In vitro antileukemia, antibacterial and antifungal activities of some 3d metal complexes: Chemical synthesis and structure – activity relationships

Abstract

The present paper describes the synthesis, characterization and in vitro biological evaluation screening of different classes (ammoniacates, dioximates, carboxylates, semi- and thiosemicarbazidates) of Co(II), Co(III), Cu(II), Ni(II), Mn(II), Zn(II) and Fe(III) complexes. Schiff bases were obtained from the reaction of some salicyl aldehydes with, respectively, furoylhydrazine, benzoylhydrazine, semicarbazide, thiosemicarbazide and S-methylthiosemicarbazide to give tridentate ligands containing ONO, ONS or ONN as donor atoms. The synthetic metal complexes are of various geometrical and electronic structures, thermodynamic and thermal stabilities, and magnetic and conductance properties. All complexes, except those of Cu, are octahedral. Some Cu, Co and Mn compounds have a dimeric or a polymeric structure. The composition and structure of complexes were analysed by elemental analysis, IR and ¹H NMR and ¹³C NMR spectroscopies, and magnetochemical, thermoanalytical and molar conductance measurements. All ligands and metal complexes were tested as inhibitors of human leukemia (HL-60) cells growth, and the most potent, the Cu(II) complexes, have been also tested for their in vitro antibacterial and antifungal activities. Structure-activity relationships were carried out.

Keywords::

• metal complexes
• organic ligand
• complex
• leukemia
• antibacterial
• antifungal

Introduction

Twenty-eight years after the first approval of cisplatin in the clinic against a number of cancer diseases, cisplatin and related compounds continue to be among the most efficient anticancer drugs used so far. Efforts are now focused to develop novel platinum- and non-platinum-based antitumor drugs to improve clinical effectiveness, to reduce general toxicity and to broaden the spectrum of activity [1]. DNA is a main target for the therapeutic treatment of various disorders and diseases. It can interact with many biomolecules and synthetic compounds including organometallic compounds and metal complexes. Therefore, investigations of the interaction of some ligands with transition metals can further provide and/or improve our understanding about rational metal based inhibitors design [2–7]. We have started a program directed toward the synthesis of different classes of anticancer, antibacterial and antifungal agents designed with complexes of a transition metal and an organic ligand [8–12]. Thiosemicarbazones and their transition metal (Cu and Co) complexes demonstrated potent cytotoxic activities against a series of murine and human tumor cells in culture [13–15]. In continuation of this approach, the present paper describes the chemical synthesis, characterization and biological in vitro evaluation of different classes (ammoniacates, dioximates, carboxylates, semi- and thiosemicarbazidates) of Co(II), Co(III), Cu(II), Ni(II), Mn(II), Zn(II) and Fe(III) complexes. Schiff bases H₂L¹ - HL⁷ obtained from interaction of salicyl aldehydes and appropriate amine were used as tridentate ligands containing ONO, ONS or ONN as donor atoms (Figure 1). Diamine L⁸, H₄EDTA and H₅DTPA were also used as ligands. The composition and structure of synthesized complexes were analysed by elemental analysis, IR and NMR spectroscopies, magnetochemical, thermoanalytical and molar conductance measurements. All complexes were tested as inhibitors of human leukemia (HL-60) cells growth. The most potent, the Cu(II) complexes, have been also tested for their in vitro antibacterial activity against Staphylococcus aureus (Wood-46, Smith, 209-P), Staphylococcus saprophyticus, Streptococcus faecalis, Escherichia coli (O-111), Salmonella typhimurium, Salmonella enteritidis, Klebsiella pneumoniaie, Pseudomonas aeruginosa, Proteus vulgaris and Proteus mirabilis and antifungal activity against laboratory stems Aspergillus niger, Aspergillus fumigatus, Candida albicans and Penicillium.

Figure 1.  The chemical structure of organic ligands used in the preparation of metal complexes I-XXVII.

Materials and methods

General

All commercially available reagents and chemicals were of analytical- or reagent-grade purity and used as received. ¹H and ¹³C nuclear magnetic resonance (NMR) spectra were recorded at room temperature with a Bruker DRX 400 spectrometer. All chemical shifts (¹H, ¹³C) are given in ppm versus SiMe₄ using DMSO–d₆ as solvent. IR spectra were recorded on a Specord M80 and are reported in cm^{− 1}. Classic methods were applied for C, H, N and Br elemental analyses, which were performed at Academy of Sciences of Moldova (Institute of Chemistry). The complexes were analysed for their metal contents by EDTA titration [16].TG/DT combined analyses were carried out using a SETARAM 92-1600 instrument. Each sample was deposited in a platinum crucible, which was heated (60°C h^{− 1}) in a current of air to allow evacuation of the products resulting from decomposition. Magnetic measurements were carried out on solid complexes using the Gouy's method [17].

Synthesis of Schiff's bases (H₂L¹-HL⁷)

General procedure with H₂L¹

A hot solution (50°C) of salicylic aldehyde (10 mmol) in ethanol (25 mL) was added to a magnetically stirred solution of furoylhydrazine (10 mmol) in warm ethanol and the mixture was refluxed for 30 min. After completion of the reaction, the mixture was cooled and the solid residue was filtered, washed with cold ethanol, then with diethyl ether, and dried. Crystallization from ethanol gave H₂L¹. The same method was applied for the synthesis of HL² - HL⁷ by using corresponding benzoylhydrazine and 5-nitrofurfural (HL²), salicylic aldehyde and semicarbazide (H₂L³), salicylic aldehyde and thiosemicarbazide (H₂L⁴), 3,5-dibromosalicylic aldehyde and thiosemicarbazide (H₂L⁵), 5-bromosalicylic aldehyde and S-methylthiosemicarbazide (HL⁶), salicylic aldehyde and S-methylthiosemicarbazide (HL⁷).

Salicilydenfuroylhydrazone (H₂L¹)

Yield: 65%. IR (KBr): 3650 (m, OH), 3058 (m, NH), 1630 (m, C = O), 1586 (w, C = N), 1535 (m, NNH). ¹H NMR (DMSO–d₆): 12.28 (s, 1H, NNH), 8.74 (s, 1H, HC = N), 8.40 (s, 1H, OH), 7.88, 7.81, 6.75 and 6.74 (m, 4H, phenyl), 7.80, 7.27 and 6.74 (m, 3H, furan). ¹³C NMR (DMSO–d₆): 152.4 (C = O), 150.5 (HC = N), 152.1 (C–OH), 135.9, 135.4, 121.2, 116.5 and 115.7 (phenyl), 151.9, 146.8, 114.5 and 114.5 (furan). Elemental analysis found for C₁₂H₁₀N₂O₃: C, 62.4; H, 4.3; N, 12.4; calculated: C, 62.6; H, 4.4; N, 12.1%.

5-Nitrofurfuroylidenbenzoylhydrazone (HL²)

Yield: 67%. IR (KBr): 3058 (m, NH), 1625 (m, C = O), 1585 (w, C = N), 1535 (m, NNH). ¹H NMR (DMSO–d₆): 12.26 (s, 1H, NNH), 8.41 (s, 1H, HC = N); 7.94, 7.92, 7.58, 7.56 and 7.64 (m, 5H, phenyl); 7.54 and 7.28 (d, 2H, J = 3 Hz, furan). ¹³C NMR (DMSO–d₆): 165.8 (C = O), 151.8 (HC = N), 135.5, 127.7, 127.3, 128.6, 128.3 and 132.2 (phenyl), 163.4, 151.9, 115.4 and 114.7 (furan). Elemental analysis found for C₁₂H₉N₃O₄: C, 55.9; H, 3.3; N, 16.2; calculated: C, 55.6; H, 3.5; N, 16.2%.

Salicylidensemicarbazone (H₂L³)

Yield: 87%. IR (KBr): 3600 (m OH), 3058 (m, NH), 1630 (m, C = O), 1586 (w, C = N), 1535 (m, NNH). ¹H NMR (DMSO–d₆): 11.20 (s, 1H, NNH), 9.98 (s, 1H, OH), 8.35 (s, 1H, HC = N), 7.93 and 8.02 (1s 2H, NH₂), 8.20, 7.21, 6.85 and 6.80 (m, 4H, phenyl). ¹³C NMR (DMSO–d₆): 197.6 (C = O), 154.4 (HC = N), 140.6 (C–OH), 116.2, 132.1, 121.4, 126.7 and 118.0 (phenyl). Elemental analysis found for C₈H₉N₃O₂: C, 59.3; H, 4.9; N, 23.4; calculated: C, 59.6; H, 5.1; N, 23.5%.

Salicylidenthiosemicarbazone (H₂L⁴)

Yield: 75%. IR (KBr): 3600 (m, OH), 3058 (m, NH), 1560 (s, C = S), 1586 (w, C = N), 1535 (m, NNH). ¹H NMR (DMSO–d₆): 11.39 (s, 1H, NNH), 9.88 (s, 1H, OH), 8.37 (s, 1H, HC = N), 7.93 and 7.91 (2s, 2H, NH₂), 8.20, 7.21, 6.85 and 6.80 (m, 4H, phenyl). ¹³C NMR (DMSO–d₆): 177.6 (C = S), 156.4 (HC = N), 139.6 (C–OH); 116.0, 131.1, 120.4, 126.7 and 118.9 (phenyl). Elemental analysis found for C₈H₉N₃OS: C, 49.4; H, 4.9; N, 21.3; calculated: C, 49.2; H, 4.9; N, 21.5%.

3,5-Dibromosalicylidenthiosemicarbazone (H₂L⁵)

Yield: 72%. IR (KBr): 3650 (m, OH), 3058 (m, NH), 1560 (s, C = S), 1586 (w, C = N), 1535 (m, NNH), ¹H NMR (DMSO–d₆): 11.45 (s, 1H, NNH), 10.55 (s, 1H, OH), 8.29 (s, 1H, HC = N), 8.10 and 8.01 (2s, 2H, NH₂), 8.20 and 7.56, (2s, 2H, phenyl). ¹³C NMR (DMSO–d₆): 178.5 (C = S), 155.4 (HC = N), 150.2 (C–OH), 118.1, 137.5, 111.2, 130.8 and 123.0 (phenyl). Elemental analysis found for C₈H₇Br₂N₃OS: C, 27.0; H, 2.1; N, 11.8; Br, 44.3; S, 9.3%; calculated: C, 27.2; H, 2.0; N, 11.9; Br, 45.3; S, 9.1%.

5-Bromosalicyliden-S-methylthiosemicarbazone (HL⁶)

Yield: 58%. IR (KBr): 3600 (m, OH), 3058 (m, NH), 1658 (s, C-S), 1586 (w, C = N). ¹H NMR (DMSO–d₆): 9.88 (s, 1H, OH), 8.34 (s, 1H, HC = N), 8.60 (s, 2H, NH₂), 8.20, 7.34 and 6.80 (m, 3H, phenyl); 2.10 (s, 3H, CH₃). ¹³C NMR (DMSO–d₆): 160.6 (C-S), 154.2 (HC = N), 137.2 (C–OH), 118.0, 133.2, 111.4, 128.7 and 122.9 (phenyl), 7.9 (S–CH₃). Elemental analysis found for C₉H₁₀BrN₃OS: C, 37.4; H, 3.6; N, 14.5; calculated: C, 37.5; H, 3.5; N, 14.6%.

Salicyliden-S-methylthiosemicarbazone (HL⁷)

Yield: 65%. IR (KBr): 3600 (m, OH), 3058 (m, NH), 1586 (w, C = N). ¹H NMR (DMSO–d₆): 9.80 (s, 1H, OH), 8.30 (s, 1H, HC = N), 8.40 (s, 2H, NH₂), 8.11, 7.21, 6.90 and 6.80 (m, 4H, phenyl), 2.11 (s, 3H, CH₃). ¹³C NMR (DMSO–d₆): 160.0 (C-S), 155.2 (HC = N), 137.0 (C–OH), 118.0, 130.2, 120.4, 131.7, 115.9 and 118.4 (phenyl), 7.9 (S–CH₃). Elemental analysis found for C₉H₁₁N₃OS: C, 51.7; H, 5.2; N, 20.3; calculated: C, 51.6; H, 5.3; N, 20.1%.

Bis(4-aminophenyl)methane (L⁸)

This ligand was commercially available.

Syntheses of metal complexes (Table I)

The reagents for the synthesis of I and II were Bi(HEDTA) · 2H₂O, obtained according to a published method [18], BaCO₃ and the two sulphates [Co(NxH)₂(An)₂]₂SO₄·5H₂O and [Co(NxH)₂(p-Tol)₂]₂SO₄·5H₂O. The dioximates were prepared by reaction of CoSO₄, NxH (1,2-cyclohexanedionedioxime) and the aromatic amine aniline (An) or para-toluidine (p-Tol) in a molar ratio of 1:2:3 in the presence of oxygen. The compounds I and II are crystalline substances stable in air, soluble in water, poorly soluble in alcohols, and insoluble in acetone and diethyl ether.

Trans-[co(nxh)₂(an)₂]₂[bi(edta)(h₂o)]₂ · 7h₂o (I)

The complex Bi(HEDTA) · 2H₂O (1.068 g, 2 mmol) was dissolved in water (50 mL) upon heating. BaCO₃ (0.197 g, 1 mmol) was added and the mixture heated with stirring for 1 h to give Ba(BiEDTA)₂ solution, then [Co(NxH)₂(An)₂]₂SO₄ · 5H₂O (0.577 g, 1 mmol) (NxH₂ = nioxime = 1,2-cyclohexanedione dioxime) in water (20 mL) was added. BaSO₄ was filtered off, and the clear solution was allowed to stand at room temperature. Transparent and brownish crystals of I were separated by filtration, washed with ethanol (2–5 mL) and diethyl ether (2–3 mL), and dried in air up to the constant weight. Yield: 65%. Water content: Exp. (TGA) 7.5%, Calc. 7.33%.

Trans-[co(nxh)₂(p-tol)₂][bi(edta)] · 4h₂o (Ii)

Red-brown crystals of complex II were obtained as above by reaction of [Co(NxH)₂(p-Tol)₂]₂SO₄·5H₂O and Ba(BiEDTA)₂. Yield: 52%. Water content: Exp. (TGA) 6.5%, Calc. 6.41%.

Trans-[co(nh₃)₄(no₂)₂][bi(edta)(h₂o)] · 2h₂o (Iii)

A solution containing trans-[Co(NH₃)₄(NO₂)₂]₂SO₄ (0.534 g, 1 mmol) dissolved in a minimum of hot water was added with vigorous stirring to an aqueous solution of Ba[Bi(EDTA)]₂ (prepared from 1.068 g, 2 mmol, of Bi(HEDTA)·2H₂O as described in literature [18]) and BaCO₃ (0.197 g, 1 mmol). After removal of precipitated BaSO₄, the solution was allowed to stand overnight. The resulting yellow crystalline product was collected by filtration, washed with ethanol and then dried in air. This complex is soluble in water but insoluble in alcohols, acetone, or diethyl ether. It can be recrystallized from water solution without change in composition. Yield: 80%.

[Co(nh₃)₅ncs][bi(edta)]₂ · 4h₂o (Iv)

The complex IV was prepared by reaction of Ba[Bi(EDTA)]₂ and [Co(NH₃)₅NCS]SO₄·2H₂O in aqueous solution. Bi(HEDTA) · 2H₂O (1.068 g, 2 mmol) was dissolved in hot water (80 mL) and BaCO₃ (0.197 g, 1 mmol) was added. After complete dissolution, a solution of [Co(NH₃)₅NCS]SO₄ ·2H₂O (0.334 g, 1 mmol) of water (20 mL) was added with stirring. The precipitated BaSO₄ was removed by filtration. Ethanol was added and the solution was allowed to stand for 24 h. [Co(NH₃)₅NCS][Bi(EDTA)]₂ · 4H₂O crystallized as orange needles from a clear solution. The compound was recrystallized from hot water and dried in air. Yield: 72%.

[Co₂(-h₂o)(-ccl₃coo)₂(ccl₃coo)₂(h₂o)₄]·h₂o (V), [Mn₂(-h₂o)(-ccl₃coo)₂ (Ccl₃coo)₂(h₂o)₄] ·H₂o (Vi) And [Zn(cf₃coo)₂(h₂o)₂] · 2H₂o (Vii)

Non-symmetric Co(II), Mn(II) trichloroacetate complexes were prepared as described in the literature [19,20].

[Fe(l⁶)(h₂o)₃]so₄• Or ·2H₂o (Viii)

This complex was prepared according to the literature [21].

Compounds Ix-xiii

The complexes of type M(HL¹)₂ · nH₂O (M = Mn^{2 +} , Co^{2 +} , Ni^{2 +} , Zn^{2 +} and Cu^{2 +} ; n = 0 – 3) were synthesised from metal acetates and H₂L¹ in the presence of ammonium hydroxide solution (pH = 8) [22,23].

[Co(l⁷)(h₂o)₃]₂so₄ · 2h₂o (Xiv) And [Cu(l²)₂] · 2H₂o) (Xv)

These complexes were obtained as described in the literature [21].

{[Cu(hl³)(h₂o)bi(edta)(h₂o)] · 4h₂o}₂ (Xvi)

A solution of [Cu(HL³)(H₂O)]₂SO₄ · 2H₂O (0.521 g, 0.8 mmol), prepared as described in literature [24], in distilled water (50 mL) was added to a solution of Ba[Bi(EDTA)]₂ (0.8 mmol) obtained upon reacting ?i(?EDTA) · 2H₂O (0.854 g, 1.6 mmol) with BaCO₃ (0.158 g, 0.8 mmol) in water (25 mL). BaSO₄ was filtered off and the resulting solution, after heating for half an hour on a water bath, was filtered hot and left for two days. The light-green crystals were collected by filtration, washed with water and ethanol prior to be dried. Yield: 75%.

[Cu(hl³)(h₂o)]₂[bi(dtpa)] · 10h₂o (Xvii)

In a 100 mL container, [Cu(HL³)(H₂O)]₂SO₄ · 2H₂O (0.521 g, 0.8 mmol) was dissolved in deionized water (50 mL). Separately, BiH₂DTPA · 2H₂O (0.508 g, 8 mmol) [18,24] was dissolved in distilled water (25 mL) under stirring and heating, and BaCO₃ (0.158 g, 8 mmol) was gradually added to this clear solution. The second solution was then added dropwise to the fist one. BaSO₄ was filtered off and the resulting solution was left for crystallization at room temperature. The light-green crystalline powder was collected by filtration, washed with water and ethanol prior to be dried. Yield: 85%.

[Cu(l⁸)₂]so₄ (Xviii)

This complex was prepared as previously described [25].

[Cu(hl⁴)(h₂o)][bi(edta)] · h₂o (Xix)

A solution containing [Cu(HL⁴)(H₂O)]₂SO₄ · 3H₂O (0.137 g, 0.2 mmol), prepared by reacting equimolar quantities of CuSO₄·5H₂O and H₂L⁴ in ethanol, in distilled water (150 mL) was added to a solution of Ba[Bi(EDTA)]₂ (0.2 mmol) obtained upon reacting Bi(HEDTA) · 2H₂O [18] (0.214 g, 0.4 mmol) with BaCO₃ (0.0395 g, 0.2 mmol) in water (25 mL). BaSO₄ was filtered off and the resulting green-bluish substance was collected by filtration, washed with water and ethanol prior to be dried. Yield: 52%.

[Cu(hl⁴)h₂o]₂[bidtpa] ·6H₂o (Xx)

A solution containing [Cu(HL⁴)(H₂O)]₂SO₄·3H₂O (0.1366 g (0.2 mmol), prepared by reacting equimolar quantities of CuSO₄ · 5H₂O and salicylidenthiosemicarbazone (H₂L⁴) in ethanol, in distilled water (150 mL) was added to a solution of Ba[Bi(DTPA)] (0.2 mmol), which was obtained upon reacting BiH₂DTPA · 2H₂O [26] (0.127 g, 0.2 mmol) with BaCO₃ (0.039 g, 0.2 mmol) in water (25 mL). BaSO₄ was filtered off and the resulting green powder was collected by filtration, washed with water and ethanol prior to be dried. Yield: 46%.

[Cu(hl⁵)cl] (Xxi)

To a solution of Cu(II) chloride (10 mmol) in ethanol (30 mL), heated (50–55°C) and mixed continuously with a magnetic agitator, was added a solution of 3,5-dibromosalicyliden-thiosemicarbazone (H₂L⁵) (10 mmol) in ethanol (120 mL) and the mixture was heated for 30–40 min. After cooling, the small green crystals formed from the reaction mixture were filtered on glass filter, washed with ethanol and diethyl ether, and dried in air. Yield: 81%.

[Cu(hl⁵)br] (Xxii), [Cu(hl⁵)no₃] (Xxiii) And [Cu(hl⁵)(h₂o)]₂so₄ (Xxiv)

According to the method reported above for XXI and using Cu(II) bromide, Cu(II) nitrate trihydrate or Cu(II) sulphate pentahydrate with 3,5-dibromosalicylic aldehyde thiosemicarbazone as initial substances in the 1:1 molar ratio, we synthesized XXII, XXIII and XXIV, respectively. Yields are 85% for XXII, 79% for XXIII and 80% for XXIV.

[Cu(hl⁴)no₃] (Xxv), [Cu(hl⁴)(thio)]₂so₄. H₂o (Xxvi) And (Cu(hl⁴)(h₂o)(₂so₄ (Xxvii)

These complexes were prepared as previously described [25].

Antileukemia bioassay (Table II)

Cell culture

Human promyelocytic leukemia cells HL-60 (ATCC, Rockville, MD, USA) were routinely grown in suspension in 90% RPMI-1640 (Sigma, Saint Louis, USA) containing L-glutamine (2 nM), antibiotics (100 IU penicillin/mL, 100 μg streptomycin/mL) and supplemented with 10% (v/v) foetal bovine serum (FBS), in a 5% CO₂ humidified atmosphere at 37°C. Cells were currently maintained twice a week by diluting the cells in RPMI 1640 medium containing 10% FBS.

Cell proliferation assay

The cell proliferation assay for complexes I-XXVII and ligands was performed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium (MTS) (Cell Titer 96 Aqueous, Promega, USA), which allowed us to measure the number of viable cells. In brief, triplicate cultures of 10,000 cells in a total of 100 μL medium in 96-well microtiter plates (Becton Dickinson and Company, Lincoln Park, NJ, USA) were incubated at 37°C, 5% CO₂. Compounds were dissolved in ethanol to prepare the stock solution of 1 × 10^{− 2} M. These compounds and doxorubicin (Novapharm, Toronto, Canada), as a positive control, were diluted at multiple concentrations with culture media, added to each well and incubated for 3 days. Following each treatment, MTS (20 μL) was added to each well and the mixture incubated for 4 h. MTS is converted to water-soluble colored formazan by dehydrogenase enzymes present in metabolically active cells. Subsequently, the plates were read at 490 nm using a microplate reader (Molecular Devices, Sunnyvale, CA).

Antibacterial bioassay (Table III)

The antibacterial activity of complexes XXI-XXIV and also of their prototype Furaciline was determined under liquid nutritive environment [2% of peptonate bullion (pH 7.0)] using successive dilutions method [11,12]. Staphylococcus aureus (Wood-46, Smith, 209-P), Staphylococcus saprophyticus, Streptococcus faecalis, Escherichia coli (O-111), Salmonella typhimurium, Salmonella enteritidis, Klebsiella pneumoniaie, Pseudomonas aeruginosa, Proteus vulgaris and Proteus mirabilis standard stems were used as reference culture for in vitro experiment. The dissolution of studied substances in dimethylformamide, microorganisms' cultivation, suspension obtaining, determination of minimal inhibition concentration (MIC) and minimal bactericide concentration (MBC) were carried out according to the method previously reported [27].

Antifungal bioassay (Table IV)

Antimycotic properties of the complexes XXI-XXIV were investigated in vitro on laboratory stems: Aspergillus niger, Aspergillus fumigatusi, Candida albicans and Penicillium. The activity was determined in liquid Sabouroud nutritive environment (pH 6.8). The inoculates were prepared from fungi stems which were harvested during 3–7 days. Their concentration in suspension is (2–4) × 10⁶ colonies forming units/mh. Sowings for levures and micelles were incubated at 37°C during 7 and 14 days, respectively.

Results and discussion

Chemistry

The heterometallic bismuth(III) complexes containing cobalt(III) dioximates (I and II) or ammoniacates (III and IV) are of cation-anion structure (Figure 2). The complex I is an unique example, which differs structurally from other Bi(EDTA)⁻ complexes by formation of a bridge between the cationic and anionic moieties through an oxime oxygen atom. The influence of the minor changes in the cationic moiety on the anionic sub-lattice is the subject of a special interest in this class of compounds. The heterometallic complexes I-IV have been obtained by an exchange reaction of barium EDTA bismuthate(III) with corresponding cobalt(III) dioximate/ ammoniacate sulphates. After removal of barium sulphate the resulting solution contains cobalt(III) nioximate/ammoniacate complex cation and bismuth(III) EDTA complex anion in molar ratio 1:1. Initial complex dioximates were prepared by passing an intense flow of air through a mixture of cobalt(II) sulphate heptahydrate, 1,2-cyclohexanedionedioxime, and corresponding amine in methanol–water solution. Complexes I-IV are brown crystalline substances readily soluble in water or dimethylsulfoxide and insoluble in acetone or diethyl ether. The complexes are stable to storage in air under normal conditions and can be recrystallized from water without change of their composition.

Figure 2.  Representation of complexes I-IV.

The ¹H NMR spectra of diamagnetic complexes I and II are similar. The following signals are characteristic for the coordinated p-toluidine: 2.24 ppm for the CH₃ group, 5.48 ppm for the NH₂ group and two doublets at 6.49 and 7.02 ppm (J_HH = 7.7 Hz) for the aromatic hydrogen atoms. A confirmation of the trans-configuration of complex cations in I and II in solution is provided by the observation of a large singlet (ΔH/2 ≈ 20 Hz) at 17.7 ppm corresponding for two symmetric hydrogen bonds [28]. These data are in concordance with X-ray analysis in solid state [29]. The signals from CH₂-groups of nioxime ligands were observed at 2.54 and 1.51 ppm (1.50 ppm for complex II) as strong singlets. The singlet signal at 3.26 ppm was identified as that of ethylene protons of EDTA ligand. The NCH₂CH₂N component of EDTA in the domain of fast exchange and the signal from this group is a singlet. The four acetate methylene protons give rise to an AB quartet system J_AB = 15.9 Hz for I, and 15.6 Hz for II in the range 3.68–3.73 ppm.

The thermogravimetric investigation of I-IV in air (5 °C/min) revealed four major steps corresponding to dehydration, deamination, ligand pyrolysis and formation of inorganic residue. The dehydration step occurs in a wide range of temperature, from 50°C up to 160–170°C. Deamination (removal of aniline or p-toluidine) begins at 180–195°C, but it is overlapped by the strong exothermic effect that begins near 220°C and finishes around 480–490°C. The investigations of pyrolysis products were carried out using X-ray powder diffraction. X-ray powder diffraction patterns of all four residues are identical. The final products were identified as the sellinite-type phase with a minor mixture of the cobalt (II, III) oxide Co₃O₄ (∼5%). X-ray diffraction data of the sellinite-type phase were indexed on the basis of a body-centred cubic structure with a = 10.190(2)Å. In accordance with literature [30] this is the Bi_{26 − x}Co_xO_{40 − δ}-phase with the metal (Bi:Co) ratio ≈ 1:1.

The complexes V and VI, (μ-aqua)di(μ-tricloracetato)-tri(aqua)cobalt(II)-aqua-bis(triclor-acetato)cobalt(II) and (μ-aqua)di(μ-tricloracetato)-tri(aqua)manganese(II)-aqua-bis(triclor-acetato)manganese(II), have an asymetric structure including two nonequivalent metal atoms (Co or Mn) up bridged by one water molecule and two μ-tricloroacetate anions [19,20]. Main IR features are in good agreement with the proposed structure. The effective magnetic moments of V and VI, 4.8 B.M. for cobalt(II) and 5.5 B.M. for manganese(II), are characteristic for an octahedral environment of central atoms, but the values are a little lower than those for a theoretical high spin state. This fact confirms a weak antiferromagnetic interaction in dimeric bimetallic complexes V and VI. The zinc trifluoracetate complex VII is diamagnetic in 3d¹⁰ ground electronic state.

The complexes IX-XIII of type [M(HL¹)₂] · nH₂O (M = Mn^{2 +} , Co^{2 +} , Ni^{2 +} , Zn^{2 +} and Cu^{2 +} ; n = 0 – 3) were synthesised from metal acetates and H₂L¹ in the presence of ammonia (pH = 8). Physico-chemical properties and composition of these metal complexes are shown in Table I and their structures are represented in Figure 3. The magnetic moment of 4.9 B.M. is indicative of three unpaired electrons for cobalt (II) compound X with pronounced spin orbital interaction in an octahedral environment. The nickel(II) complex XI showed a μ_eff of 3.1 B.M., which corresponds to two unpaired electrons per nickel(II) ion for a six-coordinated configuration.

Table I.  Physical and analytical data of the metal complexes I-XXVII.

No	Metal complex and molecular formula^a	Mr^b	μ eff^c (B.M.)	C, H, N, calc (found) (%)	M (3d)^d (%)	IR band (cm^− 1)	η^e (%)	T dec. ^f (°C)
I	trans-[Co(NxH)2(An)2]2[Bi(EDTA)(H2O)]2·7H2O C34H53BiCoN8O16	1106	dia	36.9(37.0); 4.8(4.7); 10.1(9.8)	5.3(5.5)	1750 νas(C—O)COO; 1330 νs(C—O)COO;1080; 1055 ν(C—N, CN); 460 (M-O); 400 (M-N)	65	490
II	trans-[Co(NxH)2(p-Tol)2][Bi(EDTA)]·4H2O C36H56BiCoN8O16	1125	dia	38.4(38.1); 5.0(4.6); 9.9(9.5)	5.2(5.3)	1750 νas(C—O)COO; 1330 νs(C—O)COO;1080; 1055 ν(C—N, CN); 460 (M-O);400 (M-N)	52	480
III	trans-[Co(NH3)4(NO2)2][Bi(EDTA)(H2O)]·2H2O C10H30[BiCoN8O15	770	dia	15.6(15.8), 3.9(3.9), 14.5(14.8)	7.7(7.6)	1750 νas(C—O)COO; 1330 νs(C—O)COO;1080; 1055 ν(C—N, CN), 1355 ν(N—O)NO2; 460 (M-O); 400 (M-N)	80	480
IV	[Co(NH3)5NCS](Bi(EDTA)]2·4H2O C21H47Bi2CoN10O20S	1326	dia	19.9(20.1); 3.7(3.6); 11.0(11.2)	4.6(4.7)	1750 νas(C—O)COO; 1330 νs(C—O)COO;1080; 1055 ν(C—N, CN); 460 (M-O);400 (M-N)	72	490
V	[Co2(-H2O)(μ-CCl3COO)2(CCl3COO)2(H2O)4]·H2O C8H12Co2O14Cl12	896	4.8	10.6(10.2); 1.3(1.4)	13.5(13.1)	1750 νas(C—O)COO; 1330 νs(C—O)COO;460 (M-O)	85	350
VI	[Mn2(-H2O)(μ-CCl3COO)2(CCl3COO)2(H2O)4]·H2O C8H12Mn2O14Cl12	868	5.5	11.1(11.0); 1.4(1.3)	12.7(12.9)	1750 νas(C—O)COO; 1330 νs(C—O)COO;460 (M-O)	70	360
VII	[Zn(CF3COO)2(H2O)2]·2H2O C4H8ZnO8F6	361	dia	13.2(13.4); 2.2(2,1)	17.9(18.0)	1750 νas(C—O)COO; 1330 νs(C—O)COO;460 (M-O)	92	320
VIII	[Fe(HL^6)(H2O)3]SO4·2H2O C9 H19FeN3O10SBr	496	5.8	21.8(22.0); 3.8(3.7); 8.5(8.8)	11.3(11.0)	1600 (C = N); 1510 (NNH); 460 (M-O);400 (M-N)	72	420
IX	[Mn(HL^1)2]·2H2O C24H22MnN4O8	547	5.9	52.6(52.1); 4.0(3.9); 10.2(10.5)	10.0(10.1)	1560 (C = NN); 460 (M-O); 400 (M-N)	56	445
X	[Co(HL^1)2]·3H2O C24H24CoN4O9	569	4.9	50.6(50.2); 3.9(3.7); 9.8(9.7)	10.4(10.0)	1560 (C = NN); 460 (M-O); 400 (M-N)	68	460
XI	[Ni(HL^1)2]·2H2O C24H22NiN4O8	551	3.1	52.3(52.7); 4.0(3.8); 10.2(10.5)	10.7(10.2)	1560 (C = NN); 460 (M-O); 400 (M-N)	71	450
XII	[Zn(L^1)2] C24H18 ZnN4O6	521	dia	55.3(55.6); 3.5(3.4); 10.7(10.9)	12.1(12.0)	1560 (C = NN); 460 (M-O); 400 (M-N)	85	475
XIII	[Cu2(HL^1)2]·2H2O C24H20Cu2N4O8	619	1.6	46.5(46.4); 3.2(3,1); 9.0(8.8).	20.4(20.3)	1560 (C = NN); 460 (M-O); 400 (M-N)	65	400
XIV	[CoL^7(H2O)3]2SO4·2H2O C18H36Co2N6O14S4	774	5.2	27.9(28.3); 4.7(4.5); 10.8(10.9)	15.2(15.0)	1600 (C = N); 1510 (NNH); 460 (M-O);400 (M-N)	68	380
XV	[Cu(L^2)2]·2H2O C24H22CuN6O10	617	1.9	46.7(46.9); 3.6(3.9); 13.6(13.8)	10.2(10.0)	400 (M-N)	70	410
XVI	{[Cu(HL^3)(H2O)Bi(EDTA)(H2O)]·4H2O}2C36H64Bi2Cu2N10O32	1689	1.9	25.6(25.4); 3.8(3.4); 8.3(8.2)	7.5(7.2)	1648 ν(C = O)SSA; 1628 ν(C = N)SSA;1572 νas(C—O)COO; 1391 νs(C-O)COO;1252, ν(CO) phenolic;1117, 1087 ν(C—N)EDTA	75	380
XVII	[Cu(HL^3)(H2O)]2[Bi(DTPA)]·10H2O C30H58BiCu2N9O26	1296	1.9	27.8(27.1); 4.5(4.6); 9.7(8.9)	9.8(9.3)	1661 ν(C = O)SSA; 1581 ν(C = N)SSA;1557 νas(C—O)COO; 1393 νs(C-O)COO; 1301 ν(CO)phenolic;1124; 1088 ν(C—N)EDTA	85	460
XVIII	[Cu(L^8)2]SO4 C26H28CuN4O4S	556	1.7	56.1(56.4); 5.0(5.2); 10.1(10.2)	11.3(11.1)	1600 (C = N); 1510 (NNH); 460 (M-O);400 (M-N);	60	460
XIX	[Cu(HL^4)(H2O)][Bi(EDTA)]·H2O C18H24BiCuN5O11S	790	1.8	25.5(25.4); 3.8(3.9); 8.9(8.8)	8.0(7.6)	1606 ν(C = N)TSSA; 1575 νas(C-O)COO;1359 νs(C-O)COO;1283 ν(CO)phenolic; 1108; 1090 ν(C—N)EDTA	52	480
XX	[Cu(HL^4)H2O]2[BiDTPA]·6H2O C30H50BiCu2N9O20S1	1256	1.8	28.6(27.7); 4.0(4.3); 10.0(10.3)	10.1(9.7)	1602 ν(C = N)TSSA; 1570 νas(C-O)COO;1369 νs (C-O)COO; 1294 ν(CO)phenolic; 1081 ν(C—N)EDTA	46	475
XXI	[Cu(HL^5)Cl] C8H6Br2ClCuN3OS	451	1.9	21.3(21.0); 1.3(1.1); 9.3(9.1) Br 35.4(35.6)	14.2(13.8)	1610 (C = N); 455 (Cu-O); 400 (Cu-N);320 (Cl-O); 300 (Cu-S)	79	470
XXII	[Cu(HL^5)Br] C8H8Br3CuN3O2S	513	1.8	18.7(18.5); 1.6(1.3); 8.2(8.1); Br 46.7(46.5)	12.5(12.4)	1618 (C = N); 455 (Cu-O); 400 (Cu-N);300 (Cu-S); 250 (Cu-Br)	85	460
XXIII	[Cu(HL^5)(NO3)] C8H6Br2CuN4O4S	480	2.0	20.0(19.9); 1.2(1.0); 11.7(11.6); Br 33.5(33.2)	13.4(13.2)	1612 (C = N); 455 (Cu-O); 400 (Cu-N);300 (Cu-S)	80	425
XXIV	[Cu(HL^5)(H2O)]2SO4 C16H22Br4Cu 2N6O11S	1016	2.1	18.9(18.6); 2.2(2.0); 8.3(8.0); Br 31.4(31.1)	12.6(12.3)	1612 (C = N); 455 (Cu-O); 400 (Cu-N); 300 (Cu-S)	82	430
XXV	[Cu(L^6)(NO3)] C9H10CuN4O4S	333	1.8	32.4(32.0); 3.0(3.3); 16.8(16.5)	19.1(19.5)	1612 (C = N); 455 (Cu-O); 400 (Cu-N);	81	420
XXVI	[Cu(HL^4)(thio)]2SO4·H2O C18H26Cu2N10O7S5	780	1.6	27.7(27.5); 3.3(3.1); 17.9(18.1)	16.1(16.4)	1600 (C = N); 1510 (NNH); 460 (M-O);400 (M-N);300 (M-S)	72	450
XXVII	[Cu(HL^4)(H2O)]2SO4·3H2O C16H26Cu2N6O11S3	700	1.7	27.4(27.0); 3.7(3.9); 12.0(12.3)	18.0(18.4)	1600 (C = N); 1510 (NNH); 460 (M-O);400 (M-N); 300(M-S)	85	435

^aThe chemical structure of ligands H₂L¹-L⁸, H₄EDTA and H₅DTPA used in the preparation of complexes are reported in Figure 1.

^bMr: relative molecular masse; ^cμ_eff: magnetic moment; ^dM (3d): metal 3d; ^eη: yield; ^fT_dec.: decomposition temperature

Figure 3.  General structure of complexes IX-XII (a) and XIII (b).

The manganese(II) compound IX with an effective magnetic moment of 5.9 B.M. is an example of 5 electrons in a high state of octahedral coordination. The zinc(II) complex XII is diamagnetic. The thermal stability and total decomposition temperature (t) of the metal complexes IX-XII is influenced by the nature of the central atom according to the following relation: t (Co) ≥ t (Ni) ≥ t (Zn) t > (Mn). For complex XIII, a comparison of the IR spectra of the Shiff's base H₂L¹ [31–32] to their metal chelates indicated that H₂L¹ is coordinated to the metal atom mainly in a deprotonated way acting in a tridentate ONO manner including a phenolic oxygen, an azomethine nitrogen and an amidic oxygen, then forming five and six atom rings with the central metal atom. In IR, a band that appears at 1560 cm^{− 1} due to the azomethine group was shifted to lower frequency by 28 cm^{− 1} indicating the participation of azomethine nitrogen in the complexation. A new band appearing at 460 cm^{− 1} was assigned to ν(M-O) [33] whereas the absence of the band at 1635 cm^{− 1} demonstrated that the oxygen has formed a coordinative bond with metal ions in an enolic form. A weak band at 400 cm^{− 1} was assigned to ν(M-N). The room temperature magnetic moment of the solid copper(II) complex XIII, 1.6 B.M., demonstrates the anti-ferromagnetic spin-spin interaction through a dimeric complex association.

The heterobimetalic (Bi,Cu) complexes containing the salicylic aldehyde semicarbazone (XVI or XVII) or salicylic aldehyde thiosemicarbazone (XIX or XX) were prepared by reacting [Cu(HL³)(H₂O)]₂SO₄• or ·2H₂O or [Cu(HL⁴)(H₂O)]₂SO₄• or ·3H₂O with solutions of Ba[Bi(EDTA)]₂ or Ba[Bi(DTPA)].

The coordinative compounds XXI-XXIV (figure 4) have been prepared in 79–85% yields by a reaction between hydrate of copper(II) chloride, bromide, nitrate or sulphate with 3,5-dibromosalicylic aldehyde thiosemicarbazone (H₂L⁵) in the 1:1 molar ratio. The mechanism of the given reaction is connected with the addition of this ligand, which has the role of tridentate ONS ligand, to copper(II) ion. Chloride, bromide, nitrate or water occupies the fourth place in the inner coordination sphere. At the same time, the deprotonation of the phenol takes place in the reaction mixture. Complexes XXI-XXIV are stable in contact with air, poorly soluble in water and alcohol, soluble in dimethylformamide and dimethylsulfoxide, practically insoluble in diethyl ether. The composition and each structure have been determined from the elemental analysis, IR spectroscopy, magnetochemistry and thermogravimetry methods. By determining the molar electric conductibility in dimethylformamide, it was established that XXI-XXIII are non-electrolytes ( = 4–9 Ohm^{− 1} cm² mol^{− 1}) whereas XXIV was a triple electrolyte ( = 149 Ohm^{− 1}. cm². mol^{− 1}). According to the magnetochemical research of complexes at room temperature (293 K), the calculated values of their effective magnetic moment are close to the spin value for an uncoupled electron and represent 1.93 (XXI), 1.85 (XXII), 1.99 (XXIII) and 2.09 (XXIV) B.M. This fact allows us to suppose that the studied substances have a monomeric structure [17,34,35].

The comparative analysis of the IR spectra of the synthesized compounds and the ligand (3,5-dibromosalicylic aldehyde thiosemicarbazone) was made in order to determine the coordination mode of azomethine with copper(II) ion. It was established that the thiosemicarbazone in XXI-XXIV behaves as a monodeprotonated tridentate ONS ligand, connected to the central ion by a deprotonated phenolic oxygen atom, azomethine nitrogen and sulphur, forming metalocycles of five and six members. This fact finds the explanation in the disappearing of the δ(OH) absorption band in the IR spectra, which can be observed in the free thiosemicarbazone in the range 1245–1240 cm^{− 1}. In compounds XXI-XXIV and their structural analogues, ν(C = N) absorption band is shifted by 35–30 cm^{− 1} to a smaller frequency [in starting thiosemicarbazone, ν(C = N) is observed in the range of 1620–1610 cm^{− 1}]. The mentioned coordination mode of 3,5-dibromosalicylic aldehyde thiosemicarbazone is supported by the appearance of a series of new absorption bands in the range 630–300 cm^{− 1}, bands that according to the published data are detected as ν(Cu-O), ν(Cu-N) and ν(Cu-S). Besides this, the confrontation of the absorption bands maxima, determined according to those described in [36], proves that the nitrate group in compound XXIII is coordinated to a central ion and behaves as a monodentate ligand in the interior coordination sphere [the intervals of the main oscillation frequencies coincide (ν₁(A₁) = 1295–1250; ν₂(A₁) = 1035–970; ν₄(B₁) = 1530–1480; ν₆(B₂) = 800–780 cm^{− 1}) and two weak absorption bands appear at 1780 and 1720 cm^{− 1}]. However, the sulphate-ion in XXIV is placed in the exterior sphere. In fact, a single absorption band characteristic for this non-coordinated anion is observed in the range 1110–1120 cm^{− 1}.

Thermal analysis of complexes XXI-XXIV showed that their thermolysis occurs in steps. An endothermic effect, which corresponds to the breaking of the crystallization water molecules (dehydration) of compounds XXII and XXIV in the temperature range of 75–96°C. In the case of XXIV, the process of deaquation occurs with endothermic effect at 155°C, but at 470 (XXI), 460 (XXII), 425 (XXIII) and 430 (XXIV)^oC, the complete thermooxidative destruction of the coordinated thiosemicarbazone with exothermic effect takes place.

Antileukemia activity

All 27 compounds were tested as inhibitors of HL-60 cells proliferation. These human promyelocytic leukemia cells were incubated for three days in the presence of synthetic compounds (ligands and complexes) and the number of viable cells was measured using the MTS assay. The results are expressed as the percentage of cell growth inhibition at three concentrations. The ligands have insignificant inhibitor activity (data not shown), but some metal complexes selectively act in this biological process (Table II). The nature, electronic structure and coordination number of the central atom, the geometric configuration of metal complexes and the nature of the ligands (donor atoms) appear to modulate the cell proliferation. Among all the compounds tested, the most indicative are copper(II) complexes XIX, XX, XXIV, XXVI and XXVII and cobalt(II) complex V, which efficiently inhibited the HL-60 cell proliferation at 1 μM.

Table II.  Antiproliferative activity of complexes I–XXVII on human leukemia (HL-60) cells at three concentrations.

	Inhibition of cell proliferation (%)^b
Complex^a	10 μM	1 μM	0.1 μM
I	–	17	5
II	–	18	4
III	35	30	15
IV	28	15	10
V	88	40	20
VI	–	14	16
VII	–	14	17
VIII	–	0	0
IX	–	20	10
X	–	20	7
XI	–	17	7
XII	–	4	0
XIII	–	12	0
XIV	–	5	0
XV	–	17	0
XVI	20	0	0
XVII	90	5	0
XVIII	–	15	12
XIX	97	92	10
XX	99	99	5
XXI	–	30	8
XXII	–	20	5
XXIII	–	22	6
XXIV	–	60	8
XXV	–	10	0
XXVI	87	88	10
XXVII	88	50	4
Doxorubicin	99	98	15

^aThe molecular formula of complexes are reported in Table I.

^bSEM < ± 4% of a single experiment in triplicate.

The quite essential activity of copper complexes may be a consequence of Jahn-Teller distortion effect, which takes place along the axe dz² of copper(II) [37]. The activity of copper complexes is influenced also by the nature of the ligand donor atoms that are present in coordination polyhedra. In fact, complexes with coordinated ligand containing a sulphur donor atom (ONS) are essentially more active than complexes including inner sphere oxygen (ONO) or nitrogen (ONN) (figure 5).

In the case where a sulphur donor atom is blocked by a CH₃ group (as for ONN ligands HL⁶ and HL⁷), the biological activity of copper complex became insignificant as illustrated by a comparison of XX and XXV. If copper is capsulated in a dimeric complex as for XIII (Figure 3b) or polynuclear as for XVI, the cell growth inhibition dramatically change and became minimal. In the series of complexes [Cu(HL⁵)Y] (XXI-XXIV) having the same metal (copper) and tridentate ONS ligand H₂L⁵, the second inner sphere ligand Y (), influence the biological activity in the following order: H₂O (XXIV) > Cl⁻ (XXI) > Br⁻ (XXII) ≅ NO₃⁻ (XXIII). In this case, the presence of an inner coordination sphere labile ligand, as a molecule of water, raises considerably (till 60%) the antiproliferative activity. Only one complex with a metal atom other than copper gave a significant inhibition of cell proliferation. Thus, the cobalt complex V inhibited 40% of cell growth at 1 μM. For the cobalt(II) and cobalt(III) octahedral complexes V and X with coordination number 6, the thermodynamic stability is also important as well as in the case of copper(II) complexes XXI-XXIV. In solutions, the most labile cobalt (II) complex V (Co^{2 +} has the electronic configuration 3d⁷ with an inner coordination sphere lifetime of the ligand 10^{− 4} to 10^{− 5} s) is more active in comparison with the inert cobalt(III) complex X (Co^{3 +} has the electronic configuration 3d⁶ with a lifetime 10⁴–10⁵ s) [38,39].

Figure 4.  Representation of complexes XXI-XXIV.

Figure 5.  Copper(II) complexed by different ligand donor atoms (ONS, ONO and ONN).

Antibacterial activity

Four copper(II) complexes have been screened for their in vitro antibacterial and antifungal activity. Experimental results, obtained from the study of antimicrobial activity of compounds XXI-XXIV, are given in Table III. As can be seen, they display bacteriostatic activity towards gram-positive and gram-negative bacteria in 0.018–2000 μg/mL concentration. Pseudomonas aeruginosa is an exception, for which MIC is 300–4000 μg/mL). For comparison, we also presented the antimicrobial data characteristic for Furaciline, a bactericide used in medical practice. The experimental data prove that complexes XXI-XXIV display an antimicrobial activity of 16–1052 times higher towards staphylococci and streptococci than Furaciline and outruns by 16–517 times its bacteriostatic activity towards majority of gram-negative microorganisms. At the same time the mentioned compounds are of 4–9 times more active towards gram-positive bacteria and not less of 130 times – towards gram-negative microorganisms than their structural analogue (after MIC).

Table III.  Minimum inhibitor concentration (MIC) and minimum bactericide concentration (MBC) in μg/mL for complexes XXI-XXIV in comparison with Furaciline.

		Complex^a
Microorganism strain			XXI	XXII	XXIII	XXIV	Furaciline
Staphylococcus aureus	Wood-46	MIC	0.145	0.141	0.145	0.145	9.35
		MBC	0.58	0.58	0.145	0.58	18.7
	Smith	MIC	0.145	0.018	0.036	0.072	9.35
		MBC	0.58	0.29	0.145	0.145	9.35
	209-P	MIC	0.29	0.29	0.072	0.29	18.7
		MBC	0.58	1.16	0.145	0.58	37.5
Staphylococcus saprophiticus		MIC	0.29	0.29	0.072	0.145	9.35
		MBC	0.29	0.58	0.29	0.58	18.7
Streptococcus faecalis		MIC	0.036	0.018	0.58	1.16	37.5
		MBC	0.145	0.072	0.58	4.67	75
Escherichia coli (O-111)		MIC	1.16	4.67	9.35	2.33	18.7
		MBC	37.5	4.67	9.35	0.58	37.5
Salmonella typhimurium		MIC	0.145	0.145	0.145	0.58	75
		MBC	18.7	9.35	4.67	2000	150
Salmonella enteritidis		MIC	0.145	0.145	0.145	1.16	9.35
		MBC	37.5	75	9.35	2000	9.35
Klebsiella pneumoniae		MIC	0.29	0.145	0.145	0.29	>300
		MBC	2.33	0.29	0.29	300	>300
Pseudomonas aeruginosa		MIC	2000	300	300	1000	>300
		MBC	2000	2000	2000	>4000	>300
Proteus vulgaris		MIC	2.33	4.67	75	2.33	150
		MBC	300	75	>300	1000	300
Proteus mirabilis		MIC	0.145	0.145	0.29	1.16	150
		MBC	300	75	300	2000	300

^aThe molecular formula of complexes are reported in Table I.

Antifungal activity

The experimental data obtained from the study of antimycotic properties of selected compounds XXI-XXIV are given in Table IV. They also display selective activity towards investigated fungi stems in the concentration range of 18.7–300 μg/mL. In order to make a comparison, data regarding the activity of Nistatine, an antifungal agent used in medicine for mycose treatment, are also given in Table IV. The data show that the synthesized complexes display an antimycotic activity of 6.4–1.1 times higher towards majority fungi than Nistatine. The properties found for synthesized and studied coordinative compounds are of interest from the view point of the growth of the arsenal of antimicrobial and antimycotic remedies.

Table IV.  Antimicotic activity (MIC / MBC) in μg/mL for complexes XXI-XXIV in comparison with Nistatine.

	Fungi type
Complex^a	Aspergillus niger	Aspergillus fumigatus	Candida albicans	Penicillium
Nistatine	240/240	240/240	80/80	80/80
XXI	300/300	37.5/37.5	18.7/18.7	150/150
XXII	300/300	37.5/37.5	37.5/37.5	37.5/37.5
XXIII	300/300	37.5/37.5	75/75	37.5/37.5
XXIV	150/150	37.5/37.5	300/300	18.7/18.7

^aThe molecular formula of complexes are reported in Table I.

Conclusion

Metal complexes have been efficiently elaborated by reacting a series of 3d metal with different organic ligands. They have various geometrical and electronic structure, thermodynamic and thermal stabilities, magnetic and conductance properties. All complexes are octahedral except those of Cu which are of square planar or pyramidal geometry. Some copper, cobalt and manganese compounds have dimeric or polymeric structure. From our investigations we have deducted that there are three most indicative criteria for future synthesis of biological active coordination compounds from the view point of the inhibitors of HL-60 cells proliferation:

• Use of copper (II) planar or pyramidal complexes;

• Presence of sulphur donor atom in the ligand composition;

• Use of ONS - tridentate ligands.

Our future investigations will be directed on the synthesis of copper(II) complexes with tridentate ONS containing ligands.

Acknowledgements

The authors wish to thank the CHUL Research Center at Québec City (Canada) and State University of Medicine and Pharmacy of Chisinau (Moldova) for their help in carrying out biological studies. We also thank the AUF (grant funding project 6301-PS-323) and FQRNT (grant funding project 115825) for financial support.