Journal of Chemistry

Volume 2013 (2013), Article ID 789743, 8 pages

http://dx.doi.org/10.1155/2013/789743

Research Article

Antibacterial, Cytotoxic Studies and Characterization of Some Newly Synthesized Symmetrical ,-Bis(disubstituted)isophthalyl-bis(thioureas) and Their Cu(II) and Ni(II) Complexes

Muhammad Jamil,¹ Muhammad Zubair,¹ Muhammad Asim Farid,² Umer Rashid,³ Nasir Rasool,¹ and Saiful Islam⁴

¹Department of Chemistry, Government College University, Faisalabad 38000, Pakistan

²Institute of Chemical Sciences, Bahauddin Zakariya University, Multan 60800, Pakistan

³Institute of Advanced Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

⁴Department of Chemistry, Faculty of Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

Received 4 July 2013; Revised 9 September 2013; Accepted 9 September 2013

Academic Editor: Liviu Mitu

Copyright © 2013 Muhammad Jamil 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.

Abstract.

A series of some novel ,-bis(disubstituted)isophthalyl-bis(thioureas) compounds with general formula [C₆H₄ {CONHCSNHR}₂], where R = 2-ClC₆H₄S (L¹), 3,5-(Cl)₂C₆H₃ (L²), 2,4-(Cl)₂C₆H₃ (L³), 2,5-(Cl)₂C₆H₃ (L⁴), and 2-NH₂C₆H₄ (L⁵), and their Cu(II) and Ni(II) complexes (C¹–C¹⁰) have been synthesized. These compounds (L¹–L⁵) and their metal(II) complexes (C¹–C¹⁰) have been characterized by elemental analysis, infrared spectroscopy, ¹H NMR and ¹³C NMR spectroscopy, magnetic moments, and electronic spectral measurements. The ligands are coordinated to metal atom in a bidentate pattern producing a neutral complex of the type [ML]₂. These compounds (L¹–L⁵) and their metal(II) complexes (C¹–C¹⁰) were also screened for their antibacterial and cytotoxic activities.

1. Introduction

Thioureas, an emerging class of compounds, were first synthesized by Neucki [1]. Thiourea derivatives hold broad range of applications in the field of medicine, agriculture, and analytical chemistry. These compounds show a comprehensive range of biological activities such as antiviral [2, 3], antibacterial [4], fungicidal [5–7], analgesic, herbicidal [8, 9], plant growth regulating [10], antiaggregating [11], antiarrhythmic [12], local anesthetic [13], and antihyperlipidemic activities [14]. Some thioureas have been recently described as effective antitumor and nonnucleoside inhibitors of HIV reverse transcriptase [15]. Recently reported [16] some dithiourea derivatives exhibited cytotoxicity against various cancer cells, and one of these indicated best inhibition activities against KB and CNE2 with IC₅₀ values of 10.72 and 9.91 micrometer, respectively. In view of these results, our interest increased in the synthesis of some new bis(thiourea) derivatives which were characterized by spectroscopic techniques such as FTIR, ¹H NMR, and ¹³C NMR. These derivatives are stable and contain at least two potential donor atoms as O and S. These have been found to display surprisingly rich coordination chemistry at their active sites especially with transition metals. Metalloorganic chemistry is becoming an emerging area of research due to the demand for new metal-based antibacterial and antifungal compounds [17, 18]. Many investigations have proved that binding of a drug to a metalloelement enhances its activity, and in some cases, the complex possesses even more healing properties than the parent drug [19]. Recently, a number of attempts have been made to obtain Cu(II) and Ni(II) complexes with thioureas [20–22]. In view of these observations, we became interested in the synthesis of some new bis(thiourea) derivatives and their Cu(II) and Ni(II) complexes. In present work, these compounds were synthesized, isolated, and characterized by elemental analyses, infrared spectroscopy, ¹H NMR and ¹³C NMR spectroscopy, magnetic moments, and electronic spectral measurements. These compounds were also screened for their antibacterial and cytotoxic studies.

2. Experimental

2.1. Materials and Methods

All chemicals used were of analytical reagent grade (AR) and of the highest purity available. These include isophthalic acid, thionyl chloride, potassium thiocyanate, 3-amino-5-chlorobenzenethiol, 3,5-dichloroaniline, 2,4-dichloroaniline, 2,5-dichloroaniline, and 3-aminoaniline. The organic solvents used include acetone, absolute ethyl alcohol, and dimethylformamide (DMF). These solvents were either spectroscopically pure or purified by the recommended methods and tested for their spectral purity. Deionized water collected from all-glass equipment was used wherever required.

2.2. General Procedure for Synthesis of Compounds (L¹–L⁵)

A solution of isophthalyl chloride (2) (0.1 mol), obtained by the reaction of isophthalic acid with thionyl chloride, was prepared in dry acetone solvent. Potassium thiocyanate (0.2 mol), previously dried at 80°C for two hours, was added to above solution and stirred for one hour at room temperature to obtain the isophthalyl isothiocyanate (3). This solution was mixed with a solution of primary amines (0.2 mol) and stirred for 24 hours at room temperature to get the target disubstituted bisthiourea derivatives (L¹–L⁵) in good to excellent yields (Scheme 1). The mixture was then poured into sufficient quantity of ice cold water, and the product was settled as white to yellow precipitate which was filtered, washed with cold water, and dried in vacuum desiccator. For further purification, the products were recrystallized from DMF.

Scheme 1: Synthesis of disubstituted bisthiourea derivatives (ligands L¹–L⁵).

-Bis(3-chloro-5-mercaptophenyl)isophthalyl-bis(thiourea) (L ¹). Yellow solid; Yield: 68%; m.p. 204°C; FTIR (KBr, cm⁻¹): 3345 (N–H), 756 (C–Cl), 1664 (C=O), 1600, 1530, 1459 (C=C), 1135 (C=S). ¹H NMR (DMSO-d₆, , ppm): 12.47 (s, 2H, 2CSNH), 8.56 (s, 2H, 2CONH), 8.20–7.71 (m, 4H, isophthalyl Ar-H), 7.13–6.62 (m, 6H, amine Ar-H), 3.46 (s, 2H, 2SH). ¹³C NMR (DMSO-d₆, , ppm): 125.3 (C₁), 133.9 (C₂), 132.2 (C₃), 128.5 (C₄), 165.1 (C₅), 182.4 (C₆), 138.1 (C₇), 127.2 (C₈), 129.4 (C₉), 126.7 (C₁₀), 128.2 (C₁₁), 129.8 (C₁₂). Anal. Calcd. (%) for C₂₂H₁₆Cl₂N₄O₂S₄ (567.55): C, 46.56; H, 2.84; Cl, 12.49; N, 9.87; O, 5.64; S, 22.60. Found (%): C, 46.59; H, 2.78; Cl, 12.54; N, 9.89; O, 5.61; S, 22.71.

-Bis(3,5-dichlorophenyl)isophthalyl-bis(thiourea) (L ²). Yellowish solid; Yield: 72%; m.p. 205°C; FTIR (KBr, cm⁻¹): 3334 (N–H), 759 (C–Cl), 1654 (C=O), 1607, 1545, 1490 (C=C) 1155 (C=S). ¹H NMR (DMSO-d₆, , ppm): 12.32 (m, 2H, CSNH), 8.65 (s, 2H, 2CONH), 8.26–7.714 (m, 4H, isophthalyl Ar-H) 7.43–7.08 (s, 6H, amine Ar-H). ¹³C NMR (DMSO-d₆, , ppm): 123.9 (C₁), 134.2 (C₂), 131.4 (C₃), 128.5 (C₄), 165.9 (C₅), 180.1 (C₆), 138.1 (C₇), 127.2 (C₈), 129.3 (C₉), 125.6 (C₁₀), 128.9 (C₁₁), 129.1 (C₁₂). Anal. Calcd. (%) for C₂₂H₁₄Cl₄N₄O₂S₂ (576.92): C, 46.17; H, 2.47; Cl, 24.78; N, 9.79; O, 5.59; S, 11.21. Found (%): C, 46.14; H, 2.41; Cl, 24.76; N, 9.67; O, 5.62; S, 11.33.

-Bis(2,4-chlorophenyl)isophthalyl-bis(thiourea) (L ³). Yellow solid; Yield: 70%; m.p. 196°C. FTIR (KBr, cm⁻¹): 3338 (N–H), 752 (C–Cl), 1658 (C=O), 1592, 1480, 1526 (C=C), 1161 (C=S). ¹H NMR (DMSO-d₆, , ppm): 12.31 (m, 2H, CSNH), 8.51 (s, 2H, 2CONH), 8.25–7.74 (m, 4H, isophthalyl Ar-H) 7.37–7.06 (s, 6H, amine Ar-H). ¹³C NMR (DMSO-d₆, , ppm): 125.1 (C₁), 134.2 (C₂), 132.4 (C₃), 126.5 (C₄), 164.5 (C₅), 182.3 (C₆), 138.3 (C₇), 126.7 (C₈), 129.7 (C₉), 125.9 (C₁₀). Anal. Calcd. (%) for C₂₂H₁₄Cl₄N₄O₂S₂ (576.92): C, 46.17; H, 2.47; Cl, 24.78; N, 9.79; O, 5.59; S, 11.21. Found (%): C, 46.11; H, 2.48; Cl, 24.16; N, 9.25; O, 5.54; S, 11.31.

-Bis(2,5-dichlorophenyl)isophthalyl-bis(thiourea) (L ⁴). Yellowish solid; Yield: 59%; m.p. 194°C. FTIR (KBr, cm⁻¹): 3362, (N–H), 1655 (C=O), 1600, 1516, 1474 (C=C), 1197 (C=S). ¹H NMR (DMSO-d₆, , ppm): 12.29 (m, 2H, 2CSNH), 9.12 (s, 2H, 2CONH), 8.56–7.81 (m, 4H, isophthalyl Ar-H), 7.43–6.73 (m, 6H, amine Ar-H). ¹³C NMR (DMSO-d₆, , ppm): 124.1 (C₁), 133.4 (C₂), 133.4 (C₃), 127.6 (C₄), 166.4 (C₅), 182.2 (C₆), 137.3 (C₇), 127.8 (C₈), 129.1 (C₉), 128.6 (C₁₀), 128.8 (C₁₁), 129.1 (C₁₂). Anal. Calcd. (%) for C₂₂H₁₄Cl₄N₄O₂S₂ (576.92): C, 46.17; H, 2.47; Cl, 24.78; N, 9.79; O, 5.59; S, 11.21. Found (%): C, 46.14; H, 2.43; Cl, 24.18; N, 9.23; O, 5.55; S, 11.34.

-Bis-(3-aminophenyl)isophthalyl-bis(thiourea) (L ⁵). Grey solid; Yield: 72%; m.p. 201°C; IR (KBr, max, cm⁻¹): 3330, 3211 (N–H), 1685 (C=O), 1640, 1535, 1458 (C=C), 1270, 1148 (C=S). ¹H NMR (300 MHz, DMSO-d₆, Me4Si): (ppm): 12.33 (s, 2H, 2CSNH), 9.81 (s, 2H, 2CONH), 8.25–7.64 (m, 12H, isophthalyl Ar-H), 7.14–6.47 (m, 12H, amine Ar-H), 5.22 (s, 4H, 2NH2). ¹³C NMR (DMSO-d₆, , ppm): 123.4 (C₁), 133.6 (C₂), 132.9 (C₃), 125.9 (C₄), 167.6 (C₅), 184.1 (C₆), 135.2 (C₇), 126.9 (C₈), 128.3 (C₉), 122.6 (C₁₀), 130.2 (C₁₁), 129.5 (C₁₂). Anal. Calcd. (%) for C₂₂H₂₀N₆O₂S₂ (464.56): C, 56.88; H, 4.34; O, 18.09; S, 13.80. Found (%): C, 56.93; H, 4.33; O, 18.16; S, 13.79.

2.3. General Procedure for Synthesis of Metal(II) Complexes

A solution of the bisthiourea (0.05 mol) in DMF (15 mL) was added to a solution of MCl₂ (0.05 mol) in DMF (15 mL), while M = Cu for (C¹–C⁵) and M = Ni for (C⁶–C¹⁰). The mixture was refluxed for 6 hours at room temperature and then concentrated to one-third volume and kept at room temperature for 2 hours. The solid product formed was filtered, washed with DMF, and dried.

-Bis(3-chloro-5-mercaptophenyl)isophthalyl-bis(thiourea) Copper(II) Complex (C ¹). Orange red solid; Yield: 69%; m.p. 287°C; IR (KBr, max, cm⁻¹): 3345 (N–H), 756 (C–Cl), 1600, 1531, 1459 (C=C), 1514 (C–O), 366 (M–S), 463 (M–O). Anal. Calcd. (%) for Cu₂C₄₄H₂₈Cl₄N₈O₄S₈ (1258.17): C, 42.00; H, 2.24; N, 8.91; S, 20.39; Cl, 11.27; Cu, 10.10. Found (%): C, 42.07; H, 2.28; N, 8.86; S, 20.41; Cl, 11.24; Cu, 1011.

-Bis(3,5-dichlorophenyl)isophthalyl-bis(thiourea) Copper(II) Complex (C ²). Orange red solid; Yield: 71%; m.p. 287°C; IR (KBr, max, cm⁻¹): 3333 (N–H), 750 (C–Cl), 1600, 1545, 1490 (C=C) 1604 (C–O), 365 (M–S), 445 (M–O). Anal. Calcd. (%) for Cu₂C₄₄H₂₄Cl₈N₈O₄S₄ (1257.98): C, 41.69; H, 1.91; N, 8.84; S, 10.12; Cl, 22.37; Cu, 10.03. Found (%): C, 41.64; H, 1.90; N, 8.86; S, 10.12; Cl, 22.31; Cu, 10.12.

-Bis(2,4-dichlorophenyl)isophthalyl-bis(thiourea) Copper(II) Complex (C ³). Orange red solid; Yield: 72%; m.p. 288°C; IR (KBr, max, cm⁻¹): 3338 (N–H), 752 (C–Cl), 1592, 1483, 1524 (C=C), 1508 (C–O), 363 (M–S), 432 (M–O). Anal. Calcd. (%) for Cu₂C₄₄H₂₄Cl₈N₈O₄S₄ (1257.98): C, 41.69; H, 1.91; N, 8.84; S, 10.12; Cl, 22.37; Cu, 10.03. Found (%): C, 41.65; H, 1.95; N, 8.87; S, 10.11; Cl, 22.36; Cu, 10.04.

-Bis(2,5-dichlorophenyl)isophthalyl-bis(thiourea) Copper(II) Complex (C ⁴). Orange red solid; Yield: 73%; m.p. 286°C; IR (KBr, max, cm⁻¹): 3359, (N–H), 1600, 1516, 1474 (C=C), 1459 (C–O), 357 (M–S), 429 (M–O). Anal. Calcd. (%) for Cu₂C₄₄H₂₄Cl₈N₈O₄S₄ (1257.98): C, 41.69; H, 1.91; N, 8.84; S, 10.12; Cl, 22.37; Cu, 10.03. Found (%): C, 41.62; H, 1.93; N, 8.82; S, 10.17; Cl, 22.35; Cu, 10.07.

-Bis-(3-aminophenyl)isophthalyl-bis(thiourea) Copper(II) Complex (C ⁵). Orange red solid; Yield: 69%; m.p. 285°C; IR (KBr, max, cm⁻¹): 3343 (N–H), 1682 (C=N), 1589, 1523, 1481 (C=C), 1591 (C–O), 352 (M–S), 435 (M–O). Anal. Calcd. (%) for Cu₂C₄₄H₃₆N₁₂O₄S₄ (1052.19): C, 50.23; H, 3.45; N, 15.97; S, 12.19; Cu, 12.08. Found (%): C, 50.22; H, 3.49; N, 15.89; S, 12.17; Cu, 12.11.

-Bis(3-chloro-5-mercaptophenyl)isophthalyl-bis(thiourea) Nickel(II) Complex (C ⁶). Red solid; Yield: 68%; m.p. 292°C; IR (KBr, max, cm⁻¹): 3346 (N–H), 755 (C–Cl), 1612, 1530, 1459 (C=C), 1511 (C–O), 355 (M–S), 461 (M–O). Anal. Calcd. (%) for Ni₂C₄₄H₂₈Cl₄N₈O₄S₈ (1248.46): C, 42.33; H, 2.26; N, 8.98; S, 20.55; Cl, 11.36; Ni, 9.40. Found (%): C, 42.19; H, 2.29; N, 8.97; S, 20.49; Cl, 11.24; Ni, 9.29.

-Bis(3,5-dichlorophenyl)isophthalyl-bis(thiourea) Nickel(II) Complex (C ⁷). Red solid; Yield: 73%; m.p. 292°C; IR (KBr, max, cm⁻¹): 3334 (N–H), 752 (C–Cl), 1607, 1545, 1491 (C=C) 1609 (C–O), 367 (M–S), 456 (M–O). Anal. Calcd. (%) for Ni₂C₄₄H₂₄Cl₈N₈O₄S₄ (1190.72): C, 42.01; H, 1.92; N, 8.91; S, 10.20; Cl, 22.55; Ni, 9.33. Found (%): C, 42.02; H, 1.96; N, 8.89; S, 10.32; Cl, 22.41; Ni, 9.34.

-Bis(2,4-dichlorophenyl)isophthalyl-bis(thiourea) Nickel(II) Complex (C ⁸). Red solid; Yield: 74%; m.p. 294°C; IR (KBr, max, cm⁻¹): 3336 (N–H), 751 (C–Cl), 1591, 1482, 1526 (C=C), 1504 (C–O), 354 (M–S), 437 (M–O). Anal. Calcd. (%) for Ni₂C₄₄H₂₄Cl₈N₈O₄S₄ (1190.72): C, 42.01; H, 1.92; N, 8.91; S, 10.20; Cl, 22.55; Ni, 9.33. Found (%): C, 42.04; H, 1.91; N, 8.95; S, 10.31; Cl, 22.41; Ni, 9.32.

-Bis(2,5-dichlorophenyl)isophthalyl-bis(thiourea) Nickel(II) Complex (C ⁹). Red solid; Yield: 72%; m.p. 293°C; IR (KBr, max, cm⁻¹): 3362, (N–H), 1609, 1526, 1471 (C=C), 1464 (C–O), 360 (M–S), 442 (M–O). Anal. Calcd. (%) for Ni₂C₄₄H₂₄Cl₈N₈O₄S₄ (1190.72): C, 42.01; H, 1.92; N, 8.91; S, 10.20; Cl, 22.55; Ni, 9.33. Found (%): C, 42.09; H, 1.90; N, 8.93; S, 10.29; Cl, 22.47; Ni, 9.31.

-Bis-(3-aminophenyl)isophthalyl-bis(thiourea) Nickel(II) Complex (C ¹⁰). Red solid; Yield: 75%; m.p. 297°C; IR (KBr, max, cm⁻¹): 3341 (N–H), 1678 (C=N), 1588, 1513, 1482 (C=C), 1592 (C–O), 351 (M–S), 443 (M–O). Anal. Calcd. (%) for Ni₂C₄₄H₃₆N₁₂O₄S₄ (1042.48): C, 50.69; H, 3.48; N, 16.12; S, 12.30; Ni, 11.26. Found (%): C, 50.63; H, 3.51; N, 16.09; S, 12.27; Ni, 11.30.

2.4. Characterization

Elemental microanalyses of the separated solids for C, H, N, and S and metal were performed on a PE-2400 CHNS analyzer. The analyses were repeated twice to check the accuracy of data. Infrared spectra were recorded on an Alpha Centauri FT-IR spectrophotometer in wave number region 250–4000 cm⁻¹. The spectra were recorded with the help of KBr pallets. The ¹H NMR and ¹³C NMR were recorded using FT-80 instrument, and DMSO-d₆ was used as solvent and Me₄Si as internal standard. UV visible spectra were obtained in DMF on a Hitachi U-2000 double-beam spectrophotometer. Room temperature magnetic susceptibility measurements were carried out using a Sherwood-Scientific Gouy magnetic balance (Calibrant: Hg[Co(SCN)₄]).

2.5. Pharmacology

Antibacterial activity of synthesized ligands (L¹–L⁵) and their metal(II) complexes (C¹–C¹⁰) was determined by using the disc diffusion method [18] against various gram-negative and gram-positive bacteria at a concentration of 200 μg/100 μL in DMSO solution. Ampicillin (100 μL/disc) and ciprofloxacin (100 μL/disc) were used as standard drugs. Twenty-four-hour-old cultures, containing approximately (CFU/mL), were spread on the surface of Nutrient Agar (NA) plates. The discs (6 mm diameter) were impregnated with (100 μL/disc) test samples and then placed aseptically on the inoculated agar media. Experimental plates were incubated at 37°C for 24 hours. Antibacterial activity was determined by measuring the diameter of the inhibition zone (IZ) and compared with standard drugs. The IZ values from 25 to 35 mm were taken as potent and from 20 to 25 mm as strong, and values greater than 10 mm were considered as moderate activity. Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Shigella sonnei, Salmonella typhi, and Pseudomonas aeruginosa were used as the bacterial tested organisms. In vitro cytotoxic activity of all the synthesized ligands (L¹–L⁵) and their metal(II) complexes (C¹–C¹⁰) were studied using the protocol of Meyer et al. [23]. Brine shrimp (Artemia salina Leach) eggs were hatched in a shallow rectangular plastic dish ( cm), filled with artificial sea water, which was prepared with commercial salt mixture and double-distilled water. Data were analyzed by Finney computer program to determine the LD₅₀ values [24].

3. Results and Discussion

3.1. Chemistry

The synthetic route for the newly synthesized compounds, -bis(disubstituted)isophthalyl-bis(thioureas) (L¹–L⁵), is illustrated and outlined in Scheme 1. The proposed structure for metal(II) complexes is presented in Figure 2. Isophthalyl chloride 2 was treated with anhydrous KSCN using dry acetone as solvent to give isophthalyl isothiocyanate 3 in quantitative yield. The isophthalyl isothiocyanate 3 are useful synthetic building blocks which may be efficiently used for the synthesis of N,N′-disubstituted thioureas and benzenedicarbonyl bisthioureas [15]. In the present work, the isothiocyanate 3 was not isolated and treated directly with some primary amines to give the corresponding dithiourea derivatives (L¹–L⁵) in good to excellent yields. Since the addition to –N=C=S system and nucleophilic substitution at carbonyl-carbon atom may compete with one another, it has been noticed that isothiocyanate 3 reacted additively with amines to give corresponding bisthiourea derivatives (L¹–L⁵). The target compounds (L¹–L⁵) were purified by recrystallization from DMF and characterized by IR and ¹H NMR data. Metal complexation was carried out in DMF solvent. A solution of the thiourea (0.05 mol) in DMF (15 cm³) was added to a solution of MCl₂ (0.05 mol) in DMF (15 cm³), while M = Cu for (C¹–C⁵) and M = Ni for (C⁶–C¹⁰) (Figure 2). The mixture was refluxed for 6 hours then concentrated to one-third volume and kept at room temperature for 2 hours. The solid product formed was filtered, washed with DMF, and dried. All the synthesized metal(II) complexes were characterized by IR, magnetic moments, and electronic spectral measurements.

3.2. IR Spectra

In IR spectrum, the (C=S) peak appeared in the region 1135–1197 cm⁻¹, whereas the N–H peaks appeared from 3330 to 3362 cm⁻¹. Carbonyl absorption bands were observed in the region of 1654–1685 cm⁻¹ for ligands. On comparison of the IR spectra of the ligands with their metal(II) complexes, different results were obtained. The most striking changes are that the N–H stretching frequency in the free ligands disappears completely in agreement with both ligand and complex structure and the complexation reaction. Another striking change is observed for the carbonyl stretching vibrations which shift to higher frequencies upon complexation of the thiourea ligands because the deprotonation induces delocalization of the carbonyl stretching vibration and confirming coordination through oxygen. Due to this deprotonation which induces delocalization and the (C=O) cm⁻¹ stretching vibration frequency decreases by about 150 cm⁻¹. The same trend is observed for the thiocarbonyl stretching vibration frequencies, which are observed at approximately 1135–1197 cm⁻¹ in the free ligands and shift to higher frequency after complexation; unfortunately, this vibration could not be assigned unambiguously because it is located in the fingerprint zone of the IR spectra. Moreover, in the far infrared region the bands at 335–367 cm⁻¹ and 429–463 cm⁻¹ attributed to (M–S) and (M–O) were observed for all the metal(II) complexes.

3.3. ¹H NMR Spectra

In ¹H NMR spectra, the CSN¹-H protons appeared as singlet in the range 12.29–12.47 ppm whereas CON³-H protons appeared at 8.51–9.81 ppm, depending upon the nature of the group attached to N³. The appearance of N¹-H proton at higher frequency may be attributed to the presence of carbonyl and thiocarbonyl groups which exert a strong deshielding effect. The ¹H NMR (DMSO) spectrum revealed signals at 12.29–12.47 ppm (2H, NH) in all the compounds (L¹–L⁵) which indicates the NH group between (C=O) and (C=S) group remains unaffected regardless of attached to terminal N atom. The aromatic protons of the parent isophthalyl group appeared in the range of 7.33–8.56 ppm. While the aromatic protons of amine showed peaks in the range of 6.33–7.43.

3.4. ¹³C NMR Spectra

The ¹³C NMR spectra of the ligands (L¹–L⁵) were taken in DMSO-d₆. The ¹³C NMR spectral data are reported along with their possible assignments in the experimental section, and all the carbons were found in their expected region [25]. The conclusions drawn from these studies provided further support to the modes of bonding already explained in the IR and ¹H NMR spectral data. The ¹³C NMR spectra of the ligands (L¹–L⁵) showed the carbonyl carbon (C₅) at 164.5–167.6 ppm. The spectra of same ligands displayed thiocarbonyl carbons (C₆) in the region 180.1–184.1 ppm. Furthermore, all the ligands showed central benzene ring peaks in the region 123.4–134.2 ppm. The molecular structures of ligands are given in Figure 1.

Figure 1: Bisthiourea derivatives (ligands L¹–L⁵).

Figure 2: Proposed structure for metal(II) complexes (C¹–C¹⁰) where M = Cu(II) for (C¹–C⁵) and M = Ni(II) for (C⁶–C¹⁰).

3.5. Electronic Spectra and Magnetic Susceptibility Measurements of Complexes

The electronic spectra of Ni(II) complexes display bands in the regions of 15443–16970, 18450–19876, and 23345–24340 cm⁻¹ assignable to ¹A₁g  → ¹A₂g (), ¹A₁g  → ¹B₂g (), and ¹A₁g  →¹Eg () transitions, respectively, characteristic of square planar nickel(II) complexes. The first two bands are pure d-d transitions while the band obviously was enveloped by a strong charge transfer transition. The assumed square planar geometry and diamagnetic d⁸ configuration of Ni²⁺ complexes is confirmed from the value of its room temperature magnetic moment of zero. Cu(II) complexes displayed bands at 11470–11868 and 18377–18854 cm⁻¹, which may be assigned to the transitions ²B₁g  → ²A₁g (d x 2 − y 2 → d z 2) (), ²B₁g  → ²B₂g (dx 2 − y 2 → dz 2) (). The third band at around 28600 cm⁻¹ may be due to charge transfer transitions. These observed transitions and magnetic moment values (1.7 to 1.76) B.M. suggest that Cu²⁺ complexes are square planar (Table 1).

Table 1: Electronic spectra and magnetic moments of metal(II) complexes.

No.
λmax (cm⁻¹)
Band assignment
B.M.
No.
λmax (cm⁻¹)
Band assignment
B.M.

C¹
11868

18657
2B₁g → ²A₁g

²B₁g → ²B₂g
1.72
C⁶
15987

18820

23765
1A₁g→¹A₂g

¹A₁g→¹B₂g

¹A₁g→¹Eg
dia

C²
11734

18854
2B₁g → ²A₁g

²B₁g → ²B₂g
1.73
C⁷
16560

18678

24340
1A₁g→¹A₂g

¹A₁g→¹B₂g

¹A₁g→¹Eg
dia

C³
11470

18782
2B₁g → ²A₁g

²B₁g → ²B₂g
1.76
C⁸
15443

18450

24300
1A₁g→¹A₂g

¹A₁g→¹B₂g

¹A₁g→¹Eg
dia

C⁴
11765

18377
2B₁g → ²A₁g

²B₁g → ²B₂g
1.75
C⁹
16970

19876

23345
1A₁g→¹A₂g

¹A₁g→¹B₂g

¹A₁g→¹Eg
dia

C⁵
11561

18438
2B₁g → ²A₁g

²B₁g → ²B₂g
1.76
C¹⁰
16821

19823

23347
1A₁g→¹A₂g

¹A₁g→¹B₂g

¹A₁g→¹Eg
dia

3.6. Pharmacology

3.6.1. Antibacterial Assay

In vitro antibacterial activity of all the synthesized compounds was tested against six different bacterial strains [26]. The compounds (L¹–L⁵) and their metal(II) complexes exhibited potential activity against all tested bacteria with highest inhibition zones (Table 2). L¹–L⁴ showed strong activity against B. subtilis, S. aureus, E. coli, S. sonnei, S. typhi, and P. aeruginosa. L⁵ showed no activity against all bacterial strains. However, the metal(II) complexes of L⁵ showed activity against various bacterial strains. Ampicillin and Ciprofloxacin were used as standard antibiotics studied for comparing the results. All the compounds showed less activity as compared to standard drugs. The results of the present investigation demonstrated significant () variations in the antibacterial activity of the compounds.

Table 2: In vitro antibacterial activity of ligands (L¹–L⁵) and their metal(II) complexes.

Compounds
Test microorganisms/diameter of inhibition zone (IZ in mm)

Gram-positive bacteria
Gram-negative bacteria

B. subtilis
S. aureus
E. coli
S. sonnei
S. typhi
P. aeruginosa

L¹
34 ± 0.83b
33 ± 1.67c
35 ± 1.35b
33 ± 1.64b
31 ± 1.12b
25 ± 0.76c

L²
28 ± 0.81c
32 ± 2.11bc
31 ± 0.75c
32 ± 0.63d
27 ± 1.63b
28 ± 0.81b

L³
32 ± 1.67b
29 ± 1.45c
31 ± 1.23b
30 ± 1.63b
29 ± 0.0b
22 ± 0.70c

L⁴
34 ± 0.83b
33 ± 1.42c
32 ± 1.63b
32 ± 0.63d
27 ± 1.63b
35 ± 1.35b

L⁵
—
—
—
—
—
—

C¹
37 ± 1.48b
36 ± 1.34c
37 ± 0.78b
35 ± 1.34b
34 ± 1.7b
29 ± 0.74c

C²
29 ± 0.86c
30 ± 2.11bc
24 ± 0.75c
21 ± 0.68d
30 ± 1.65b
31 ± 0.84b

C³
33 ± 1.67b
29 ± 1.45c
32 ± 1.63b
34 ± 1.63b
29 ± 0.0b
22 ± 0.70c

C⁴
35 ± 0.81e
36 ± 1.45c
32 ± 1.63b
33 ± 0.66d
35 ± 1.20a
32 ± 0.70c

C⁵
34 ± 0.41c
35 ± 1.25b
31 ±1.20a
34 ± 0.45a
36 ± 1.0f
30 ± 0.50b

C⁶
35 ± 0.83b
34 ± 1.67c
36 ± 1.35b
37 ± 1.64b
31 ± 1.12b
31 ± 0.76c

C⁷
30 ± 0.85c
31 ± 2.37bc
29 ± 0.87c
27 ± 0.66d
31 ± 1.69b
32 ± 0.83b

C⁸
34 ± 0.45f
28 ± 0.21f
33 ± 0.91b
31 ± 1.30e
30 ± 1.21c
26 ± 0.81e

C⁹
34 ± 2.11bc
35 ± 0.85c
33 ± 0.0b
34 ± 1.45c
30 ± 1.44c
31 ± 1.69b

C¹⁰
33 ± 0.01b
34 ± 0.25c
31 ± 0.10c
33 ± 1.05a
30 ± 1.04c
31 ± 1.09b

S¹
38 ± 0.41a
36 ± 1.82a
38 ± 1.61a
36 ± 1.63a
35 ± 1.63a
34 ± 1.47a

S²
38 ± 0.81a
35 ± 1.41a
38 ± 1.20a
37 ± 0.0a
34 ± 0.0a
33 ± 0.0a

S¹: Ampicillin, S²: Ciprofloxacin, —: NIL.

Values are mean SD of three separate experiments.

Letters in superscript show the significance of the results against single strain.

3.6.2. Cytotoxic Bioassay

Cytotoxicity (brine shrimp bioassay) was determined for all the ligands and their metal(II) complexes. The cytotoxicity is expressed as LD₅₀, that is, concentration, at which 50% of the viable cells were killed under the assay conditions. From the data recorded in (Table 3), it is evident that compound (C⁴) displayed highest cytotoxic activity ( moles/mL) against Artemia salina. Similarly compounds C³, C⁵ showed potent cytotoxic activity. All other synthesized compounds were almost inactive in this assay. It was interesting to note that complexation with copper increased cytotoxicity. These findings may help to serve as a basis for future direction towards the development of bacteriostatic agents of lower cytotoxicity.

Table 3: Brine shrimp bioassay of ligands (L¹–L⁵) and their metal(II) complexes (C¹–C¹⁰).

Compounds
LD₅₀ (M)
Compounds
LD₅₀ (M)
Compounds
LD₅₀ (M)

L¹
>2.05 10⁻⁴
C¹
>5.32 10⁻³
C⁶
>4.05 10⁻³

L²
>1.93 10⁻³
C²
>5.43 10⁻³
C⁷
>3.53 10⁻³

L³
>1.87 10⁻³
C³
>1.76 10⁻⁴
C⁸
>1.65 10⁻⁵

L⁴
>2.01 10⁻³
C⁴
>5.67 10⁻⁴
C⁹
>4.39 10⁻⁴

L⁵
>3.05 10⁻³
C⁵
>4.21 10⁻⁴
C¹⁰
>4.12 10⁻⁴

4. Conclusion

Some novel -bis(disubstituted)isophthalyl-bis(thioureas) and their metal(II) complexes have been synthesized and characterized by analytical and spectral (IR, ¹H NMR and ¹³C NMR, electronic) techniques. Antibacterial activity of these compounds was studied against bacterial strains. Some compounds showed potential activity against some bacterial strains and others exhibited strong antibacterial activity. These compounds were also screened for their cytotoxic inhibition activities. The outcomes of these studies also show the transition metal(II) complexes to be more antibacterial against one or more species as compared to the uncomplexed ligands. It was concluded that these compounds may be the potential source of active antibacterial agents.

Conflict of Interests

All the authors of this paper have no conflict of interests in publishing this material. All the coauthors agreed to publish this work.

Acknowledgments

The authors thank the Department of Chemistry, Government College University, Faisalabad, Pakistan, for providing research facilities. They also thank QAU, Islamabad, for providing spectroscopic services. Finally, Department of Veterinary Microbiology, University of Agriculture, Faisalabad, Pakistan, is also acknowledged for undertaking the biological assays.

References

M. Neucki, “Zur kenntniss des sulfoharnstoffs,” Berichte der Deuschen Chemischen Gesellschaft, vol. 6, no. 1, pp. 598–600, 1873.

J. Sun, S. Cai, H. Mei et al., “Molecular docking and QSAR studies on substituted acyl(thio)urea and thiadiazolo [2,3-α] pyrimidine derivatives as potent inhibitors of influenza virus neuraminidase,” Chemical Biology and Drug Design, vol. 76, no. 3, pp. 245–254, 2010.

C. Sun, X. Zhang, H. Huang, and P. Zhou, “Synthesis and evaluation of a new series of substituted acyl(thio)urea and thiadiazolo [2,3-a] pyrimidine derivatives as potent inhibitors of influenza virus neuraminidase,” Bioorganic and Medicinal Chemistry, vol. 14, no. 24, pp. 8574–8581, 2006.

Z. Zhong, R. Xing, S. Liu, L. Wang, S. Cai, and P. Li, “Synthesis of acyl thiourea derivatives of chitosan and their antimicrobial activities in vitro,” Carbohydrate Research, vol. 343, no. 3, pp. 566–570, 2008.

F. H. Wang, Z. L. Qin, and Q. Front, “Synthesis and fungicidal activity of 1,3,4-oxadiazole substituted acylthioureas,” Frontiers of Chemistry in China, no. 1, p. 112, 2006.

S. Y. Ke and S. J. Xue, “Synthesis and herbicidal activity of N(o)thioureas derivatives and related fused heterocyclic compounds,” Arkivoc, vol. 10, pp. 63–68, 2006.

S. J. Xue, S. Y. Ke, T. B. Wei, L. P. Duan, and Y. L. Guo, “Ultrasonic irradiated synthesis of N(5-aryl-2-furoyl)thiourea derivatives containing substituted pyrimidine ring under phase transfer catalysis,” Journal of Chinese Chemical Society, vol. 51, no. 5A, pp. 1013–1018, 2004.

L. Xiao, C. J. Liu, and Y. P. Li, “Ultrasound promoted synthesis of bis(substituted pyrazol-4-ylcarbonyl)-substituted thioureas,” Molecules, vol. 14, no. 4, pp. 1423–1428, 2009.

J. H. Hua, L. C. Wang, H. Liu, and T. B. Wei, “Biological activities studies and phase transfer catalysts promoting the one-pot synthesis of N-Aryl-N′-(4-ethyloxy benzoyl)-thiourea derivatives,” Phosphorus, Sulfur, and Silicon and the Related Elements, vol. 181, no. 12, pp. 2691–2698, 2006.

A. Ranise, F. Bondavalli, O. Bruno et al., “1-acyl-,3-acyl- and 1,3-diacyl-3-furfuryl-1-phenylthioureas with platelet antiaggregating and other activities,” Farmaco, vol. 46, no. 10, pp. 1203–1216, 1991.

A. Ranise, A. Spallarossa, O. Bruno et al., “Synthesis of N-substituted-N-acylthioureas of 4-substituted piperazines endowed with local anaesthetic, antihyperlipidemic, antiproliferative activities and antiarrythmic, analgesic, antiaggregating actions,” II Farmaco, vol. 58, no. 9, pp. 765–780, 2003.

S. Claridge, F. Raeppel, M.-C. Granger et al., “Discovery of a novel and potent series of thieno[3,2-b]pyridine-based inhibitors of c-Met and VEGFR2 tyrosine kinases,” Bioorganic and Medicinal Chemistry Letters, vol. 18, no. 9, pp. 2793–2798, 2008.

S. N. Manjula, N. Malleshappa Noolvi, K. Vipan Parihar et al., “Synthesis and antitumor activity of optically active thiourea and their 2-aminobenzothiazole derivatives: a novel class of anticancer agents,” European Journal of Medicinal Chemistry, vol. 44, no. 7, pp. 2923–2929, 2009.

R. Vig, C. Mao, T. K. Venkatachalam, L. Tuel-Ahlgren, E. A. Sudbeck, and F. M. Uckun, “Rational design and synthesis of phenethyl-5-bromopyridyl thiourea derivatives as potent non-nucleoside inhibitors of HIV reverse transcriptase,” Bioorganic and Medicinal Chemistry, vol. 6, no. 10, pp. 1789–1797, 1998.

H. Peng, Y. Liang, L. Chen, L. Fu, H. Wang, and H. He, “Efficient synthesis and biological evaluation of 1,3-benzenedicarbonyl dithioureas,” Bioorganic and Medicinal Chemistry Letters, vol. 21, no. 4, pp. 1102–1104, 2011.

Y.-M. Zhang, T.-B. Wei, and L.-M. Gao, “Synthesis and biological activity of N-aroyl-N′-substituted thiourea derivatives,” Synthetic Communications, vol. 31, no. 20, pp. 3099–3105, 2001.

W. Henderson, B. K. Nicholson, M. B. Dinger, and R. L. Bennett, “Thiourea monoanion and dianion complexes of rhodium(III) and ruthenium(II),” Inorganica Chimica Acta, vol. 338, pp. 210–218, 2002.

M. Dominguez, E. Antico, L. Beyer, A. Aguirre, S. García-Granda, and V. Salvadó, “Liquid-liquid extraction of palladium(II) and gold(III) with N-benzoyl-N′N′-diethylthiourea and the synthesis of a palladium benzoylthiourea complex,” Polyhedron, vol. 21, no. 14–15, pp. 1429–1437, 2002.

H. Arslan, N. Kulcu, and U. Florke, “Synthesis and characterization of copper(II), nickel(II) and cobalt(II) complexes with novel thiourea derivatives,” Transition Metal Chemistry, vol. 28, no. 7, pp. 816–819, 2003.

H. Arslan, U. Florke, N. Kulcu, and M. F. Emen, “Crystal structure and thermal behaviour of copper(II) and zinc(II) complexes with N -pyrrolidine-N′-(2-chlorobenzoyl)thiourea,” Journal of Coordination Chemistry, vol. 59, no. 2, pp. 223–228, 2006.

D. Ugur, H. Arslan, and N. Kulcu, “Synthesis, characterization and thermal behaviour of 1,1-dialkyl-3-(4-(3,3-dialkylthioureidocarbonyl)-benzoyl)thiourea and its Cu(II), Ni(II), and Co(II) complexes,” Russian Journal of Coordination Chemistry, vol. 32, no. 9, pp. 669–675, 2006.

D. S. Mansuroglu, H. Arslan, U. Florke, and N. Kulcu, “Synthesis and characterization of nickel and copper complexes with 2,2-diphenyl-N-(alkyl(aryl)carbamothioyl)acetamide: the crystal structures of HL¹and cis-[Ni(L¹)₂],” Journal of Coordination Chemistry, vol. 61, no. 19, pp. 3134–3146, 2008.

B. N. Meyer, N. R. Ferrigni, and J. E. Putnam, “Brine shrimp: a convenient general bioassay for active plant constituents,” Planta Medica, vol. 45, no. 1, pp. 31–34, 1982.

D. J. Finney, Probit Analysis, Cambridge University Press, Cambridge, UK, 3rd edition, 1971.

R. Freeman, A Handbook of Nuclear Magnetic Resonance, Longman, London, UK, 2nd edition, 1997.

Z. H. Chohan, M. S. Iqbal, and S. K. Aftab, “Design, synthesis, characterization and antibacterial properties of copper(II) complexes with chromone-derived compounds,” Applied Organometallic Chemistry, vol. 24, no. 1, pp. 47–56, 2010.