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

Introduction

Cancer is one of the most prominent diseases worldwide which represents the second cause of human mortality after cardiovascular diseases¹. Drug resistance to cancer chemotherapy is considered a serious trouble².  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 antipsychotic⁵, expectorant⁶, anticonvulsant^7,8, analgesic⁹ and anticancer activities^10-12. 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^13-15. 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^16-19.

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 profile²⁰.

Figure 1: (A) Structures of Etoposide (I) and Teniposide (II); (B) Antitumor biocandidates bearing aminoacyl moieties; (C) target compounds containing the pharmacophoric features. Click here to View figure

 

Results and Discussion

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.

The penultimate intermediate 4 was achieved by acetylation of compound 3. Subsequently, compound 4 was subjected to the reaction with different amines to afford 5a-m, while compound 3 was reacted with formaldehyde and different amines to afford 6a-f.

Scheme 1 Click here to View scheme

 

Reagents and conditions: i) Cyclohexanone, potassium cyanide, acetic acid glacial, r.t, 24 h   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 Evaluation

Antiproliferation Assay

The antitumor activity for compounds 5a-m and 6a-f were evaluated against the four cancer cell lines HepG2 (liver), MCF-7 (breast), A549 (lung) and Caco-2 (colorectal). Results are illustrated in (Table 1). Most of the compounds are selective and having potential cytotoxicity towards MCF-7 adenocarcinoma with IC₅₀ values range 3.25-36.8 μM as compared with doxorubicin (IC₅₀ value of 6.77 μM). Compound 5i showed the most potent biological activity with IC₅₀ value = 3.25 μM.

Additionally, compound 5i showed high potential activity toward human HepG2 hepatocellular carcinoma with IC₅₀ 11.5 μM, A549 lung adenocarcinoma with IC₅₀ 6.95 μM and Caco-2 colorectal adenocarcinoma with IC₅₀ 8.98 μM as compared with doxorubicin (3.07, 0.887, 2.78 μM respectively). Examining selectivity, the test compounds showed high activity against MCF-7.

Table 1: Antiproliferative activity for compounds 5a-m and 6a- f.

Compound	MCF-7 IC50 μM	HepG2 IC50 μM	A549 IC50 μM	Caco-2 IC50 μM
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

(-): No activity

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.

Table 2: Cell cycle analysis for the control and compound 5i at concentration of 3.25 μM for 48 h on MCF-7 cell line.

Sample data	Result
	%G0-G1	%S	% G2-M	% Apoptosis	Comment
5i	7.66	24.77	46.17	21.4	PreG1 apoptosis &cell growth arrest@G2/M
Cont. MCF-7	71.7	22.45	5.23	0.62	Control pattern

 

Figure 2: Graphical view of % of DNA content in G0/G1, S, and G2/M phase for control and compound 5i   Click here to View figure

 

Figure 3: Cell cycle analysis and apoptotic assay graphs for control and compound 5i at concentration 3.25 μM for 48 h on MCF-7 cell line. Click here to View figure

 

Materials and Methods

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 (¹H-NMR as well as ¹³C-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.

Synthesis of 1-(phenylamino)cyclohexanecarbonitrile (2)

Compound 2 was prepared as showed in literatures⁸.

 Synthesis of (phenylamino)cycloakanecarboxamide (3)

Compound 3 was prepared as showed in literatures⁸.

Synthesis of 1-(2-chloro-N-phenylacetamido)cyclohexane-1-carboxamide (4)

Chloroacetyl chloride (1.317g, 0.95 mL, 0.01166 mol) was added to a solution of 3 (2.1g, 0.0097 mol) in chloroform (30 mL). The mixture was allowed for stirring at room temperature for 24h, then adding an aqueous solution of 10% NaOH (2 x 30 mL) then separation of the organic layer, drying over anhydrous Na₂SO₄ and evaporation under reduced pressure to afford 2.5 g of 4 as pale yellow oil in 89.2% yield which is solidified upon standing at room temperature, m.p.170 33/°C. IR (KBr, cm^-1) 2926.23, 2851.17 (NH₂), 1645, 1625 (2 x C=O) ; ¹H NMR (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, H_ar.); ¹³C NMR (CDCl₃) δ ppm 20.79, 22.91, 32.82 (3 x CH₂, cyclohexyl), 40.41 (CH₂-Cl), 66.60 (Cq), 128.60, 128.77, 130.23 (3CH_ar.), 139.16 (C_ar.), 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 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 (3×30 mL) then separation of the organic layer, drying over anhydrous Na₂SO₄ and evaporation under 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); ¹H NMR: 1.12 (s, 10H, 5 x CH₂, cyclohexyl), 1.78 (s, 10H, 5 x CH₂, piperidine), 3.16 (s, 2H, CO-CH₂), 6.53 (s, 2H, NH₂), 7.18-7.53 (m, 5H, H_ar.); ¹³C NMR: 20.96, 22.34, 22.87 (3 x CH₂, cyclohexyl), 25.08, 25.66 (2 x CH₂-piperidine), 58.81 (CH₂-piperidine), 54.44 (CO-CH₂), 118.48, 128.85, 129.67 (3 x CH_ar.), 144.17 (C_ar.), 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)

Yellow viscous  oil, yield 85%, IR (KBr, cm^-1) 2935.13, 2860.88 (NH₂), 1742.37, 1660.41 (2 x C=O); ¹H NMR: 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 Hz, CH-pyridine.), 6.82 (d, 1H, J = 7.6 Hz, CH-pyridine), 6.91 (d, 1H, J = 7.6 Hz, CH-pyridine), 7.10 (s, 1H, CH-pyridine), 7.37-7.91 (m, 5H, H_ar.); ¹³C NMR: 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 CH_ar.), 143.86 (C_ar.), 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)

Yellow viscous oil, yield 90%, m.p. 92 °C, IR (KBr, cm^-1) 2932, 2859 (NH₂), 1718, 1655 (2 x C=O); ¹H NMR: 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, H_ar.), 8.33 (s, 2H, H_ar.), 10.04 (br. s, 1H, OH); ¹³C NMR: 22.53, 25.73, 33.21 (3 x 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 CH_ar.), 134.71 (C_ar.), 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); ¹H NMR: 1.51-2.09 (m, 10H, 5 x CH₂, cyclohexyl), 2.51 (t, 2H, J = 6 Hz, CH₂-morpholine), 3.71 (t, 2H, J = 6 Hz, CH₂-morpholine), 3.88 (s, 2H, CO-CH₂), 6.93 (s, 2H, NH₂), 7.15-7.19 (m, 3H, H_ar.), 7.33 (s, 2H, H_ar.); (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)        

Yellow viscous oil, yield 90%, IR (KBr, cm^-1) 2933, 2852 (NH₂), 1739, 1669 (2 x C=O); ¹H NMR: 1.17-1.54 (s, 10H, 5 x CH₂, cyclohexyl), 3.63 (s, 2H, CO-CH₂), 6.61 (s, 2H, NH₂), 7.03 (s, 3H, CH-imidazole), 7.67 (s, 5H, H_ar.); ¹³C NMR: 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 (3 x CH-imidazole, 3 x CH_ar.), 144.17 (C_ar.), 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)

Brown viscous oil, yield 79%, IR (KBr, cm^-1) 2935, 2857 (NH₂), 1739.48, 1649.81 (2 x C=O); ¹H NMR: 1.24-1.60 (m, 11H, 5 x CH₂, cyclohexyl, NH), 1.96 (s, 4H, 2 x CH₂-piperazine), 2.07 (s, 4H, 2 x CH₂-piperazine), 3.02 (s, 2H, CO-CH₂), 6.93 (s, 2H, NH₂), 7.15-7.42 (m, 5H, H_ar.); ¹³C NMR: 21.04, 25.13, 31.20 (3 x 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 CH_ar.), 144.15 (C_ar.), 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)

Yellow viscous oil, yield 75%, IR (KBr, cm^-1) 2935, 2855 (NH₂), 1739, 1668 (2 x C=O); ¹H NMR: 1.25-1.61 (m, 10H, 5 x CH₂, cyclohexyl), 1.97 (s, 3H, CH₃), 2.29 (s, 4H, 2 x CH₂-piperazine), 2.68 (s, 4H, 2 x CH₂-piperazine), 3.21( s, 2H, CH₂-Me-piperazine),  (s, 2H, CO-CH₂), 6.81 (s, 2H, NH₂), 6.91-7.52 (m, 5H, H_ar.); ¹³C NMR: 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 CH_ar.), 144.12 (C_ar.), 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)

Brown viscous oil, yield 75%, IR (KBr, cm^-1) 2935, 2860 (NH₂), 1739, 1654 (2 x C=O); ¹H NMR: 1.26-1.49 (m, 10H, 5 x CH₂, cyclohexyl), 2.18 (s, 4H, 2 x CH₂-piperazine), 3.64 (s, 4H, 2 x CH₂-piperazine), 3.81 (s, 2H, CO-CH₂), 6.89 (br. s, 2H, NH₂), 7.14 (d, 1H, J = 6.75 Hz, CH quinoline), 7.19 (d, 1H, J = 7.5 Hz, CH quinoline), 7.22 (s, 5H, H_ar.), 7.35 (s, 2H, CH quinoline), 7.46 (d, 1H, J = 5, CH quinoline); ¹³C NMR: 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 x CH_ar.), 135.61, 137.82, 152.81, 153.53, 154.22 (5 x C_ar.), 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)

Brown viscous oil, yield 80%, IR (KBr, cm^-1) 2933, 2856 (NH₂), 1745, 1647 (2 x C=O); ¹H NMR: 1.02-1.18 (m, 10H, 5 x CH₂, cyclohexyl), 1.45-1.85 (m, 10H, 5 x CH₂, cyclohexyl), 2.78 (s, 2H, CO-CH₂), 5.51 (br. s, 1H, NH), 6.56 (s, 2H, NH₂); ¹³C NMR: 21.73, 24.53, 25.18, 29.61, 30.21, 32.40 (6 x CH₂, cyclohexyl), 50.23 (CO-CH₂), 59.78, 63.50 (2 x Cq), 115.04, 115.83, 129.77 (3 x CH_ar.), 144.19 (C_ar.), 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)

Brown viscous oil, yield 85%, IR (KBr, cm^-1) 2933, 2862 (NH₂), 1747, 1647 (2 x C=O); ¹H NMR: 1.92 (s, 10H, 5 x CH₂, cyclohexyl), 2.83 (t, 2H, J = 7.45 Hz , CH₂-C₆H₅), 2.99 (t, 2H, J = 7.25 Hz, NH-CH₂-CH₂), 4.43 (s, 2H, CO-CH₂), 6.82 (s, 2H, NH₂), 7.17-7.27 (m, 10H, H_ar.); ¹³C NMR: 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 CH_ar.), 138.82, 142.51 (2 x C_ar.), 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)

Brown viscous oil, yield 78%, IR (KBr, cm^-1) 2935, 2862 (NH₂), 1747, 1647 (2 x C=O); ¹H NMR: 1.63-2.21 (m, 10H, 5 x CH₂, 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, H_ar.); (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)

Brown solid, m.p. 100 °C, yield 75%, IR (KBr, cm^-1) 2933, 2858 (NH₂), 1743, 1662 (2 x C=O; ¹H NMR: 1.13-1.34 (m, 10H, 5 x CH₂, cyclohexyl), 2.01 (s, 4H, 2 x CH₂-pyrrolidine), 2.19 (s, 4H, 2 x CH₂-pyrrolidine), 3.67 (s, 2H, CO-CH₂), 6.84 (s, 2H, NH₂), 7.20-7.47 (m, 5H, H_ar.); ¹³C NMR: 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 CH_ar.), 130.49 (C_ar.), 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)

Brown viscous oil, yield 85%, IR (KBr, cm^-1) 2933, 2862 (NH₂), 1747, 1658 (2 x C=O); ¹H NMR: 0.891 (t, 3H, J = 7  , CH₃), 1.25 (s, 2H, CH₂-CH₃), 1.32-1.39 (m, 10H, 5 x CH₂, cyclohexyl), 2.04 (s, 2H, CH₂-CH₂-CH₃), 2.17 (t, 2H, J = 7 Hz, 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, H_ar.); ¹³C NMR: 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 (2 x Cq), 115.12, 116.10, 129.04 (3 x CH_ar.), 144.07 (C_ar.), 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 procedure for the synthesis of 1-(phenyl(heteroalicyclic-1-ylmethyl)amino) cyclohexane-1-carboxamides (6a-f)

To a solution of compound 3 (2.0 g, 0.01 mol) in 96% ethanol, 40% solution of formaldehyde (0.3 g, 0.01 mol) and the appropriate amine was added. The reaction mixture was refluxed for 12 h, then allowed to evaporate under reduced pressure and purified using column chromatography [chloroform (9): ethyl acetate (1)] to afford compounds 6a-f.

1-(phenylamino)-N-(piperidin-1-ylmethyl)cyclohexane-1-carboxamide (6a)

White solid, yield 90%, m.p. 110 °C, IR (KBr, cm^-1) 2924, 1681 (C=O); ¹H NMR: 1.27-1.71 (m, 10H, 5 x CH₂, cyclohexyl), 1.74-2.07 (m, 10H, 5 x CH₂, piperidine), 4.86 (s, 2H, CH₂-piperidine), 6.91 (s, 3H, H_ar.), 7.30 (s, 2H, H_ar.);¹³C NMR: 22.12, 24.73, 29.59, (3 x CH₂, cyclohexyl), 29.83, 30.51, 31.22, (3 x CH₂, piperidine), 66.23 (CH₂-N), 67.88 (Cq), 116.02, 119.16, 129.18 (3 x CH_ar.), 143.37 (C_ar.), 175.57 (CO-NH), (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.

N-((1H-Imidazol-1-yl)methyl)-1-(phenylamino)cyclohexane-1-carboxamide (6b)

Yellow viscous oil, yield 92%; IR (KBr, cm^-1) 2941, 1600 (C=O); ¹H NMR: 1.27-1.29 (m, 10H, 5 x CH₂, cyclohexyl), 5.37 (s, 2H, CH₂-Imidazole), 6.87-6.94 (m, 3H, 3 x CH-imidazole), 7.00-7.49 (m, 5H, H_ar.); ¹³C NMR: 20.68, 27.22, 31.00 (3 x CH₂, cyclohexyl), 61.71 (CH₂-imidazole), 66.22 (Cq), 113.32, 117.65, 118.73, 123.10, 128.37, 130.10 (3 x CH_ar, 3 x CH-imidazole), 145.15 (C_ar.), 175.23 (CO-NH), (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-(Phenylamino)-N-(piperazin-1-ylmethyl)cyclohexane-1-carboxamide (6c)

White solid, m.p. 130 °C, yield 80%; IR (KBr, cm^-1) 2924, 1600 (C=O); ¹H NMR: 1.21 (s, 1H, NH piperazine), 1.71 (t, 2H, J = 10 Hz, CH₂-piperazine), 1.92-2.55 (m, 10H, 5 x CH₂, cyclohexyl), 2.74 (t, 2H, J = 10 Hz, CH₂-piperazine), 4.82 (s, 2H, CH₂-piperazine), 6.88-7.29 (m, 5H, H_ar.); ¹³C NMR: 22.07, 25.10, 29.88 (3 x CH₂, cyclohexyl), 49.13, 54.53 (2 x CH₂-piperazine), 60.58 (CH₂-piperazine), 62.87 (Cq), 116.15, 119.12, 129.14 (3 x CH_ar.), 143.40 (C_ar.), 175.37 (CO-NH), (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.

N-((4-Methylpiperazin-1-yl)methyl)-1-(phenylamino)cyclohexane-1-carboxamide (6d)

Yellow viscous oil, yield 85%; IR (KBr, cm^-1) 2921, 1621 (C=O); ¹H NMR: 1.24-1.25 (m, 10H, 5 x CH₂, cyclohexyl), 2.31 (s, 3H, CH₃), 2.64 (s, 4H, 2 x CH₂– piperazine), 3.51 (s, 4H, 2 x CH₂-piperazine), 4.79 (s, 2H, CH₂-Methyl piperazine), 7.02-7.22 (m, 5H, H_ar.); ¹³C NMR: 15.16, 18.46, 20.12 (3 x CH₂, cyclohexyl), 45.88 (CH₃), 51.72, 54.55 (2 x CH₂-Me-piperazine), 62.27 (CH₂-Methyl piperazine), 68.60 (Cq), 114.57, 120.92, 129.02 (3 x CH_ar.), 146.53 (C_ar.), 177.53 (CO-NH), (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.

N-((4-Ethylpiperazin-1-yl)methyl)-1-(phenylamino)cyclohexane-1-carboxamide (6e)

Brown viscous oil, yield 75%; IR (KBr, cm^-1) 2935, 1621 (C=O); ¹H NMR: 1.09 (t, 3H, J = 4.5 Hz, CH₃), 1.11-1.24 (m, 10H, 5 x CH₂, cyclohexyl), 2.32 (s, 2H, CH₂-CH₃), 2.47 (t, 2H, J = 8.9 Hz, CH₂– piperazine), 2.66 (t, 4H, J = 5.8 Hz, CH₂-piperazine), 4.79 (s, 2H, CH₂-Methyl piperazine), 7.00-7.09 (m, 3H, H_ar.), 7.27 (s, 2H, H_ar); (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.

N-((Cyclohexylamino)methyl)-1-(phenylamino)cyclohexane-1-carboxamide (6f)

Brown solid, m.p.130 °C, yield 80%; IR (KBr, cm^-1) 2929, 1600 (C=O); ¹H NMR: 1.25-1.77 (m, 10H, 5 x CH₂, cyclohexyl), 1.78-2.94 (m, 10H, 5 x CH₂, cyclohexyl), 4.64 (s, 2H, CH₂-NH),  6.92-7.28 (m, 5H, H_ar); ¹³C NMR: 20.09, 25.61, 28.75, 36.87, 43.42, 48.13 (6 x CH₂, 2cyclohexyl), 56.13 (CH₂-NH), 58.15, 62.46 (2 x Cq), 113.08, 128.97, 129.49 (3 x CH_ar.), 145.22 (C_ar.), 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.

Biological Evaluation

The human tumor cell lines were obtained from NCI, MD, USA. All chemicals and solvents were purchased from Sigma-Aldrich.

Cell proliferation assay

The anticancer activities for compounds 5a-m and 6a-f were performed using a standard (MTT)-based colorimetric assay²¹.

Cell Cycle Analysis and Detection of Apoptosis in MCF-7 cell line

Studying the effect of compound 5i on the cell cycle progression using BD FASCCalibur after treatment with 3.25 μM of 5i for 48 h. Then, cell staining with an annexin V-FITC antibody and propidium iodide by FACS²².

Conclusion

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) have been synthesized and screened or their anticancer activity. Compound 5i exhibited the most potent anticancer activity. It inhibited the MCF-7 cell line with IC₅₀ value of 3.25 μM inducing apoptosis at PreG1 and arrested of the cell cycle at G2/M phase.

Conflict of interest

The authers having no conflict.

Acknowledgement

The authors thank the department of pharmaceutical chemistry, College of Pharmaceutical Science and Drug Manufacturing, Misr University for Science and Technology.

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