MULTI-COMPONENT REACTIONS OF CYCLOHEXAN-1,3-DIONE TO SYNTHESIZE HETEROCYCLIC DERIVATIVES WITH c-MET ENZYMATIC ACTIVITY, ANTI-PROSTATE, ANTI-PROLIFERATIVE AND TYROSINE KINASE ACTIVITIES

We are aiming in this work to synthesize target molecules not only possess anti-tumor activities but also kinase inhibitors. The target molecules were obtained starting from aryl hydrazones of cyclohexan-1,3-dione followed by its heterocyclization reactions to produce anticancer molecules. The multi-component reactions of the arylhydrazocyclohexan-1,3-dione derivatives 3a-c produced the 1,4,5,6,7,8-hexahydroquinoline derivatives 6a-r and the 4,5,6,8-tetrahydrochromeno[2,3-c]pyrazole derivatives 10a-c. Other multi-component reactions were demonstrated. The anti-proliferative activity of the synthesized compounds toward the six cancer cell lines namely A549, H460, HT-29, MKN-45, U87MG, and SMMC-7721 was studied. In addition the c-Met enzymatic activities and inhibition toward the prostate cancer cell PC-3 were measured. The results obtained in most cases, indicated that the presence of electronegative Cl group through the molecule favour the inhibitions.


INTRODUCTION
Heterocycles constitute the largest diversity of organic molecules of chemical, biomedical, and industrial significance [1,2]. They are also among the most frequently encountered scaffolds in numerous drugs and pharmaceutically relevant substances [3][4][5][6]. In the past several decades, a significant number of efforts have been made on the discovery and development of more efficient pharmaceuticals, pesticides, insecticides, rodenticides, and weed killers by following well studied natural models and biochemical pathways in living cells [7,8]. In addition, a series of libraries consisting of heterocycles have been successfully established for the structure activityrelationship studies (SAR) for drug design and synthesis [9]. Meanwhile, the diversity-oriented synthesis (DOS) continues to be an area of importance at the interface of organic synthesis and chemical biology [10][11][12]. While DOS plays an important role in searching for new bioactive small molecules with functional and stereochemical diversity [13], more efficient multicomponent domino reactions (MDRs) for the synthesis of a series of heterocycles, particularly functionalized multi-heterocycles, have been in high demand. In the past several years, the development of new multi-component domino reactions has become an active and challenging topic in modern organic chemistry [14], they can readily provide greater atom-economic access to a diverse spectrum of compounds and their libraries for screening. In addition, hydrazones and their derivatives constitute a versatile class of compounds in organic chemistry. These compounds showed biological properties, such as anti-inflammatory, analgesic, anticonvulsant, antituberculous, antitumor, anti-HIV and antimicrobial activity [15,16]. Hydrazones are important compounds for heterocyclic synthesis due to the presence of C=O, C-N and N-N bonding where the carbon atom of the hydrazone group has both electrophilic and nucleophilic [17]. Due to the large mentioned applications of multi-component reactions together with the chemical reactivity of hydrazones in this context, herein we report on the synthesis and the spectroscopic, structural, and physicochemical characterization of new heterocyclic derivatives incorporating cyclohexanone moiety starting from the arylhydrazono derivatives of cyclohexan-1,3-diones. The antiproliferative activity of the synthesized compounds toward different cancer cell lines was also explored. This was followed by studying the inhibitions of the most active compounds toward tyrosine kinases and Pim-1 kinase.

RESULTS AND DISCUSSION
As a continued work through the uses of cyclohexan-1,3-dione to produce heterocyclic compounds characterized by their high anti-proliferative activities. In the present work, we demonstrated the use cyclohexan-1,3-dione to synthesis arylhydrazone derivatives. Thus, the reaction of cyclohexan-1,3-dione (1) with either benzene diazoniumchloride (2a), 4-methylbenzene diazonium chloride (2b) or 4-chlorobenzene diazonium chloride (2c) gave the corresponding arylhydrazone derivatives 3a-c [18]. Initially 2-arylhydrazonocyclohexan-1,3dione was chosen as the model substrate for the synthesis of fused heterocyclic compounds through studying its multi-component reactions with aromatic aldehydes and cyanomethylene reagents to give biologically active fused pyridine derivatives. The multi-component reactions of either 3a, 3b or 3c with either of benzaldehyde (4a), 4-chlorobezaldehyde (4b) or 4-methoxybenzaldehyde (4c) and either malononitrile (5a) or ethyl cyanoacetate (5b) in 1,4-dioxane solution containing ammonium acetate gave the 1,4,5,6,7,8-hexahydroquinoline derivatives 6a-r (Scheme 1). The chemical structures of new compounds were assured by spectral data (IR, 1 H, 13 C-NMR, MS). Thus, the 1 H NMR spectrum of compound 6a (as an example) showed (beside the expected signals) a singlet at  4.58 (D2O exchangeable) confirming the presence of the NH2 group and a singlet at  5.13 ppm corresponding to the pyridine H-4 In addition, the 13 C NMR spectrum showed signals at  38.3, 41.6 for the two CH2 groups, a signal at  48.8 for the pyridine C-4, a signal at  117.0 corresponding to the CN group and two signals at  166.3, 167.5 equivalent to the C=N and C=O groups, respectively.
Next, we studied the multi-component reactions of the arylhydrazone derivatives 3a-c with benzaldehyde (4a) and ethyl benzoylacetate (7) in ethanol solution containing triethylamine gave the 5,6,7,8-tetrahydro-4H-chromene derivatives 8a-c. Moreover, the multi-component reactions of the arylhydrazone derivatives 3a-c with benzaldehyde (4a) and 3-methyl-1H-pyrazol-5(4H)one (9) in ethanol solution containing triethylamine gave the 4,5,6,8-tetrahydrochromeno [2,3c]pyrazole derivatives 10a-c (Scheme 2). The structures of the latter compounds were based on their respective analytical and spectral data. Thus the 1 H NMR spectrum of compound 10a showed (beside the expected signals), a singlet at  2.80 ppm for the CH3 group and a singlet at  5.13 ppm indicating the pyran H-4. Moreover, the 13 C NMR spectrum revealed the presence of a signal at  35.8 corresponding for the CH3 group, two signals at  37.4, 41.5 corresponding to the two CH2 groups, a signal at  50.7 assigning to the pyran C-4, four signals at  130.0, 130.6, 131.4, 132.7 for the pyran carbons and three signals at  164.5, 165.2, 168.9 for the two C=N and C=O groups.

Cell proliferation assay
The anti-proliferative activities of the newly synthesized compounds (Table 1) were evaluated against the six cancer cell lines A549, HT-29, MKN-45, U87MG, and SMMC-7721 and H460 using the standard MTT assay in vitro, with foretinib as the positive control [19][20][21]. The cancer cell lines were cultured in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS). Approximate 4 x 10 3 cells, suspended in MEM medium, were plated onto each well of a 96-well plate and incubated in 5% CO2 at 37 o C for 24 h. The compounds tested at the indicated final concentrations were added to the culture medium and the cell cultures were continued for 72 h. Fresh MTT was added to each well at a terminal concentration of 5 µg/mL, and incubated with cells at 37 o C for 4 h. The formazan crystals were dissolved in 100 µL of DMSO each well, and the absorbency at 492 nM (for absorbance of MTT formazan) and 630 nM (for the reference wavelength) was measured with an ELISA reader. All of the compounds were tested three times in each cell line and the results expressed as IC50 (inhibitory concentration 50%) were the averages of three determinations and calculated by using the Bacus Laboratories Incorporated Slide Scanner (Bliss) software.
The mean values of three independent experiments, expressed as IC50 values, were presented in Table 1. Most of the synthesized compounds exhibited potent anti-proliferative activity with IC50 values less than 30 µM. Generally, the variations of substituent's within the heterocyclic ring being attached have a notable influence on the anti-proliferative activity. Table 1 showed the cytotoxicity of most of the synthesized compounds toward the six cancer cell lines A549, H460, HT-29, MKN-45, U87MG, and SMMC-7721. The reaction of cyclohexan-1,3-   Table 1 that compounds 3b, 3c, 6b, 6d, 6f, 6h, 6i, 6l, 6o, 6p, 8c, 10c, 12c, 14e and 14f were the most cytotoxic among the tested compounds toward the six cancer cell line.

HTRF kinase assay
The c-Met kinase activity of all compounds was evaluated using homogeneous time-resolved fluorescence (HTRF) assay as previously reported [22,23]. In addition, the most active compounds were further evaluated against other five tyrosine kinase (c-Kit, Flt-3, VEGFR-2, EGFR, and PDGFR) using the same screening method. The experimental procedure applied for the HTRF kinase tests were as reported procedure [24].

Inhibitions of the most active compounds towards tyrosine kinases
The most active compounds 3c, 6c, 6d, 6e, 6f, 6j, 6n, 6r, 8c, 10a, 10c, 12c, 14a, 14b, 14c, 14d, 14e and 14f towards c-Met enzymatic activity were further evaluated against the five tyrosine kinases (c-Kit, Flt-3, VEGFR-2, EGFR, and PDGFR) using the same screening method (Table 3). These receptor tyrosine kinases (RTKs) have been implicated in vascular development by affecting the proliferation and migration of endothelial cells or pericytes. Among them, VEGF is a major regulator of tumor angiogenesis via endothelial cell proliferation and blood vessel permeability [25,26]. It is clear from   , compounds 6n, 10c, 12c, 14c, 14e and 14f were less effective (IC50 > 10 µM). SGI-1776 was used as the positive control with IC50 0.048 µM in the assay. These profiles in combination with cell growth inhibitions data of the selected compounds listed in Table  4 indicated that Pim-1 kinase was a potential target of these compounds.         -1,4,5,6,7,8- General procedure for the synthesis of the 1,4,5,6,7,8-hexahydroquinoline derivatives 14a-f. Each of benzaldehyde (1.06 g, 0.01 mol) and either 2-cyanoacetamide (0.84 g, 0.01 mol) or 2cyanoethanethioamide (1.00 g, 0.01 mol) were added to a solution of either 3a (2.16 g, 0.01 mol), 3b (2.30 g, 0.01 mol) or 3c (2.50 g, 0.01 mol) in 1,4-dioxane (50 mL) containing triethylamine (2.00 mL). The whole reaction mixture was heated under reflux for 2 h then poured onto ice/water mixture containing a few drops of hydrochloric acid and the formed solid product was collected by filtration. compounds were the most common potent compounds toward the cancer cell lines, c-Met kinase, PC-3 cell line, tyrosine kinases. Whereas, eight compounds were the most common potent compounds toward Pim-1 kinase. The results obtained in this work encourage further work in the future since many compounds were considered as promising anticancer agents.