USES OF -DIKETONES FOR THE SYNTHESIS OF NOVEL HETEROCYCLIC COMPOUNDS AND THEIR ANTITUMOR EVALUATIONS

The reaction of the 3-oxo-N,3-diphenylpropan-amide (3) with either malononitrile or ethyl cyanoacetate in ammonium acetate gave the 1,2-dihydropyridine derivatives 6a or 6b, respectively. On the other hand, carrying the same reaction in the presence of triethylamine gave the 1,6-dihydropyridine derivatives 7a and 7b, respectively. Moreover, compound 3 reacted with 2-aminoprop-1-ene-1,1,3-tricarbonitrile to give the pyridine derivative 9. Compound 7b reacted with the active methylene derivatives 10a,b and 4a,b to give the naphthyridine derivatives 11a,b and 12a,b; respectively. Compound 3 was also used for the synthesis of thiophene derivatives 13a,b and 16a,b. In addition, the reaction of ethyl benzoylacetate (1) with o-phenylene diamine gave the benzimidazole derivative 18. The reactivity of the latter product towards different reagents was studied to give different products. The cytotoxicity of the newly synthesized products was studied towards some cancer and normal cell lines, in addition toxicity of compounds was measured and docking of the most active compounds was done. Compounds 6b, 7b, 9, 13a, 13b, 16a, 20b, 20c, 24b, 25 and 26b exhibited optimal cytotoxic effect against cancer tested cell lines. These active compounds were evaluated against c-Met kinase using foretinib as the reference drug where all compounds expressed higher activity than the reference drug.


INTRODUCTION
It is of great interest to note that pyridine and pyrimidine derivatives were one of the most classes of compounds due to their high pharmaceutical and biological values. Especially fused pyridine derivatives have varieties of biological uses. Among such activities, they are known to exhibit high pharmacological, CNS depressant [1,2], neuroleptic [3] and tuberculostatic [4] activities. In addition, a largenumber of pyridines were used as antimicrobial agents [5], inhibitors of glycogen syntheses kinase-3 (GSK-3) [6] and potent antitumor agents [7]. Moreover, benzimidazoles represent a group of compounds with a large number of pharmaceutical applications and many of them were basic nucleus of drugs structures. Some of them were naturally occurring nucleotides that capable to interact with biopolymers to enhance better biological properties. It was noticed that some 2-aminobenzimidazoles showed interesting antimicrobial effect, especially, their corresponding carbamate derivatives were obtained in good yields and exhibited significant in vivo antifilarial activity [8]. Within the field of drug designing such group of compounds have a great affinity towards different enzymes and protein receptors [9]. Optimization of benzimidazole-based structures has resulted in marketed drugs, e.g. within the field of chemotherapy it was found that omeprazole [10] and pimobendan [11] they were found not only as good therapeutically active drugs but also for treatment of heart feeler problems. Moreover, a large number of benzimidazoles derivatives are well known for their antimicrobial [12][13][14][15], anthelmintic [16], antiviral [17][18] and antifungal [19][20][21] activities. Many benzimidazole containing compounds are known as anticancer agents [22][23][24][25][26][27][28]. Within the field of topoisomerase inhibitors it was found that some benzimidazole was used as topoisomerase inhibitory drugs, e.g. Hoechst 33258 and Hoechst 33342 ( Figure 1) [29,30]. Within the field of DNA binders, it was also found that bis-benzimidazole derivatiives are used as head to head binders [31]. Within the field of cancer chemotherapy, many drugs containing benzimidazole nucleus are known through the market like RAF265 (CHIR-265; Novartis Pharmaceuticals, Basel, Switzerland) and AZD6244 (ARRY-142886; AstraZeneca, London, England). A very active known drug is RAF265 resulted in a reduction in tumor cell growth and in tumor cell apoptosis [32]. Due to such high importance of pyridine and benzimidazole derivatives was focused on the efficient synthesis of new pyridine and benzimidazole derivatives starting from ethyl benzoylacetate followed by their cytotoxic evaluations against human cancer and normal cell lines. 13 C NMR and 1 H NMR spectra were recorded on Bruker DPX200 instrument in CDCl 3 and DMSO with TMS as internal standard for protons and solvent signals as internal standard for carbon spectra. Chemical shift values are mentioned in δ (ppm). Mass spectra were recorded on EIMS (Shimadzu) and ESI-esquire 3000 Bruker Daltonics instrument. Elemental analyses were carried out by the Microanalytical Data Unit at Ludwig-Maximilians-Universität-München, Germany. The progress of all reactions was monitored by TLC on 2 x 5 cm pre-coated silica gel 60 F254 plates of thickness of 0.25 mm (Merck).

EXPERIMENTAL
General procedure for the synthesis of the 1,2-dihydropyridine derivatives 6a,b Either of malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol) together with ammonium acetate (0.50 g) were added to a dry solid of compound 3 (2.16 g, 0.01 mol). The reaction mixture, in each case, was heated in an oil bath at 120 o C for 15 min. The solid product formed after boiling with ethanol was collected by filtration. General procedure for the synthesis of the 1,6-dihydopyridine-2-carboxylate 7a,b

General procedure for the synthesis of the thiophene derivatives 13a,b
Each of elemental sulfur (0.32 g, 0.01 mol) and either malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol) was added to a solution of compound 3 (2.16 g, 0.01 mol) in 1,4-dioxane (40 mL) containing triethylamine (0.50 mL). The reaction mixture, in each case, 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 4-(phenylcarbamoyl)thiophene derivatives 16a,b
To a solution of compound 2 (2.39 g, 0.01 mol) in dimethylformamide (30 mL) containing potassium hydroxide (0.56 g, 0.01 mol) phenylisothiocyanate was added. The reaction mixture was stirred at room temperature for 24 h then to the reaction mixture either ethyl chloroacetate (1.22 g, 0.01 mol) or (0.92 g, 0.01 mol) was added. The reaction mixture was stirred at room temperature for an additional 24 h then poured onto ice/water containing few drops of hydrochloric acid (till pH 6) and the formed solid product was collected by filtration.

General procedure for the synthesis of the benzo[d]imidazol derivatives 20a-c
Either benzaldehyde (1.06 g, 0.01 mol) 4-methoxybanzaldehyde (1.36 g, 0.01 mol) or 4chlorobenzaldehyde (1.40 g, 0.01 mol) was added to a solution of compound 18 (2.36 g, 0.01 mol) in 1,4-dioxane (40 mL) containing piperidine (0.50 mL). The reaction mixture, in each case, was heated under reflux for 3 h then poured onto ice/water containing few drops of hydrochloric acid. The formed oily product was triturated with ethanol and the precipitated product was collected by filtration.

Chemicals
Chemical used through the anticancer evaluations of the synthesised compounds were obtained from Sigma Chemical Co. (Saint Louis, USA).

Cell cultures
The cancer cell lines namely the human gastric cancer (NUGC and HR), human colon cancer (DLD1), human liver cancer (HA22T and HEPG2), human breast cancer (MCF), nasopharyngeal carcinoma (HONE1) and normal fibroblast cells (WI38) were kindly provided by the National Cancer Institute (NCI, Cairo, Egypt). The cytotoxicity of the newly synthesised compounds against the six cancer cell lines and the normal fibroblast were demonstrated through Table 1.

c-Met enzymatic activity of the most active compounds
Using homogeneous time-resolved fluorescence (HTRF) assay as previously reported the c-Met kinase activities of compounds 6b, 7b, 9, 13a, 13b, 16a, 20b, 20c, 24b, 25 and 26b were evaluated. The IC 50 's were expressed through Table 2 using foretinib as the positive control. The data revealed that the three compounds expressed high enzymatic activity toward c-Met with IC 50 's much higher than that of the reference foretinib.

RESULTS AN DISCUSSION
The present investigation emphasized mainly on two important things, of these one is to the synthesis of molecules having nitrogen and or sulfur heterocyclic and the other is to determine Scheme 1 . Synthesis of compounds 6a,b; 7a,b and 9.
their cytotoxicity against cancer and normal cell lines. The synthetic strategies adopted for the synthesis of the intermediates and target compounds are depicted in Schemes 1-4. Nitrogen containing heterocyclic organic compounds having extra keto group show interesting chemical properties as well as biological activity [33]. The reaction of ethyl benzoylacetate (1) with aniline (2) gave the 3-oxo-N,3-diphenylpropanamide (3). Compound 3 reacted with either malononitrile (4a) or ethyl cyanoacetate (4b) to give the 1,2-dihydropyridinederivatives 6a and 6b, respectively, through the acyclic intermediates 5a,b. The structure of the latter products was based on analytical and spectral data. Thus, the 1 H NMR spectrum of 6a showed the presence of a singlet at  4.15 ppm indicating the presence of the CH 2 group, a multiplet at 7.30-7.99 ppm The high yield of compound 7b encouraged us to make further work in order to produce pharmaceutically active fused pyridine derivatives. Thus, the reaction of compound 7b with either acetylacetone (10a) or ethyl acetoacetate (10b) gave the pyrido[2,3-c]pyridine derivatives 11a and 11b, respectively. Furthermore, compound 7b reacted with either malononitrile or ethyl cyanoacetate to give the pyrido[2,3-c]pyridine derivatives 12a and 12b, respectively. The analytical and spectral data of 11a,b and 12a,b were in agreement with their proposed structures. of compounds 11a,b; 12a,b and 13a The benzoylbenzanilide was studied to synthesis thiophene through the well known Gewald's thiophene synthesis [34,35]. Thus, the reaction of compound 3 with elemental sulfur and either malononitrile (4a) and ethyl cyanoacetate (4b) gave the thiophene derivatives 13a and 13b, respectively (Scheme 2). On the other hand, compound 3 reacted with phenyl isothiocyanate in basic dimethylformamide gave the intermediate potassium sulphide salt 14. The latter intermediate reacted with either ethyl chloroacetate (15a) or chloroacetone (15b) to give the thiophene derivatives16a and 16b, respectively. The structures of the latter products were established on the basis of analytical and spectral data. Thus, the 1 H NMR spectrum of 16a showed a triplet at 0.71 ppm indicating the presence of the ester CH 3 group, a quartet at 3.87 for the ester CH 2 group, a mutiplet at  7.43-7.49 equivalent to the three phenyl protons and two singlets at  8.20 and 10.39 for the two NH groups. The 13 C NMR spectrum showed the   Compound 20a reacted with hydrazine hydrate to give the pyrazole derivative 21. On the other hand, 20b reacted with malononitrile (3a) to give the 1-amino-4-benzoyl-3-(4methoxyphenyl)benzo [4,5]imidazo[1,2-a]pyridine-2-carbonitrile (22).

Structure activity relationship
From Table 1 the newly synthesized compounds were tested against the six cancer cell lines the human gastric cancer (NUGC), human colon cancer (DLD1), human liver cancer (HA22T and HEPG2), human breast cancer (MCF), nasopharyngeal carcinoma (HONE1) and a normal fibroblast cells (WI38). Compounds 6b, 7b, 9, 13a, 13b, 16a, 20b, 20c, 24b, 25 and 26b exhibited optimal cytotoxic effect against cancer cell lines, with IC 50 's in the nM range. Comparing the cytotoxicity of the 1,2-dihydropyridine derivatives 6a and 6b, it is obvious that the ethyl ester group present in 6b is responsible for its high cytotoxicity. On the other hand considering the 1,6-dihydropyridine 7a and 7b; the latter compound with the OH group showed high cytotoxicity against the NUGC, DLDI, HA22T and HONE1 cell lines. The 2-(amino(6hydroxy-2-imino-1,4-diphenyl-1,2-dihydropyridin-3-yl)methylene)-malononitrile (9) showed high cytotoxicity against NUGC, DLDI and HEPG2 cell lines. Such high potency of compound 9 is attributed to the presence of the dicyanethylidene moiety. Comparing the cytotoxicity of the 1,6-naphthridine derivatives 11a and 11b, it is obvious that the presence of the OH group in compound 11b is responsible for its high cytotoxicity against HA22T and HEPG2 cell lines. Such finding was confirmed through the comparison of the cytotoxicity of compounds 12a and 12b, where the last one showed higher cytotoxicity towards the six cancer cell lines. The significant cytotoxicity of 12b appeared against the HEPG2 cell line with 870 nM.
For the thiophene derivatives 13a,b and 16a,b, it is clear from Table 1 that such compounds showed significant cytotoxicity against the cancer cell lines. Compounds 13b and 16a with the special optimal cytotoxicity which is attributed to the presence of the oxygen rich COOEt and OCH 3 moeties in both compound, respectively. It is of great interest to compare the cytotoxicity of the un-substituted benzimidazole derivative 18 and the benzylidene derivatives 20a-c. It is obvious that compound 18 showed lower cytotoxicity relative to 20a-c. In addition going through the latter compounds, one can notice that the presence of the electronegative groups OCH 3 and Cl in 20b and 20c, respectively are responsible for the high cytotoxicity of such compounds. Moreover, the Cl group showed more potency than the OCH 3 group as cleared from the greater cytotoxicity of 20c over 20b. The annulated compound 22 showed higher cytotoxicity than the pyrazole derivative 21. The reaction of the benzimidazole derivative 18 with arylidenediazonium salts 23a and 23b gave the arylhydrazone derivatives 24a and 24b leading to a remarkable increase of cytotoxicity of 24a,b. In addition compound 24b with Cl group showed an optimal cytotoxicity against NUGC, DLDI, HA22T, HEPG2 and MCF cell lines with IC 50 's 380, 122, 33, 669 and 890 nM, respectively. It is of great value to note that compound 24b with the Cl group showed more potency than 24a. Considering the imidazo[1,2a]pyridine derivatives 25 and 26a,b compound 26b showed the highest cytotoxticity among the three compounds. Moreover, the nitrogen rich compound 25 showed higher cytotoxicity than 26a. It is of good worthy to notice that the ethyl 6-hydroxy-2-imino-1,4-diphenyl-1,2dihydropyridine-3-carboxylate (6b) and the (1-hydroxy-3-methylbenzo [4,5]imidazo[1,2a]pyridine-4-yl)(phenyl)methanone (26b) showed the maximum inhibitory effect towards the six cancer cell lines among the tested compounds.
It is very clear from our present finding that the heterocyclic systems with halogen substituted pattern OCH 3 , Cl or COOEt show greater cytotoxic property. In every case it was observed that molecules with electronegative substitutions as compounds 6b, 7b, 13a,b; 16a,b; 20b, 20c, 25, and 26b showed higher cytotoxicity because they were bearing either oxygen or chlorine substituted as well as comprised with similar structural features.

Toxicity
It is well known that bioactive compounds are often toxic to shrimp larvae. Thus, in order to monitor these chemicals' in vivo lethality to shrimp larvae (Artemia salina), results are given in Table 3 for the compounds which exhibited optimal cytotoxic effect against cancer cell lines which are the ten compounds 6b, 7b, 13a, 13b, 16a, 16b, 20b, 20c, 25 and 26b. The shrimp lethality assay is considered as a useful tool for preliminary assessment of toxicity, and it has been used for the detection of fungal toxins, plant extract toxicity, heavy metals, cyanobacteria toxins, pesticides, and cytotoxicity testing of dental materials, natural and synthetic organic compounds [36]. In order to prevent the toxicity results from possible false effects originated from solubility of compounds and DMSO's possible toxicity effect, compounds were prepared by dissolving in DMSO in the suggested DMSO volume ranges. It is clear from Table 3 that compounds 6b and 26b showed non toxicity against the tested organisms.

Molecular docking
The molecular studies were carried out using Molecular Operating Environment (MOE 2014). All the minimizations were performed with MOE until a RMSD gradient of 0.01 kcal/mol Å with MMFF94X force field and the partial charges were automatically calculated. Docking simulations were performed using the crystal structure of NUGC (PDB ID: 4ASD) which obtained from Protein Data Bank. Enzyme structure was checked for missing atoms, bonds and contacts. Water molecules was removed. Protonate 3D application of MOE was used to add the missing hydrogens and properly assign the ionization states. The ligand molecules were constructed using the builder molecule and were energy minimized. The active site was generated using the MOE-Alpha site finder. Ligands were docked within the active sites using the MOE-Dock The generated poses were energy minimized using the MMFF94x force field. Finally, the optimized poses were ranked using the GBVI/WSA DG free-energy estimates. Docking poses were visually inspected and interactions with binding pocket residues were analyzed.
Docking simulation was carried out to illustrate the binding mode and the interaction of the active compounds 6b, 7b, 13a and 16b with the amino acids in the active site of the NUGC. Docking study was performed using the crystal structure of NUGC (PDB ID: 4ASD) [20] which has co-crystallized ligand (sorafenib, BAX) as inhibitor inside its active site. In the beginning, docking study was validated by re-docking of co-crystallized ligand (BAX) inside the active site of NUGC. The re-docking of the co-crystallized ligand, BAX, was carried out to indicate the suitability of the used protocol for the planned docking study. The validation method was achieved by removing the bound ligand from the complex followed by its docking back into the binding site, which yielded root mean square deviation values RMSD of 0.88 Å with energy score (S) -9.94 kcal/mol. The top pose obtained from the MOE docking simulation showed the interactions of the co-crystallized ligand, BAX, with the key amino acids inside the active site. Docking ligand interaction and electrostatic map of compounds 6b, 7b, 13a and 16b were indicated through

CONCLUSION
The present work, through simple synthetic approaches, led to the development of novel pyridine, thiophene and imidazole derivatives that exhibited remarkable antitumor activities against six tumor cell lines. As new class of heterocyclic compounds, eleven of the obtained new compounds showed remarkable cytotoxicity against most of the six tumor cell lines. Most of the active compounds were devoid of the typical nitrogen and or sulfur feature of heterocyclic derivatives and so the activity could be attributed to some sort of electronegative substituents through the synthesized compounds. In addition c-Met kinase inhibitions for the most active compounds showed that all compounds exhibited inhibitions higher than the reference drug foretinib. The results of the biological screening will encourage future work of heterocyclic compounds derived from -diketones.