SYNTHESIS AND BIOLOGIC PROPERTIES OF SOME 1-( ALCHYL ) PHENYL-3-( 4-( 3-( PYRIDIN-2-IL ) ACRYLOYL ) PHENYLTHIOUREA

This paper describe the synthesis of some 1-(alchyl)aril-3-(4-(3-pyridin-2-il) acryloyl)phenylthiourea obtained by condensation of 2-pyridincarboxaldehide with some derivatives of 4-acetylphenilthioureas in basic medium or by addition of aliphatic and aromatic amines to the correspondingisothiocyanatopropenones. 12 new compounds were obtained and their biological properties were analysed. The substituted thioureas by pyridine radicals, morpholine and phenol show a maximum bacteriostatic activity for Gram positive microorganisms like: Staphylococcus Aureus and Enterococcus Faecalis at the minimum inhibitory concentration 9.37-37.5 μM. Antifungal activity for Candida Albicans, Aspergillus Niger, AspergillusFumigatus, Penicillium is weak, in minimum inhibitory concentration 600->600 μM. The leukemia activity like inhibitor (HL-60), is 84-96.9% at the concentration 10-5mol/l and 1520% and at the concentrations 10-6, 10-7mol/l.

Some derivatives, obtained from chalcones through chemical transformations are also biologically active compounds.The modifi cation of the chalcones on the carbonyl group with some hydrazine derivatives, followed by cyclisation [16], leads to 1,3,5-substituted pyrazolines with anticonvulsant and antidepressant properties.Were identifi ed bacterial species which can modify and cyclisedchalcones in biologically active fl avonoids [17].
In the literary sources mentioned above, the chalcones are obtained through the condensation of the aromatic and heterocyclic aldehides with acetyl arenes or by modifying the functional groups [18].1,3-Pyridylphenylpropenones with thiourea groups are less studies and became our object of study.
The condensation of the thioureas 2a-k with 2-pyridincaboxaldehide1 in alkaline catalysis lead to 1,3-arilpyridil-propenones3a-k with thioureas groups.Silofol thin layer chromatography showed that the reactions take place easy, with good yields, but with small quantities of secondary products which can be isolated by recristallisation from ethanol.
Alternative method of synthesis of thiourea 3b was investigated following the transformations: By heating the thiourea 3awithaceticanhydride is obtained the 1-(4-isothiocyanatophenyl)-3-(pyridin-2-il)prop-2-en-1-one4a with 53% of yield.The addition of the monoethanolamine to the isothiocyanate 4a, lead to thiourea 3b with 92% of yield.The synthesis of thioureas 3b-k in this way is less convenient because of low yield (53%) of the isothiocyanate 4a.The solvents and reagents were purifi ed in the usual manners where necessary.The structure of the compounds3a-k and 4a were confi rmed by the elemental and spectral analysis ( 13 C, 1 H-NMR).The NMR ( 13 C, 1 H-NMR) spectra were recorded on a Bruker DRX-400spectrometer at room temperature.All chemical shifts ( 1 H, 13 C) are given in ppm versus SiMe 4 using DMSO -d 6 as solvent.Elemetal analyses (C, H, N) were performed on a Elemental Analyza Vario EL (III).
The melting points were determined with a Melting point meter A. KRUSS OPTRONIC Germania KSP-1N 90-26V/Al.
For sowing were used cultures of indicated microorganisms, grown on agar during 18 hours and washed with isotonic solution of sodium chloride.Insemination dose is 500 thousand copies for 1 mL of nutrient medium.The tubes were shaken and thermostated at 37°C during 24 hours.As control were used nutrients media inoculated with the same strains but without investigated substances.Evaluation of bacteriostatic activity (CMI) was carried out visually, as lack of growths of microorganisms in the broth.Bacterial activity (CMB) was determined based on the lack of growth of microorganisms after repeated seeding on peptone agar with subsequent thermostating for 24, 48 hours.
The obtained results are presented in Table 2.

Table 2 Antibacterial activity of compounds3a-k
Nr.

Staphylococcus Aureus
Enterococcus Faecalis Escherichia Coli Proteus Vulgaris Pseudomonas Aeruginosa The investigation results show that the substance 3c posses bacteriostatic activity for Gram-positive microorganisms: Staphylococcus Aureus and Enterococcus Faecalis at concentration of 37.5 mcg/mL.The substance 3j show bacteriostatic activity for S. Aureus (minimum inhibitory concentration is 9.37 μM) which prevail furaciline activity 2 times; minimum antibacterial concentration of this substance for Staphylococcus Aureus and for the other test bacterial cultures (Table 2) investigated is more than 300 μM.

Antifungal activity
Antifungal activity of compounds 3a-k was investigated for fungi: Candida Albicans, Aspergillus Niger, Aspergillus Fumigatus, Penicillium.Initially, the substances were dissolved in dimethylformamide (concentration 10 mg/mL) and subsequent concentrations were obtained using serial dilution method in broth (broth Saburo).The inoculates were prepared from cultures of fungi.After mixing the inoculates with the solutions of investigated substances, the tubes were exposed in thermostat at 28°C during 14 days (Candida Albicans during 48 hours).Antifungal activity was determined by the absence of the fungal growth in a repeated sowing on Saburo agar with incubation during 7 days (Candida albicans during 48 hours).
The investigation results show that the substances 3a-k possess antifungal activity for Candida albicans, Aspergillus Niger, Aspergillus Fumigatus, Penicillium in minimum inhibitory concentration of 600 and…>600 μM.
Antileukemia activity (HL-60) 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 mg streptomycin/mL) and supplemented with 10% (v/v) foetal bovine serum (FBS), in a 5% CO 2 humidifi ed atmosphere at 37 o C. Cells were currently maintained twice a week by diluting the cells in RPMI 1640 medium containing 10% FBS.Cellproliferation assay.The cell proliferation assay for compounds 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 mL medium in 96-well microtiter plates (Becton Dickinson and Company, Lincoln Park, NJ, USA) were incubated at 37 o C, 5% CO 2 .Compounds were dissolved in ethanol to prepare the stock solution of 1 Ј 1022 M.These compounds and doxorubicin (Novapharm, Toronto, Canada), as a positive control, were diluted at multiple concentrations (1 and 10 μM) 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 hours.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).

Nr. Compound Inhibition of cell proliferation ( %)
Concentrates, mol/l 10 -5 10 -6 10 - If we look at the compounds 3a, 3g, 3f, 3h, 3k like derivatives of thiourea, we observe that their anticancer activity depends strongly on structure and varies from 0...96.9% for the compound concentration 10 -5 mol/L and from 0...23.8% for concentrations 10 -6 , 10 -7 mol/L.The introduction of a 2 pyridincarbonil radical in the thiourea molecule (compound 3a) increase suddenly the activity from 0 to 84%, which is also mentioned for the other inhibitors with the Chemistry Journal of Moldova. General, Industrial and Ecological Chemistry. 2013, 8 (1), 83-89 enon structure [11].Replacing methyl groups in thiourea 3a with the rest of phenol compound3g increases activity, but is reduced by ~8% for thiourea 3f, when the hydroxyl group is methylated.The compounds 3g and 3h have almost the same activity, which is maximal for thiourea 3k (96.9%), which contain in his structure two pyridine residues.A. Gulea et all. [21] explain the activity of anticancer inhibitors by the formation of hydrogen bonds with ADN cancerous cells.Indeed this type of interaction can be maximum for thiourea 3k with two pyridine nuclei.For compound 3k, when the structural fragment of 2-pyridil acryloyl is replaced by 4-dimethylaminophenyl-(thiourea6a), the activity decrease to 24%.In the case of compounds 7a and 8a with more pronounced hydrophobic character when the hydrogen bonds with the substrate are weaker, the activity is zero.
The biological research has shown: The thioureas with the rest of morpholine -3c and o-bromfenil 3jpossess bacteriostatic activity more pronounced for microorganisms Staphylococcus Aureus and Enterococcus Faecalis.
The antifungal activity of compounds -3a-k is weak.