Synthesis and Biological Evaluation of Some Novel Dithiocarbamate Derivatives

Sağlık, Begüm Nurpelin; Özkay, Yusuf; Demir Özkay, Ümide; Karaca Gençer, Hülya

doi:https://doi.org/10.1155/2014/387309

Journal of Chemistry

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Research Article | Open Access

Volume 2014 | Article ID 387309 | https://doi.org/10.1155/2014/387309

Synthesis and Biological Evaluation of Some Novel Dithiocarbamate Derivatives

Begüm Nurpelin Sağlık,¹Yusuf Özkay,¹Ümide Demir Özkay,²and Hülya Karaca Gençer³

Academic Editor: Xinyong Liu

Received29 Jul 2014

Accepted09 Sept 2014

Published13 Oct 2014

Abstract

18 novel dithiocarbamate derivatives were synthesized in order to investigate their inhibitory potency on acetylcholinesterase enzyme and antimicrobial activity. Structures of the synthesized compounds were elucidated by spectral data and elemental analyses. The synthesized compounds showed low enzyme inhibitory activity. However, they displayed good antimicrobial activity profile. Antibacterial activity of compounds 4a, 4e, and 4p (MIC = 25 μg/mL) was equal to that of chloramphenicol against Klebsiella pneumoniae (ATCC 700603) and Escherichia coli (ATCC 35218). Most of the compounds exhibited notable antifungal activity against Candida albicans (ATCC 10231), Candida glabrata (ATCC 90030), Candida krusei (ATCC 6258), and Candida parapsilosis (ATCC 7330). Moreover, compound 4a, which carries piperidin-1-yl substituent and dimethylthiocarbamoyl side chain as variable group, showed twofold better anticandidal effect against all Candida species than reference drug ketoconazole.

1. Introduction

Alzheimer’s disease (AD) is a an age associated neurodegenerative syndrome with clinical characteristic and pathological properties as loss of neurons in certain brain regions leading to deficiency of memory, cognitive dysfunction, behavioral disturbances, and deficits in activities of daily living, which eventually leads to death [1–3]. Although the underlying pathophysiological mechanisms are not clear, AD is firmly associated with impairment in cholinergic pathway, which results in reduced level of acetylcholine (Ach) that is hydrolysed by cholinesterases (ChE) in certain areas of brain [1, 2, 4, 5]. It is well known that there are two major forms of ChE in the brain of mammals. These are acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) found in neurons and glial cells and in neuritic plaques and tangles in Alzheimer’s disease (AD) patients [6].

Since the late 1970s the treatment of AD has proceeded and trended to a transmitter replacement strategy. Elevation of acetylcholine levels in brain through the use of AChE inhibitors has been established as the most effective treatment strategy against AD [3, 7]. Therefore, AChE and/or BuChE inhibitors have been recognised as the drug of choice in management of AD [8]. Several AChE inhibitors called as “cognitive enhancers” are being investigated for the symptomatic treatment of Alzheimer’s disease [2, 7, 8]. These drugs increase the concentration of acetylcholine at the neurotransmitter sites or acts by regulating activity at nicotinic receptors [2, 4].

There are three important subsites in ChE; anionic site, oxyanion hole, and acyl pocket [9]. Carbamates as pyridostigmine, rivastigmine, and physostigmine constitute a class of ChE inhibitors. These compounds have been reported to direct their carbamate region towards the acyl pocket that includes esteratic site of the enzyme. This inhibition is subsequently reversed upon decarbamylation (Figure 1). However, carbamates have a relatively short duration of action and limited penetration to blood-brain barrier [10].

(a)

(b)

Figure 1

(a) Ligand binding sites in acetylcholinesterase. Hydrophobic residues in the gorge are shown as orange sticks. The surface of the protein is shown in cadetblue. Acetylcholine (green sticks) is shown in the catalytic anionic site (CAS). π-Cation interactions and hydrogen bonds are shown as white dotted lines. The acyl pocket residues are shown as brown sticks. (b) Interaction between rivastigmine and acetylcholinesterase. After hydrolysis, rivastigmine, shown as coral sticks, releases the carbamate residue towards the acyl pocket and enzyme inhibition occurs. Images were taken from the official web site of European Bioinformatics Institute.

Dithiocarbamates have attracted a great deal of interest in medicinal chemistry due to the fact that new effective compounds can be gained by the bioisosteric replacement of carbamate moiety with dithiocarbamate moiety. They are also important pharmacophores because of their lipophilicity, which is essential for the delivery of central nervous system (CNS) drugs to their site of action through the blood-brain barrier [11–18]. Thus, evaluation of novel dithiocarbamate derivatives as potential AChE inhibitors will be rational.

The treatment of infectious diseases still remains a crucial problem because of the increasing number of multidrug resistant microbial pathogens. In spite of a large number of antibiotics and chemotherapeutics existing for therapeutic use, the emergence of antibiotic resistance developed in the last decades has created a substantial medical need for new classes of antibacterial agents. A potential approach to overcome the resistance problem is to design novel agents with a different mode of action [19, 20].

In addition to their potential AChE inhibitory activity, dithiocarbamate derivatives are important compounds in antimicrobial chemotherapy due to their antibacterial and antifungal properties. First, Miller and Elson [21] and Kligman and Rosensweig [22] determined the activity of dimethyl dithiocarbamate salts against several pathogenic fungi and commented on their possible application in human therapy. In the last decade, dithiocarbamate moiety combined heterocyclic ring systems were studied widely, and now these compounds form a promising group of novel antifungal agents [23].

Cyclic amines as piperidine, piperazine, and morpholine possess antimicrobial importance and thus they are often subjected to new antimicrobial agents development studies. For instance, quinoline drugs as norfloxacin and ciprofloxacin carry piperazine nucleus and show broad antibacterial activity spectrum [24]. Linezolid, used for the treatment of infections caused by gram-positive bacteria, includes morpholine group [25–27]. Piperidine based chemical entities with aryl substituents have been documented as potent microbial agents [28, 29].

On the basis of above findings, in the present study we report the synthesis and biological evaluation of some novel dithiocarbamate derivatives as probable anticholinesterase and antimicrobial agents.

2. Experiment

2.1. Materials

All reagents were purchased from commercial suppliers and were used without further purification. Melting points were determined on an Electrothermal 9100 melting point apparatus (Weiss-Gallenkamp, Loughborough, UK) and were uncorrected. IR spectra were recorded on Shimadzu 8400 FT-IR spectrophotometer (Shimadzu, Tokyo, Japan). ¹H-NMR spectra were recorded on a Bruker 500 MHz spectrometer (Bruker, Billerica, USA). Mass spectra were recorded on a VG Quattro mass spectrometer (Agilent, Minnesota, USA). Elemental analyses were performed on a Leco CHNS-932 analyser (Leco, Michigan, USA). The TLC was performed to monitor reactions on silica gel 60 F₂₅₄ (Merck) layer using petroleum ether : ethyl acetate (3 : 1 v/v) for the first and third reaction steps and petroleum ether : ethyl acetate (1 : 1 v/v) for the second and fourth reaction steps (Scheme 1).

2.2. General Procedure for 4-Substituted-1-nitrobenzene Derivatives (1a–1c)

4-Fluoro-1-nitrobenzene (4.24 mL, 0.04 mol), K₂CO₃ (5.52 g 0.04 mol), appropriate cyclic secondary amine (0.04 mol), and DMF (10 mL) were added into a vial (30 mL) of microwave synthesis reactor (Anton-Paar Monowave 300, Graz, Austria). The reaction mixture was heated under conditions of 200°C and 10 bars for 15 min. After cooling, the mixture was poured into iced-water; precipitated product was washed with water, dried, and recrystallized from ethanol.

2.3. General Procedure for 4-Substituted Aniline Derivatives (2a–2c)

Corresponding 4-substituted-1-nitrobenzene derivative (1a–1c) (0.035 mol) was dissolved in ethanol (100 mL) and 25% HCl (100 mL) mixture. Zinc powder (22.75 g, 0.35 mol) was divided into ten equal portions (2.275 g × 10) and each portion was added to the stirring solution in 15 min intervals. Once the addition of the zinc was completed reaction mixture was refluxed for 1 h. Hot solution was allowed to cool down, poured into ice water, and then neutralized with 10% NaOH solution. The precipitate was extracted with chloroform (3 × 100 mL). The extracts were combined and filtered over anhydrous Na₂SO₄. The solvent was evaporated and the residue was recrystallized from ethanol to give the 4-substituted aniline derivatives (2a–2c).

2.4. General Procedure for 2-Chloro-N-(4-substituted-phenyl)acetamide (3a–3c)

Appropriate 4-substituted aniline (2a–2c) (0.022 mol) and triethylamine (3.2 mL, 0.22 mol) were dissolved in THF (100 mL). This mixture was allowed to stir on an ice bath. Chloroacetyl chloride (1.8 mL, 0.022 mol) in THF (10 mL) was added drop by drop. After this stage, the content was stirred for 1 h at room temperature. THF was evaporated and the product was recrystallized from ethanol.

2.5. N-[(4-Substituted-phenyl)-2-substitutedthiocarbonylthio]acetamide Derivatives (4a–4s)

The compounds 3a–3c (0.001 mol) were stirred with appropriate sodium salt of dithiocarbamic acid (0.0011 mol) in acetone (30 mL) for 3 h. The precipitated product was filtered, washed with water, and recrystallized from ethanol to gain the title products 4a–4s.

2.5.1. N-[4-(Piperidin-1-yl)phenyl]-2-(dimethylaminothiocarbonylthio)acetamide (4a)

IR (KBr) (cm⁻¹): 3288 (N–H), 1651 (C=O), 1217 (C=S), 821 (1,4-disubstituted benzene). ¹H-NMR (500 MHz, DMSO-d6): 1.51–1.62 (m, 6H, piperidine, C_3,4,5–H), 3.05 (s, 6H, –N (CH₃)₂), 3.41–3.47 (m, 4H, piperidine, C_2,6–H), 4.17 (s, 2H, –COCH₂), 6.87 (d, 2H, Hz, phenyl, C_3,5–H), 7.40 (d, 2H, Hz, phenyl, C_2,6–H), 10.00 (s, 1H, –NHCO). MS (ESI) (m/z): [M + 1]⁺ 338.16. Anal. Calcd. for C₁₆H₂₃N₃OS₂: C, 56.94; H, 6.87; N, 12.45; Found: C, 56.63; H, 6.85; N, 12.49.

2.5.2. N-[4-(Piperidin-1-yl)phenyl]-2-(diethylaminothiocarbonylthio)acetamide (4b)

IR (KBr) (cm⁻¹): 3289 (N–H), 1659 (C=O), 1236 (C=S), 824 (1,4-disubstituted benzene). ¹H-NMR (500 MHz, DMSO-d6): 1.31 (t, 6H, Hz, –N(CH₂CH₃)₂), 1.51–1.63 (m, 6H, piperidine, C_3,4,5–H), 3.03 (q, 4H, Hz, –N(CH₂CH₃)₂), 3.39–3.49 (m, 4H, piperidine, C_2,6–H), 4.18 (s, 2H, –COCH₂), 6.88 (d, 2H, Hz, phenyl, C_3,5–H), 7.41 (d, 2H, Hz, phenyl, C_2,6–H), 10.05 (s, 1H, –NHCO). MS (ESI) (m/z): [M + 1]⁺ 366.18. Anal. Calcd. for C₁₈H₂₇N₃OS₂: C, 59.14; H, 7.44; N, 11.49; Found: C, 59.07; H, 7.42; N, 11.50.

2.5.3. N-[4-(Piperidin-1-yl)phenyl]-2-(pyrrolidin-1-yl-thiocarbonylthio)acetamide (4c)

IR (KBr) (cm⁻¹): 3291 (N–H), 1659 (C=O), 1236 (C=S), 822 (1,4-disubstituted benzene). ¹H-NMR (500 MHz, DMSO-d6): 1.51–1.62 (m, 6H, piperidine, C_3,4,5–H), 1.91–2.06 (m, 4H, pyrrolidine, C_3,4–H), 3.04–3.09 (m, 4H, pyrrolidine, C_2,5–H), 3.41–3.47 (m, 4H, piperidine, C_2,6–H), 4.20 (s, 2H, –COCH₂), 6.87 (d, 2H, Hz, phenyl, C_3,5–H), 7.40 (d, 2H, Hz, phenyl, C_2,6–H), 9.99 (s, 1H, –NHCO). MS (ESI) (m/z): [M + 1]⁺ 364.11. Anal. Calcd. for C₁₈H₂₅N₃OS₂: C, 59.47; H, 6.93; N, 11.56; Found: C, 59.22; H, 6.91; N, 11.59.

2.5.4. N-[4-(Piperidin-1-yl)phenyl]-2-(piperidin-1-yl-thiocarbonylthio)acetamide (4d)

IR (KBr) (cm⁻¹): 3287 (N–H), 1659 (C=O), 1234 (C=S), 822 (1,4-disubstituted benzene). ¹H-NMR (500 MHz, DMSO-d6): 1.51–1.66 (m, 12H, 2 × piperidine, C_3,4,5–H), 3.04–3.08 (m, 4H, piperidine, C_2,6–H), 3.41–3.47 (m, 4H, piperidine, C_2,6–H), 4.20 (s, 2H, –COCH₂), 6.87 (d, 2H, Hz, phenyl, C_3,5–H), 7.40 (d, 2H, Hz, phenyl, C_2,6–H), 10.01 (s, 1H, –NHCO). MS (ESI) (m/z): [M + 1]⁺ 378.19. Anal. Calcd. for C₁₉H₂₇N₃OS₂: C, 60.44; H, 7.21; N, 11.13; Found: C, 60.70; H, 7.19; N, 11.15.

2.5.5. N-[4-(Piperidin-1-yl)phenyl]-2-(4-methylpiperidin-1-yl-thiocarbonylthio)acetamide (4e)

IR (KBr) (cm⁻¹): 3291 (N–H), 1659 (C=O), 1234 (C=S), 822 (1,4-disubstituted benzene). ¹H-NMR (500 MHz, DMSO-d6): 0.93 (d, 3H, Hz, –CH₃), 1.50–1.64 (m, 11H, 2 × piperidine, C_3,4,5–H), 3.04–3.09 (m, 4H, piperidine, C_2,6–H), 3.41–3.46 (m, 4H, piperidine, C_2,6–H), 4.19 (s, 2H, –COCH₂), 6.87 (d, 2H, Hz, phenyl, C_3,5–H), 7.40 (d, 2H, Hz, phenyl, C_2,6–H), 10.02 (s, 1H, –NHCO). MS (ESI) (m/z): [M + 1]⁺ 392.14. Anal. Calcd. for C₂₀H₂₉N₃OS₂: C, 61.34; H, 7.46; N, 10.73; Found: C, 61.42; H, 7.45; N, 10.70.

2.5.6. N-[4-(Piperidin-1-yl)phenyl]-2-(4-benzylpiperidin-1-yl-thiocarbonylthio)acetamide (4f)

IR (KBr) (cm⁻¹): 3289 (N–H), 1659 (C=O), 1234 (C=S), 822 (1,4-disubstituted benzene). ¹H-NMR (500 MHz, DMSO-d6): 1.44–1.63 (m, 11H, 2 × piperidine, C_3,4,5–H), 2.54 (d, 2H, Hz, –CH₂C₆H₅), 3.04–3.10 (m, 4H, piperidine, C_2,6–H), 3.42–3.48 (m, 4H, piperidine, C_2,6–H), 4.19 (s, 2H, –COCH₂), 6.87 (d, 2H, Hz, phenyl, C_3,5–H), 7.19–7.31 (m, 5H, –CH₂C₆H₅), 7.41 (d, 2H, Hz, phenyl, C_2,6–H), 10.02 (s, 1H, –NHCO). MS (ESI) (m/z): [M + 1]⁺ 468.17. Anal. Calcd. for C₂₆H₃₃N₃OS₂: C, 66.77; H, 7.11; N, 8.98; Found: C, 66.59; H, 7.09; N, 9.00.

2.5.7. N-[4-(Morpholin-4-yl)phenyl]-2-(dimethylaminothiocarbonylthio)acetamide (4g)

IR (KBr) (cm⁻¹): 3287 (N–H), 1659 (C=O), 1234 (C=S), 822 (1,4-disubstituted benzene). ¹H-NMR (500 MHz, DMSO-d6): 3.04 (s, 6H, –N(CH₃)₂), 3.41–3.47 (m, 4H, morpholine, C_3,5–H), 3.72–3.74 (m, 4H, morpholine, C_2,6–H), 4.18 (s, 2H, –COCH₂), 6.89 (d, 2H, Hz, phenyl, C_3,5–H), 7.44 (d, 2H, Hz, phenyl, C_2,6–H), 10.01 (s, 1H, –NHCO). MS (ESI) (m/z): [M + 1]⁺ 340.15. Anal. Calcd. for C₁₅H₂₁N₃OS₂: C, 53.07; H, 6.24; N, 12.38; Found: C, 53.33; H, 6.25; N, 12.36.

2.5.8. N-[4-(Morpholin-4-yl)phenyl]-2-(diethylaminothiocarbonylthio)acetamide (4h)

IR (KBr) (cm⁻¹): 3289 (N–H), 1659 (C=O), 1235 (C=S), 822 (1,4-disubstituted benzene). ¹H-NMR (500 MHz, DMSO-d6): 1.23 (t, 6H, Hz, –N(CH₂CH₃)₂), 3.04 (q, 4H, Hz, –N(CH₂CH₃)₂), 3.39–3.49 (m, 4H, morpholine, C_3,5–H), 3.72–3.76 (m, 4H, morpholine, C_2,6–H), 4.19 (s, 2H, –COCH₂), 6.89 (d, 2H, Hz, phenyl, C_3,5–H), 7.45 (d, 2H, Hz, phenyl, C_2,6–H), 10.06 (s, 1H, –NHCO). MS (ESI) (m/z): [M + 1]⁺ 368.12. Anal. Calcd. for C₁₇H₂₅N₃O₂S₂: C, 55.56; H, 6.86; N, 11.43; Found: C, 56.62; H, 6.85; N, 11.41.

2.5.9. N-[4-(Morpholin-4-yl)phenyl]-2-(pyrrolidin-1-yl-thiocarbonylthio)acetamide (4i)

IR (KBr) (cm⁻¹): 3287 (N–H), 1659 (C=O), 1234 (C=S), 822 (1,4-disubstituted benzene). ¹H-NMR (500 MHz, DMSO-d6): 1.93–2.07 (m, 4H, pyrrolidine, C_3,4–H), 3.03–3.06 (m, 4H, pyrrolidine, C_2,5–H), 3.41–3.48 (m, 4H, morpholine, C_3,5–H), 3.73–3.82 (m, 4H, morpholine, C_2,6–H), 4.21 (s, 2H, –COCH₂), 6.89 (d, 2H, Hz, phenyl, C_3,5–H), 7.44 (d, 2H, Hz, phenyl, C_2,6–H), 10.05 (s, 1H, –NHCO). MS (ESI) (m/z): [M + 1]⁺ 336.17. Anal. Calcd. for C₁₇H₂₃N₃O₂S₂: C, 55.86; H, 6.34; N, 11.50; Found: C, 55.94; H, 6.37; N, 11.51.

2.5.10. N-[4-(Morpholin-4-yl)phenyl]-2-(piperidin-1-yl-thiocarbonylthio)acetamide (4j)

IR (KBr) (cm⁻¹): 3289 (N–H), 1651 (C=O), 1217 (C=S), 821 (1,4-disubstituted benzene). ¹H-NMR (500 MHz, DMSO-d6): 1.56–1.64 (m, 6H, piperidine, C_3,4,5–H), 3.03–3.06 (m, 4H, piperidine, C_2,6–H), 3.41–3.48 (m, 4H, morpholine, C_3,5–H), 3.73–3.86 (m, 4H, morpholine, C_2,6–H), 4.20 (s, 2H, –COCH₂), 6.89 (d, 2H, Hz, phenyl, C_3,5–H), 7.44 (d, 2H, Hz, phenyl, C_2,6–H), 10.05 (s, 1H, –NHCO). MS (ESI) (m/z): [M + 1]⁺ 380.15. Anal. Calcd. for C₁₈H₂₅N₃O₂S₂: C, 56.96; H, 6.64; N, 11.07; Found: C, 57.10; H, 6.67; N, 11.08.

2.5.11. N-[4-(Morpholin-4-yl)phenyl]-2-(4-methylpiperidin-1-yl-thiocarbonylthio)acetamide (4k)

IR (KBr) (cm⁻¹): 3288 (N–H), 1659 (C=O), 1234 (C=S), 822 (1,4-disubstituted benzene). ¹H-NMR (500 MHz, DMSO-d6): 0.94 (d, 3H, Hz, –CH₃), 1.50–1.65 (m, 5H, piperidine, C_3,4,5–H), 3.03–3.09 (m, 4H, piperidine, C_2,6–H), 3.40–3.46 (m, 4H, morpholine, C_3,5–H), 3.71–3.77 (m, 4H, morpholine, C_2,6–H), 4.19 (s, 2H, –COCH₂), 6.88 (d, 2H, Hz, phenyl, C_3,5–H), 7.42 (d, 2H, Hz, phenyl, C_2,6–H), 10.02 (s, 1H, –NHCO). MS (ESI) (m/z): [M + 1]⁺ 394.18. Anal. Calcd. for C₁₉H₂₇N₃O₂S₂: C, 57.98; H, 6.91; N, 10.68; Found: C, 58.08; H, 6.92; N, 10.69.

2.5.12. N-[4-(Morpholin-4-yl)phenyl]-2-(4-benzylpiperidin-1-yl-thiocarbonylthio)acetamide (4l)

IR (KBr) (cm⁻¹): 3289 (N–H), 1659 (C=O), 1233 (C=S), 822 (1,4-disubstituted benzene). ¹H-NMR (500 MHz, DMSO-d6): 1.51–1.62 (m, 5H, piperidine, C_3,4,5–H), 2.52 (d, 2H, Hz, –CH₂C₆H₅), 3.05–3.08 (m, 4H, piperidine, C_2,6–H), 3.41–3.47 (m, 4H, morpholine, C_3,5–H), 3.72–3.78 (m, 4H, morpholine, C_2,6–H), 4.18 (s, 2H, –COCH₂), 6.89 (d, 2H, Hz, phenyl, C_3,5–H), 7.19–7.31 (m, 5H, –CH₂C₆H₅), 7.41 (d, 2H, Hz, phenyl, C_2,6–H), 10.04 (s, 1H, –NHCO). MS (ESI) (m/z): [M + 1]⁺ 470.21. Anal. Calcd. for C₂₅H₃₁N₃O₂S₂: C, 63.93; H, 6.65; N, 8.95; Found: C, 63.75; H, 6.63; N, 8.98.

2.5.13. N-[4-(4-Methylpiperazin-1-yl)phenyl]-2-(dimethylaminothiocarbonylthio)acetamide (4m)

IR (KBr) (cm⁻¹): 3273 (N–H), 1654 (C=O), 1221 (C=S), 822 (1,4-disubstituted benzene). ¹H-NMR (500 MHz, DMSO-d6): 2.22 (s, 3H, –NCH₃), 3.06 (s, 6H, –N(CH₃)₂), 3.39–3.57 (m, 8H, piperazine, C_2,3,5,6–H), 4.18 (s, 2H, –COCH₂), 6.88 (d, 2H, Hz, phenyl, C_3,5–H), 7.42 (d, 2H, Hz, phenyl, C_2,6–H), 10.02 (s, 1H, –NHCO). MS (ESI) (m/z): [M + 1]⁺ 353.11. Anal. Calcd. for C₁₆H₂₄N₄OS₂: C, 54.51; H, 6.86; N, 15.89; Found: C, 54.38; H, 6.84; N, 15.84.

2.5.14. N-[4-(4-Methylpiperazin-1-yl)phenyl]-2-(diethylaminothiocarbonylthio)acetamide (4n)

IR (KBr) (cm⁻¹): 3292 (N–H), 1654 (C=O), 1234 (C=S), 820 (1,4-disubstituted benzene). ¹H-NMR (500 MHz, DMSO-d6): 1.23 (t, 6H, Hz, –N(CH₂CH₃)₂), 2.24 (s, 3H, –NCH₃), 3.06 (q, 4H, Hz, –N(CH₂CH₃)₂), 3.39–3.62 (m, 8H, piperazine, C_2,3,5,6–H), 4.18 (s, 2H, –COCH₂), 6.88 (d, 2H, Hz, phenyl, C_3,5–H), 7.42 (d, 2H, Hz, phenyl, C_2,6–H), 10.03 (s, 1H, –NHCO). MS (ESI) (m/z): [M + 1]⁺ 381.16. Anal. Calcd. for C₁₈H₂₈N₄OS₂: C, 56.81; H, 7.42; N, 14.72; Found: C, 56.58; H, 7.44; N, 14.75.

2.5.15. N-[4-(4-Methylpiperazin-1-yl)phenyl]-2-(pyrrolidin-1-yl-thiocarbonylthio)acetamide (4o)

IR (KBr) (cm⁻¹): 3287 (N–H), 1654 (C=O), 1234 (C=S), 820 (1,4-disubstituted benzene). ¹H-NMR (500 MHz, DMSO-d6): 2.06–2.10 (m, 4H, pyrrolidine, C_3,4–H), 2.22 (s, 3H, –NCH₃), 3.04–3.08 (m, 4H, pyrrolidine, C_2,5–H), 3.42–3.65 (m, 8H, piperazine, C_2,3,5,6–H), 4.20 (s, 2H, –COCH₂), 6.88 (d, 2H, Hz, phenyl, C_3,5–H), 7.41 (d, 2H, Hz, phenyl, C_2,6–H), 10.00 (s, 1H, –NHCO). MS (ESI) (m/z): [M + 1]⁺ 379.17. Anal. Calcd. for C₁₈H₂₆N₄OS₂: C, 57.11; H, 6.92; N, 14.80; Found: C, 57.42; H, 6.94; N, 14.75.

2.5.16. N-[4-(4-Methylpiperazin-1-yl)phenyl]-2-(piperidin-1-yl-thiocarbonylthio)acetamide (4p)

IR (KBr) (cm⁻¹): 3291 (N–H), 1661 (C=O), 1234 (C=S), 820 (1,4-disubstituted benzene). ¹H-NMR (500 MHz, DMSO-d6): 1.56–1.68 (m, 6H, piperidine, C_3,4,5–H), 2.24 (s, 3H, –NCH₃), 3.03–3.08 (m, 4H, piperidine, C_2,6–H), 3.41–3.68 (m, 8H, piperazine, C_2,3,5,6–H), 4.19 (s, 2H, –COCH₂), 6.88 (d, 2H, Hz, phenyl, C_3,5–H), 7.42 (d, 2H, Hz, phenyl, C_2,6–H), 10.03 (s, 1H, –NHCO). MS (ESI) (m/z): [M + 1]⁺ 393.13. Anal. Calcd. for C₁₉H₂₈N₄OS₂: C, 58.13; H, 7.19; N, 14.27; Found: C, 58.42; H, 7.21; N, 14.25.

2.5.17. N-[4-(4-Methylpiperazin-1-yl)phenyl]-2-(4-methylpiperidin-1-yl-thiocarbonylthio)acetamide (4r)

IR (KBr) (cm⁻¹): 3293 (N–H), 1661 (C=O), 1234 (C=S), 820 (1,4-disubstituted benzene). ¹H-NMR (500 MHz, DMSO-d6): 0.96 (d, 3H, Hz, –CH₃), 1.52–1.65 (m, 5H, piperidine, C_3,4,5–H), 3.04–3.08 (m, 4H, piperidine, C_2,6–H), 3.41–3.75 (m, 8H, piperazine, C_2,3,5,6–H), 4.19 (s, 2H, –COCH₂), 6.88 (d, 2H, Hz, phenyl, C_3,5–H), 7.41 (d, 2H, Hz, phenyl, C_2,6–H), 10.01 (s, 1H, –NHCO). MS (ESI) (m/z): [M + 1]⁺ 407.16. Anal. Calcd. for C₂₀H₃₀N₄OS₂: C, 59.08; H, 7.44; N, 13.78; Found: C, 59.22; H, 7.46; N, 13.71.

2.5.18. N-[4-(4-Methylpiperazin-1-yl)phenyl]-2-(4-benzylpiperidin-1-yl-thiocarbonylthio)acetamide (4s)

IR (KBr) (cm⁻¹): 3275 (N–H), 1659 (C=O), 1221 (C=S), 822 (1,4-disubstituted benzene). ¹H-NMR (500 MHz, DMSO-d6): 1.51–1.62 (m, 5H, piperidine, C_3,4,5–H), 2.61 (d, 2H, Hz, –CH₂C₆H₅), 3.05–3.09 (m, 4H, piperidine, C_2,6–H), 3.44–3.75 (m, 8H, piperazine, C_2,3,5,6–H), 4.18 (s, 2H, COCH₂), 6.88 (d, 2H, Hz, phenyl, C_3,5–H), 7.17–7.30 (m, 5H, –CH₂C₆H₅), 7.42 (d, 2H, Hz, phenyl, C_2,6–H), 10.04 (s, 1H, NHCO). MS (ESI) (m/z): [M + 1]⁺ 483.15. Anal. Calcd. for C₂₆H₃₄N₄OS₂: C, 64.69; H, 7.10; N, 11.61; Found: C, 64.77; H, 7.12; N, 11.58.

2.6. Enzymatic Assay

All compounds were subjected to a slightly modified method of Ellman’s test [30, 31] in order to evaluate their potency to inhibit the AChE. Donepezil hydrochloride was used as a positive control (Table 2). Enzyme solutions were prepared in gelatin solution (1%), at a concentration of 2.5 units/mL. AChE and compound solution (50 μL) which is prepared in 2% DMSO at 0.1 and 1 mM concentrations were added to 3.0 mL phosphate buffer (pH ) and incubated at 25°C for 5 min. The reaction was started by adding DTNB (50 μL) and ATC (10 μL) to the enzyme-inhibitor mixture. The production of the yellow anion was recorded for 10 min at 412 nm. As a control, an identical solution of the enzyme without the inhibitor is processed following the same protocol. The blank reading contained 3.0 mL buffer, 50 μL 2% DMSO, 50 μL DTNB, and 10 μL substrate. All processes were assayed in triplicate. The inhibition rate (%) was calculated by the following equation: where is the absorbance in the presence of the inhibitor, is the absorbance of the control, and is the absorbance of blank reading. Both of the values are corrected with blank-reading value. SPSS for Windows 15.0 was used for statistical analysis. Student’s -test was used for all statistical calculations. Data were expressed as mean ± SD inactive in culture medium.

2.7. Broth Microdilution Assay

The antimicrobial activities of compounds were tested using the microbroth dilution method [32]. MIC readings were performed twice for each chemical agent. Final products were tested for their in vitro growth inhibitory activity against Enterococcus faecalis (ATCC 29212), Pseudomonas aeruginosa (ATCC 27853), Klebsiella pneumoniae (ATCC 700603), Escherichia coli (ATCC 35218), Escherichia coli (ATCC 25923), Candida albicans (ATCC 10231), Candida glabrata (ATCC 90030), Candida krusei (ATCC 6258), and Candida parapsilosis (ATCC 7330). Chloramphenicol and ketoconazole were used as control drugs.

The cultures were obtained from Mueller-Hinton broth (Difco) for the bacterial strains after overnight incubation at 35 ± 1°C. The yeasts were maintained in Sabouraud dextrose broth (Difco) after overnight incubation 35 ± 1°C. The inocula of test microorganisms were adjusted to match the turbidity of a MacFarland 0.5 standard tube as determined with a spectrophotometer and the final inoculum size was 0.5–2.5 × 10⁵ cfu/mL for antibacterial and antifungal assays. Testing was carried out in Mueller-Hinton broth and Sabouraud dextrose broth (Difco) at pH 7 and the twofold serial dilution technique was applied. The last well on the microplates containing only inoculated broth was kept as controls and the last well with no growth of microorganism was recorded to represent the MIC expressed in μg/mL. For both the antibacterial and antifungal assays the compounds were dissolved in DMSO. Further dilutions of the compounds and standard drugs in test medium were prepared at the required quantities of 800, 400, 200, 100, 50, 25, 12.5, 6.25, 3.13, and 1.63 μg/mL concentrations with Mueller-Hinton broth and Sabouraud dextrose broth [32, 33]. Each experiment in the antimicrobial assays was replicated twice in order to define the MIC values given in Table 1.

3. Results and Discussion

In the present study some N-[(4-substituted-phenyl)-2-substitutedthiocarbonylthio]acetamide derivatives (4a–4s) were synthesized in order to evaluate their biological activities. Target compounds were prepared in four steps. Initially, 4-fluoro-1-nitrobenzene in DMF was reacted with appropriate cyclic secondary amine under microwave irradiation to obtain 4-substituted-1-nitrobenzene derivatives (1a–1c). In the second step, nitro group reduction of 1a–1c gave 4-substituted aniline derivatives (2a–2c), which were acetylated in further step by chloroacetyl chloride to gain 2-chloro-N-(4-substituted-phenyl)acetamides (3a–3c). Finally, compounds 3a–3c were reacted with appropriate dithiocarbamic acid sodium salt to obtain target compounds (4a–4s). Synthetic route for the preparation of target compounds is outlined in Scheme 1. Some physicochemical properties of the compounds are given in Table 1.

Structure elucidations of the final compounds (4a–4s) were performed with IR, ¹H-NMR, and ES-MS spectroscopic methods and elemental analysis. Characteristic stretching absorptions of C=O groups and N–H bonds were observed at 1651–1661 cm⁻¹ and 3273–3493 cm⁻¹, respectively. The stretching absorptions at about 1230 cm⁻¹ were assigned to C=S bond. In the ¹H-NMR spectra, all of the aromatic and aliphatic protons were observed at the estimated chemical shifts. The N–H proton of the acetylamino group gave a singlet signal at 9.99–10.06 ppm. The aromatic protons of 1,4-disubstituted phenyl ring were found at 6.87–7.45 ppm. The –COCH₂ protons appeared as singlet signals at 4.17–4.21 ppm. The M + 1 peaks in ES-MS spectra were in agreement with the calculated molecular weight of the target compounds (4a–4s). Elemental analysis results for C, H, and N elements were satisfactory within 0.4% calculated values of the compounds.

The anticholinesterase effects of the compounds (4a–4s) were determined by modified Ellman’s spectrophotometric method (Table 2). Donepezil was used as a standard AChE inhibitor. The tested compounds showed low enzyme inhibitory activity. However, the compounds 4c and 4e, which carry piperidine ring on fourth position of phenyl, displayed better inhibitory activity than other compounds.

Contrary to their enzyme inhibitory potency synthesized compounds displayed good antimicrobialy activity profile. Antibacterial activity of the compounds 4a, 4e, and 4p (MIC = 25 μg/mL) was equal to that of chloramphenicol against Klebsiella pneumoniae (ATCC 700603) and Escherichia coli (ATCC 35218). Most of the compounds (4b–4d, 4f, 4h, 4k–4o, 4r, and 4s) showed moderate antibacterial activity against these two bacterial strains (Table 3).

When compared with bacterial strain, Candida species were found to be more sensitive to synthesized compounds. Compound 4a (Figure 2) which carries piperidin-1-yl substituent and dimethylthiocarbamoyl side chain as variable groups exhibited twofold better anticandidal effect than reference drug ketoconazole. Furthermore, the other piperidin-1-yl substituted compounds 4b–4f showed significant antifungal activity (Table 3). Although morpholin-4-yl and 4-methylpiperazin-1-yl substituted compounds 4g–4l and 4m–4s showed equal activity to reference drug against Candida glabrata (ATCC 90030), their potency against the other Candida species was not the same with reference. This finding suggests that piperidin-1-yl substitution has good influence on antifungal activity. Besides, dimethylthiocarbamoyl side chain raises the antifungal activity significantly.

4. Conclusion

In an effort to develop potent anticholinesterase and antimicrobial agents, we described the synthesis of a series of N-[(4-substituted-phenyl)-2-substitutedthiocarbonylthio]acetamide derivatives (4a–4s) and focused on their biological activity evaluation. Anticholinesterase activity of the synthesized compounds was not as notable as antimicrobial activity. When compared with antibacterial activity, target compounds displayed better antifungal effect profile. Among these derivatives, compound 4a can be identified as the most promising antifungal agent against all tested Candida species with a twofold lower MIC value (25 μg/mL) than ketoconazole. This result may have a good impact on medicinal chemists to synthesize more active compounds that contain similar chemical structure.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgment

This study was financially supported by Anadolu University Scientific Research Projects Fund, project no. 1205S88.

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Copyright © 2014 Begüm Nurpelin Sağlık et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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