Anticancer Activity of Some New Synthesized Tetrahydroquinoline and Tetrahydrochromene Carbonitrile Derivatives

Problem statement: In continuation to our search for new heterocyclic hemistry based anticancer, the suggestion, synthesis, structure el ucidation of some naphthalene, chromene and quinoline derivatives 3-7 were realized herein usin g 3-methylcyclohexanone 1 as a starting material. Approach: The antitumor activities of the newly synthesized compounds 4-7 were evaluated utilizing 60 different human tumor cell lines, representing l eukemia, melanoma, lung, colon, brain, ovary, breast and prostate as well as kidney. Results: Some of the tested compounds exhibited better in v itro antitumor activities at low concentration (log10 GI 50 = -4.7) against the used human tumor cell lines. Conclusion: From the obtained results, we can conclude that cy nopyridine and pyrane moieties fused to 3-methycyclohexane ring are essential for antitu mor activities. In the present work, we can suggest that the anticancer activity is due to the presence of nitrogen heterocyclic rings and the presence of the nitrile groups (CN) generally enhancing the activit y.


INTRODUCTION
The pyridine scaffold is a widespread structural motif that can be found in many natural products and in several pharmacologically interesting compounds. Therefore the synthesis of pyridine derivatives, aiming to develop new drugs, is an active research area. Therefore, cyanopyridyl derivatives are a potent inhibitor of dihydrouracil dehydrogenase and its coadministration with 1-ethoxymethyl-5-fluorouracil enhances the antitumor effect (Cocco et al., 2005). Dicyanopyridines derivatives have been described as intermediates in the synthesis of pyrido [2,3d]pyrimidines as antihistaminic agents (Quintela et al., 1997), pyridothieno-and pyridodithienotriazines endowed with antihistaminic and cytotoxic activity (Quintela et al., 1998), triazabenz[d,e]anthracene and tetrazabenz [d,e]anthracene that are DNA intercalating agents (Quintela and Peinador, 1996) and acyclo-3deazapyrimidine S-nucleosides that are active toward HIV (Attia and Ismail, 2003). In a previous work we reported that certain of our newly substituted heterocyclic compounds exhibited antitumor (Amr et al., 2006;Hammam et al., 2003;2001, Velusamy andPalaniappan, 2011;Covyeou et al., 2011), antiparkinsonian , antimicrobial Attia et al., 2000) and antiinflammatory (Abou-Ghalia et al., 2003) activities. Heterocyclic derivatives present an interesting group of compounds many of which possess widespread pharmacological properties (Borgio et al., 2011;Ngoy et al., 2011), specially as anticancer, analgesic, antipyretic and antirheumatic activities (Mohamed et al., 2010;Bryzgalov et al., 2006;Rezvani and Shariati, 2010). In addition, the pharmacological and antitumor activities of many compounds containing heterocyclic ring have been reviewed (Amr, 2000;Hammam et al., 2000, Mohamed et al., 2011. Also, the heterocyclic nitrogen derivatives exhibited a general ionophoric potency for divalent cations (Hassan et al., 2003a) and used a novel thiocyanate-selective membrane sensor (Hassan et al., 2003b). In view of these reports and in continuation of our previous works in heterocyclic chemistry; we have herein synthesized some new heterocyclic ring fused with substituted cyclohexene structure for the evaluation of their anticancer activity.

MATERIALS AND METHODS
Chemistry: All melting points were determined on open glass capillaries using an Electrothermal IA 9000 digital melting point apparatus. Elemental analyses were performed on Elementar, Vario EL, Microanalytical Unit, National Research Centre, Cairo Egypt and were found within ±0.4% of the theoretical values. Infrared (IR) spectra were recorded on Carlzeise Spectrophotometer model "UR 10" spectrophotometer using the KBr disc technique. 1 H-NMR spectra were recorded on Varian Gemini 270 MHz spectrometer (DMSO-d 6 ), 13 C-NMR spectra were recorded on Varian Gemini 67.5 MHz spectrometer and the chemical shifts are given in δ (ppm) downfield from Tetramethylsilane (TMS) as an internal standard. The mass spectra (MS) were measured using a Finnigan SSQ 7000 mass spectrometer. Follow up of the reactions and checking the purity of the compounds were made by TLC on silica gel-precoated aluminum sheets (Type 60 F 254 , Merck, Darmstadt, Germany) and the spots were detected by exposure to UV lamp at λ 254 nm for few seconds. The anticancer screening occurred in United States National Institute of Health (NIH)/National Cancer Institute (NCI).
The reaction mixture was refluxed for 6 h. and concentrated under reduced pressure, the obtained solid was filtered off, washed with water, dried and crystallized from acetic acid/water to give the title compounds 4a-c.  Synthesis of 2,6-bisarylmethelene-3-methylcyclohexanones (5a-c). To a mixture of 3-methylcyclohexanone 1 (0.01 mole) and the appropriate aromatic aldehyde, namely, p-chloro-, or 3,4-dimethoxy-or 3,4,5-trimethoxybenzaldehyde (0.02 mole) in 50 ml ethanol, KOH (0.01 mole in 5 ml H 2 O) was added. The reaction mixture was stirred at room temperature for 2 hr, the yellow solid formed was collected by filtration and crystallized from the proper solvent to give compounds 5a-c, respectively.

Method B:
A mixture of compound 6a-c (5 mmol), ammonium acetate (40 mmol) and few drops of triethylamine in absolute ethanol 100 mL was refluxed for 8 h. The solvent was concentrated under reduced pressure, the formed solid was filtered off, dried and crystallized from the proper solvent to give products identified as 7a-c, by their mp and R f -values in comparison with authentic samples previously obtained by Method A.     Anticancer activity: Some of the synthesized compounds were selected and screened for their anticancer activity. Each compound was tested at five different concentrations against 60 cell lines of nine types of human cancers, namely, leukemia, lung, colon, CNS, melanoma, ovarian, renal, prostate and breast cancer. Results are expressed as log 10 GI 50 , which the drug concentration (M) is causing a 50% reduction in the net protein increase in control cells during the drug incubation (Lomox and Naryanan, 1981) Table 1. Some of the synthesized compounds showed good anticancer activity at low concentration compared with 5fluorodeoxyuridine log 10 GI 50 = -4.7 as reference control.

RESULTS
In continuation to our search for new heterocyclic chemistry based anticancer, the suggestion, synthesis, structure elucidation of some naphthalene, chromene and quinoline derivatives 3-7 were realized herein using 3-methylcyclohexanone 1 as a starting material. Some of the synthesized compounds were selected and screened for their anticancer activity. Each compound was tested at five different concentrations against 60 cell lines of nine types of human cancers, namely, leukemia, lung, colon, CNS, melanoma, ovarian, renal, prostate and breast cancer. Some of the tested compounds were better exhibited in vitro antitumor activities at low concentration (log 10 GI 50 = -4.7) against the used human tumor cell lines. From the in vitro observed data it has been noticed that, the selected compounds 4a, 4b, 4c, 5c, 6a, 6b, 6c, 7a, 7b and 7c seem to be the most active prepared derivatives against all the tested cell lines.
Antitumor screening: Antitumor activity screening for the synthesized compounds utilizing 59 different human tumor cell lines, representing leukemia, melanoma and cancers of the lung, colon, brain, ovary, breast, prostate as well as kidney, was carried out according to the previously reported standard procedure (Xiu et al., 2010;Fylaktakidou et al., 2004;Jung et al., 2005;Ally et al., 1988). The obtained results (Table 1) represent concentrations of the used investigated compounds resulting in growth inhibition of 50% (GI 50 ) for the tested human tumor cell lines. From the in vitro observed data it has been noticed that, the selected compounds 4a, 4b, 4c, 5c, 6a, 6b, 6c, 7a, 7b and 7c seem to be the most active prepared derivatives against all the tested cell lines.

Structural-Activity Relationship (SAR):
From the above-obtained results (Table 1), we can conclude that cyanopyridine and pyrane moieties fused to 3methycyclohexane ring are essential for antitumor activities. In the present work, we can suggest that the anticancer activity is due to: • The presence of nitrogen heterocyclic rings • The most active compounds being 5c, 6a, 6b, 6c and 7a against leukemia cell lines • The presence of the nitrile groups (CN) generally enhancing the activity • The difference in activity between the compounds which is due to the indicated subsistents in the phenyl group of the molecule

CONCLUSION
In our previous works, we reported that fused pyrimidine derivatives were proved to be active anticancer agents. In the present work, a series of naphthalene, chromene and quinoline derivatives were synthesized using 3-methylcyclohexanone 1 as a starting material. The antitumor activities of the newly synthesized compounds were evaluated utilizing 60 different human tumor cell lines, representing leukemia, melanoma, lung, colon, brain, ovary, breast, prostate as well as kidney. Some of the tested compounds were better exhibited in vitro antitumor activities at low concentration (log 10 GI 50 = -4.7) against the used human tumor cell lines. From the obtained results, we can conclude that cyanopyridine and pyrane moieties fused to 3-methycyclohexane ring are essential for antitumor activities. In the present work, we can suggest that the anticancer activity is due to the presence of nitrogen heterocyclic rings and the presence of the nitrile groups (CN) generally enhancing the activity.