AbstractSeries of 1-(N-phenyl-2-(heteroalicyclic-1-yl)acetamido)cyclohexane-1-carboxamidederivatives 5a-m and1-(phenyl(heteroalicyclic-1-ylmethyl)amino)cyclohexane-1-carboxamide 6a-fwere designed and synthesized with biological interest through coupling of 1-(2-chloro-N-phenylacetamido)cyclohexane-1-carboxamide(4) and (phenylamino)cycloakanecarboxamides (2) with differentamines.

The structures of the target compounds were elucidated via IR, 1HNMR,13CNMR, MS, and microanalysis. In-vitro antitumor activityfor compounds 5a-m and 6a-f against four different cancer celllines MCF-7, HepG2, A549, and Caco-2 was evaluated. Compound 5idisplayed promising activity against breast cancer cell lines (IC50 value3.25 ?M,) compared with doxorubicin (IC50 value 6.77 ?M). Resultsfrom apoptosis and cell cycle analysis for compound 5i revealed goodantitumor activity against MCF-7 cancer cell line and potent inhibition, so itmay be a promising antitumor agent.                 Introduction     Cancer is the most prominent diseaseworldwide which represents the second cause of human mortality aftercardiovascular diseases 1. Drug resistance to cancer chemotherapy is considereda serious trouble2 .

Thus, there are a critical need for newchemotherapeutic agents 3. The cyclohexane core generally has enriched themedicinal chemistry armamentarium with several bioactive candidates havingdiverse biological activities such as antibacterial, antidiabetic,antipsychotic4, expectorant5 and anticancer activities6. Etoposide (I) and tenopside (II) are containingcyclohexane moiety in their structures and are used in cancer chemotherapy forthe treatment of lung cancer, acute leukemia and lymphoma through a cytotoxic mechanismof DNA- topoisomerase II inhibition. 7,8Moreover,the aminoacyl pharmacophoric chain and amide moiety was included in thestructural frame of different antitumor compounds and was found to possessantitumor activity III – IV 9-11 (Fig. 1). These findings have encouraged us to prepare the targetcompounds 5a-m and 6a-f through molecular hybridization tactic oftwo or more pharmacophore in one molecule aiming to increase thepharmacological profile.12              Fig.1.

Structures of Etoposide andTenopside (A), antitumor biocandidates bearing aminoacyl moieties (B), targetcompounds (C) containing the pharmacophoric features.       Results and discussionChemistryThe Preparation of ultimate compounds 5a–m and 6a–fin addition to the intermediates 1–4 is illustrated in Scheme 1. Cyclohexanonewas reacted with potassium cyanide and aniline in glacial acetic acid to producethe nitrile compound 1 which was hydrolyzed using sulfuric acid at roomtemperature and producing the amidic compound 3.Thepenultimate intermediate 4 was achieved by acetylation and subsequently,reaction with different amines to afford 5a-m or reaction withformaldehyde and different amines to afford 6a-m.                            Reagents and conditions: i) Cyclohexanone, Potassium cyanide, acetic acid glacial, r.

t, 24h   ii) H2SO4, r.t,  48 h, iii) ClCOCH2Cl, CHCl3,r.t , 24 h, iv) ethanol, appropriate amine, 12 h, reflux  v) HCHO, ethanol, appropriate amine, 12 h,reflux.Biological EvaluationAntiproliferation assayThe antitumor activity for compounds 5a-m and 6a-fwere evaluated against the four cancer cell lines HepG2 (liver), MCF-7(breast), A549 (lung) and Caco-2 (colorectal). Results are illustrated in (Table1). Most of the compounds are selective and having potential cytotoxicity towardsMCF-7 adenocarcinoma with IC50 values range 3.

25-36.8 ?M as comparedwith doxorubicin (IC50 value of 6.77 ?M). Compound 5i showedthe most potent biological activity with IC50 value = 3.25 ?M. Additionally, compound 5i showed high potentialactivity toward human HepG2 hepatocellular carcinoma with IC50 11.

5?M, A549 lung adenocarcinoma with IC50 6.95 ?M and Caco-2 colorectaladenocarcinoma with IC50 8.98 ?M as compared with doxorubicin (3.

07,0.887, 2.78 ?M respectively). Examining selectivity, the test compounds showedhigh activity against MCF-7.                   Table1. Antiproliferative activity for compounds 5a-mand 6a f.   Compound MCF-7 IC50 ?M HepG2 IC50 ?M A549 IC50 ?M Caco-2 IC50 ?M 5a 26.15 50.

2 35.4 30.21 5b 3.68 10.61 7.98 5.46 5c 7.

21 15.02 9.97 14.12 5d 28.41 60.

3 42.7 52.40 5e 31.

22 56.4 40.6 50.22 5f 20.08 43.32 39.74 4.

33 5g 9.13 33.88 30.3 25.43 5h 22.68 58.3 31.

29 47.23 5i 3.25 11.5 6.95 8.98 5j 13.59 31.

28 15.64 22.34 5k 5.48 20.71 14.

59 13.66 5l            4.58 12.08 8.51 11.25 5m 6.

48 13.82 11.83 10.53 6a             36.8 – – – 6b 23.51 16.46 31.

21 28.67 6c – – – – 6d 17.05 15.91 26.22 39.

52 6e 26.13 21.74        42.09 51.43 6f 22.

89 17.54 31.6 44.12 Doxorubicin 6.77 3.

07 0.887 2.78 2.     (-): No activity Cell cycle analysis and apoptosisinduction    Compound 5i wasthe most potent against MCF-7 cancer cell line. Consequently, we examined itseffect on the cell cycle progression using BD FASCCalibur after treatment with3.

25 ?M of 5i for 48 h. Then, cell was stained with an annexin V-FITCantibody and propidium iodide by FACS (Table 2). For the cell cycle, compound 5irevealed induction of apoptosis at pre G1 phase and arresting at G2/M phase.    Table 2.  Cell cycle analysisfor control and compound 5i at concentration 3.

25 ?M for 48 h on MCF-7 cellline. Sample data          Result %G0-G1 %S % G2-M % Apoptosis Comment 5i 7.66 24.77 46.17 21.4 PreG1 apoptosis &cell growth [email protected]/M Cont. MCF-7 71.

7 22.45 5.23 0.62 Control pattern                Fig.2.Graphical view of % of DNA content in G0/G1, S, and G2/M phase for control andcompound 5i         Fig.3. Cell cycle analysis and apoptotic assaygraphs for control and compound 5i at concentration 3.

25 ?M for 48 h on MCF-7 cell line           Material and methodsChemistryGeneral    Melting points for allcompounds were uncorrected through Electrothermal Capillary apparatus and the infrared(IR) spectra were recorded on JASCO FT/IR-6100 spectrometer. Spectral data 1H-NMRas well as 13C-NMR were performed on Jeol ECA 500 MHz spectrometerand their values of the chemical shift are recorded as ppm on ? scale. Massspectra data gained using the technique of electron impact (EI) ionization.Column chromatography using silica gel 60 was performed. The mobile phaseconsisted of chloroform/methanol 9/1 v/v. Synthesis of 1- (phenylamino)cyclohexanecarbonitrile (2)      Was prepared as showed in literatures. 13 Synthesisof (phenylamino) cycloakanecarboxamides (3)        Was prepared as showedin literatures.

14Synthesis of1-(2-chloro-N-phenylacetamido)cyclohexane-1-carboxamide (4)Chloroacetyl chloride (1.317g, 0.95 mL, 0.01166 mol) was added to asolution of 3 (2.1g, 0.0097 mol) in chloroform (30 mL).

The mixture was allowedfor stirring at room temperature for 24h, then adding an aqueous solution of10% NaOH (2 x 30 mL) then separation of the organic layer, drying overanhydrous Na2SO4 and evaporation under reduced pressureto afford 2.5 g of 4 as pale yellow oil in 89.2% yield which is solidifiedupon standing at room temperature, m.p.170 33/°C.

IR(KBr, cm-1) 2926.23,2851.17 (NH2), 1645, 1625 (2 x C=O) ; 1HNMR (CDCl3) 1.

34-1.52 (m, 10H, 5 x CH2, cyclohexyl), 3.26(s, 2H, CH2-Cl), 6.99 (s, 2H,NH2), 7.25-7.32 (m, 5H, Har.

);13C NMR (CDCl3) ? ppm 20.79, 22.91, 32.82 (3 x CH2,cyclohexyl), 40.41 (CH2-Cl), 66.

60 (Cq), 128.60, 128.77, 130.23 (3CHar.

),139.16 (Car.), 167.16 (CO-CH2), 178.

60 (CO-NH2);MS (EI) m/z (%): 294.78 (M+, 1.60, ), 250 (100). (EI) m/z(%): 294.78 (M+, 5), 250 (100).

Anal.calcd. for C15H19ClN2O2: C,61.12;H,6.

50; Cl, 12.03; N, 9.50.

Found: C, 61.23; H, 6.51; Cl, 12.23; N, 9.49.

General procedures for synthesis of1-(N-phenyl-2-(heteroalicyclic-1-yl)acetamido)- cyclohexane-1-carboxamide (5a-m )        To a solution of 4(1.32 g, 0.0045 mol) in ethanol (30 mL), the appropriate amine derivative (0.0135mol) was added. Then the reaction was refluxed under stirring for 12 h, andthen ethanol was evaporated under reduced pressure. The residual was dissolvedin ethyl acetate (30 mL) and washed with water (3×30 mL) then separation of theorganic layer, drying over anhydrous Na2SO4 and evaporationunder the reduced pressure to produce 5a-m.     1-(N-phenyl-2-(piperidin-1-yl)acetamido)cyclohexane-1-carboxamide(5a)     White solid, m.

p. 120 °C, yield 75%; IR(KBr, cm-1) 2924, 2841 (NH2),1635, 1621 (2 x C=O). 1HNMR: 1.12 (s, 10H, 5CH2,cyclohexyl), 1.

78 (s, 10H, 5CH2, piperidine), 3.16 (s, 2H, CO-CH2),6.53 (s, 2H, NH2), 7.18-7.53 (m, 5H, Har.); 13CNMR:20.96, 22.34, 22.

87 (3 x CH2, cyclohexyl), 25.08. 25.66 (2CH2-piperidine),58.81 (CH2-piperidine), 54.44 (CO-CH2), 118.

48,128.85, 129.67 (3 x CHar.), 144.17 (Car.), 168.

55 (CO-CH2),179.90 (CO-NH2); MS (EI) m/z (%):343.47 (M+, 1.83), 47.

9 (100). Anal.calcd. for C20H29N3O2: C, 69.94; H,8.51; N,12.

23; O, 9.32. Found: C, 69.74; H, 8.72; N, 12.41; O, 9.33. 1-(N-phenyl-2-(pyridin-1(2H)-yl)acetamido)cyclohexane-1-carboxamide(5b)       Yellowish oil, yield 85% IR(KBr, cm-1) 2935.

13,2860.88 (NH2), 1742.37, 1660.41 (2 x C=O); 1HNMR: 1.14-1.35(s, 10H, 5 x CH2, cyclohexyl), 3.62 (s, 2H, CH2-pyridine),3.64 (s, 2H, CO-CH2), 6.

03 (s, 2H, NH2), 6.63 (d,1H, J=7.6, CH-pyridine.), 6.82 (d, 1H, J=7.

6, CH-pyridine), 6.91 (d, 1H, J=7.6,CH-pyridine), 7.10 (s, 1H, CH-pyridine), 7.37-7.91 (m, 5H, Har.); 13CNMR:21.

07, 22.20, 25.11 (3 x CH2, cyclohexyl), 52.

21 (CH2-pyridine),60.22 (CO-CH2), 64.43 (Cq), 118.98, 125.94, 131.54, (3 x CHar.

),143.86 (Car.), 167.79 (CO-CH2), 179.66 (CO-NH2),MS(EI) m/z (%): 339.41 (M+, 1.

19), 174 (100). Anal. calcd. for C20H25N3O2:C, 70.

77; H, 7.42; N,12.38; O, 9.43. Found: C, 70.

57; H, 7.44; N, 12.33. 1-(2-(3-Hydroxypyridin-1(2H)-yl)-N-phenylacetamido)cyclohexane-1-carboxamide(5c)    Yellowish solid, yield 90%, m.

p. 92 °C,IR (KBr, cm-1) 2932, 2859 (NH2),1718, 1655 (2 x C=O). 1HNMR: 1.34-2.19 (m, 10H, 5 x CH2,cyclohexyl), 3.

71 (s, 2H, CH2-pyridine), 3.92 (s, 2H, CO-CH2),6.65 (s, 2H, NH2), 6.67 (s, 1H, CH-pyridine), 7.13 (s, 1H,CH-pyridine), 7.49 (s, 1H, CH-pyridine), 8.06 (s, 3H, Har.), 8.

33(s, 2H, Har.), 10.04 (br.

s, 1H, OH); 13CNMR: 22.53,25.73, 33.21 (3 CH2, cyclohexyl), 48.91 (CH2-pyridine),55.

34 (CO-CH2), 68.71 (Cq), 94.23 (CH-pyridine), 116.12,125.63, 127.

11 (3 x CHar.), 134.71 (Car.), 136.44 (CH-N), 156.11(C-OH), 167.21 (CO-CH2), 181.42 (CO-NH2); (EI)m/z (%): 355.

44 (M+,2.5), 336 (100). Anal. calcd.

for C20H25N3O3:C,67.58; H,7.09; N, 11.82; O, 13.50. Found: C, 67.

48; H, 7.12; N, 11.71.   1-(2-Morpholino-N-phenylacetamido)cyclohexane-1-carboxamide(5d)Pale yellow oil, yield 90%, IR (KBr, cm-1) 2906, 2860 (NH2), 1716, 1658 (2 x C=O); 1HNMR:1.51-2.

09 (m, 10H, 5CH2, cyclohexyl), 2.51 (t, 2H, J=6, CH2-morpholine),3.71 (t, 2H, J=6, CH2-morpholine), 3.88 (s, 2H, CO-CH2),6.93 (s, 2H, NH2), 7.15-7.

19 (m, 3H, Har.), 7.33 (s, 2H,Har.); (EI)m/z (%): 345.44 (M+,0.

81), 174 (100). Anal. calcd. for C19H27N3O3:C,66.06; H,7.88; N, 12.

16. Found: C, 66.21; H, 7.75; N, 12.24.1-(2-(1H-Imidazol-1-yl)-N-phenylacetamido)cyclohexane-1-carboxamide(5e)          Yellowish oil, yield 90%, IR (KBr, cm-1) 2933, 2852 (NH2), 1739, 1669 (2 x C=O).1HNMR: 1.17-1.

54 (s, 10H, 5CH2, cyclohexyl), 3.63 (s, 2H,CO-CH2), 6.61 (s, 2H, NH2), 7.03 (s, 3H,CH-imidazole), 7.67 (s, 5H, Har.); 13CNMR: 20.

97, 25.12,31.24 (3 x CH2, cyclohexyl), 53.12 (CO-CH2), 59.99(Cq), 115.19, 115.98, 121.51, 129.

02, 129.24, 130.13 (3x CH-imidazole, 3x CHar.),144.17 (Car.), 168.

21 (CO-CH2), 180.13 (CO-NH2),(EI)m/z (%): 326.40 (M+,15), 174 (100). Anal. calcd.for C18H22N4O2: C,66.24; H,7.79; N,17.

17. Found: C, 66.21; H, 7.81; N, 17.25.1-(N-Phenyl-2-(piperazin-1-yl)acetamido)cyclohexane-1-carboxamide(5f)      Brownish oil, yeild 79%, IR (KBr, cm-1) 2935, 2857 (NH2), 1739.

48, 1649.81 (2 x C=O)1HNMR: 1.24-1.60 (m, 11H, 5CH2, cyclohexyl, NH), 1.96 (s,4H, 2CH2-piperazine), 2.

07 (s, 4H, 2CH2-piperazine), 3.02(s, 2H, CO-CH2), 6.93 (s, 2H, NH2), 7.15-7.42 (m,5H, Har.

); 13CNMR: 21.04, 25.13, 31.20 (3 CH2,cyclohexyl), 41.96, 44.45 (2 x CH2-piperazine), 59.

99 (CO-CH2),60.38 (Cq), 116.04, 118.85, 129.01 (3 x CHar.), 144.15 (Car.),171.

15 (CO-CH2), 179.61 (CO-NH2), (EI)m/z (%): 344.46 (M+,3.03), 174 (100).

Anal. calcd. for C19H28N4O2:C,66.25; H,8.

19; N, 16.27. Found: C, 66.33; H, 8.22; N, 16.22.1-(2-(4-Methylpiperazin-1-yl)-N-phenylacetamido)cyclohexane-1-carboxamide(5g)       Yellowish oil, yield 75%, IR(KBr, cm-1) 2935, 2855 (NH2),1739, 1668 (2 x C=O).

1HNMR: 1.25-1.61 (m, 10H, 5CH2, cyclohexyl), 1.97 (s, 3H,CH3), 2.29 (s, 4H, 2CH2-piperazine), 2.68 (s, 4H, 2CH2-piperazine),3.21( s, 2H, CH2-Me-piperazine), (s, 2H, CO-CH2),6.81 (s, 2H, NH2), 6.

91-7.52 (m, 5H, Har.); 13CNMR:21.

05, 25.12, 31.19 (3 x CH2, cyclohexyl), 45.75 (CH3),52.70, 54.52 (2 x CH2-piperazine), 54.

65 (CO-CH2),66.02 (Cq), 118.90, 129.02, 130.

71 (3 x CHar.), 144.12 (Car.),169.

51 (CO-CH2), 179.57 (CO-NH2), (EI)m/z (%): 358.49 (M+,1.8), 113 (100). Anal. calcd. for C20H30N4O2:C,67.01; H,8.

44; N, 15.63. Found: C, 67.22; H, 8.

52; N, 15.73.1-(2-(4-(7-Chloroquinolin-4-yl)piperazin-1-yl)-N-phenylacetamido)cyclohexane-1-carboxamide(5h)      Brownish oil, yield 75%, IR (KBr, cm-1) 2935, 2860 (NH2), 1739, 1654 (2 x C=O).1HNMR: 1.26-1.

49 (m, 10H, 5CH2, cyclohexyl), 2.18 (s, 4H,2CH2-piperazine), 3.64 (s, 4H, 2CH2-piperazine), 3.81 (s,2H, CO-CH2), 6.89 (br.s, 2H, NH2), 7.

14 (d, 1H, J=6.75, CH quinoline), 7.19 (d, 1H, J= 7.

5   , CH quinoline), 7.22(s, 5H, Har.), 7.35 (s, 2H, CH quinoline), 7.46(d, 1H, J=5, CH quinoline); 13CNMR: 22.35, 25.10, 33.58 (3 x CH2,cyclohexyl), 51.

21, 54.32 (2 x CH2-piperazine), 56.91 (CO-CH2),67.81 (Cq), 128.87, 129.

11, 129.51, 129.61, 129.87, 130.33, 133.42, 133.63 (6 xCHar.), 135.

61, 137.82, 152.81, 153.53, 154.

22 (5 x Car.),166.21 (CO-CH2), 178.93 (CO-NH2); (EI) m/z (%): 506 (M+,7.2), 288 (100). Anal. calcd.for C28H32ClN5O2: C,66.

46; H,6.37;Cl, 7.01; N, 13.84. Found: C, 66.42; H, 6.36; Cl, 7.11; N, 13.82.1-(2-(Cyclohexylamino)-N-phenylacetamido)cyclohexane-1-carboxamide(5i)       Brownish oil, yield 80%, IR (KBr, cm-1) 2933, 2856 (NH2), 1745, 1647 (2 x C=O), 1HNMR:1.02-1.18 (m, 10H, 5CH2, cyclohexyl), 1.45-1.85 (m, 10H, 5CH2,cyclohexyl), 2.78 (s, 2H, CO-CH2), 5.51 (br.s, 1H, NH), 6.56 (s, 2H,NH2); 13CNMR: 21.73, 24.53, 25.18, 29.61, 30.21, 32.40 (6x CH2, cyclohexyl), 50.23 (CO-CH2), 59.78, 63.50(2Cq), 115.04, 115.83, 129.77 (3 x CHar.), 144.19 (Car.),162.81 (CO-CH2), 179.88 (CO-NH2), (EI)m/z (%): 357.50 (M+, 0.77), 174 (100). C21H31N3O2: C, 70.55;H, 6.74; N, 11.75. Found: C, 70.43; H, 6.72; N, 11.72.1-(2-(Phenethylamino)-N-phenylacetamido)cyclohexane-1-carboxamide(5j)        Brownish oil, yield 85%, IR (KBr, cm-1) 2933, 2862 (NH2), 1747, 1647 (2 x C=O).1HNMR: 1.92 (s, 10H, 5CH2, cyclohexyl), 2.83 (t, 2H, J=7.45 , CH2-C6H5), 2.99 (t, 2H, J= 7.25   , NH-CH2-CH2),4.43 (s, 2H, CO-CH2), 6.82 (s, 2H, NH2), 7.17-7.27 (m,10H, Har.); 13CNMR: 22.81, 23.17, 25.16 (3 x CH2,cyclohexyl), 35.52 (CH2-C6H5), 42.46(NH-CH2-CH2) 52.51 (CO-CH2),73.12 (Cq), 115.15, 116.06, 126.44, 127.51, 128.58, 128.84 (6 x CHar.),138.82, 142.51 (2 x Car.), 161.64 (CO-CH2), 181.71(CO-NH2), (EI) m/z (%): 379.50 (M+,5.29),174 (100). C23H29N3O2:C, 72.79; H, 7.70; N, 11.07. Found: C, 72.81; H, 7.85; N, 11.15. 1-(N-Phenyl-2-(1H-pyrrol-1-yl)acetamido)cyclohexane-1-carboxamide(5k)       Brownish oil, yield 78%, IR (KBr, cm-1) 2935, 2862 (NH2), 1747, 1647 (2 x C=O).1HNMR: 1.63-2.21 (m, 10H, 5CH2, cyclohexyl), 3.77 (s, 2H,CO-CH2), 5.61 (s, 2H, NH2), 6.63-6.80 (m, 4H,CH-pyrrole), 7.28-7.46 (m, 5H, Har.); (EI) m/z(%): 325.41 (M+, 3.45), 174 (100). C19H23N3O2:C, 70.13; H, 7.12; N, 12.91. Found: C, 70.25; H, 7.22; N, 12.85.1-(N-Phenyl-2-(pyrrolidin-1-yl)acetamido)cyclohexane-1-carboxamide(5l)         Brownish solid, m.p100°C, yield 75%, IR(KBr, cm-1) 2933, 2858 (NH2),1743, 1662 (2 x C=O). 1HNMR: 1.13-1.34 (m, 10H, 5CH2,cyclohexyl), 2.01 (s, 4H, 2CH2-pyrrolidine), 2.19 (s, 4H, 2CH2-pyrrolidine),3.67 (s, 2H, CO-CH2), 6.84 (s, 2H, NH2), 7.20-7.47(m, 5H, Har.); 13CNMR: 21.07, 22.41, 25.08, 33.63, 30.21(3 x CH2, cyclohexyl, CH2-pyrrolidine), 52.32 (CH2-pyrrolidine),56.93 (CO-CH2), 65.61 (Cq), 129.10, 129.41, 129.56 (3 x CHar.),130.49 (Car.), 162.54 (CO-CH2), 179.81 (CO-NH2),(EI)m/z (%): 329.44 (M+,0.75), 174 (100). C19H27N3O2: C, 69.27;H, 8.26; N, 12.76. Found: C, 69.22; H, 8.31; N, 12.82.1-(2-(Butylamino)-N-phenylacetamido)cyclohexane-1-carboxamide (5m)           Brownish oil, yield 85%, IR (KBr, cm-1) 2933, 2862 (NH2), 1747, 1658 (2 x C=O).1HNMR: 0.891 (t, 3H,J=7  , CH3),1.25 (s, 2H, CH2-CH3), 1.32-1.39 (m, 10H, 5CH2,cyclohexyl), 2.04 (s, 2H, CH2-CH2-CH3),2.17 (t, 2H, J=7, NH-CH2),  2.89 (br.s, 1H, NH), 3.21 (s, 2H, CO-CH2),6.81 (s, 2H, NH2), 7.31-7.62 (m, 5H, Har.); 13CNMR:21.04 (CH3), 13.54, 22.47, 24.97, 29.50, 30.93 (3 x CH2,cyclohexyl, -CH2-CH2-CH3), 45.84(NH-CH2), 51.32 (CO-CH2), 60.39 (2Cq),115.12, 116.10, 129.04 (3 x CHar.), 144.07 (Car.), 167.33(CO-CH2), 178.91 (CO-NH2), (EI)m/z (%): 367.95 (M+,1.68), 174 (100). C19H30ClN3O2: C, 62.03;H, 8.22; Cl, 9.64; N, 11.42. Found: C, 62.21; H, 8.32; N, 11.35.General procedures for synthesis of1-(phenyl(heteroalicyclic-1-ylmethyl)amino) cyclohexane-1-carboxamide (6a-f)           To a solution of compound 2 (2.0g, 0.01 mol) in 96% ethanol, 40% solution of formaldehyde (0.3 g, 0.01 mol) andthe corresponding amine were added. The reaction mixture was refluxed for 12 h,then allowed to evaporate under reduced pressure, drying over Na2SO4and purified using column chromatography chloroform (9): ethyl acetate (1) toafford compounds 6a-f. 1-(Phenyl(piperidin-1-ylmethyl)amino)cyclohexane-1-carboxamide(6a)       White solid, yield 90%, m.p110°C, IR(KBr, cm-1) 2924, 2866 (NH2),1681 (C=O).1HNMR: 1.27-1.29 (m, 4H, 2CH2, cyclohexyl), 1.74 (t, 2H, J=9.15, CH2-piperidine), 2.05 (s, 2H, CH2-piperidine), 2.30(s, 6H, 3CH2, cyclohexyl), 4.86 (s, 2H, CH2-N), 6.91 (m, 5H, NH2,3 Har.), 7.30 (s, 2H, Har.);13CNMR: 22.12,24.73, 29.59, (3 x CH2, cyclohexyl), 67.88 (Cq), 87.26 (CH2-N),116.02, 119.16, 129.18 (3 x CHar.), 143.37 (Car.), 177.20(CO-NH2), (EI) m/z (%): 315.23 (M+,5.3), 189 (100). C19H29N3O:C, 72.34; H, 9.27; N, 13.32. Found: C, 72.41; H, 9.25; N, 13.45.1-(((1H-Imidazol-1-yl)methyl)(phenyl)amino)cyclohexane-1-carboxamide(6b)      Yellowish oil, yield 92%; IR (KBr, cm-1) 2941, 2899 (NH2), 1600 (C=O).1HNMR: 1.27-1.29 (m, 10H, 5CH2, cyclohexyl), 5.37 (s, 2H,CH2-N), 6.39 (br.s, 2H, NH2), 6.87-6.94 (m, 3H, 3CH-imidazole), 7.00-7.49 (m, 5H, Har.); 13CNMR: 20.68,27.22, 31.00 (3 x CH2, cyclohexyl), 70.09 (Cq), 81.22 (CH2-N),113.32, 117.65, 118.73, 123.10, 128.37, 130.10 (3 x CHar, 3 xCH-imidazole), 145.15 (Car.), 177.23 (CO-NH2), (EI)m/z (%): 298.39 (M+,1.38). C17H22N4O:C, 68.43; H, 7.43; N, 18.78. Found: C, 68.38; H, 7.55; N, 18.91.1-(Phenyl(piperazin-1-ylmethyl)amino)cyclohexane-1-carboxamide(6c)White solid, m.p130°C, yield 80%; IR (KBr, cm-1) 2924, 2852 (NH2), 1600 (C=O).1HNMR: 1.21 (s, 1H, NH), 1.71 (t, 2H, J=10, CH2-piperazine), 1.92-2.55 (m, 10H, 5CH2,cyclohexyl), 4.28 (t, 2H,J=10, CH2-piperazine), 4.14 (s, 2H, CH2-N), 6.06 (s, 2H,NH2), 6.88-7.29 (m, 5H, Har.); 13CNMR: 22.07, 25.10,29.88 (3 x CH2, cyclohexyl), 49.13, 54.53 (2CH2-piperazine),62.87 (CH2-N), 78.24 (Cq), 116.15, 119.12, 129.14 (3 x CHar.),143.40 (Car.), 177.45 (CO-NH2), (EI)m/z (%): 345.44 (M+,0.81), (EI) m/z (%): 316.45 (M+,20.23), 99 (100). C18H28N4O:C, 68.32; H, 8.92; N, 17.71. Found: C, 68.35; H, 8.85; N, 17.75.1-(((4-Methylpiperazin-1-yl)methyl)(phenyl)amino)cyclohexane-1-carboxamide(6d)       Yellowish oil, yield 85%; IR (KBr, cm-1) 2921, 2854 (NH2), 1621 (C=O).1HNMR: 1.24-1.25 (m, 10H, 5CH2, cyclohexyl), 2.31 (s, 3H,CH3), 2.64 (s, 4H, 2CH2- piperazine), 3.51 (s, 4H,2CH2-piperazine), 4.79 (s, 2H, CH2-N), 6.87 (s, 2H, NH2),7.02-7.22 (m, 5H, Har.); 13CNMR: 15.16, 18.46, 20.12 (3 xCH2, cyclohexyl), 45.88 (CH3), 51.72, 54.55 (2x CH2-Me-piperazine),68.60 (Cq), 82.75 (CH2-N), 114.57, 120.92, 129.02 (3 x CHar.),146.53 (Car.), 177.53 (CO-NH2), (EI)m/z (%): 330.48 (M+,11.4), 76.93 (100). C19H30N4O: C, 69.05; H, 9.15; N,16.95. Found: C, 69.12; H, 9.23; N, 16.85.1-(((4-Ethylpiperazin-1-yl)methyl)(phenyl)amino)cyclohexane-1-carboxamide(6e)      Brownish oil, yield 75%; IR (KBr, cm-1) 2935, 2823 (NH2), 1621 (C=O); 1HNMR: 1.09 (t,3H, J= 4.5, CH3), 1.11-1.24 (m, 10H, 5CH2,cyclohexyl), 2.32 (s, 2H, CH2-CH3), 2.47 (t, 2H,J= 8.9, CH2- piperazine), 2.66 (t, 4H, J= 5.8, CH2-piperazine),4.79 (s, 2H, CH2-N), 5.30 (br.s, 2H, NH2), 7.00-7.09 (m,3H, Har.), 7.27 (s, 2H, Har); (EI)m/z (%): 344.50 (M+,0.75), 127 (100). C20H32N4O: C, 69.73; H, 9.36; N,16.26. Found: C, 69.77; H, 9.42; N, 16.35.1-(((Cyclohexylamino)methyl)(phenyl)amino)cyclohexane-1-carboxamide(6f) Brownish solid, m.p130°C,yield 80%; IR (KBr, cm-1)2929, 2854 (NH2), 1600 (C=O).1HNMR: 1.21 (s, 1H, NH), 1.25-1.77 (m, 10H, 5CH2,cyclohexyl), 1.78-2.94 (m, 10H, 5CH2, cyclohexyl), 3.45 (s, 1H, NH),4.64 (s, 2H, CH2-NH), 6.81 (s, 2H, NH2), 6.92-7.28 (m,5H, Har); 13CNMR: 20.09, 25.61, 28.75, 36.87, 43.42,48.13 (6 x CH2, 2cyclohexyl), 65.13, 66.23 (2 x Cq), 88.75 (CH2-NH),113.08, 128.97, 129.49 (3 x CHar.), 145.22 (Car.), 177.97(CO-NH2), (EI) m/z (%): 329.49 (M+,5.75), 106 (100). C20H31N3O:C, 72.91; H, 9.48; N, 12.75. Found: C, 72.85; H, 9.37; N, 12.83.BiologicalEvaluation       The humantumor cell lines were obtained from NCI, MD, USA. All chemicals and solventswere purchased from Sigma-Aldrich.   Cellproliferation assay       The anticanceractivities for compounds 5a-m and 6a-f were performed using astandard (MTT)-based colorimetric assay. Cell Cycle Analysis and Detection of Apoptosis in MCF-7 cell line      Studying the effect ofcompound 5i on the cell cycle progression using BD FASCCalibur aftertreatment with 3.25 ?M of 5i for 48 h. Then, cell staining with anannexin V-FITC antibody and propidium iodide by FACS.  Conclusion    Series of 1-(N-phenyl-2-(heteroalicyclic-1-yl)acetamido)cyclohexane-1-carboxamidederivatives 5a-m and 1-(phenyl(heteroalicyclic-1-ylmethyl)amino)cyclohexane-1-carboxamide6a-f have been synthesized and screened ortheir anticancer activity. Compound 5i was shown to have the most potentanticancer activity which inhibited the MCF-7 cell line with the IC50value of 3.25 ?M inducing apoptosis at PreG1 and arresting of the cellcycle at G2/M phase. Conflict of interestThe authers having no conflict. 

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