SUBSTITUTED 1,3-PHENYL(PYRIDYL) PROPENONES AND DERIVATIVES WITH THIOSEMICARBAZIDIC GROUPS. STRUCTRURE – (HL-60) ANTILEUKEMIA ACTIVITY RELATIONSHIP

3-(4-(Dimethylamino)phenyl-1-(4-isothiocyanatophenyl)prop-2-en-1-one was obtained from the corresponding N,N-dimethylthyoureas by elimination of dimethylamine at heating with gaseous hydrogen chloride in chloroform and 1-(4-isothiocyanatophenyl)-3-(pyridin-2-il)prop-2-en-1-one by treating 1,1-dimethyl-3-(4-(3(pyridin-2-il)-acryloyl)-phenyl)thyourea with acetic anhydride. The difference in the reactivity of the groups >C=O and NCS in the synthesis with hydrazine hydrate and its derivatives allows the synthesis of some 1,3-disubstituted propenones with thiosemicarbazide groups (4and 1,4-disubstituted) in good yields. From 4-substituted thiosemicarbazides and 2-formilpyridine thiosemicarbazones were obtained. In the case of some derivatives, the propenone group in the reaction with hydrazine hydrate allows the formation of pyrazole derivatives. All obtained compounds were investigated for antileukemia activity. It was found that this activity is more pronounced for thiosemicarbazide derivatives with two pyridine nuclei at concentrations 10-10 mol/L.

Thiosemicarbazides 4-and 1,4-disubstituited can be obtained at the addition of hydrazine and its derivatives to isothiocyanates [5,6], or directly from N-aril-N,N-dimethylthiourea [7].The purpose of this study was to obtain biologically active compounds.
In the paper [8], the anticancer activity (for 5 types of cancer) was investigated for a class of chalcones with different substituents (H, CH 3 , OH, OCH 3 , N(CH 3 ) 2 , Cl) for both aromatic nuclei (A and B).In some cases the chalcones with OCH 3 and N(CH 3 ) 2 groups show higher anticancer activity.The thiosemicarbazones of some chalcones show antitumor activity for HepG2 cell line [9].The authors [10] investigated the anticancer activity for some thiosemicarbazones with the structures depicted in Figure 1.It was demonstrated that the anticancer activity depends on the nature and the position of the substituents in the structure.The 1,3-aril(heteryl-2-prop-1-ones chalcones at heating in a basic medium allow the formation of 1-thiocarbamoil-3-phenyl-5-heteroaril-2-pyrazoline with anticonvulsant and antidepressant properties [11]. The (1,3-aril(heteryl)propen-2-one) chalcones with thiosemicarbazidic groups (4-and 1,4-disubstituted) and respectively thiosemicarbazones are lacking in the literature and they became our object of study.

Chemistry
Introduction of groups 4-and 1,4-thiosemicarbazide in chalcones structure was performed by treating isothiocianato-1,3-prop-2-one 1a, b with hydrazine or with their derivatives 2a-d following the Scheme 1. Taking into account the high activity of NCS group in the reaction with nucleophile agents, the synthesis was realized at room temperature with a molar ratio of reagents of 1:1, to exclude the participation of the carbonyl group at condensation with hydrazine hydrate and its derivatives.Benzene was used as solvent, from which the hydrazinecarbothioamides 3a-c and 4a,b crystallized during the synthesis.Hydrazinecarbothioamides 3a and 4a which condense at heating were fi ltrated and washed fi rst with benzene and after with water.They were used without further recrystallization.The other compounds can be recrystallized from corresponding solvents in a yield of 60-92%.
4,5-Dihydro-1H-pyrazol-3-yl)phenyl)hydrazinecarbothioamides 7a and 8a were obtained through the sequence of reactions that is indicated in Scheme 3. First the N,N-dimethylthioureas 1,2 with hydrazine at room temperature transforms in hydrazones in pyridine, which without being isolated at heating it will cyclise in pyrazole derivatives [7].In parallel, dimethylamine is substituted by hydrazine [11] with the formation of compounds 7a and 8a in good yields.Compound 7a was also obtained by an alternative method, from isothiocyanatophenylprop-2-en-1-one 1a, pyridine and hydrazine at room temperature, after that the mixture was heated.It could be possible that the same intermediate is formed, which is transformed into the fi nal product 7a.
Compounds 9a and 10a (Figure 2) were obtained from 7a and 8a at heating in ethanol with 2-formylpyridine.

Biological Activity Antiproliferative Activity of Human Leukemia HL-60 Cells
For all obtained compounds the antileukemia activity was investigated.It is more pronounced for thiosemicarbazide derivatives with two pyridine nuclei at concentrations 10 -5 -10 -7 mol/L (see Table 1).
The aryl isothiocyanates with different substituents in their structure are inactive against leukemia [12].Isothiocyanatochalcone 1a and 1-(4-isothiocyanatophenyl)-3-(pyridine-2-yl)prop-2-en-1-one 1b are also inactive.Similarly, the N-phenylhydrazinecarbothioamide 11a is inactive.It was found that the modifi cation of the -NCS fragment in compounds 1a and 1b by the addition of the hydrazine and its derivatives, leads to anticancer activity for all the chalcones 3a-d.Chalcone 3c, which contain residues of pyridine in the fi rst position of the tiosemicarbazidic fragment, shows an inhibition of the antileukemia of 92% at concentration of 10 -5 mol/L.It can be concluded that the introduction of the fragment (CH 3 ) 2 N-C 6 H 4 -CH=CH-CO-or Py-CH=CH-CO-in the molecule of N-phenylhydrazinecarbothioamide 11a plays an important role to increase the anticancer activity.This activity increases when the fragment (CH 3 ) 2 N-C 6 H 4of the chalcone 3c is replaced by rest of pyridine, propenone 4b (100%, C =10 -5 mol/L).
To highlight the role of the propenonic group on the anticancer activity on the propenones 3a, 4a, 5a and 6a the fragment -CH=CH-CO-was transformed in pyrazole heterocycle.For all obtained and studied samples 7a, 8a, 9a and 10a a sudden decrease of anticancer activity was observed (see Table 1).It was identifi ed that for compound 10a with two pyridine nuclei the inhibitor activity decrease slower when is diluted and achieves ~60% at concentration of 10 -7 mol/L.The high anticancer activity for compound 10a can be explained by the formation of the strong hydrogen bonds between the inhibitor and the nucleic acid of the cancer cells [13].

Table 1
Antiproliferative activity of compounds on human leukemia (HL-60) cells at three concentrations.

Conclusions
Ten new propenones and thiosemicarbazidic groups have been synthesized and characterized.The IR, 1 H-NMR and 13 C-NMR data were successfully used to elucidate the formation of the structure of compounds.All obtained compounds were investigated for antileukemia activity.It was found that this activity is more pronounced for thiosemicarbazide derivatives with two pyridine nuclei at concentrations 10 -5 -10 -7 mol/L.

Experimental
The structure of compounds 1a, b, 3a-c, 4a, b, 5a, 6a, 7a, 8a, 9a and 10a was confi rmed by elemental and spectral analysis ( 1 H and 13 C NMR).The 1 H and 13 C NMR spectra were recorded on a Bruker Avance III-400 spectrometer at room temperature.All chemical shifts ( 1 H, 13 C) are given in ppm versus SiMe 4 using DMSO-d 6 as solvent.Elemental analyses (C, H and N) were performed on an Elemental Analyzer Vario EL (III).Compounds 3a and 4a form insoluble in organic solvents polycondensed compounds at heating until the melting point.The melting points were determined with a Melting point meter A. KRUSS OPTRONIC Germany KSP-1N 90-26V/Al.
Cell proliferation 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 .All compounds were dissolved in ethanol to prepare the stock solution of 1 Ј 1022 M.These compounds and doxorubicin (Novapharm, Toronto, Canada) which was used as a positive control were diluted at multiple concentrations (1 and 10 μM) with culture media and added to each well and incubated for 3 days.Following each treatment, MTS (20 μL) was added to each well and the mixture was 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).