounds 37 and 38 which also have electron withdrawing fluoro and chloro functional group at the para position of the phenyl rings at positions 2 and 6 of the piperidone ring respectively, were potent against M. luteus and exhibited a MIC of 6.25 μg/mL whereas dimethyl substitution at position 3 of the piperidone ring for compounds 42 and 43 were inactive against M. luteus. Compound 34, 35, methyl substituted compound 39, 40 and dimethyl substituted compound 44, 45 which all have an electron donating methoxy and methyl functional group at the para position of the phenyl rings at positions 2 and 6 of the piperidone ring exhibited moderate antibacterial activity against M. luteus. Against S. typhii, compounds 31, 36 and 41 were inactive. The electron withdrawing substituted compounds of 32, 33, 37, 38, 42 and 43 showed potent activity against S. typhii at a MIC value of 12.5–6.25 μg/ mL. The electron donating substituted compounds of 34, 35, 39, 40, 44 and 45 showed moderate activity against S. typhii at a MIC value of 100–25 μg/ mL. The electron withdrawing substituted compounds of 37, 38, 42 and 43 were active against S. paratyphii B. The electron donating substituted compounds of 34, 35, 39, 40, 44 and 45 were active against S. felxneri. Against P.vulgaris all the compounds exerted activity at a MIC value of 100–6.25 μg/mL.

Table 2.  In vitro antibacterial activity of compounds 31–45 against clinically isolated bacterial strains.

Compound	Minimum inhibitory concentration (MIC) in µg/mL
	B. subtilis	M. luteus	S. typhii	S. paratyphii B	S. felxneri	P. vulgaris
31	100	200	‘-‘	200	50	100
32	200	12.5	12.5	25	200	50
33	200	12.5	25	25	100	50
34	25	50	100	100	12.5	25
35	25	100	100	50	12.5	25
36	12.5	200	‘-‘	200	50	200
37	100	6.25	12.5	12.5	100	25
38	100	6.25	12.5	12.5	100	25
39	200	25	50	200	12.5	12.5
40	200	50	50	100	25	12.5
41	6.25	200	‘-‘	‘-‘	100	50
42	200	25	6.25	6.25	200	12.5
43	‘-‘	25	6.25	6.25	200	12.5
44	100	50	25	100	6.25	6.25
45	100	50	25	100	12.5	6.25
Penicillin	25	12.5	25	25	25	12.5

‘-‘ No inhibition even at a higher concentration of 200 µg/mL

Antifungal activity

The in vitro antifungal activity of the 3-(3-alkyl-2,6-diarylpiperin-4-ylidene)-2-thioxoimidazolidin-4-ones 31-45 was studied against clinically isolated the following fungal strains: A. niger, Mucor, Rhizopus and M. gypsuem. Fluconazole was used as a standard drug. Minimum inhibitory concentration (MIC) in μg/mL values are reproduced in Table 3. Compound 36 which doesn’t have a substitutent at the para position of the phenyl rings at positions 2 and 4 of the piperidone ring but does have an electron donating methyl substituent at position 3 of the piperidone ring did not exert any antifungal activity against A. niger and M. gypsuem even at a high concentration of 200 μg/ mL. The methyl substituted compounds of 37 and 38 with electron withdrawing fluoro and chloro functional groups at the para position of the phenyl rings at positions 2 and 6 of the piperidone ring exerted good activity at a MIC of 12.5 μg/ mL against A. niger. The dimethyl substituted compounds of 42 and 43 with electron withdrawing fluoro and chloro functional groups at the para position of the phenyl rings at positions 2 and 6 of the piperidone ring exerted good activity at a MIC of 6.25 and 12.5 μg/ mL respectively against A. niger. Against Mucor, the electron withdrawing fluoro and chloro substituted compounds 32, 33, 37, 38, 42 and 43 exhibited good antifungal activity at a concentration of 12.5-6.25 μg/ mL. Compounds 34, 35, 39, 40, 44 and 45 which all have electron donating methoxy and methyl functional group at the para position of the phenyl rings at positions 2 and 6 of the piperidone ring exerted good activity at a MIC of 6.25 and 12.5 μg/mL against Rhizopus. Similarly compounds 34 and 35, which have electron donating methoxy and methyl functional group at the para position of the phenyl rings at positions 2 and 6 of the piperidone ring exerted excellent antifungal activity against M. gypsuem at a MIC of 6.25 and 12.5 μg/mL.

Table 3.  In vitro antifungal activity of compounds 31–45 against clinically isolated fungal strains.

Compound	Minimum inhibitory oncentration (MIC) in µg/mL
	A. niger	Mucor	Rhizopus	M. gypsuem
31	200	100	200	200
32	25	6.25	100	100
33	25	12.5	200	100
34	50	100	6.25	12.5
35	50	100	6.25	6.25
36	‘-‘	100	50	50
37	12.5	6.25	200	‘-‘
38	12.5	6.25	200	200
39	50	200	12.5	25
40	50	200	25	25
41	100	100	100	100
42	6.25	12.5	100	50
43	12.5	12.5	100	50
44	100	50	200	100
45	100	50	200	100
Amphotericin B	25	25	50	25

‘-‘, No inhibition even at a higher concentration of 200 µg/mL

Conclusion

The 3-(3-Alkyl-2,6-diarylpiperin-4-ylidene)-2-thioxoimidazolidin-4-ones, a class of novel hybrid heterocyclic compounds can be synthesised from various 3-alkyl-2,6-diphenylpiperidin-4-one thiosemicarbazones, anhydrous sodium acetate and chloroethyl acetate in refluxing ethanol and a comparative study was also carried out under microwave irradiation. The synthesised compounds were characterised by mps, elemental analysis, MS, FT-IR, one-dimensional NMR (¹H, D₂O exchanged ¹H & ¹³C), two dimensional HOMOCOSY and NOESY spectroscopic data. The in vitro microbiological screening studies carried out to evaluate the antibacterial and antifungal potencies of the newly synthesised 3-(3-alkyl-2,6-diarylpiperin-4-ylidene)-2-thioxoimidazolidin-4-ones 31–45 are shown in Table 2 and Table 3. A close inspection of the in vitro antibacterial and antifungal activity profile in differently electron withdrawing (F and Cl) and electron donating (OCH₃ and CH₃) functional groups substituted phenyl rings of novel 3-(3-alkyl-2,6-diarylpiperin-4-ylidene)-2-thioxoimidazolidin-4-ones 31–45 exerted strong antibacterial activity against all the clinically isolated tested bacterial and fungal strains namely: B. subtilis, M. luteus, S. typhii, S. paratyphii B, S. felxneri, P. vulgaris, A. niger, Mucor, Rhizopus and M. gypsuem. These observations may promote further development of our research into this group of pyrido 2-thioxoimidazolidin-4-ones and may lead to the development of compounds with a better pharmacological profile than the standard antibacterial and antifungal drugs.

Acknowledgements

The authors would like to thank the NMR Research Centre, Indian Institute of Science, Bangalore for recording spectra.

Declaration of interest

V. Kanagarajan is grateful to the Council of Scientific and Industrial Research (CSIR), New Delhi, Republic of India for providing financial support in the form of CSIR-Senior Research Fellowship (SRF) in Organic Chemistry. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.