Synthesis and biological evaluation of certain new cyclohexane-1-carboxamides as apoptosis inducers

Article abstract of DOI:10.13005/ojc/340228

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.

Full text of DOI:10.13005/ojc/340228

ISSN: 0970-020 X
CODEN: OJCHEG
018, Vol. 34, No.(2):
Pg. 825-833
ORIENTAL JOURNAL OF CHEMISTRY
An International Open Free Access, Peer Reviewed Research Journal
2
www.orientjchem.org
Synthesis and Biological Evaluation of Certain new
Cyclohexane-1-carboxamides as Apoptosis Inducers
1
2
WALAA HAMADA ABD-ALLAH * and MOHAMED FATHY ELSHAFIE
1
Pharmaceutical Chemistry Department, Collage of Pharmaceutical Science and Drug Manufacturing,
th
Misr University for Science & Technology, P.O. 77, 6 of October City, Giza, Egypt.
2
Pharmacology, Toxicology Department, Faculty of Pharmacy, Al-Azhar University,
Nasr City Cairo, Egypt.
*
Corresponding author E-mail: Walaa.abdalla@must.edu.eg
http://dx.doi.org/10.13005/ojc/340228
(Received: January 20, 2018; Accepted: March 28, 2018)
ABSTRACT
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
(
1
13
compounds were elucidated via IR, H and C 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.
Keywords: 1,1-Disubstituted cyclohexane, Amides, Synthesis,
Cytotoxic evaluation, Apoptosis.
INTRODUCTION
medicinal chemistry armamentarium with several
bioactive candidates having diverse biological
5
6
Cancer is one of the most prominent
diseases worldwide which represents the second
cause of human mortality after cardiovascular
activities such as antipsychotic , expectorant ,
7
,8
9
anticonvulsant , analgesic and anticancer
activities
1
0-12
. Etoposide (I) and Teniposide (II)
1
diseases . Drug resistance to cancer chemotherapy
contain cyclohexane moiety in their structures and
are used in cancer chemotherapy for the treatment
of lung cancer, acute leukemia and lymphoma
2
is considered a serious trouble . Thus, there is a
3
,4
critical need for new chemotherapeutic agents .
The cyclohexane core generally has enriched the
through
a
cytotoxic
mechanism
of
This is an
.0 International License (https://creativecommons.org/licenses/by-nc-sa/4.0/ ), which permits unrestricted
NonCommercial use, distribution and reproduction in any medium, provided the original work is properly cited.
Open Access article licensed under a Creative Commons Attribution-NonCommercial-ShareAlike
4

8
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ABD-ALLAH, Elshafie et al., Orient. J. Chem., Vol. 34(2), 825-833 (2018)
1
3-15
DNA- topoisomerase II inhibition . Moreover, the
These findings have encouraged us to
aminoacyl pharmacophore chain and amide moiety prepare the target compounds 5a-m and 6a-f
were included in the structural frame of different through molecular hybridization tactic of two or more
antitumor compounds, III and IV (Fig. 1), which were pharmacophore moieties in one molecule aiming
1
6-19
20
found to possess antitumor activity
.
to improve the pharmacological profile .
OH
H
O
OH
O
S
O
O
H
O
HO
O
O
H
H
H
O
O
O
O
O
A
H
O
O
O
O
O
OH
Etopside (I)
Tenopside (II)
O
O
B
N
N
O
N
N
N
N
N
O
N
N
(
III)
N
N
(
IV)
O
O
O
C
N
NH
H
2
N
R
1
R
2
N
H
O
5a-m
6a-f
Fig. 1. (A) Structures of Etoposide (I) and Teniposide (II); (B) Antitumor biocandidates bearing aminoacyl
moieties; (C) target compounds containing the pharmacophoric features
RESULTS AND DISCUSSION
sulfuric acid at room temperature to produce the
amidic compound 3.
Chemistry
The preparation of the ultimate
The penultimate intermediate 4 was
compounds 5a–m and 6a–f as well as the achieved by acetylation of compound 3.
intermediates 1–4 is illustrated in Scheme 1. Subsequently, compound 4 was subjected to the
Cyclohexanone was reacted with potassium reaction with different amines to afford 5a-m, while
cyanide and aniline in glacial acetic acid to produce compound 3 was reacted with formaldehyde and
the nitrile derivative 1 which was hydrolyzed using different amines to afford 6a-f.

ABD-ALLAH, Elshafie et al., Orient. J. Chem., Vol. 34(2), 825-833 (2018)
827
Reagents and conditions: i) Cyclohexanone,
potassium cyanide, acetic acid glacial, r.t, 24 h ii)
H SO , r.t, 48 h, iii) ClCOCH Cl, CHCl , r.t , 24 h, iv)
selective and having potential cytotoxicity towards
MCF-7 adenocarcinoma with IC50 values range
3
.25-36.8 μM as compared with doxorubicin
2
4
2
3
(
IC50 value of 6.77 μM). Compound 5i showed the
ethanol, appropriate amine, 12 h, reflux v) HCHO,
ethanol, appropriate amine, 12 h, reflux.
most potent biological activity with IC value = 3.25 μM.
50
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
Biological Evaluation
Antiproliferation assay
The antitumor activity for compounds
5
a-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
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-7IC50 μM
HepG2IC50 μM
A549IC50 μM
Caco-2IC50 μM
5
5
5
5
5
a
b
c
d
e
26.15
3.68
50.2
10.61
15.02
60.3
56.4
43.32
33.88
58.3
11.5
31.28
20.71
12.08
13.82
-
35.4
7.98
9.97
42.7
40.6
39.74
30.3
31.29
6.95
15.64
14.59
8.51
11.83
-
30.21
5.46
7.21
28.41
31.22
20.08
9.13
22.68
3.25
14.12
52.40
50.22
4.33
5f
5
5
5
5
5
5
5
6
6
6
6
6
g
h
i
25.43
47.23
8.98
j
13.59
5.48
4.58
22.34
13.66
11.25
10.53
-
k
l
m
a
b
c
d
e
6.48
36.8
23.51
-
16.46
-
31.21
-
28.67
-
17.05
26.13
22.89
6.77
15.91
21.74
17.54
3.07
26.22
42.09
31.6
0.887
39.52
51.43
44.12
2.78
6f
Doxorubicin
(-): No activity
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.

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ABD-ALLAH, Elshafie et al., Orient. J. Chem., Vol. 34(2), 825-833 (2018)
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
%S
%
G0-G1
% G2-M
% Apoptosis
Comment
5
i
7.66
71.7
24.77
22.45
46.17
5.23
21.4
0.62
PreG1 apoptosis & cell growth
arrest@G2/M Control pattern
Cont. MCF-7
Fig. 2. Graphical view of % of DNA content in G0/G1, S, and G2/M phase
for control and compound 5i
Fig. 3. Cell cycle analysis and apoptotic assay graphs for control and compound 5i at concentration
.25 μM for 48 h on MCF-7 cell line
3

ABD-ALLAH, Elshafie et al., Orient. J. Chem., Vol. 34(2), 825-833 (2018)
829
MATERIALS AND METHODS
General procedure for the synthesis of
-(N-phenyl-2-(heteroalicyclic-1-yl)acetamido)-
1
Chemistry
General
cyclohexane-1-carboxamides (5a-m)
To a solution of 4 (1.32 g, 0.0045 mol) in
ethanol (30 mL), the appropriate amine derivative
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 ( H-NMR as well as 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
(
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 SO
1
13
2
4
and evaporation under the reduced pressure to
produce 5a-m.
(
v/v) as a mobile phase.
1
-(N-Phenyl-2-(piperidin-1-yl)acetamido)cyclohexane
1-carboxamide (5a)
White solid, m.p. 120 °C, yield 75%; IR
-
Synthesisof1-(phenylamino)cyclohexanecarbonitrile(2)
Compound 2 was prepared as showed in
8
-1
(
KBr, cm ) 2924, 2841 (NH ), 1635, 1621 (2 x C=O);
2
literatures .
1
H NMR: 1.12 (s, 10H, 5 x CH , cyclohexyl), 1.78 (s,
2
1
6
0H, 5 x CH , piperidine), 3.16 (s, 2H, CO-CH ),
2
2
Synthesis of (phenylamino)cycloakanecarboxamide (3)
13
.53 (s, 2H, NH ), 7.18-7.53 (m, 5H, H .); C NMR:
Compound 3 was prepared as showed in
2
ar
8
20.96, 22.34, 22.87 (3 x CH , cyclohexyl), 25.08,
2
literatures .
2
5
1
5.66 (2 x CH -piperidine), 58.81 (CH -piperidine),
2
2
4.44 (CO-CH ), 118.48, 128.85, 129.67 (3 x CH ),
Synthesis of 1-(2-chloro-N-phenylacetamido)
cyclohexane-1-carboxamide (4)
2
ar.
44.17 (C ), 168.55 (CO-CH ), 179.90 (CO-NH );
ar.
2
2
+
MS (EI) m/z (%): 343.47 ([M] , 1.83), 47.9 (100).
Anal. calcd. for C H N O : C, 69.94; H, 8.51;
Chloroacetyl chloride (1.317g, 0.95 mL,
0
.01166 mol) was added to a solution of 3
20 29
3
2
N,12.23; O, 9.32. Found: C, 69.74; H, 8.72; N, 12.41;
O, 9.33.
(
2.1g, 0.0097 mol) in chloroform (30 mL). The
mixture was allowed for stirring at room temperature
for 24 h, then adding an aqueous solution of 10%
NaOH (2 x 30 mL) then separation of the organic
layer, drying over anhydrous Na SO4 and
evaporation under reduced pressure to afford
1
-(N-Phenyl-2-(pyridin-1(2H)-yl) acetamido)
cyclohexane-1-carboxamide (5b)
Yellow viscous oil, yield 85%, IR
2
-
1
2
.5 g of 4 as pale yellow oil in 89.2% yield which is
(KBr, cm ) 2935.13, 2860.88 (NH ), 1742.37,
2
solidified upon standing at room temperature, m.p.
1
1
1660.41 (2 x C=O); H NMR: 1.14-1.35 (s, 10H, 5 x
-
1
70 33/°C. IR (KBr, cm ) 2926.23, 2851.17 (NH ),
2
CH , cyclohexyl), 3.62 (s, 2H, CH -pyridine), 3.64
2 2
1
1
645, 1625 (2 x C=O) ; H NMR (CDCl ) 1.34-1.52
3
(s, 2H, CO-CH ), 6.03 (s, 2H, NH ), 6.63 (d, 1H, J =
2 2
(
m, 10H, 5 x CH , cyclohexyl), 3.26 (s, 2H, CH -Cl),
2
2
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),
13
6
.99 (s, 2H,NH ), 7.25-7.32 (m, 5H, H .); C NMR
2
a
r
(
CDCl ) δ ppm 20.79, 22.91, 32.82 (3 x CH ,
3
2
7.10 (s, 1H, CH-pyridine), 7.37-7.91 (m, 5H, H );
ar.
cyclohexyl), 40.41 (CH -Cl), 66.60 (Cq), 128.60,
13
2
C NMR: 21.07, 22.20, 25.11 (3 x CH , cyclohexyl),
2
1
28.77, 130.23 (3CH .), 139.16 (C .), 167.16
52.21 (CH -pyridine), 60.22 (CO-CH ), 64.43 (Cq),
ar
ar
2
2
(
CO-CH ), 178.60 (CO-NH ); MS (EI) m/z (%):
118.98, 125.94, 131.54, (3 x CH ), 143.86 (C ),
2
2
ar. ar.
+
2
94.78 ([M] , 1.60, ), 250 (100). (EI) m/z (%): 294.78
167.79 (CO-CH ), 179.66 (CO-NH ), MS (EI) m/z
2 2
+
+
([M] , 5), 250 (100). Anal. calcd. for C H ClN O :
(%): 339.41 ([M] , 1.19), 174 (100). Anal. calcd. for
C H N O : C, 70.77; H, 7.42; N, 12.38; O, 9.43.
15
19
2
2
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.
20
25
3
2
Found: C, 70.57; H, 7.44; N, 12.33.

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ABD-ALLAH, Elshafie et al., Orient. J. Chem., Vol. 34(2), 825-833 (2018)
1
1-(2-(3-Hydroxypyridin-1(2H)-yl)-N-phenylacetamido)
H NMR: 1.24-1.60 (m, 11H, 5 x CH , cyclohexyl,
2
cyclohexane-1-carboxamide (5c)
NH), 1.96 (s, 4H, 2 x CH -piperazine), 2.07 (s, 4H, 2
2
Yellow viscous oil, yield 90%, m.p. 92 °C, x CH -piperazine), 3.02 (s, 2H, CO-CH ), 6.93
2
2
-
1
13
IR (KBr, cm ) 2932, 2859 (NH ), 1718, 1655 (s, 2H, NH ), 7.15-7.42 (m, 5H, H .); C NMR: 21.04,
2
2
ar
1
(
2 x C=O); H NMR: 1.34-2.19 (m, 10H, 5 x CH , 25.13, 31.20 (3 x CH , cyclohexyl), 41.96, 44.45
2
2
cyclohexyl), 3.71 (s, 2H, CH -pyridine), 3.92 (s, 2H, (2 x CH -piperazine), 59.99 (CO-CH ), 60.38 (Cq),
2
2
2
CO-CH ), 6.65 (s, 2H, NH ), 6.67 (s, 1H, 116.04, 118.85, 129.01 (3 x CH .), 144.15 (C .),
2
2
ar
ar
CH-pyridine), 7.13 (s, 1H, CH-pyridine), 7.49 171.15 (CO-CH ), 179.61 (CO-NH ), (EI) m/z (%):
2
2
+
(
s, 1H, CH-pyridine), 8.06 (s, 3H, H ), 8.33 (s, 2H, 344.46 ([M] , 3.03), 174 (100). Anal. calcd. for
H ), 10.04 (br. s, 1H, OH); C NMR: 22.53, 25.73, C H N O : C, 66.25; H, 8.19; N, 16.27. Found: C,
ar.
13
ar.
19 28
4
2
3
5
1
1
1
3
7
3.21 (3 x CH , cyclohexyl), 48.91 (CH -pyridine), 66.33; H, 8.22; N, 16.22.
2
2
5.34 (CO-CH ), 68.71 (Cq), 94.23 (CH-pyridine),
2
16.12, 125.63, 127.11 (3 x CH ), 134.71 (C ), 1-(2-(4-Methylpiperazin-1-yl)-N-phenylacetamido)
ar.
ar.
36.44 (CH-N), 156.11 (C-OH), 167.21 (CO-CH ), cyclohexane-1-carboxamide (5g)
2
+
81.42 (CO-NH ); (EI) m/z (%): 355.44 ([M] , 2.5),
Yellow viscous oil, yield 75%, IR (KBr,
2
36 (100). Anal. calcd. for C H N O : C, 67.58; H,
-1
20
25
3
3
cm ) 2935, 2855 (NH ), 1739, 1668 (2 x C=O);
2
.09; N, 11.82; O, 13.50. Found: C, 67.48; H, 7.12;
1
H NMR: 1.25-1.61 (m, 10H, 5 x CH , cyclohexyl),
2
N, 11.71.
1.97 (s, 3H, CH ), 2.29 (s, 4H, 2 x CH -piperazine),
3 2
2
.68 (s, 4H, 2 x CH -piperazine), 3.21( s, 2H, CH -
2
2
1
-(2-Morpholino-N-phenylacetamido)cyclohexane
Me-piperazine), (s, 2H, CO-CH ), 6.81 (s, 2H, NH ),
2
2
-
1-carboxamide (5d)
13
6.91-7.52 (m, 5H, H ); C NMR: 21.05, 25.12, 31.19
-
1
ar.
Pale yellow oil, yield 90%, IR (KBr, cm )
1
(3 x CH , cyclohexyl), 45.75 (CH ), 52.70, 54.52
2 3
2
1
906, 2860 (NH ), 1716, 1658 (2 x C=O); H NMR:
2
(
2 x CH -piperazine), 54.65 (CO-CH ), 66.02 (Cq),
2 2
.51-2.09 (m, 10H, 5 x CH , cyclohexyl), 2.51 (t, 2H,
2
1
1
3
18.90, 129.02, 130.71 (3 x CH ), 144.12 (C .),
J = 6 Hz, CH -morpholine), 3.71 (t, 2H, J = 6 Hz,
CH -morpholine), 3.88 (s, 2H, CO-CH ), 6.93 (s, 2H,
NH ), 7.15-7.19 (m, 3H, H ), 7.33 (s, 2H, H ); (EI)
m/z (%): 345.44 ([M] ,0.81), 174 (100). Anal. calcd.
for C H N O : C, 66.06; H, 7.88; N, 12.16. Found:
ar. ar
2
69.51 (CO-CH ), 179.57 (CO-NH ), (EI) m/z (%):
2
2
2
2
+
58.49 ([M] , 1.8), 113 (100). Anal. calcd. for
2
ar.
ar.
+
C H N O : C, 67.01; H, 8.44; N, 15.63. Found: C,
20 30 4 2
67.22; H, 8.52; N, 15.73.
19
27
3
3
C, 66.21; H, 7.75; N, 12.24.
1
-(2-(4-(7-Chloroquinolin-4-yl)piperazin-1-yl)-N-
phenylacetamido)cyclohexane-1-carboxamide (5h)
1
-(2-(1H-Imidazol-1-yl)-N-phenylacetamido)
cyclohexane-1-carboxamide (5e)
Brown viscous oil, yield 75%, IR
-
1
Yellow viscous oil, yield 90%, IR (KBr, cm ) 2935, 2860 (NH ), 1739, 1654 (2 x C=O);
2
-
1
1
(
KBr, cm ) 2933, 2852 (NH ), 1739, 1669 (2 x C=O);
H NMR: 1.26-1.49 (m, 10H, 5 x CH , cyclohexyl),
2
2
1
H NMR: 1.17-1.54 (s, 10H, 5 x CH , cyclohexyl),
2
2.18 (s, 4H, 2 x CH -piperazine), 3.64 (s, 4H, 2 x
2
3
.63 (s, 2H, CO-CH ), 6.61 (s, 2H, NH ), 7.03 (s, 3H,
2
2
CH -piperazine), 3.81 (s, 2H, CO-CH ), 6.89 (br. s,
2
2
13
CH-imidazole), 7.67 (s, 5H, H ); C NMR: 20.97,
ar.
2H, NH ), 7.14 (d, 1H, J = 6.75 Hz, CH quinoline),
2
2
5
1
1
3
5.12, 31.24 (3 x CH , cyclohexyl), 53.12 (CO-CH ),
9.99 (Cq), 115.19, 115.98, 121.51, 129.02, 129.24,
30.13 (3 x CH-imidazole, 3 x CH ), 144.17 (C ),
2
2
7.19 (d, 1H, J = 7.5 Hz, CH quinoline), 7.22 (s, 5H,
H ), 7.35 (s, 2H, CH quinoline), 7.46 (d, 1H, J = 5,
ar.
ar.
ar.
13
CH quinoline); C NMR: 22.35, 25.10, 33.58 (3 x
68.21 (CO-CH ), 180.13 (CO-NH ), (EI) m/z (%):
2
2
+
CH , cyclohexyl), 51.21, 54.32 (2 x CH -piperazine),
26.40 ([M] , 15), 174 (100). Anal. calcd. for
2
2
5
1
1
1
5
6.91 (CO-CH ), 67.81 (Cq), 128.87, 129.11, 129.51,
C H N O : C, 66.24; H, 7.79; N, 17.17. Found: C,
2
18
22
4
2
29.61, 129.87, 130.33, 133.42, 133.63 (6 x CH ),
66.21; H, 7.81; N, 17.25.
ar.
35.61, 137.82, 152.81, 153.53, 154.22 (5 x C ),
ar.
66.21 (CO-CH ), 178.93 (CO-NH ); (EI) m/z (%):
1
-(N-Phenyl-2-(piperazin-1-yl)acetamido)
2
2
+
06 ([M] , 7.2), 288 (100). Anal. calcd. for
cyclohexane-1-carboxamide (5f)
Brown viscous oil, yield 79%, IR (KBr, C H ClN O : C, 66.46; H, 6.37; Cl, 7.01; N, 13.84.
28
32
5
2
-1
cm ) 2935, 2857 (NH ), 1739.48, 1649.81 (2 x C=O); Found: C, 66.42; H, 6.36; Cl, 7.11; N, 13.82.
2

ABD-ALLAH, Elshafie et al., Orient. J. Chem., Vol. 34(2), 825-833 (2018)
831
1
-(2-(Cyclohexylamino)-N-phenylacetamido)
cyclohexane-1-carboxamide (5i)
Brown viscous oil, yield 80%, IR
CH -pyrrolidine), 52.32 (CH -pyrrolidine), 56.93
2
2
(CO-CH ), 65.61 (Cq), 129.10, 129.41, 129.56 (3 x
2
CH ), 130.49 (C ), 162.54 (CO-CH ), 179.81 (CO-
ar.
ar.
2
-1
+
(
KBr, cm ) 2933, 2856 (NH ), 1745, 1647 (2 x C=O);
NH ), (EI) m/z (%): 329.44 ([M] ,0.75), 174 (100).
2
2
1
H NMR: 1.02-1.18 (m, 10H, 5 x CH , cyclohexyl),
C H N O : C, 69.27; H, 8.26; N, 12.76. Found: C,
2
19 27
3
2
1
.45-1.85 (m, 10H, 5 x CH , cyclohexyl), 2.78 (s,
69.22; H, 8.31; N, 12.82.
2
2
H, CO-CH ), 5.51 (br. s, 1H, NH), 6.56 (s, 2H, NH );
2
2
13
C NMR: 21.73, 24.53, 25.18, 29.61, 30.21, 32.40
6 x CH , cyclohexyl), 50.23 (CO-CH ), 59.78, 63.50
1-(2-(Butylamino)-N-phenylacetamido)
cyclohexane-1-carboxamide (5m)
(
2
2
-
1
(
2 x Cq), 115.04, 115.83, 129.77 (3 x CH ), 144.19
Brown viscous oil, yield 85%, IR (KBr, cm )
ar.
1
(C ), 162.81 (CO-CH ), 179.88 (CO-NH ), (EI) m/z
2933, 2862 (NH ), 1747, 1658 (2 x C=O); H NMR:
ar.
2
2
2
+
(%): 357.50 ([M] , 0.77), 174 (100). C H N O : C,
0.891 (t, 3H, J = 7 , CH ), 1.25 (s, 2H, CH -CH ),
21
31
3
2
3
2
3
70.55; H, 6.74; N, 11.75. Found: C, 70.43; H, 6.72;
1.32-1.39 (m, 10H, 5 x CH , cyclohexyl), 2.04 (s,
2
N, 11.72.
2H, CH -CH -CH ), 2.17 (t, 2H, J = 7 Hz, NH-CH ),
2
2
3
2
2
.89 (br. s, 1H, NH), 3.21 (s, 2H, CO-CH ), 6.81 (s,
2
13
1
-(2-(Phenethylamino)-N-phenylacetamido)
2H, NH ), 7.31-7.62 (m, 5H, H ); C NMR: 21.04
2
a
r
.
cyclohexane-1-carboxamide (5j)
(CH ), 13.54, 22.47, 24.97, 29.50, 30.93 (3 x CH ,
3
2
-1
Brown viscous oil, yield 85%, IR (KBr, cm )
cyclohexyl, -CH -CH -CH ), 45.84 (NH-CH ), 51.32
2 2 3 2
1
2
1
7
933, 2862 (NH ), 1747, 1647 (2 x C=O); H NMR:
(CO-CH ), 60.39 (2 x Cq), 115.12, 116.10, 129.04
2
2
.92 (s, 10H, 5 x CH , cyclohexyl), 2.83 (t, 2H, J =
(3 x CH ), 144.07 (C ), 167.33 (CO-CH ), 178.91
2
ar.
ar.
2
+
.45 Hz , CH -C H ), 2.99 (t, 2H, J = 7.25 Hz, NH-
(CO-NH ), (EI) m/z (%): 367.95 ([M] ,1.68), 174
2
6
5
2
CH -CH ), 4.43 (s, 2H, CO-CH ), 6.82 (s, 2H, NH ),
(100). C H ClN O : C, 62.03; H, 8.22; Cl, 9.64; N,
11.42. Found: C, 62.21; H, 8.32; N, 11.35.
2
2
2
2
19 30
3
2
13
7
2
4
1
.17-7.27 (m, 10H, H ); C NMR: 22.81, 23.17,
ar.
5.16 (3 x CH , cyclohexyl), 35.52 (CH -C H ),
2
2
6
5
2.46 (NH-CH -CH ) 52.51 (CO-CH ), 73.12 (Cq),
General procedure for the synthesis of 1-(phenyl
2
2
2
15.15, 116.06, 126.44, 127.51, 128.58, 128.84 (6
(
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
x CH ), 138.82, 142.51 (2 x C ), 161.64 (CO-CH ),
ar.
ar.
2
+
1
1
81.71 (CO-NH ), (EI) m/z (%): 379.50 ([M] , 5.29),
2
74 (100). C H N O : C, 72.79; H, 7.70; N, 11.07.
23
29
3
2
Found: C, 72.81; H, 7.85; N, 11.15.
(
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
-(N-Phenyl-2-(1H-pyrrol-1-yl)acetamido)
cyclohexane-1-carboxamide (5k)
-1
Brown viscous oil, yield 78%, IR (KBr, cm )
1
2
1
2
935, 2862 (NH ), 1747, 1647 (2 x C=O); H NMR:
.63-2.21 (m, 10H, 5 x CH , cyclohexyl), 3.77 (s,
2
2
H, CO-CH ), 5.61 (s, 2H, NH ), 6.63-6.80 (m, 4H,
2
2
1
-(phenylamino)-N-(piperidin-1-ylmethyl)
cyclohexane-1-carboxamide (6a)
White solid, yield 90%, m.p. 110 °C, IR
CH-pyrrole), 7.28-7.46 (m, 5H, H ); (EI) m/z (%):
ar.
+
325.41 ([M] , 3.45), 174 (100). C H N O : C, 70.13;
19
23
3
2
H, 7.12; N, 12.91. Found: C, 70.25; H, 7.22; N, 12.85.
-
1
1
(
KBr, cm ) 2924, 1681 (C=O); H NMR: 1.27-1.71
1
-(N-Phenyl-2-(pyrrolidin-1-yl)acetamido)
cyclohexane-1-carboxamide (5l)
Brown solid, m.p. 100 °C, yield 75%, IR
(m, 10H, 5 x CH , cyclohexyl), 1.74-2.07 (m, 10H, 5
2
x CH , piperidine), 4.86 (s, 2H, CH -piperidine), 6.91
2
2
13
(s, 3H, H ), 7.30 (s, 2H, H ); C NMR: 22.12, 24.73,
ar.
ar.
-
1
(
1
KBr, cm ) 2933, 2858 (NH ), 1743, 1662 (2 x C=O;
29.59, (3 x CH , cyclohexyl), 29.83, 30.51, 31.22,
2
2
H NMR: 1.13-1.34 (m, 10H, 5 x CH , cyclohexyl),
(3 x CH , piperidine), 66.23 (CH -N), 67.88 (Cq),
2
2
2
2
.01 (s, 4H, 2 x CH -pyrrolidine), 2.19 (s, 4H, 2 x
116.02, 119.16, 129.18 (3 x CH ), 143.37 (C ),
2
ar. ar.
+
CH -pyrrolidine), 3.67 (s, 2H, CO-CH ), 6.84 (s, 2H,
NH ), 7.20-7.47 (m, 5H, H ); C NMR: 21.07, 22.41,
175.57 (CO-NH), (EI) m/z (%): 315.23 ([M] , 5.3),
189 (100). C H N O: C, 72.34; H, 9.27; N, 13.32.
2
2
13
2
ar.
19 29
3
2
5.08, 33.63, 30.21 (3 x CH , cyclohexyl,
Found: C, 72.41; H, 9.25; N, 13.45.
2

8
32
ABD-ALLAH, Elshafie et al., Orient. J. Chem., Vol. 34(2), 825-833 (2018)
N-((1H-Imidazol-1-yl)methyl)-1-(phenylamino) 3H, H ), 7.27 (s, 2H, H ); (EI) m/z (%): 344.50
ar.
ar
+
(
[M] ,0.75), 127 (100). C H N O: C, 69.73; H, 9.36;
cyclohexane-1-carboxamide (6b)
20 32 4
-1
N, 16.26. Found: C, 69.77; H, 9.42; N, 16.35.
Yellow viscous oil, yield 92%; IR (KBr, cm )
1
2
941, 1600 (C=O); H NMR: 1.27-1.29 (m, 10H, 5 x
N-((Cyclohexylamino)methyl)-1-(phenylamino)
cyclohexane-1-carboxamide (6f)
CH , cyclohexyl), 5.37 (s, 2H, CH -Imidazole), 6.87-
2
2
6
.94 (m, 3H, 3 x CH-imidazole), 7.00-7.49 (m, 5H,
1
3
Brown solid, m.p. 130 °C, yield 80%; IR
H ); C NMR: 20.68, 27.22, 31.00 (3 x CH2,
ar.
-
1
1
(
KBr, cm ) 2929, 1600 (C=O); H NMR: 1.25-1.77
cyclohexyl), 61.71 (CH -imidazole), 66.22 (Cq),
2
(
m, 10H, 5 x CH , cyclohexyl), 1.78-2.94 (m, 10H, 5
1
13.32, 117.65, 118.73, 123.10, 128.37, 130.10
2
x CH , cyclohexyl), 4.64 (s, 2H, CH -NH), 6.92-7.28
(
(
3 x CH , 3 x CH-imidazole), 145.15 (C ), 175.23
CO-NH), (EI) m/z (%): 298.39 ([M] ,1.38).
2
2
ar
ar.
13
+
(m, 5H, H ); C NMR: 20.09, 25.61, 28.75, 36.87,
ar
4
3.42, 48.13 (6 x CH , 2cyclohexyl), 56.13 (CH -
2 2
C H N O: C, 68.43; H, 7.43; N, 18.78. Found: C,
17
22
4
NH), 58.15, 62.46 (2 x Cq), 113.08, 128.97, 129.49
68.38; H, 7.55; N, 18.91.
(
3 x CH ), 145.22 (C ), 177.97 (CO-NH ), (EI) m/z
ar. ar. 2
+
(%): 329.49 ([M] , 5.75), 106 (100). C H N O: C,
20 31 3
1
-(Phenylamino)-N-(piperazin-1-ylmethyl)
72.91; H, 9.48; N, 12.75. Found: C, 72.85; H, 9.37;
cyclohexane-1-carboxamide (6c)
White solid, m.p. 130 °C, yield 80%; IR
N, 12.83.
-
1
1
(
KBr, cm ) 2924, 1600 (C=O); H NMR: 1.21 (s, 1H,
Biological Evaluation
NH piperazine), 1.71 (t, 2H, J = 10 Hz, CH2-
piperazine), 1.92-2.55 (m, 10H, 5 x CH , cyclohexyl),
The human tumor cell lines were obtained
2
2
.74 (t, 2H, J = 10 Hz, CH -piperazine), 4.82 (s, 2H, from NCI, MD, USA. All chemicals and solvents were
2
13
CH -piperazine), 6.88-7.29 (m, 5H, H ); C NMR: purchased from Sigma-Aldrich.
2
ar.
2
5
6
1
2.07, 25.10, 29.88 (3 x CH , cyclohexyl), 49.13,
2
4.53 (2 x CH -piperazine), 60.58 (CH -piperazine),
2
2
Cell proliferation assay
The anticancer activities for compounds
a-m and 6a-f were performed using a standard
2.87 (Cq), 116.15, 119.12, 129.14 (3 x CH ),
ar.
43.40 (C ), 175.37 (CO-NH), (EI) m/z (%): 345.44
ar.
5
+
+
([M] ,0.81), (EI) m/z (%): 316.45 ([M] , 20.23), 99
100). C H N O: C, 68.32; H, 8.92; N, 17.71. Found:
21
(MTT)-based colorimetric assay .
(
18
28
4
C, 68.35; H, 8.85; N, 17.75.
Cell Cycle Analysis and Detection of Apoptosis
in MCF-7 cell line
N-((4-Methylpiperazin-1-yl)methyl)-1-(phenylamino)
cyclohexane-1-carboxamide (6d)
Studying the effect of compound 5i on the
cell cycle progression using BD FASC Calibur after
treatment with 3.25 μM of 5i for 48 h. Then, cell
staining with an annexin V-FITC antibody and
-1
Yellow viscous oil, yield 85%; IR (KBr, cm )
1
2
921, 1621 (C=O); H NMR: 1.24-1.25 (m, 10H, 5 x
CH , cyclohexyl), 2.31 (s, 3H, CH ), 2.64 (s, 4H, 2 x
2
3
22
propidium iodide by FACS .
CH - piperazine), 3.51 (s, 4H, 2 x CH -piperazine),
2
2
4
5
.79 (s, 2H, CH -Methyl piperazine), 7.02-7.22 (m,
2
CONCLUSION
13
H, H ); C NMR: 15.16, 18.46, 20.12 (3 x CH2,
ar.
cyclohexyl), 45.88 (CH ), 51.72, 54.55 (2 x CH -
3
2
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
Me-piperazine), 62.27 (CH -Methyl piperazine),
2
6
1
8.60 (Cq), 114.57, 120.92, 129.02 (3 x CH ),
ar.
46.53 (C ), 177.53 (CO-NH), (EI) m/z (%): 330.48
ar.
+
([M] , 11.4), 76.93 (100). C H N O: C, 69.05; H, synthesized and screened for their anticancer
19
30
4
9
.15; N, 16.95. Found: C, 69.12; H, 9.23; N, 16.85. activity. Compound 5i exhibited the most potent
anticancer activity. It inhibited the MCF-7 cell line
N-((4-Ethylpiperazin-1-yl)methyl)-1-(phenylamino) with IC50 value of 3.25 μM inducing apoptosis at
PreG1 and arrested of the cell cycle at G2/M phase.
ACKNOWLEDGEMENT
cyclohexane-1-carboxamide (6e)
-1
Brown viscous oil, yield 75%; IR (KBr, cm )
1
2
935, 1621 (C=O); H NMR: 1.09 (t, 3H, J = 4.5 Hz,
CH ), 1.11-1.24 (m, 10H, 5 x CH , cyclohexyl), 2.32
3
2
The authors thank the department of
(
s, 2H, CH -CH ), 2.47 (t, 2H, J = 8.9 Hz, CH - pharmaceutical chemistry, College of Pharmaceutical
2
3
2
piperazine), 2.66 (t, 4H, J = 5.8 Hz, CH -piperazine), Science and Drug Manufacturing, Misr University
2
4
.79 (s, 2H, CH -Methyl piperazine), 7.00-7.09 (m, for Science and Technology.
2

ABD-ALLAH, Elshafie et al., Orient. J. Chem., Vol. 34(2), 825-833 (2018)
833
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