USES OF CHALCONE ACETOPHENONE TO SYNTHESIS HETEROCYCLIC COMPOUNDS WITH CYTOTOXIC AND C-MET KINASE ACTIVITIES

The aim of present study was the uses of a series of α,β-unsaturated carbonyl compounds (chalcones), in the synthesis of pyridine, pyran, thiophene, thiazole, together with their uses in heterocyclic synthesis. The work has resulted in the synthesis of a variety of 2,5-dihydropyridine, hydrazide-hydrazone, thiophene derivatives, coumarin, pyran and thiazolo[4,5-d]thiazole derivatives. The antitumor activities of the newly synthesized products were carried out against three cancer cell lines namely MCF-7, NCI-H460 and SF-268 and normal human cell line WI38. In addition, the inhibitions of most of the synthesized compounds against c-Met kinase were studied and results showed that many compounds were of high inhibitions, and these are considered as promising anticancer agents. The results obtained encouraged further work in the future.


INTRODUCTION
Chalcones constitute an important group of natural products and their pharmacological values received much interest in recent years. Chemically, they consist of an open chain flavanoids in which the two aromatic rings are joined by a three carbon α,β-unsaturated carbonyl system. The presence of a reactive α,β-unsaturated keto function in chalcones is found to be responsible for their antimicrobial activity [1]. In recent years a variety of chalcones have been reviewed for their cytotoxic, anticancer, chemopreventive, mutagenic as well as antiviral, insecticidal and enzyme inhibitory properties [2,3].
A number of chalcones having hydroxy, alkoxy groups in different position have been reported to possess anti-bacterial [4], antiulcer [5], antifungal [6], antioxidant [7], vasodilatory [8], antimitotic [9], antimalarial [10], antileishmanial [11] and inhibition of chemical mediators release. In addition of their inhibition of leukotriene B4 [12], inhibition of tyrosine kinase [13,14] and inhibition of aldose reductase [15] activities. Appreciation of these findings motivated us to synthesize chalcones as a potential template for anticancer agents as a continuation for our previous work [16][17][18]. It must be noted that this scaffold provides substitution pattern on benzylideneacetophenones nucleus. In this work, we present the synthesis of a series of α,βunsaturated carbonyl compounds (chalcones), and report the cytotoxic evaluations of the newly synthesized together with the c-Met kinase inhibitions.

RESULTS AND DISCUSSION
In the present work, we demonstrate the uses of some chalcones of acetophenone for different heterocyclization reactions to produce compounds that showed cytotoxic and c-Met kinase activities. Thus, chalcones 3a,b (Scheme 1) were synthesized via Claisen-Schmidt condensation reaction of either acetophenone (1a) with benzaldehyde (2a) or 4-chloroacetophenone (1b) with 4-methoxybenzaldehyde (2b), in aqueous NaOH (0.05 M) and ethanol, at room temperature (r.t.). After completion of the reaction, the mixture was filtered to collect the precipitates and purification by re-crystallization affords the pure chalcones 3a and 3b, respectively. Compounds 3a and 3b were the key starting compounds for different heterocyclic derivatives. Thus, the reaction of either compound 3a or 3b with 2-aminoprop-1-ene-1,1, 3-tricarbonitrile (4) in the presence of a catalytic amount of ammonium acetate gave the 2,5-dihydropyridine derivatives 6a and 6b, respectively. Formation of 6a,b took place through the intermediate formation of the acyclic intermediates 5a,b followed by ring closure. The structures of compounds 6a,b were established on the basis of analytical and spectral data. Thus, the 1 H NMR spectrum of 6b showed the presence of a singlet at  3.09 ppm corresponding to the OCH3 group, a singlet at  6.87 ppm Scheme 3. Synthesis of compounds 15a,b; 16a,b; 17a,b and 19a,b.
The reaction of either compound 13a or 13b with acetophenone (14) in the presence of ammonium acetate in an oil bath at 120 o C gave the condensation products 15a and 15b, respectively. Moreover, the reaction of either compound 15a or 15b with elemental sulfur, as a method of Gewald's thiophene synthesis [22][23][24], in 1,4-dioxane containing triethylamine gave the thiophene derivatives 16a and 16b, respectively. The analytical and spectral data were in agreement with their respective structures. The same products were obtained through the reaction of either of compound 13a or 13b with acetophenone and elemental sulfur in 1,4-dioxane containing triethylamine (m.p., mixed m.p. and fingerprint IR). The reaction of either of 13a or 13b with benzaldehyde (2a) gave the benzylidene derivatives 17a and 17b, respectively. On the other hand, the reaction of either of compound 13a or 13b with salicylaldehyde (18) gave the coumarin derivatives 19a and 19b, respectively (Scheme 3).
The reaction of either compound 13a or 13b with malononitrile (20) and elemental sulfur gave the thiophene derivatives 21a and 21b, respectively. On the other hand, the reaction of either 13a or 13b with malononitrile in 1,4-dioxane containing a catalytic amount of triethylamine gave the pyridine-6-one derivatives 23a and 23b, respectively. The reaction took place through the intermediate formation of 22a and 22b followed by ring closure (Scheme 4).
Next, we moved towards studying the reactivity of compounds 13a,b via the multi-component reactions through their reactions with malononitrile and aromatic aldehydes to afford biologically active polyfunctionally substituted pyran derivatives. Thus, the reaction of either 13a or 13b with malononitrile (20) and either benzaldehyde (2a), 4-methoxybenzaldehyde (2b) or 4-chlorobenzaldehyde (24) in ethanol containing triethylamine gave the pyran derivatives 25a-f, respectively. The analytical and spectral data of the latter products were consistent with their respective structures. Thus, the 1 H NMR spectrum of 25a (as an example) showed a singlet at  4.72 ppm (D2O exchangeable) equivalent to the NH2 group, two singlets at  6.11, 6.24 ppm confirming the CH=CH moiety, a singlet  6.93 confirming the pyran H-4, a multiplet at  7.27-7.37 equivalent to the three C6H5 groups and a singlet at  8.25 (D2O exchangeable) for the NH group. In addition, the 13 C NMR spectrum showed a signal at  88.3 for the pyran C-4, two signals at  90.4, 92.4 for the CH=CH moiety, signals at  119. 3, 119.5, 120.6, 120.9, 121.3, 121.5, 121.8, 122.0, 122.3, 123.4, 123.6, 125.8 equivalent to the three C6H5 groups, four signals at  128. 6, 129.2, 130.8, 131.7 for the pyran C-2, C-3, C-5, C-6 and a signal at  168.8 for the C=N group. The reaction of either compound 13a or 13b with thioglycollic acid (26) gave the thiazol-4-one derivatives 27a and 27b, respectively (Scheme 5).
Our trials for the reaction of either compound 13a or 13b with elemental sulfur and phenylisothiocyanate to form the thiazol-5-hydrazidohydrazone derivatives 32a and 32b in a similar manner like the reaction of compounds 27a and 27b were unsuccessful; for that reason we tried to synthesis them through another reaction root. Thus, the reaction of cyanoacetylhydrazine (12) with elemental sulfur and phenylisothiocyanate gave the thiazole-2-thione derivative 33 which intern reacted with either compound 13a or 13b to give the thiazole-2-hydrazidohydrazone derivatives 32a and 32b, respectively (Scheme 7). The structures of the latter products were confirmed on the basis of their respective analytical and spectral data (see experimental section). The presence of the α-carbonylmethylene moiety in compound 27b showed interesting reactivity towards the Gewald's thiophene synthesis. Thus, the reaction of compound 27b with elemental sulfur and either malononitrile (20) or ethyl cyanoacetate (34) gave the thieno [2,3-b] thiazole derivatives 35a and 35b, respectively. The structures of the latter compounds were elucidated on the basis of their respective analytical and spectral data (see experimental section) Cell cultures. Three human tumor cell lines, MCF-7 (breast adenocarcinoma), NCI-H460 (nonsmall cell lung cancer), and SF-268 (CNS cancer) were used. MCF-7 was obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK), NCI-H460, SF-268 and normal fibroblast cells (WI 38) were kindly provided by the National Cancer Institute (NCI, Cairo, Egypt). They grow as monolayer and routinely maintained in RPMI-1640 medium supplemented with 5% heat inactivated FBS, 2 mM glutamine and antibiotics (penicillin 100 U/mL, streptomycin 100 µg/mL), at 37 o C in a humidified atmosphere containing 5% CO2. Exponentially growing cells were obtained by plating 1.5 x 105 cells/mL for MCF-7 and SF-268 and 0.75 x 104 cells/mL for NCI-H460, followed by 24 h of incubation. The effect of the vehicle solvent (DMSO) on the growth of these cell lines was evaluated in all the experiments by exposing untreated control cells to the maximum concentration (0.5 %) of DMSO used in each assay.

Tumor cell growth assay
The effects of the synthesized compounds on the in vitro growth of human tumor cell lines were evaluated according to the procedure adopted by the National Cancer Institute (NCI, USA) in the 'In vitro Anticancer Drug Discovery Screen' that uses the protein-binding dye sulforhodamine B to assess cell growth. Briefly, exponentially, cells growing in 96-well plates were then exposed for 48 h to five serial concentrations of each compound, starting from a maximum concentration of 150 µM. Following this exposure period adherent cells were fixed, washed, and stained. The bound stain was solubilized, and the absorbance was measured at 492 nm in a plate reader (Bio-Tek Instruments Inc., Power wave XS, Wincoski, USA). For each test compound and cell line, a dose-response curve was obtained and the inhibition of 50% (IC50), corresponding to the concentration of the compounds that inhibited 50% of the net cell growth. Doxorubicin was used as a positive control and tested in the same manner.
Results are given in cconcentrations that were able to cause 50% of cell growth inhibition (IC50) after a continuous exposure of 48 h and show means ± SEM of three-independent experiments performed in duplicate.
The effect of the newly synthesized compounds on the in vitro growth of the three human tumor cell lines representing different tumor namely (MCF-7), (NCI-H460), (SF-268) and normal cell line (WI 38) after continuous exposure for 48h was demonstrated through Table 1.

Structure activity relationship
Compounds 19a and 29c showed the highest inhibitory effect against the three human tumor cell lines. Compounds 6a, 11b, 16b, 25e, 25b, 25c, 25f, 35a and 35b showed high inhibitory effect against the three human cancer cell lines. Compound 11b showed high inhibitory effect against (MCF-7), (NCI-H460) and compounds 23a, 25e and 35a moderate inhibitor effect. Compounds 11a and 29a showed high inhibitory effect against (SF-268). Compounds 6b, 8b, 11a, 13a, 13b, 15a, 15b, 16a, 17a, 17b, 19b, 21a, 21b, 23b, 25a, 25d, 27a, 27b, 28a, 28b, 28c, 29a, 29b, 31b, 32a and 33 showed lowest inhibitory effect against the three human tumor cell lines. The highest inhibitory effect of compound 29c against the three human tumor cell lines was attributed to the presence of pyridine heterocyclic ring, thiazole ring, 4-methoxy and chlorine groups. Considering coumarin derivative 19a showed that highest inhibitory effect against all three human tumor cell lines. High inhibitory effect of compound 6a against all three human tumor cell lines was attributed to the presence of pyridine heterocyclic ring. Also compounds 25b, 25c, 25e and 25f have high inhibitory effects against the three human tumor cell lines this was attributed to the presence of pyran heterocyclic ring, 4-methoxy and chlorine groups. Compounds 35a and 35b showed high inhibitory effects against the three human tumor cell lines and this was attributed to the presence of thiazole ring, 4-methoxy and chlorine groups. Compound 11b showed high inhibitory effect against (MCF-7), (NCI-H460) due to the presence of pyridine heterocyclic ring, OH and chlorine groups, respectively. Compound 28b showed high inhibition against  and this was attributed to the presence of coumarin, thiazole ring and the hydrazide-hydrazone moiety. Table 2. c-Met enzymatic activity of the newly synthesized compounds.
Compound Number

c-Met kinase inhibition
Most of the newly synthesized compounds were evaluated for their inhibitions toward c-Met enzyme using a homogeneous time-resolved fluorescence (HTRF) assay. Taking foretinib as the positive control, the results expressed as IC50 were summarized in Table 2. The IC50 values are the average of at least three independent experiments. As illustrated in Table 2, all the tested compounds displayed potent c-Met enzymatic activity with IC50 values ranging from 0.14 to 10.24 nM. Compared with foretinib (IC50 = 1.16 nM), fifteen of them 6b, 8b, 11b, 13b, 15b, 16b, 17b, 21b, 23b, 25b, 25f, 27b, 28c, 29c and 35b exhibited higher potency than the reference foretinib (IC50 = 1.16 nM). In addition, compounds 6a, 11a, 13a, 21a, 23a, 25a, 25e, 27a, 28b, 31b and 32a exhibited low inhibitions toward c-Met kinase. To a dry solid of either of compound 3a (2.08 g, 0.01 mol) or 3b (2.72 g, 0.01 mol) 2-aminoprop-1-ene-1,1,3-tricarbonitrile (4) (1.32 g, 0.01 mol) and ammonium acetate (0.50 g) was added. The reaction mixture was heated in an oil bath at 120 o C for 30 min then left to cool. The remaining product was boiled in ethanol (40 mL) and formed solid product was collected by filtration.  General procedure for the synthesis of the tetrahydropyridine 8a,b
2- (4, General procedure for the synthesis of the pyridine derivatives 11a,b To a dry solid of either of compound 3a (2.08 g, 0.01 mol) or 3b (2.72 g, 0.01 mol) acetoacetanilide (9) (1.77 g, 0.01 mol) and ammonium acetate (0.50 g) was added. The reaction mixture was heated in an oil bath at 120 o C for 45 min then left to cool. The remaining product was boiled in ethanol (50 mL) and formed solid product was collected by filtration.

General procedure for the synthesis of the hydrazide-hydrazone derivatives 13a,b
To a solution of either of compound 3a (2.08 g, 0.01 mol) or 3b (2.72 g, 0.01 mol) in 1,4-dioxane (50 mL) cyanoacetylhydrazine (1.0 g, 0.01 mol) was added. The reaction mixture, in each case, was heated under reflux for 3 h and the formed solid product, upon cooling, was collected by filtration. (1,3-diphenylallylidene)

General procedure for the synthesis of the hydrazide-hydrazone derivatives 15a,b
To a dry solid of either of compound 13a (2.89 g, 0.01 mol) or 13b (3.53 g, 0.01 mol) acetophenone (14) (1.20 g, 0.01 mol) and ammonium acetate (0.50 g) was added. The reaction mixture was heated in an oil bath at 120 o C for 1 h then left to cool. The remaining product was boiled in ethanol (40 mL) and formed solid product was collected by filtration.

General procedure for the synthesis of the thiophene derivatives 16a,b
Method (A). To a solution of either compound 15a (3.91 g, 0.01 mol) or 15b (4.55 g, 0.01 mol) in 1,4-dioxane (50 mL) containing triethylamine (1.0 mL) elemental sulfur (0.32 g, 0.01 mol) was added. The reaction mixture was heated under reflux for 2 h then poured onto ice/water containing few drops of hydrochloric acid and the formed solid product was collected by filtration.

General procedure for the synthesis of the coumarin derivatives 19a,b
To a solution of compound 13a (2.89 g, 0.01 mol), or 13b (3.35 g, 0.01 mol) in 1,4-dioxane (40 mL) containing piperidine (0.50 mL) salicylaldehyde (1.22 g, 0.01 mol) was added. The reaction mixture was heated under reflux for 2 h then poured onto ice/water containing few drops of hydrochloric acid and the formed solid product was collected by filtration.

General procedure for the synthesis of the thiophene derivatives 21a,b
To a solution of compound 13a (2.89 g, 0.01 mol), or 13b (3.35 g, 0.01 mol) in 1,4-dioxane (40 mL) containing triethylamine (0.50 mL) both of elemental sulfur (0.32 g, 0.01 mol) and malononitrile (0.66 g, 0.01 mol) were added. The reaction mixture was heated under reflux for 2 h then poured onto ice/water containing few drops of hydrochloric acid and the formed solid product was collected by filtration.

General procedure for the synthesis of the pyridine derivatives 23a,b
To a solution of compound 13a (2.89 g, 0.01 mol), or 13b (3.35 g, 0.01 mol) in 1,4-dioxane (40 mL) containing triethylamine (0.50 mL) malononitrile (0.66 g, 0.01 mol) was added. The reaction mixture was heated under reflux for 4 h then poured onto ice/water containing few drops of hydrochloric acid and the formed solid product was collected by filtration.

General procedure for the synthesis of the pyran derivatives 28a-c
To a solution of compound 27b (4.13 g, 0.01 mol) in 1,4-dioxane (40 mL) containing triethylamine (0.50 mL) any of benzaldehyde (1.08 g, 0.01 mol), 4-methoxybenzaldehyde (1.36 g, 0.01 mol) or 4-chlorobenzaldehyde (1.40 g, 0.01 mol) and malononitrile (0.66 g, 0.01 mol) were added. The reaction mixture was heated under reflux for 3 h then poured onto ice/water containing few drops of hydrochloric acid and the formed solid product was collected by filtration.

General procedure for the synthesis of the pyran derivatives 29a-c
To a solution of compound 27b (4.13 g, 0.01 mol) in 1,4-dioxane (40 mL) containing ammonium acetate (0.50 g) any of benzaldehyde (1.06 g, 0.01 mol), 4-methoxybenzaldehyde (1.36 g, 0.01 mol) or 4-chlorobenzaldehyde (1.40 g, 0.01 mol) and malononitrile (0.66 g, 0.01 mol) were added. The reaction mixture was heated under reflux for 3 h then poured onto ice/water containing few drops of hydrochloric acid and the formed solid product was collected by filtration.