Synthesis and Biological Evaluation of Certain new Cyclohexane-1-carboxamides as Apoptosis Inducers

Series of 1-(N-phenyl-2-(heteroalicyclic-1-yl)acetamido)cyclohexane-1-carboxamide derivatives (5a-m) and 1-(phenyl(heteroalicyclic-1-ylmethyl)amino)cyclohexane-1-carboxamide (6a-f) were designed and synthesized with biological interest through coupling of 1-(2-chloro-N-phenylacetamido)cyclohexane-1-carboxamide (4) and (phenylamino)cycloakanecarboxamide (2) with different amines. The structures of the target compounds were elucidated via IR, 1H and 13C NMR, MS, and microanalysis. Compounds 5a-m and 6a-f were evaluated for their in vitro antitumor activity against four different cancer cell lines, MCF-7, HepG2, A549, and Caco-2. Compound 5i exhibited a promising activity against breast cancer cell line (IC50 value = 3.25 μM) compared with doxorubicin (IC50 value = 6.77 μM). Results from apoptosis and cell cycle analysis for compound 5i revealed good antitumor activity against MCF-7 cancer cell line and potent inhibition.


INTRODUCTION
Cancer is one of the most prominent diseases worldwide which represents the second cause of human mortality after cardiovascular diseases 1 .Drug resistance to cancer chemotherapy is considered a serious trouble 2 .Thus, there is a critical need for new chemotherapeutic agents 3,4 .The cyclohexane core generally has enriched the medicinal chemistry armamentarium with several bioactive candidates having diverse biological activities such as antipsychotic 5 , expectorant 6 , anticonvulsant 7,8 , analgesic 9 and anticancer activities [10][11][12] .Etoposide (I) and Teniposide (II) contain cyclohexane moiety in their structures and are used in cancer chemotherapy for the treatment of lung cancer, acute leukemia and lymphoma through a cytotoxic mechanism of DNA-topoisomerase II inhibition [13][14][15] .Moreover, the aminoacyl pharmacophore chain and amide moiety were included in the structural frame of different antitumor compounds, III and IV (Fig. 1), which were found to possess antitumor activity [16][17][18][19] .
These findings have encouraged us to prepare the target compounds 5a-m and 6a-f through molecular hybridization tactic of two or more pharmacophore moieties in one molecule aiming to improve the pharmacological profile 20 .

Chemistry
The preparation of the ultimate compounds 5a-m and 6a-f as well as the intermediates 1-4 is illustrated in Scheme 1. Cyclohexanone was reacted with potassium cyanide and aniline in glacial acetic acid to produce the nitrile derivative 1 which was hydrolyzed using sulfuric acid at room temperature to produce the amidic compound 3.

Cell cycle analysis and apoptosis induction
Compound 5i was the most potent against MCF-7 cancer cell line.Consequently, we examined its effect on the cell cycle progression using BD FASCCalibur after treatment with 3.25 μM of 5i for 48 h.Then, cell was stained with an annexin V-FITC antibody and propidium iodide by FACS (Table 2).For the cell cycle, compound 5i revealed induction of apoptosis at pre G1 phase and arresting at G2/M phase.

Chemistry General
Melting points were measured through Electrothermal Capillary apparatus and are uncorrected.The infrared (IR) spectra were recorded on JASCO FT/IR-6100 spectrometer.Spectral data ( 1 H-NMR as well as 13 C-NMR) were performed on Jeol ECA 500 MHz spectrometer and their values of the chemical shift are recorded as ppm on δ scale.Mass spectral data were gained using the technique of electron impact (EI) ionization.Column chromatography was conducted using silica gel 60 and chloroform/methanol 9/1 (v/v) as a mobile phase.

General procedure for the synthesis of 1-(N-phenyl-2-(heteroalicyclic-1-yl)acetamido)cyclohexane-1-carboxamides (5a-m)
To a solution of 4 (1.32 g, 0.0045 mol) in ethanol (30 mL), the appropriate amine derivative (0.0135 mol) was added.Then the reaction was refluxed under stirring for 12 h, and then ethanol was evaporated under reduced pressure.The residual was dissolved in ethyl acetate (30 mL) and washed with water (3x30 mL) then separation of the organic layer, drying over anhydrous Na 2 SO 4 and evaporation under the reduced pressure to produce 5a-m.

Table 1 : Antiproliferative activity for compounds 5a-m and 6a-f
).Most of the compounds are selective and having potential cytotoxicity towards MCF-7 adenocarcinoma with IC 50 values range 3.25-36.8μM as compared with doxorubicin (IC 50 value of 6.77 μM).Compound 5i showed the most potent biological activity with IC 50 value = 3.25 μM.