New Approaches for the Synthesis of Heterocyclic ... New Approaches for the Synthesis of Heterocyclic Compounds Derived from Cyclohexan-1,3-dione with Anti-proliferative Activities

In the present work a series of heterocyclization reactions were adopted using cyclohexan-1,3-dione through its reaction with either furan-2-carbaldehyde or thiophene-2-carbaldehyde to give the corresponding ylidene derivatives 3a , b . The latter compounds underwent heterocyclization reactions to give thiophene and pyran derivatives 5a – d and 6a – d , respectively. Moreover, compounds 3a , b reacted with elemental sulfur and phenyl isothiocyanate to give the fused thiazole derivatives 8a , b . In addition, the reaction with either of hydrazine hydrate or phenylhydrazine has given the 4-hydrazo-no-4,5,6,7-tetrahydro-2 H -indazole derivatives 10a – d , respectively. Similarly, the reaction of either 3a or 3b with hydroxylamine hydrochloride gave the 6,7-dihydrobenzo[ c ]isoxazol-4(5 H )-one oxime derivatives 12a and 12b , respectively. Other fused heterocyclic compounds were produced and their structures were elucidated. Evaluation of the synthesized compounds against selected cancer cell lines was performed. The most active compounds were further evaluated against tyrosine kinases Pim-1


Introduction
Within the last few years the synthesis of heterocyclic compounds attracted the attention due to the wide spectrum of their high biological activities. In addition, many compounds were considered as good synthons for fused systems that were characterized by different pharmaceutical applications. 1-10 Therefore, organic chemists have been making extensive efforts to produce heterocyclic compounds by developing new and efficient synthetic transformations. Within the field of pharmaceutical chemistry, many pyrazoles, thiophenes and thiazoles were reported with a wide spectrum of biological activities that included potent analgesic, anti-convulsant, anti-inflammatory and anti-bacterial, anti-pyretic, anti-tumor, anti-parasitic, anti-microbial, anti-histaminic (H1), anti-anexiety activities in tests in mice, anti-arrhythmic and as serotonin antagonists. [11][12][13][14][15][16][17][18][19][20][21][22][23] The present work is dealing with the current application of pyrazole, thio-phene, pyrimidine and oxazine cores in the designing of anticancer agents within tumor progression. In our research it was possible to verify that such compounds are readily applicable to provide new insights and valuable inspiration in the research of new drugs and in their development as well as to contribute to the management of cancer. This encouraged our group to be attracted toward the synthesis of pyran derivatives research through the uses of b-diketones. The produced heterocyclic compounds showed high anti-proliferative activities against cancer cell lines together with high inhibitions toward tyrosine kinases. [24][25][26] Based on such importance of hete rocyclic compounds, therefore, we studied the reaction of cyclohexane-1,3-dione with some heterocyclic aldehydes and cyanomethylene reagents followed by heterocyclization of the products. Additionally, the anti-tumor evaluations of the resulting compounds towards cancer cell lines are reported. Mohareb

1. 3. General Procedure for the Synthesis of the 2H-Chromen-5-one Derivatives 6a-d
Either malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol) was added to a solution of either compound 3a (1.90 g, 0.01 mol) or 3b (2.06 g, 0.01 mol) in absolute ethanol (40 mL) containing triethylamine (1.0 mL). The reaction mixture, in each case, was heated under reflux for 3 h then the excess solvent was removed under vacuum. The remaining product was triturated with diethyl ether and the formed solid product was collected by filtration.

1. 4. General Procedure for the Synthesis of 2-Thioxohexahydrobenzo[d]thiazole Derivatives 8a,b
Each of elemental sulfur (0.32 g, 0.01 mol) and phenyl isothiocyanate (1.30 g, 0.01 mol) were added to a solution of either compound 3a (1.90 g, 0.01 mol) or 3b (2.06 g, 0.01 mol) in 1,4-dioxane (40 mL) containing triethylamine (1.0 mL). The reaction mixture was heated under reflux for 2 h then was left to cool and the formed solid product, in each case, was collected by filtration. Either hydrazine hydrate (0.1 mL, 0.02 mol) or phenylhydrazine (2.16 g, 0.02 mol) was added to a solution of either of compound 3a (1.90 g, 0.01 mol) or 3b (2.06 g, 0.01 mol) in 1,4-dioxane (40 mL). The reaction mixture was heated under reflux for 3 h then poured onto ice/water mixture containing a few drops of hydrochloric acid and the formed solid product was collected by filtration.

1. Cell Proliferation Assay
Foretinib was used as the positive control 27-29 during measuring the anti-proliferative activities of the newly synthesized compounds (Table 1). The newly synthesized compounds 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.
The IC 50 values were measured through three independent experiments and the data are shown in Table  1. It is clear that many of the tested compounds showed potent anti-proliferative activity with IC 50 values less than 6.00 µM. Generally, the variations of substituents within the aryl moiety together with the heterocyclic ring being attached had a notable effect and a positive impact on the anti-proliferative activity.

Structure Activity Relationship
It is clear from Table 1

Results and Discussion
The synthesis of the 2-(hetero-2-ylmethylene)cyclohexane-1,3-dione derivatives 3a,b has been accomplished as outlined in Scheme 1 starting from cyclohexan-1,3-dione (1). Compounds 3a and 3b were obtained through the reaction of 1 with either of furan-2-carbaldehyde or thiophene-2-carbaldehyde. The reaction of either of compound 3a or 3b with elemental sulfur and either of malononitrile (4a) or ethyl cyanoacetate (4b) gave the 6,7-dihydrobenzo[b]thiophen-5(4H)-one derivatives 5a-d, respectively. The structures of the latter products were established on the analytical and spectral data. Thus, the 1 H NMR spectrum of compound 5a (as an example) showed the presence of two triplets at δ 2.62-2.78 ppm for the two CH 2 groups, a singlet at δ 4.73 ppm (D 2 O exchangeable) indicating the presence of the NH 2 group, a singlet at δ 6.84 for the pyran H-4 and a multiplet at δ 6.82-7.86 ppm for the furan protons. In addition, the 13 C NMR spectrum revealed three signals at δ 16.6, 36.3 and 39.5 equivalent to the three CH 2 groups, two signals at δ 112.6 and 158.4 for the C=CH group, a signal at δ 116.8 for the CN group, eight signals at δ 135.4, 140.6, 141.4, 142.2, 142.7, 144.8, 145.6, 146.5 for the thiophene and furan carbons and a signal at δ 179.3 indicating the CO group. The reaction of either compound 3a or 3b with either of malononitrle (4a) or ethyl cyanoacetate (4b) in ethanol containing a catalytic amount of triethylamine gave the 2H-chromen-5-one derivatives 6a-d, respectively (Scheme 1).  of compounds 3b, 5d, 6b, 6d, 8a, 8b,  10c, 12a, 12b, 16c, 17e, 17f and 19c  took place to form the 4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)acetamide derivatives 20a-c, respectively. On the other hand, the reaction of either compounds 19a, 19b or 9c with phenyl isothiocyanate gave the corresponding N-phenylthiourea derivatives 21a-c, respectively. The analytical and spectral data of compounds 21a-c were consistent with their respective structures. Compounds 20a-c were ready for further cyclization to form biologically active annulated compounds. Thus, heating of either compound 21a, 21b or 21c in sodium ethoxide solution in a boiling water bath afforded the hexahydrobenzo [4,5] thieno [2,3-d]pyrimidine derivatives 22a-c, respectively (Scheme 4). Their structures were based on the analytical and spectral data (see experimental section).

Conclusion
Forty novel heterocyclic compounds bearing cyclohexanone moiety were designed and synthesized. Their structures were confirmed by multiple techniques. The synthesized compounds were screened for cytotoxic activity against a panel of six human cancer cell lines using MTT assay. Some intriguing structure-activity relationships were found and discussed and the most active compounds were selected for further screening against tyrosine kinases, Pim-1 kinase and the results indicated that these compounds are good condidates as anti-cancer agents that will encourange further work in the future.

Human and Animal Rights
No Animals/Humans were used for studies that are basis of this research.

Consent for Publication
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