Introduction

Thiourea derivatives are flexible ligands efficient in binding with broad range of metal ions in diverse coordination modes. Based on the number of donor functionalities in the thiourea ligand, they have potency to bind in either as monodentate or bidentate chelating fashion and in a range of protonation states as neutral ligands, or mono- or di-anions [1–3]. Thiourea ligands containing N, O and S donor atoms show a broad spectrum of biological activities, and the metal ions, bonded to bioactive compounds, may enhance/modify their activities. The substituted acyl thioureas help to establish a structure–activity relationship as certain reports manifest [4, 5]. These compounds have found extensive range of practices in heterocyclic chemistry, metal complexes and also exhibit a wide range of biological activities [6, 7]. It has been acknowledged that the activity of the metal centres toward the target bio-molecules can be tuned through a suitable choice of ligands. Recently, many of the acyl thioureas and their metal complexes have been screened as antitumoral, antimicrobial and enzyme inhibitors [8–11], parasiticidal [12], fungicidal, bactericidal [13] and pesticidal agents [14–16], and have shown excellent results.

Urease has a pivotal role in nitrogen metabolism of plants and microbes and acts as a virulence factor for human and animal pathogens [17–21]. It accounts for the excess quantity of ammonia and increase in pH which has negative effects in domains of agriculture and health. Urease serves as a malignant factor in microbes that are the main cause of peptic ulcers, kidney stone formation, pyelonephritis and other malfunctions [15, 16]. Urease can also decrease the effectiveness of urea fertilizers with the release of large amounts of ammonia and further enhances plant damage by ammonia toxicity and soil pH increase [17]. These adverse effects can be averted and the rate at which urea hydrolyzes using urease inhibitors that would enhance the efficiency of urea fertilizers and development of improved therapeutic strategies for the treatment of infections caused by ureolytic bacteria. Recently, urease inhibition studies for polyphenols, quinones, phosphoramides, hydroxamic acids and thiols have attracted attention to address these issues [18–31]. The inhibitory effects of metal ions upon the urease raise the possibility of metals being used as modulators and inhibitors of urease activities and this inhibition might involve blockage of thiol groups in the protein [32]. The serendipitous discovery of the metal-based drugs like antitumor-cisplatin, antileishmanial-antimonials and antirheumatic-aurafin has triggered the research in the field of inorganic medicinal chemistry. Most significant investigations on inorganic medicinal chemistry have been made to the development of anti-tumor compounds of various metals like Pt, Rh, Ru, Os, Sn, Ga, Au, Ti, Sn, Pd, Re, etc., that can exhibit improved therapeutic indexes and broader activity spectra [33–42].

The complexes of gold(I) and gold(III), palladium(II), rhenium(V), ruthenium(II), vanadium(IV) have shown great potential as inhibitors of different cysteine proteases in American trypanosomiasis and for Leishmaniasis as well [43–45]. A variety of gold(I) and gold(III) complexes have shown cytotoxic effects related to the inhibition of mitochondrial thioredoxin reductase and mitochondrial function [46–48]. In 1987, sodium aurothiomalate was used in the treatment of kala-azar and showed excellent clinical results. The gold(III), palladium(II) and rhenium(V) complexes have been tested against Leishmania species (L. major, L. mexicana, L. donovani) and Trypanosoma cruzi, along with evaluation of the inhibitory effect against parasite cysteine proteases. All the tested compounds were inhibitors of this parasite enzyme, and one of the palladium complexes assayed was the most active against these parasites. The preliminary data indicate that the metal complexes target parasite cysteine proteases [43, 49–51].

Baiocco et al. [52, 53] recently reported the crystal structure for Leishmania trypanothione reductase disclosing the actual mechanism of enzyme inhibition by antimonials. The observed antimony–protein interaction is consistent with the usual modalities of cysteine binding of thiophilic metals such as As(III), Sb(III) and Bi(III). Such metal-dependent inhibition of thiol reductases opens the way to combined metal therapy of parasitic diseases like leishmaniasis and the parasitic disease such as trypanosomiasis and malaria. It is very likely that this enzyme is similarly inhibited by other classes of metal complexes, like that of soft transition metals (Ni, Pd, Au, Cu, Ag, etc.). To the best of our knowledge literature related to antiparasitic studies of nickel complexes is scarcely available.

Keeping in view of the above aspects and to further explore the biological activities of acyl thiourea derivatives and their metal complexes with thiophilic metals such as nickel, palladium and platinum, we report here synthesis, characterization, urease inhibition, cytotoxicity and antileishmanial studies of nickel(II) complexes with N,N,N′-trisubstituted thioureas as depicted in Scheme 1.

Scheme 1
scheme 1

The reaction scheme for the synthesis of complexes (1a–12a)

Full size image

Experimental

Materials and measurements

All reagents and solvents were purchased from commercial sources (Sigma-Aldrich Chemicals) and were used without further purification. Infrared spectra were recorded on a FT-IR IR Nicolet Avatar 320 spectrophotometer (4,000–400 cm−1, KBr pellets). Elemental analyses (C, H, N, S) were carried out on a Perkin-Elmer-2400 elemental analyzer. Melting points were determined on a Kofler melting point apparatus and were uncorrected. The 1H and 13C NMR were recorded on a Bruker 300 MHz, internally referenced to TMS, chemical shift (δ), multiplicity (m), spin–spin coupling constant (J), integral (I). CDCl3 was used as solvent, unless mentioned otherwise. LC–MS was recorded in CH3CN solvent at ambient temperature on an Agilent 6310 ion trap instrument.

Synthesis of N,N,N′-trisubstituted thioureas (1–12)

The syntheses of N,N,N′-trisubstituted thiourea ligands were carried out according to the reported methodology [8–11]. To a solution of an appropriate acyl isothiocyanate (20 mmol) in acetone (30 mL) was added dropwise a solution of corresponding amine (20 mmol) dissolved in acetone with constant stirring. The reaction mixture was stirred for 8–9 h at room temperature until the reaction was completed (the progress of the reaction was monitored by TLC). The precipitation of the pure product was done in acidified water. The solid N,N,N′-trisubstituted thioureas were collected by filtration and finally purified by re-crystallization from ethanol.

Synthesis of Ni(II) complexes (1a–12a)

A solution of the nickel acetate dihydrate (1.0 mmol), in a minimal amount of methanol, was added dropwise to a solution of the corresponding N,N,N′-trisubstituted thiourea (2.0 mmol), dissolved in 30 mL of methanol, at 45–50 °C. The resulting mixture was stirred for 2–4 h. The precipitated complexes were filtered and washed with dry acetone. Crystals suitable for X-ray diffraction measurements were obtained by slow evaporation of methanol/chloroform (1:1) solution of the complexes. The 1H and 13C NMR, FT-IR, the elemental analyses, melting point data for the ligands (1–12) and complexes (1a–12a) are as follows:

(1) N, N-methylphenyl-N′-benzoylthiourea

Yield 84 %; white solid; MP, 139.5–140.0 °C; FT-IR (KBr, cm−1) 3,182 (N–H), 3,020 (C–H), 2,935 (C–H), 1,693 (C=O), 1,599, 1,513, 1,435, 1,380, 1,255, 1,167 (C=S), 1,112, 1,021, 906, 769, 708, 661, 546; 1H NMR (300 MHz, CDCl3) δ 3.76 (s, 3H, N–CH 3 ), 7.16–7.78 (m, 10H, Ar–H), 8.64 (s, CONH); 13C NMR (75 MHz, CDCl3) δ 45.7 (N–CH3), 125.5, 127.5 (2C), 127.9 (2C), 128.7 (2C), 129.4 (2C), 132.7, 132.1, 145.1 (Ar–C), 162.8 (C=O), 180.7 (C=S); Anal. calcd. for C15H14N2OS; C, 66.64 H, 5.22 N, 10.36 S, 11.86 Found: C, 66.63 H, 5.25 N, 10.39 S, 11.88.

(2) N, N-methylphenyl-N′-(2-chlorobenzoyl)thiourea

Yield 87 %; white solid; MP, 116–118 °C; FT-IR (KBr, cm−1) 3,209 (N–H), 3,028 (C–H), 2,929 (C–H), 1,699 (C=O), 1,596, 1,518, 1,435, 1,389, 1,270, 1,242, 1,179 (C=S), 1,113, 1,048, 908, 831, 763, 746, 689, 650, 552, 464; 1H NMR (300 MHz, CDCl3) δ 3.74 (s, 3H, N–CH 3 ), 7.22–7.48 (m, 9H, Ar–H), 8.8 (s, CONH); 13C NMR (75 MHz, CDCl3) δ 45.3 (N–CH3), 125.6, 126.0 (2C), 128.2, 129.7, 130.6, 130.4 (2C), 130.6, 131.2, 133.4, 142.1 (Ar–C), 164.6 (C=O), 178.8 (C=S); Anal. calcd. for C15H13N2OSCl; C, 59.11 H, 4.30 N, 9.19 S, 10.52 Found: C, 59.12 H, 4.23 N, 9.21 S, 10.53.

(3) N, N-methylphenyl-N′-(3-chlorobenzoyl)thiourea

Yield 82 %; white solid; MP, 131–132 °C; FT-IR (KBr, cm−1) 3,184 (N–H), 3,026 (C–H), 2,932 (C–H), 1,696 (C=O), 1,573, 1,514, 1,439, 1,389, 1,272, 1,244, 1,174 (C=S), 1,119, 1,058, 915, 763, 734, 637, 555, 466; 1H NMR (300 MHz, CDCl3) δ 3.77 (s, 3H, N–CH 3 ), 7.25–8.42 (m, 9H, Ar–H), 8.7 (s, CONH); 13C NMR (75 MHz, CDCl3) δ 45.6 (N–CH3), 125.3, 125.4, 127.7 (2C), 127.8 (2C), 128.1 (2C), 129.6 (2C), 130.1, 132.5, 133.5, 134.8, 134.0 (Ar–C), 161.58 (C=O), 175.73 (C=S); Anal. calcd. for C15H13N2OSCl; C, 59.11 H, 4.30 N, 9.19 S, 10.52 Found: C, 59.10 H, 4.28 N, 9.21 S, 10.50.

(4) N, N-methylphenyl-N′-(4-chlorobenzoyl)thiourea

Yield 83 %; white solid; MP, 127–129 °C; FT-IR (KBr, cm−1) 3,172 (N–H), 3,071 (C–H), 2,941 (C–H), 1,698 (C=O), 1,592, 1,515, 1,428, 1,375, 1,316, 1,251, 1,181 (C=S), 1,108, 1,099, 911, 834, 743, 752, 696, 541, 471, 412; 1H NMR (300 MHz, CDCl3) δ 3.76 (s, 3H, N–CH 3 ), 7.30–7.51 (m, 9H, Ar–H), 8.46 (s, CONH); 13C NMR (75 MHz, CDCl3) δ 45.6 (N–CH3), 125.1, 128.2 (2C), 129.1 (2C), 129.1 (2C), 129.4 (2C), 133.6, 139.0, 145.1 (Ar–C), 162.8 (C=O), 180.2 (C=S); Anal. calcd. for C15H13N2OSCl; C, 59.11 H, 4.30 N, 9.19 S, 10.52 Found: C, 59.15 H, 4.26 N, 9.26 S, 10.50.

(5) N, N-methylphenyl-N′-(2-flourobenzoyl)thiourea

Yield 83 %; white solid; MP, 129–130 °C; FT-IR (KBr, cm−1) 3,183 (N–H), 3,071 (C–H), 2,942 (C–H), 1,693(C=O), 1,592, 1,513, 1,428, 1,376, 1,318, 1,253, 1,181 (C=S), 1,108, 1,091, 913, 834, 747, 752, 695, 547, 473, 413; 1H NMR (300 MHz, CDCl3) δ 3.76 (s, 3H, N–CH 3 ), 7.32–7.57 (m, 9H, Ar–H), 8.57 (s, CONH); 13C NMR (75 MHz, CDCl3) δ 45.8 (N–CH3),125.2, 128.1 (2C), 129.6 (2C), 129.9 (2C), 129.5 (2C), 133.1, 139.8, 145.6 (Ar–C), 162.4 (C=O), 180.0 (C=S); Anal. calcd. for C15H13N2OSF; C, 62.48 H, 4.54 N, 9.72 S, 11.12 Found: C, 62.46 H, 4.55 N, 9.75 S, 11.13.

(6) N, N-methylphenyl-N′-(3-flourobenzoyl)thiourea

Yield 85 %; white solid; MP, 127–129 °C; FT-IR (KBr, cm−1) 3,176 (N–H), 3,073 (C–H), 2,949 (C–H), 1,698 (C=O), 1,599, 1,513, 1,420, 1,379, 1,311, 1,258, 1,195 (C=S), 1,108, 1,094, 918, 834, 749, 751, 697, 548, 475, 412; 1H NMR (300 MHz, CDCl3) δ 3.78 (s, 3H, N–CH 3 ), 7.34–7.57 (m, 9H, Ar–H), 8.57 (s, CONH); 13C NMR (75 MHz, CDCl3) δ 45.3 (N–CH3), 125.6, 128.1 (2C), 129.6 (2C), 129.9 (2C), 129.0 (2C), 133.2, 138.0, 147.0 (Ar–C), 163.7 (C=O), 181.1 (C=S); Anal. calcd. for C15H13N2OSF; C, 62.48 H, 4.54 N, 9.72 S, 11.12 Found: C, 62.49 H, 4.50 N, 9.74 S, 11.12.

(7) N, N-methylphenyl-N′-(4-flourobenzoyl)thiourea

Yield 89 %; white solid; MP, 126–129 °C; FT-IR (KBr, cm−1) 3,172 (N–H), 3,071 (C–H), 2,943 (C–H), 1,690 (C=O), 1,598, 1,511, 1,428, 1,379, 1,316, 1,254, 1,155 (C=S), 1,101, 1,099, 911, 837, 747, 752, 695, 547, 473, 413; 1H NMR (300 MHz, CDCl3) δ 3.77 (s, 3H, N–CH 3 ), 7.31–7.50 (m, 9H, Ar–H), 8.47 (s, CONH); 13C NMR (75 MHz, CDCl3) δ 45.6 (N–CH3),125.0, 127.0 (2C), 128.0 (2C), 128.3 (2C), 129.1 (2C), 135.2, 137.0, 147.0 (Ar–C), 161.7 (C=O), 176.1 (C=S); Anal. calcd. for C15H13N2OSF; C, 62.48 H, 4.54 N, 9.72 S, 11.12 Found: C, 62.47 H, 4.52 N, 9.73 S, 11.11.

(8) N, N-methylphenyl-N′-(2-methylbenzoyl)thiourea

Yield 86 %; white solid; MP, 126–127 °C; FT-IR (KBr, cm−1) 3,177 (N–H), 3,093 (C–H), 2,993 (C–H), 1,660 (C=O), 1,591, 1,524, 1,439, 1,367, 1,327, 1,254, 1,178 (C=S), 1,119, 1,080, 917, 845, 758, 763, 686, 558, 494, 423; 1H NMR (300 MHz, CDCl3) δ 2.45 (s, 3H, –CH 3 ), 3.47 (s, 3H, N–CH 3 ), 6.31–7.70 (m, 9H, Ar–H), 8.40 (s, CONH); 13C NMR (75 MHz, CDCl3) δ, 29.0 (CH3), 44.7 (N–CH3),127.4, 129.0 (2C), 130.0 (2C), 132.3 (2C), 135.5 (2C), 137.2, 142.0, 149.0 (Ar–C), 167.7 (C=O), 185.1 (C=S); Anal. calcd. for C16H16N2OS; C, 67.58 H, 5.67 N, 9.85 S, 11.28 Found: C, 67.57 H, 5.67 N, 9.86 S, 11.27.

(9) N, N-methylphenyl-N′-(3-methylbenzoyl)thiourea

Yield 88 %; white solid; MP, 128–129 °C; FT-IR (KBr, cm−1) 3,191 (N–H), 3,063 (C–H), 3,001 (C–H), 1,694 (C=O), 1,583, 1,514, 1,449, 1,378, 1,327, 1,259, 1,174 (C=S), 1,108, 1,091, 922, 834, 747, 751, 690, 540, 471, 409; 1H NMR (300 MHz, CDCl3) δ 2.55 (s, 3H, –CH 3 ), 3.76 (s, 3H, N–CH 3 ), 6.33–7.55 (m, 9H, Ar–H), 8.27 (s, CONH); 13C NMR (75 MHz, CDCl3) δ, 29.9 (CH3), 43.7 (N–CH3),124.4, 127.0 (2C), 128.0 (2C), 129.3 (2C), 130.5 (2C), 137.2, 139.0, 144.0 (Ar–C), 166.7 (C=O), 183.1 (C=S); Anal. calcd. for C16H16N2OS; C, 67.58 H, 5.67 N, 9.85 S, 11.28 Found: C, 67.56 H, 5.66 N, 9.86 S, 11.27.

(10) N, N-methylphenyl-N′-(4-methylbenzoyl)thiourea

Yield 90 %; white solid; MP, 129–130 °C; FT-IR (KBr, cm−1) 3,181 (N–H), 3,077 (C–H), 2,999 (C–H), 1,676 (C=O), 1,583, 1,524, 1,428, 1,378, 1,337, 1,274, 1,168 (C=S), 110, 1,091, 916, 845, 798, 713, 656, 538, 484, 463; 1H NMR (300 MHz, CDCl3) δ 2.55 (s, 3H, –CH 3 ), 3.47 (s, 3H, N–CH 3 ), 6.78–7.50 (m, 9H, Ar–H), 8.17 (s, CONH); 13C NMR (75 MHz, CDCl3) δ, 28.9 (CH3),45.9 (N–CH3),125.2, 128.6 (2C), 131.0 (2C), 132.3 (2C), 133.5 (2C), 134.2, 139.0, 148.0 (Ar–C), 168.7 (C=O), 186.1 (C=S); Anal. calcd. for C16H16N2OS; C, 67.58 H, 5.67 N, 9.85 S, 11.28 Found: C, 67.55 H, 5.65 N, 9.87 S, 11.27.

(11) N, N-methylphenyl-N′-pivaloylthiourea

Yield 87 %; white solid; MP, 135–137 °C; FT-IR (KBr, cm−1) 3,290 (N–H), 3,072 (C–H), 3,021 (C–H), 1,667 (C=O), 1,583, 1,524, 1,428, 1,397, 1,337, 1,244, 1,171 (C=S), 1,119, 1,080, 911, 834, 746, 733, 686, 546, 478, 453; 1H NMR (300 MHz, CDCl3) δ, 1.34 (s, 9H, –CH 3 ), 3.67 (s, 3H, N–CH 3 ), 7.01–7.40 (m, 5H, Ar–H), 8.27 (s, CONH); 13C NMR (75 MHz, CDCl3) δ, 28.0 (–CH3), 39.6 (quat-C), 42.7 (N–CH3), 123.4, 125.0 (1C), 128.0 (1C), 129.3 (1C), 129.5 (1C), 135.2, 138.0, 142.0 (Ar–C), 161.7 (C=O), 183.1 (C=S); Anal. calcd. for C13H18N2OS; C, 62.37 H, 7.25 N, 11.19 S, 12.81 Found: C, 62.36 H, 7.23 N, 11.21 S, 12.79.

(12) N, N-methylphenyl-N′-isobutyrylthiourea

Yield 89 %; white solid; MP, 139–141 °C; FT-IR (KBr, cm−1) 3,181 (N–H), 3,083 (C–H), 3,039 (C–H), 1,663 (C=O), 1,573, 1,517, 1,427, 1,337, 1,337, 1,274, 1,187 (C=S), 1,100, 1,070, 922, 833, 747, 743, 676, 540, 470, 443; 1H NMR (300 MHz, CDCl3) δ, 1.18 (d, 6H, –CH 3 ), 2.77 (m, IH, –CH), 3.67 (s, 3H, N–CH 3 ), 7.21–7.60 (m, 5H, Ar–H), 8.27 (s, CONH); 13C NMR (75 MHz, CDCl3) δ 21.0 (–CH3), 35.3 (ter-C), 44.7 (N–CH3), 127.4, 130.0 (1C), 132.0 (1C), 133.3 (1C), 134.5 (1C), 137.2, 139.0, 148.0 (Ar–C), 164.7 (C=O), 187.1 (C=S); Anal. calcd. for C12H16N2OS; C, 60.99 H, 6.82 N, 11.85 S, 13.57 Found: C, 60.97 H, 6.81 N, 11.87 S, 13.56.

(1a) cis-bis[N,N-methylphenyl-N′-benzoylthioureato-κ2 O,S]nickel(II)

Yield 77 %; reddish pink solid; MP, 224–226 °C; FTIR (cm−1): 3,050 (C–H), 2,939 (C–H), 1,585 (C=O), 1,511, 1,490, 1,413, 1,371, 1,270, 1,106 (C=S), 1,070, 688, 464, 402. 1H NMR (300 MHz, CDCl3) δ 3.63 (s, 6H, N–CH 3 ), 7.21-7.72 (m, 20H, ArH). 13C NMR (75 MHz, CDCl3) δ 33.4 (N–CH3), 113.5, 117.5, 127.5, 128.8, 129.3, 129.6, 136.5, 149.4, 167.6 (C=O), 175.1 (C=S). LC–MS (EI) m/z: 596.2 (M+), 595.6, 433.4, 293.6 (base peak), 150.9. Anal. Calc. For C30H26N4NiO2S2: C, 60.32; H, 4.39; N, 9.38; S, 10.74; Found: C, 60.35; H, 4.37; N, 9.40; S, 10.71.

(2a) cis-bis[(N,N-methylphenyl-N′-(2-chlorobenzoyl)thioureato-κ2 O,S]nickel(II)

Yield 89 %; pink; MP, 216–218 °C; FT-IR (KBr, cm−1) 3,047 (C–H), 2,925 (C–H), 1,589 (C=O), 1,569, 1,514, 1,490, 1,409, 1,374, 1,277, 1,112 (C=S), 909, 786, 658, 562, 454, 402; 1H NMR (300 MHz, CDCl3) δ 3.52(s, 6H, N–CH 3 ), 7.02-7.82 (m, 18H, ArH). 13C NMR (75 MHz, CDCl3) δ 42.25 (N–CH3), 126.3, 127.3, 128.2, 128.9, 129.7, 130.6, 134.8, 138.1, 145.7, 171.5 (C=O), 175.3 (C=S); LC–MS (EI) m/z: 666.2 (M+), 631.4, 550.2, 477.4, 368.4, 327.5 (base peak), 220.1, 150.1; Anal. Calcd. for C30H24Cl2N4NiO2S2; C, 54.08 H, 3.63 N, 8.41 S, 9.63 Found: C, 54.09 H, 3.64 N, 8.42 S, 9.62.

(3a) cis-bis[N,N-methylphenyl-N′-(3-chlorobenzoyl)thioureato-κ2 O,S]nickel(II)

Yield 78 %; reddish pink solid; MP, 234–236 °C, FTIR (cm−1): 3,063 (C–H), 3,022 (C–H), 1,596 (C=O), 1,518, 1,467, 1,408, 1,371, 1,220, 1,179, 1,135 (C=S), 1,068, 901, 828, 799, 674, 555, 452, 402. 1H NMR (300 MHz, CDCl3) δ 3.66 (s, 6H, N–CH 3 ), 7.19–7.67 (m, 18H, ArH). 13C NMR (75 MHz, CDCl3) δ 33.4 (N–CH3), 114.2, 119.1, 125.9, 131.0, 138.3, 145.8, 152.6, 171.0 (C=O), 179.9 (C=S). LC–MS (EI) (m/z): 667.2 (M+), 631.4 (base peak), 550.2, 477.4, 368.4, 327.5, 220.1, 150.1. Anal. Calc. For C30H24Cl2N4NiO2S2: C, 54.08; H, 3.63; N, 8.41; S, 9.63; Found: C, 54.10; H, 3.65; N, 8.43; S, 9.61.

(4a) cis-bis[N,N-methylphenyl-N′-(4-chlorobenzoyl)thioureato-κ2 O,S]nickel(II)

Yield 75 %; reddish pink solid; MP, 244–246 °C, FTIR (cm−1): 3,060 (C–H), 2,961 (C–H), 1,577 (C=O), 1,540, 1,489, 1,418, 1,373, 1,270, 1,168, 1,141 (C=S), 1,013, 905, 845, 752, 629, 578, 454, 405. 1H NMR (300 MHz, CDCl3) δ 3.63 (s, 6H, N–CH 3 ), 7.21–7.59 (m, 18H, ArH). 13C NMR (75 MHz, CDCl3) δ 33.0 (N–CH3), 112.9, 126.3, 127.3, 129.8, 130.6, 134.8, 138.1, 145.7, 171.5 (C=O), 175.3 (C=S). LC–MS (EI) m/z: 667.3 (M+), 631.1, 551.0, 477.3, 367.42, 327.4 (base peak), 220.8, 150.3. Anal. Calc. For C30H24Cl2N4 Ni O2S2: C, 54.08; H, 3.63; N, 8.41; S, 9.63; Found: C, 54.12; H, 3.66; N, 8.44; S, 9.59.

(5a) cis-bis[N,N-methylphenyl-N′-(2-flourobenzoyl)thioureato-κ2 O,S]nickel(II)

Yield 75 %; reddish pink solid; MP, 226–228 °C; FTIR (cm−1): 3,066 (C–H), 2,978 (C–H), 1,584 (C=O), 1,520, 1,492, 1,410, 1,373, 1,270, 1,115 (C=S), 1,016, 908, 756, 569, 454, 403. 1H NMR (300 MHz, CDCl3) δ 3.50 (s, 6H, N–CH 3 ), 6.93–7.39 (m, 18H, ArH). 13C NMR (CDCl3) (ppm): 31.0 (N–CH3), 116.3, 120.3, 124.1, 128.3, 132.2, 158.4, 170.7 (C=O), 178.8 (C=S). LC–MS (EI) m/z: 633.2 (M+), 599.3, 553.5, 452.3, 414.2, 373.2, 311.2 (base peak), 150.0. Anal. Calc. For C31H27F2N4NiO2S2: C, 57.42; H, 4.20; N, 8.64; S, 9.89; Found: C, 57.43; H, 4.23; N, 8.66; S, 9.90.

(6a) cis-bis[N,N-methylphenyl-N′-(3-flourobenzoyl)thioureato-κ2 O,S]nickel(II)

Yield 75 %; reddish pink solid; MP, 236–238 °C; FTIR (cm−1): 3,051 (C–H), 2,970 (C–H), 1,597 (C=O), 1,519, 1,492, 1,371, 1,301, 1,275, 1,117 (C=S), 1,020, 909, 763, 559, 460, 404. 1H NMR (300 MHz, CDCl3) δ 3.65 (s, 6H, N–CH 3 ), 7.21–7.92 (m, 18H, ArH). 13C NMR (75 MHz, CDCl3) δ 31.0 (N–CH3), 114.8, 119.2, 129.4, 130.3, 134.9, 144.9, 170.1 (C=O), 176.2 (C=S). LC–MS (EI) (m/z): 633.1 (M+), 599.0, 553.1, 451.3, 414.0, 373.8, 311.2 (base peak), 150.8. Anal. Calc. For C31H27F2N4NiO2S2: C, 57.42; H, 4.20; N, 8.64; S, 9.89; Found: C, 57.46; H, 4.24; N, 8.68; S, 9.93.

(7a) cis-bis[N,N-methylphenyl-N′-(4-flourobenzoyl)thioureato-κ2 O,S]nickel(II)

Yield 76 %; reddish pink solid; MP, 225–227 °C; FTIR (cm−1): 3,054 (C–H), 2,959 (C–H), 1,598 (C=O), 1,501, 1,417, 1,368, 1,272, 1,208, 1,148, 1,128 (C=S), 1,012, 906, 843, 756, 551, 451, 404. 1H NMR (300 MHz, CDCl3) δ 3.64 (s, 6H, N–CH 3 ), 6.89–7.67 (m, 18H, ArH). 13C NMR (75 MHz, CDCl3) δ 31.0 (N–CH3), 114.9, 116.6, 127.9, 129.3, 131.1, 145.0, 167.0 (C=O), 180.5 (C=S). LC–MS (EI) (m/z): 633.2 (M+), 599.3, 553.5, 452.3, 414.2, 373.2, 311.1 (base peak), 150.6. Anal. Calc. For C31H27F2N4NiO2S2: C, 57.42; H, 4.20; N, 8.64; S, 9.89; Found: C, 57.44; H, 4.22; N, 8.68; S, 9.91.

(8a) cis-bis[N,N-methylphenyl-N′-(2-methylbenzoyl)thioureato-κ2 O,S]nickel(II)

Yield 76 %; red solid; MP, 212–216 °C; FT-IR (KBr, cm−1) 3,053 (C–H), 2,955 (C–H), 1,596 (C=O), 1,516, 1,496, 1,362, 1,282, 1,117 (C=S), 939, 748, 655, 453, 402; 1H NMR (300 MHz, CDCl3) δ 2.31 (s, 6H, –CH 3 ), 3.33 (s, 6H, N–CH 3 ), 6.85–7.49 (m, 18H, ArH); 13C NMR (75 MHz, CDCl3) δ 26.6 (–CH3), 35.4 (N–CH3), 116.9, 119.8, 128.0, 129.4, 131.3, 134.9, 136.8, 153.6, 171.8 (C=O), 177.2 (C=S); LC–MS (EI) m/z: 625.0 (M+), 591.9, 509.2, 446.2, 307.0 (base peak), 150.5; Anal. Calcd. for C32H30N4 NiO2S2; C, 61.45 H, 4.83 N, 8.96 S, 10.25 Found: C, 61.44 H, 4.83 N, 8.97 S, 10.24.

(9a) cis-bis[N, N-methylphenyl-N′-(3-methylbenzoyl)thioureato-κ2 O,S]nickel(II)

Yield 78 %; brick red solid; MP, 210–213 °C; FT-IR (KBr, cm−1) 3,066 (C–H), 2,984 (C–H), 1,590 (C=O), 1,512, 1,491, 1,372, 1,272, 1,101 (C=S), 933, 744, 650, 454, 403; 1H NMR (300 MHz, CDCl3) δ 2.32 (s, 6H, –CH 3 ), 3.43 (s, 6H, N–CH 3 ), 6.95–7.69 (m, 18H, ArH); 13C NMR (75 MHz, CDCl3) δ 24.6 (–CH3), 33.4 (N–CH3), 113.9, 117.8, 127.0,128.4, 129.3, 129.9, 136.8, 151.6, 170.7 (C=O), 178.1 (C=S); LC–MS (EI) m/z: 625.2 (M+), 591.3, 509.4, 446.3, 307.2 (base peak), 150.7; Anal. Calcd. for C32H30N4 Ni O2S2; C, 61.45 H, 4.83 N, 8.96 S, 10.25 Found: C, 61.42 H, 4.87 N, 8.99 S, 10.21.

(10a) cis-bis[N, N-methylphenyl-N′-(4-methylbenzoyl)thioureato-κ2 O,S]nickel(II)

Yield 75 %; red solid; MP, 255–258 °C; FT-IR (KBr, cm−1) 3,060 (C–H), 2,973 (C–H), 1,607 (C=O), 1,581, 1,488, 1,407, 1,372, 1,274, 1,102 (C=S), 903, 688, 453, 402; 1H NMR (300 MHz, CDCl3) δ 2.38 (s, 6H, –CH 3 ), 3.60 (s, 6H, N–CH 3 ), 7.23–7.75 (m, 18H, ArH); 13C NMR (75 MHz, CDCl3) δ 24.3 (–CH3), 33.4 (N–CH3), 113.9, 117.8, 127.5, 128.8, 129.3, 129.6, 136.5, 149.6, 169.6 (C=O), 176.1 (C=S); LC–MS (EI) m/z: 625.4 (M+), 591.0, 509.2, 446.9, 307.7 (base peak), 150.9; Anal. Calcd. for C32H30N4NiO2S2; C, 61.45 H, 4.83 N, 8.96 S, 10.25 Found: C, 61.43 H, 4.86 N, 8.98 S, 10.27.

(11a) cis-bis[N, N-methylphenyl-N′-pivaloylthioureato-κ2 O,S]nickel(II)

Yield 89 %; pink solid; MP, 188–189 °C; FT-IR (KBr, cm−1) 3,073 (C–H), 2,978 (C–H), 1,541 (C=O), 1,491,1,410, 1,360, 1,233, 1,112 (C=S), 1,020, 760, 452, 402; 1H NMR (300 MHz, CDCl3) δ 2.02 (s, 18H, –CH 3 ), 3.34 (s, 6H, N–CH 3 ), 7.13–7.54 (m, 10H, ArH), 13C NMR (75 MHz, CDCl3) δ 20.5 (–CH3), 32.9 (quat-C), 36.4 (N–CH3), 113.5, 117.5, 129.3, 149.4, 168.0 (C=O), 174.9 (C=S); LC–MS (EI) m/z: 557.29 (M+), 523.3, 395.3, 314.2, 273.2 (base peak), 150.0; Anal. Calcd. for C26H34N4NiO2S2; C, 56.52 H, 6.15 N, 10.05 S, 11.51 Found: C, 56.49 H, 6.19 N, 10.11 S, 11.57.

(12a) cis-bis[N, N-methylphenyl-N′-isobutyrylthioureato-κ2 O,S]nickel(II)

Yield 77 %; pink solid; MP, 176–178 °C; FT-IR (KBr, cm−1) 3,078 (C–H), 3,018 (C–H), 1,542 (C=O), 1,481, 1,412, 1,375, 1,230, 1,117 (C=S), 1,090, 769, 452, 402; 1H NMR (300 MHz, CDCl3) δ 1.12 (d, 12H, –CH 3 ), 2.67 (m, 2H, –CH), 3.36 (s, 6H, N–CH 3 ), 7.03–7.44 (m, 10H, ArH); 13C NMR (75 MHz, CDCl3) δ 24.5 (–CH3), 31.0 (ter-C), 39.4 (N–CH3), 112.5, 116.5, 128.3, 148.4, 169.0 (C=O), 176.9 (C=S); LC–MS (EI) m/z: 529.2 (M+), 495.2, 459.1, 374.3, 259.2 (base peak), 150.0; Anal. Calcd. for C24H30 N4 Ni O2S2; C, 54.46 H, 5.71 N, 10.58 S, 12.11 Found: C, 54.49 H, 5.73 N, 10.59 S, 12.07.

Crystal structure determination

Single crystals of 12, 7, 4a, 5a and 1a were mounted on a thin glass fiber at room temperature and the reflection data were collected on a Bruker kappa APEXII CCD diffractometer equipped with graphite mono-chromated Mo-Kα radiation (λ = 0.71, 073 Å). Data were also corrected to Lorentz and polarization effect. The structures were solved using SHELXS-97. Final refinement on F 2 was carried out by full-matrix least-squares techniques using SHELXL-97 [54]. The crystallographic data and structure refinement parameters are summarized in Table 1.

Table 1 Crystallographic data and structure refinements for compounds 7, 12, 4a, 5a and 1a
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Table 2 The intermolecular and intramolecular hydrogen bonds for compounds 12, 7, 4a, 5a and 1a. Hydrogen-bond geometry: distance (Å), angle (°)
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Urease inhibition assay

The synthesized compounds were screened at a concentration of 1 mM on Jack bean urease for the inhibition potencies (Table 1). Thiourea was used as a standard inhibitor in urease inhibitory assay. The indophenol method described by Weatherburn [55] was used for the determination of urease activity. The assay mixture consisted of 40 μL of buffer (100 mmol/L urea, 0.01 mol/L K2HPO4, 1 mmol/L EDTA and 0.01 mol/L LiCl2, pH 8.2), 10 μL of enzyme (5 U/mL) and 10 μL of test compound (1 mM). The mixture was pre-incubated at 37 °C for 30 min in 96-well plates. Following the addition of 40 μL of phenol reagent (1 %, w/v phenol, 0.005 %, w/v sodium nitroprusside) and 40 μL of alkali reagent (0.5 %, w/v NaOH, 0.1 % active chloride NaOCl) to each well and after 10 min of incubation at 37 °C, the absorbance was measured at 630 nm using a Microplate reader (Bio-Tek ELx 800™, Instruments, Inc. USA). All the experiments were carried out in triplicate. For calculation of percentage inhibition following equation was used.

$${\text{Inhibition}}\; ( {\% )}\;{ = }\; 1 0 0- \left[ {\frac{{{\text{absorbance}}\;{\text{of}}\;{\text{the}}\;{\text{compounds}}}}{{{\text{absorbance}}\;{\text{of}}\;{\text{the}}\;{\text{control}}}}} \right] \times 100$$

The results were analyzed using PRISM 5.0 (GraphPad, San Diego, CA, USA).

Cytotoxicity assay

Cell lines and cultures

Lung carcinoma (H-157), (ATCC CRL-5,802), kidney fibroblast (BHK-21), (ATCC CCL-10) and African green monkey kidney normal cell line (Vero), (ATCC CCL-81) were kept in RPMI-1,640, having heat-inactivated fetal bovine serum (10 %) glutamine (2 mM), Pyruvate (1 mM), 100 U/mL penicillin and 100 µg/mL streptomycin, in T-75 cm2 sterile tissue culture flasks in a 5 % CO2 incubator at 37 °C [56]. For experiment, 96-well plates were used for growing H-157, BHK-21 and Vero cells by inoculating 5 × 104 cells per 100 µL per well and plates were incubated at 37 °C for 24 h in a humidified atmosphere containing 5 % CO2. Within 24 h, a uniform monolayer was formed which was used for experiments.

Cytotoxicity analysis by sulforhodamine B (SRB) assays

To perform cytotoxicity assay, with H-157, BHK-21 and Vero cells, a previously described method by Skehan et al. [57] was adopted with minor modifications. Briefly, cells were cultured in different 96-well plates for 24 h. The compounds in different concentrations (100, 10, 1 and 0.1 µM) were inoculated in test wells. Furthermore, positive controls (vincristine and cisplatin) were prepared in DMSO. The well containing culture media with cells having no compound or drug was taken as blank. Vero cells were treated at 100 µM test compounds to check the toxicity against normal cell lines. The plates were then incubated for 48 h. After that, cells were fixed with 50 µL of 50 % ice-cold trichloroacetic acid solution (TCA) and plates were incubated at 4 °C for 1 h. Subsequently, plates were washed five times with phosphate-buffered saline (PBS) and air dried. Fixed cells were further treated with 0.4 % w/v sulforhodamine B dye (prepared in 1 % acetic acid solution) and left at room temperature for 30 min. After that the plates were rinsed with 1 % acetic acid solution and allowed to dry. In order to solubilize the dye, the dried plates were treated with 10 mM Tris base solution for 10 min at room temperature. The absorbance was measured at 490 nm subtracting the background measurement at 630 nm [58]. All experiments were repeated at least three times. Results reported are the mean value of three independent experiments (±SEM) and expressed as percent inhibitions calculated by the following formula:

$${\text{Inhibition}}\; ( {\% )}\;{ = }\; 1 0 0- \left[ {\frac{{{\text{absorbance}}\;{\text{of}}\;{\text{the}}\;{\text{test}}\;{\text{compounds}}}}{{{\text{absorbance}}\;{\text{of}}\;{\text{the}}\;{\text{control}}}}} \right] \times 100$$

In vitro antileishmanial assay

Parasite and culture

Leishmania major promastigotes were cultured at 25 ± 1 °C to logarithmic phase in D-MEM/F-12 medium (Gibco BRL) without phenol red, supplemented by 10 % heat inactivated fetal bovine serum (FBS), 100 IU/mL penicillin and 100 μg/mL streptomycin and then washed 3 times with phosphate-buffered saline (PBS). The cells were centrifugated at 1,500 rpm, for 10 min at room temperature and re-suspended at a concentration of 2.5 × 106 parasites/mL in medium.

Antileishmanial activity assays

In vitro antileishmanial activity of the compounds was evaluated against the promastigote forms of Leishmania major using a MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide). The MTT assay used was originally described by Mosmann [59] and later on modified by Niks and Otto [60]. A stock solution of MTT (Sigma Chemical Co., St. Louis, Mo.) was prepared by dissolving in PBS at 5 mg/mL and storing in the dark at 4 °C for up to 2 weeks before use. For the antileishmanial activity assay, 100 μL/well of the culture which contained 2.5 × 106 cells/mL promastigotes was seeded in 96-well flat-bottom plates. Then 10 μL/well from various concentrations of compounds were added to triplicate wells and plates were incubated for 72 h at 25 ± 1 °C. The well containing only 100 μL culture medium without any compound, drug or parasite, was taken as blank. Amphotericin B was used as a standard drug. At the end of incubation, 10 μL of MTT was added to each well and plates were again incubated for 3 h at 25 ± 1 °C. Enzyme reaction was then stopped by the addition of 100 μL of 50 % isopropanol and 10 % sodium dodecyl sulfate(0.1 N HCl). The plates were incubated for an additional 30 min under agitation at room temperature. Relative optical density (OD) was then measured at a wavelength of 570 nm using a 96-well microplate reader (Bio-Tek ELx 800™, Instruments, Inc. USA). The background absorbance of plates was measured at 690 nm and subtracted from 570 nm measurement. The absorbance of the formazan produced by the action of mitochondrial dehydrogenases of metabolically active cells was correlated with the number of viable cells [59, 60]. All experiments were repeated at least three times. Results reported were the mean of three independent experiments (±SEM) and expressed as per cent inhibitions calculated by the following formula:

$${\text{Inhibition}}\; ({\% )}\;{ = }\; 1 0 0- \left[ {\frac{{{\text{absorbance}}\;{\text{of}}\;{\text{the}}\;{\text{test}}\;{\text{compounds}}}}{{{\text{absorbance}}\;{\text{of}}\;{\text{the}}\;{\text{control}}}}} \right] \times 100$$

Results and discussion

Synthesis and spectroscopic studies

The N,N,N′-trisubstituted thiourea ligands (HL) afford Ni(L-O, S)2 type complexes in methanol at room temperature with nickel salts. Regarding the infrared spectra, N,N,N-trisubstituted thioureas (1–12) display strong bands in the region of 1,700–1,100 cm−1. The bands that center near 1,699–1,660, 1,500–1,400 and 1,195–1,155 cm−1, can be attributed to absorptions of the C=O, C–N and C=S groups, respectively. The N–H stretching vibration is observed in the region 3,290–3,172 cm−1 as a broad band. In the Ni(L-O, S)2 complexes (1a–12a), the C=O and C=S bands were shifted to 1,607–1,541 and 1,141–1,101 cm−1, respectively. Deprotonation induces delocalization and the C=O and C=S stretching vibration frequencies decrease by ca. 110–100 and 50–55 cm−1, respectively. These band shifts confirm coordination through the oxygen and sulfur atoms. A comparative absorption pattern of the complexes with the values of the free ligands demonstrate that the coordination of thiourea ligands to nickel atom has significant effect on vibrational frequencies of N–H, C–N, C=S and C=O.

The 1H NMR data for the synthesized N,N,N′-tisubstituted thioureas (1–12) showed that the NH hydrogen resonates considerably downfield from other resonances in the spectra. The proton chemical shifts were found in range 8.17–8.80 ppm for NH and aromatic protons mostly appeared downfield near 6.31–8.42 ppm as multiplets. The protons associated with aliphatic groups appeared upfield at 3.47–3.78, 2.77, 2.45–2.55, 1.34 and 1.18 ppm for N–CH 3 , –CH(CH3)2, Caromatic–CH 3 , –C(CH 3 )3 and –CH(CH 3 )2, respectively. The data for the synthesized Ni(II) complexes (1a–12a) have shown a similar pattern except the disappearance of NH protons, that is, the NH protons deprotonated when acyl thiourea ligands behave as bidentate chelators [8–11].

The 13C NMR data revealed that all the signals, due to the unique carbons present in ligands, thioureas (1–12) and the complexes (1a–12a), are in good agreement with the proposed structures. The aromatic carbon resonances of the thioureas were assigned on the basis of signal intensities and then were compared with the reported values [8–11]. The chemical shifts of the carbons pertaining to CONH and CSNH groups of the thioureas resonate around 161–168 and 175–187 ppm, respectively. The substituent, –CH3, attached to aromatic ring resonate around 29 ppm whereas in case of N–CH3 group it resolved around 45 ppm in the compounds. The distinct carbons of –C(CH3)3 and –CH(CH3)2 groups show resonances around 28, 39 and 21, 35 ppm, respectively. However, on complexation there is a slight in δ-values associated with CONH and CSNH groups. There is a clear shift in values of CONH and CSNH groups present in the ligand when they are coordinated with the metal as enunciated in downfield trend for CONH and a slight upfield trend in CSNH group (~5–6 ppm in both cases) owing to the electronegative effect of oxygen and sulfur as well as to the coordination modes the atoms.

For the N,N,N′-trisubstituted thiourea complexes, (CS–N) bond cleavage with the retention of charge on the thionyl group is the major fragmentation pattern noticed in these complexes, as a result of getting the [CH3NPh(CS)] cation (m/z = 150). In the N,N, N′-trisubstituted thiourea complexes (1a–12a) retention of charge on the free ligand after the cleavage of chelate mostly gives the base peak. Comparatively low relative intensity peak was observed for the Ar–CO+ fragment.

X-ray structure analysis

The asymmetric units of (12) and (7) contain two molecules having different conformations. The thiocarbonyl and carbonyl groups are not coplanar with the fluorophenyl ring (12) and isopropyl group (7), as reflected by the torsion angles: 122.5° for C7–N1–C8–N2, −3.6 (3)° for O1–O7–N1–C8 (I) and −2.2 (3)° for O2–C22–N3–C23, 58.2 (2)° for C22–N3–C23–N4 (II); −3.3 (2)° for O2–C21–N4–C20, 105.3 (2)° for C21–N4–C20–N3 (I) and 1.5 (3)° for O1–C9–N1–C8, 1.5 (3)° for C9–N1–C8–N2 (II), respectively. These torsion angles indicate that all the molecules have very similar conformations. The dihedral angles formed by the two benzene ring planes in (12) and the dihedral angles formed by the benzene ring plane and isopropyl group in (7) are 64.41 (5) (I), 87.02 (6) (II), and 45.32 (6) (I), 56.36 (5) (II), respectively, for the two molecules. The other geometric parameters present no unusual features compared with previously determined related structures. In the crystal structure of 12 intermolecular N1/4–H…O interactions connect the molecules into endless chains. The structure 7 (Fig. 1) exhibits N1/2–H…S (Table 2) bonds that link molecules into centro-symmetric di mmers. Crystallographic and refinement details are shown in Table 1. Selected bond lengths and angles are given in Table 3. Figure 1 shows ORTEP diagrams of [Ni(tu)2] complexes 1a, 4a and 5a, crystallize in monoclinic (P 21/c), orthorhombic (P na21) and triclinic (P -1) crystal systems, respectively. The Ni(II) cation is four-coordinated with a slight distorted square planar geometry, completed by oxygen and sulfur atoms from two thiourea ligands. Slightly distorted cis square planar geometry is being observed in these complexes as reflected by the angles: S1–Ni–O2 = 178.10 (6) and S2–Ni–O1 = 179.16 (6), S1–Ni–O2 = 179.4 (2) and S2–Ni–O1 = 178.8 (2), and S1–Ni–O2 = 177.70 (5) and S2–Ni–O1 = 178.93 (5), for 1a, 4a and 5a, respectively. As compared to thiourea free ligand, bond lengths of C–O and C–S are relatively larger in complexes than the corresponding lengths in ligands. Other related complexes also show similar features as reported earlier [8–11]. The bond lengths of C–N in non-coordinated ligand are longer and or different than those found in the Ni(II) complexes (Fig. 2).

Fig. 1
figure 1

ORTEP view of (7) and (12) showing 30 % probability ellipsoids. Some labels are omitted for clarity

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Table 3 Selected bond lengths (Å) and angles (°) for compounds 12, 7, 4a, 5a and 1a
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Fig. 2
figure 2

ORTEP view of (4a), (5a) and (1a) showing 30 % probability ellipsoids. Some labels are omitted for clarity

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Biological activities

Urease inhibition assay

All the synthesized Ni complexes were screened for their urease inhibitory potential. The IC50 values were calculated for compounds (1–12) and (1a–12a) by in vitro testing against Jack bean urease. The IC50 values given in the Table 4 reveal that many compounds possess potent urease inhibition. All the synthesized derivatives exhibit functional groups at phenyl ring. The screened compounds present excellent urease inhibition in micromolar range. Among the series originally designed, compound 1a (IC50 = 1.17 ± 0.12 µM), without any substituent in the phenyl ring, was found ~20-fold more potent as compared to thiourea (IC50 = 23.0 ± 11 µM). Same was the case with 3a having 3-chloro group at phenyl ring, which exhibits IC50 = 1.19 ± 0.41 µM, ~20-fold more active than thiourea. The halogen derivatives also exhibit significant potency against urease activity. Upon further modification, moderate activity was exhibited by 2a and 5a–10a ranging from 3.32 to 16.4 µM. Among the synthesized analogs, 4a, 11a and 12a were least potent with IC50 values greater than 26.8 µM. In the case of N,N,N′-trisubstituted thioureas, compounds 1–12 show less inhibition against urease as compared to Ni complexes. The % age inhibition possessed by these compounds is shown in Table 4.

Table 4 Urease inhibition activity of N,N,N′-trisubstituted thioureas (1–12) and Ni(II) complexes (1a–12a) against Jack bean urease
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Cytotoxicity assay

Antiproliferative activity of Ni(II) complexes 1a–12a was measured in vitro at four different concentrations (100, 10, 1 and 0.1 µM) by the cell growth inhibition against kidney fibroblast (BHK-21) and lung carcinoma (H157). The activity was then compared with vincristine and cisplatin, the standard anticancer drugs, to get percent inhibition of each compound. Vero cells were treated with same Ni(II) complexes 1a–12a to find the effect of these on normal cells (Table 5). N,N,N′-trisubstituted thiourea compounds (1–12) were also tested against BHK-21 and H157 cell lines at 100 µM, and result revealed that they possess less inhibition. So the compounds were not further tested at lower concentrations.

Table 5 Anticancer activity of Ni(II) complexes (1a–12a) against kidney fibroblast (BHK-21), lung carcinoma (H157) and Vero cell lines
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Table 6 Antileishmanial activity of Ni(II) complexes (1a–12a)
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Most of the compounds exhibited less than 50 % activity for both cell lines, having somewhat variable capacity due to their structure range in terms of attached functional groups (Table 5). Among all the screened compounds, 5a show the highest inhibition (80.5 %) for BHK-21 cells, which is more as compared to standard drugs, vincristine (74.5 %) and cisplatin (79.5 %) at 100 µM concentrations. The compounds 6a and 9a proved to be potent inhibitors for cancer therapy and when were tested against BHK-21 cells showed 72.6 and 67.1 % inhibition, respectively. In case of H157, from the series of Ni(II) complexes, compound 5a was the most potent, with 85.5 % inhibition at 100 µM. Other potent inhibitors of H157 cells were 4a, 9a and 10a, with 70.5, 68.9 and 68.2 % inhibition, respectively. Compound 12a showed less than 50 % activity against H157 cells. Among the series, least active compound was 11a with inhibition value 45.3 % and 32.0 % at 100 µM for BHK-21 and H157, respectively. When the N,N,N′-trisubstituted thiourea compounds (1–12) were tested against kidney fibroblast and lung carcinoma cell lines at 100 µM, they presented less inhibition as compared to their Ni complexes. The results are shown in Table 7.

Table 7 % inhibition activity of N,N,N′-trisubstituted thioureas (1–12) against kidney fibroblast (BHK-21) and lung carcinoma (H157) cell lines and Leishmania major
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The ability of any drug to prevent or retard the spread of malignant cells determines its role in the therapy. The anticancer drugs used in chemotherapy are fundamental antiproliferative agents. The results of present work suggested that these compounds may be a good choice for treatment of cancer after in vivo and other clinical studies. However, the exact mechanism of apoptosis of the investigated compounds in cancer cells versus normal cells still has to be determined.

Antileishmanial assay

The antileishmanial activity of Ni(II) complexes (1a–12a) was measured by MTT method and the investigated compounds produce significant reduction in viable promastigotes as shown in Table 6. Amphotericin B was used as a standard drug for anti-leishmanial activity. The results in Table 6 reveal that the tested compounds show significant inhibition against leishmania major in a dose-dependent manner. Almost all the compounds showed remarkable in vitro antileishmanial activity when checked at different concentrations (100, 10, 1, 0.1 µM). Antileishmanial activity of the Ni(II) complexes (1a–12a) proved significant inhibitory potential as compared to N,N,N′-trisubstituted thiourea compounds (1–12) at 100 µM, which exhibited less inhibition as shown in Table 7.

Among all the compounds screened in the series, 5a and 9a show excellent inhibition comparable with Amphotericin B used as a standard drug. Compounds 6a, 8a and 10a also revealed significant inhibition against leishmania major. Other complexes like 1a, 2a, 3a, 4a and 7a also exhibited good inhibition. Least active compounds among the series were 11a and 12a, which displayed less than 50 % inhibition. These results may support the probable use of these compounds, as a source of advance antileishmanial agents.

Conclusions

A series of N,N,N′-trisubstituted thioureas (1–12) and their Ni(II) complexes (1a–12a) were synthesized. The inhibition ability of these compounds against urease was evaluated. The results presented above clearly demonstrate that N,N,N′-trisubstituted thioureas showed little inhibitory activity against Jack bean urease as compared to their Ni(II) complexes. The compounds 1a, 2a, 3a, 5a, 6a, 7a, 8a, 9a and 10a showed excellent urease inhibition, more than the standard drug. The inhibitors 1a and 3a were most potent having IC50 value 1.17 ± 0.12 and 1.19 ± 0.41 µM, respectively. Therefore, these drugs are beneficial and potential alternatives and hence should be investigated further for the control of associated diseases. In case of cytotoxic and antileishmanial activity, Ni complexes proved to have significant inhibitory potential against both the cell lines when compared to N,N,N′-trisubstituted thioureas. Complexes represented remarkable ability to inhibit the growth of cancer cells and had little toxic effect on normal cell lines. Moreover, complexes revealed potent inhibition against leishmaniasis. Due to synthetic and biological versatility of tested compounds, they may be used as therapeutic agents having antiproliferative and antileishmanial activities after further in vivo studies.

Supplementary data

Crystallographic data (excluding structure factors) for the structures reported in this paper have been deposited in the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC-1010230 for (7), CCDC-1010231 for (12), CCDC-1010227 for (1a), CCDC-1010228 for (4a) and CCDC-1010229 for (5a). Copies of available materials can be obtained, free of charge, on application to the director, CCDC, 12 Union Road, Cambridge CB21EZ, UK, (Fax: +44-(0)-1223-336033 or e-mail: deposit@ccdc.cam.ac.uk).