Anticancer activity of newly synthesized 1,1-disubstituted cyclohexane-1-carboxamides: in vitro caspases mediated apoptosis activators in human cancer cell lines and their molecular modeling

Article abstract of DOI:10.1002/ddr.21573

Novel 1,1-disubstituted cyclohexane-1-carboxamides 6a-h, 7a-e, and 8a-b were designed and synthesized as apoptotic inducers. Cytotoxicity test revealed that some compounds have strong to moderate effect, while others displayed weak action against different cancer cell lines including, MCF-7, HepG2, A549, and HTC-116. A549 carcinoma cell line exhibited higher sensitivity toward all synthesized candidates especially compounds 6a and 8a which offered the lowest IC₅₀ values 3.03 and 5.21 μM, respectively, relative to the positive control doxorubicin with IC₅₀ value of 3.01 μM. Compared to doxorubicin treatment, compounds 6a and 8a induced caspases-3, -8, and -9 activities and G2/M growth arrest in A549 carcinoma cell line. The expression levels of p53 (tumor suppressor protein that in humans is encoded by the TP53 gene), Bax (apoptosis regulator protein in humans that is encoded by bax gene), and the Bax/Bcl-2 ratio were all higher than those in doxorubicin-treated cells (Bcl-2, B-cell lymphoma 2, encoded in humans by the Bcl-2 gene). Additionally, compounds 6a and 8a appeared to exhibit higher selectivity against MCF-10 human breast normal cell line. The synthesized congeners could be considered as potent apoptotic inducers interfering with extrinsic and intrinsic apoptotic pathways. Moreover, compound 6a was able to form complex with zinc ions as indicated by UV spectrophotometry which revealed its ability for being caspase activator. Molecular docking studies expected the interactions and binding modes of the synthesized inhibitors in the caspase-3 active site.

Full text of DOI:10.1002/ddr.21573

Received: 26 April 2019
DOI: 10.1002/ddr.21573
Revised: 26 June 2019
Accepted: 4 July 2019
R E S E A R C H A R T I C L E
Anticancer activity of newly synthesized 1,1-disubstituted
cyclohexane-1-carboxamides: in vitro caspases mediated
apoptosis activators in human cancer cell lines and their
molecular modeling
Walaa Hamada Abd-Allah1
| Asmaa Salman2 | Samah Sabry Saad3
1Pharmaceutical Chemistry Department,
College of Pharmaceutical Science and Drug
Manufacturing, Misr University for Science &
Technology, Giza, Egypt
Abstract
Novel 1,1-disubstituted cyclohexane-1-carboxamides 6a-h, 7a-e, and 8a-b were
designed and synthesized as apoptotic inducers. Cytotoxicity test revealed that some
compounds have strong to moderate effect, while others displayed weak action
against different cancer cell lines including, MCF-7, HepG2, A549, and HTC-116.
A549 carcinoma cell line exhibited higher sensitivity toward all synthesized candi-
dates especially compounds 6a and 8a which offered the lowest IC50 values 3.03 and
5.21 μM, respectively, relative to the positive control doxorubicin with IC50 value of
3.01 μM. Compared to doxorubicin treatment, compounds 6a and 8a induced
caspases-3, -8, and -9 activities and G2/M growth arrest in A549 carcinoma cell line.
The expression levels of p53 (tumor suppressor protein that in humans is encoded by
the TP53 gene), Bax (apoptosis regulator protein in humans that is encoded by bax
gene), and the Bax/Bcl-2 ratio were all higher than those in doxorubicin-treated cells
(Bcl-2, B-cell lymphoma 2, encoded in humans by the Bcl-2 gene). Additionally,
compounds 6a and 8a appeared to exhibit higher selectivity against MCF-10 human
breast normal cell line. The synthesized congeners could be considered as potent
apoptotic inducers interfering with extrinsic and intrinsic apoptotic pathways.
Moreover, compound 6a was able to form complex with zinc ions as indicated by
UV spectrophotometry which revealed its ability for being caspase activator.
Molecular docking studies expected the interactions and binding modes of the
synthesized inhibitors in the caspase-3 active site.
2Department of Medicinal and Pharmaceutical
Chemistry (Pharmacology Group), National
Research Centre, Giza, Egypt
3Pharmaceutical Analytical Chemistry
Department, College of Pharmaceutical
Science and Drug Manufacturing, Misr
University for Science & Technology, Giza,
Egypt
Correspondence
Walaa Hamada Abd-Allah, Pharmaceutical
Chemistry Department, College of
Pharmaceutical Science and Drug,
Manufacturing, Misr University for Science &
Technology, P.O. 77, 6th of October City,
Giza, Egypt.
Email: walaa.abdalla@must.edu.eg
K E Y W O R D S
1,1-disubstituted cyclohexane, apoptosis, cytotoxic evaluation, synthesis
death receptor (Zapata et al., 2001), and the mitochondrial pathway
(intrinsic) that upon activation results in cytochrome-C release from the
1
| INTRODUCTION
mitochondria and activation of the death signal (Zapata et al., 2001).
The two pathways join to a final common pathway including the activa-
tion of a cascade of cysteine aspartate protease enzymes known as
caspases which cleave regulatory and structural molecules, causing cell
death (Zapata et al., 2001). Caspases are a set of enzymes that involved
Several chemotherapeutic agents destroy cancer via inducing apoptosis
which is considered the main mechanism in cell death if DNA damage
is not repaired (Lowe & Lin, 2000). There are two main pathways for
apoptosis: the cytoplasmic pathway (extrinsic), which is activated
through a member of the TNF receptor super family known as the Fas
Drug Dev Res. 2019;1–15.
wileyonlinelibrary.com/journal/ddr
© 2019 Wiley Periodicals, Inc.
1

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ABD-ALLAH ET AL.
in the initiation and execution of apoptosis; the executioner caspase-3,
-6, and -7, and the initiator caspase-8 and -9 are the most associated
with apoptosis. Additionally, caspase-3 is the most important member
of this family which is critical for the last stages of cellular dismantling
during apoptosis. Failure of chemotherapy is frequently accompanied
with apoptosis faults (Johnstone, Ruefli, & Lowe, 2002), while cancer
development and progression is directly connected to dysregulation of
apoptosis (Storey, 2008). Therefore, apoptosis pathways are attractive
goals for the progress of novel selective anticancer drugs. Among these
apoptosis targets, procaspase-3/caspase-3 activation pathway gained
an outstanding interest due to elevated procaspase-3 level in various
tumors (Botham et al., 2014; Simpson et al., 2013; Yang et al., 2017).
Moreover, the Bcl-2 family is most prominent in regulating the
intrinsic apoptotic pathway which consists of members that either
stimulate apoptosis as Bax, or inhibit apoptosis, such as Bcl-2
(Thornberry & Lazebnik, 1998). MK-0557 (Figure 1) is an orally active
anticancer candidate that has been selected for proof of concept
studies in human clinical trials. It has proved potent activity against
numerous tumor cells, namely breast cancer, prostate cancer, lung
cancer, brain cancer, colon cancer, ovarian cancer, and lymphoma.
Chemically, it is a cyclohexane carboxamide derivative and is directly
inducing apoptosis (Pan, 2014, 2016).
(Wolan, Zorn, Gray, & Wells, 2009). Additional caspase-3 activators
have also been introduced with a subtle difference in pharmacophoric
groups and SAR (Aliabadi et al., 2010; Cai, Kasibhatla, Drewe, Reddy, &
Zhang, 2006; Tai et al., 2006; Zhang, Drewe, Tseng, Kasibhatla, & Cai,
2004; Zhang et al., 2005; Zhang, Kasibhatla, et al., 2004) (Figure 1).
On the other hand, further series of small molecules (like PAC-1
[Peterson, Hsu, et al., 2009; Putt et al., 2006] and WF-208 [Wang
et al., 2014; Wang et al., 2015], Figure 2) was reported to activate
procaspase-3 and caspases-3 through chelating the inhibitory zinc
ions (Peterson, Goode, et al., 2009).
Recent work has utilized caspases-3 activation pathways by com-
bination therapy via administering both compounds PAC-1 and
1541B which demonstrated substantial in vitro synergy in activating
procaspase-3 (Botham et al., 2014). On the contrary of combination
therapy, in pharmacy practice, it is preferable to administer single drug
to avoid patient incompliance to polypharmacy and unexpected con-
sequences of concurrent intake of different drugs.
In the present work, hybrid structure of MK-0557, bearing
cyclohexane-carboxamide moiety, and acylhydrazone scaffold of
caspases-3 activators has been designed (Figure 3), prepared, and
tested for their antiproliferative activity. Addition of the metal chelat-
ing acylhydrazone/thiourea pharmacophore to the cyclohexane-
carboxamide backbone of the apoptotic MK-0557 is expected to
afford synergetic effect and potentiates the anticancer profile of the
novel candidates. Moreover, the most successful compound 6a was
investigated as Zn chelator using continuous variation method,
whereas the modification of the Job's (Job, 1928) continuous variation
method, which was performed by Vesburge and Cooper (Ali, 1999),
Indeed, the activation of caspase-3 has been accomplished where
the small molecule reorganizes and binds procaspase-3 active site for
catalysis and render its internal cleavage sites more accessible to the
initiator caspases (caspase-6, -8, -9, and -10, their primary function is to
activate the executioner caspases-3, -6 and -7) (Chu et al., 2005). Com-
pound 1541 and its analogues (Figure 1) were the first in this class
FIGURE 1 (a) Structure of MK-0557
and (b) structures of direct caspases-3
activators

ABD-ALLAH ET AL.
3
FIGURE 2 (a) Structures of Zn
chelators as caspases-3 activators and
(b) bi- and monodentated chelation
FIGURE 3 Rational design of new
lead compounds based on symbiotic
approach

4
ABD-ALLAH ET AL.
was applied to find the stoichiometry and formation constant of the
complex.
5 hr. The obtained solid was filtered off and recrystallized from etha-
nol to afford 6a-h.
1-(2-(2-Benzylidenehydrazinyl)-N-phenylacetamido)cyclohexane-
1-carboxamide 6a
2
| EXPERIMENTAL
White solid, yield 90%, IR (KBr, cm−1) 3446, 3,423 (NH2), 1672, 1666
(2 × C O), 1HNMR (CDCl3) 1.26–1.65 (m, 10H, 5CH2, cyclohexyl),
4.68 (s, 2H, CO─CH2), 7.28 (s, 2H, NH2), 7.41–7.77 (m, 10H, Har.),
8.56 (s, 1H, CH─C6H5), 10.05 (s, 1H, NH); 13CNMR (CDCl3) 20.81,
20.93, 30.21 (3 × CH2, cyclohexyl), 51.21 (CO─CH2), 60.22 (Cq),
127.85, 128.62, 128.81, 129.01, 129.76, 131.27 (6 × CHar.), 135.91,
2.1 | Chemistry
All chemicals were commercially available. Infrared (IR) spectra were
recorded with JASCO FT/IR-6100 spectrometer as thin film for oils in
NaCl discs or as KBr pellets for solids and values are expressed in
cm−1. Proton nuclear magnetic resonance (1HNMR) and carbon NMR
13CNMR) spectra were determined on Jeol ECA 500 MHz spectrom-
136.72 (2
Car.), 144.21 (CH─C6H5), 165.92 (CO─CH2), 181.21
(
(CO─NH2). (EI) m/z (%): 378.4 ([M]+, 1.4%), C22H26N4O2: C,69.82;
eter and signals are expressed as (s [singlet], d [doublet], brs [broad
singlet]).
H,6.92; N, 14.80 Found: C, 69.75; H, 6.93; N, 14.75.
Melting points were measured on a Walden Precision Electro ther-
mal 9300 apparatus (Stone, Staffordshire, England) and are uncorrected.
1-(2-(2-(3-Chlorobenzylidene)hydrazinyl)-N-phenylacetamido)
cyclohexane-1-carboxamide 6b
Yellowish white solid, m.p. 220 ꢀC yield 93%, IR (KBr, cm−1) 3446,
3423 (NH2), 1637, 1624 (2 × C O), 1HNMR(CDCl3) 1.54–2.21 (m,
10H, 5CH2, cyclohexyl), 4.68 (s, 2H, CO─CH2), 7.17 (br.s, 2H, NH2),
7.18–7.84 (m, 9H, Har.). 8.56 (s, 1H, CH─C6H5); 13CNMR (CDCl3)
22.82, 24. 47, 34.53 (3 × CH2, cyclohexyl), 52.38 (CO─CH2), 63.58
(Cq), 126.27, 127.37, 128.86, 129.56, 129.91, 130.03, 130.77
(7 × CHar.), 134.97, 135.77, 136.72 (3Car.), 148.74 (CH─C6H5), 165.83
(CO─CH2), 190.89 (CO─NH2). (EI) m/z (%): 412.9 ([M]+, 14%), C22H25
ClN4O2: C,63.99; H,6.10; N, 13.57 Found: C, 63.82; H, 6.14; N, 13.62.
2.1.1 | Synthesis and spectral data
Synthesis of 1-(phenylamino)cyclohexanecarbonitrile 2
Compound 2 was synthesized as illustrated in the literature (Abd-
Allah, Aboutabl, Aboul-Enein, & El-Azzouny, 2017).
Synthesis of (phenylamino)cycloakanecarboxamide 3
Compound 3 was synthesized as illustrated in literature (Abd-Allah
et al., 2017).
1-(2-(2-(4-Bromobenzylidene)hydrazinyl)-N-phenylacetamido)
cyclohexane-1-carboxamide 6c
Synthesis of 1-(2-chloro-N-phenylacetamido)cyclohexane-
1-carboxamide 4
White solid, m.p. 132 ꢀC yield 90%, IR (KBr, cm−1) 3460, 3446(NH2),
1697, 1666 (2 × C O), 1HNMR (CDCl3) 1.41–1.62 (m, 10H, 5CH2,
cyclohexyl), 4.41 (s, 2H, CO─CH2), 6.69 (br.s, 2H, NH2), 7.22–7.43 (m,
5H, Har.), 7.62 (d, 2H, J = 6.9, Har.), 7.72 (d, 2H, J = 6.9, Har.), 8.62 (s,
1H, CH─C6H5); 13CNMR (CDCl3) 21.11, 22.76, 31.75 (3 × CH2,
cyclohexyl), 54.47 (CO─CH2), 60.67 (Cq), 116.81, 120.20, 129.26,
129.95, 130.03 (5 × CHar.), 132.13, 132.88, 134.63 (3Car.), 148.74
(CH─C6H5), 161.23 (CO─CH2), 177.76 (CO─NH2). (EI) m/z (%):
457.37 ([M]+, 20%), C22H25 BrN4O2: C,57.77; H,5.51; N, 12.25
Found: C, 57.60; H, 5.39; N, 12.45.
Compound 4 was synthesized as illustrated in literature (Abd-Allah &
Elshafie, 2018).
Synthesis of 1-(2-hydrazinyl-N-phenylacetamido)cyclohexane-
1-carboxamide 5
To a solution of 4 (3.04 g, 0.0105 mol) in absolute ethanol (10 mL),
hydrazine hydrate 99% 0.02 mol) was added. The reaction mixture
was refluxed under stirring for 8 hr. Then the precipitate was filtered
off and recrystallized from ethanol.
Buff solid, yield 85%, IR (KBr, cm−1) 3435, 3340 (NH2), 1675, 1650
(2 × C O), 1HNMR (CDCl3) 1.10–1.92 (m, 10H, 5CH2, cyclohexyl),
3.31 (s, 2H, NH2), 3.54 (s, 2H, CO─CH2), 6.91 (s, 2H, NH2), 7.10–7.31
(m, 5H, Har.), 10.05 (br.s, 1H, NH); 13CNMR (CDCl3) 20.59, 20.75, 27.25
(3 × CH2, cyclohexyl), 52.53 (CO─CH2), 66.21 (Cq), 126.21, 128.55,
128.81 (3 × CHar.), 135.22 (Car.), 166.22 (CO─CH2), 180.91 (CO─NH2).
(EI) m/z (%): 290 ([M]+, 15%), C15H22N4O2: C,62.05; H,7.64; N, 19.30
Found: C, 62.32; H, 7.91; N, 19.53.
1-(2-(2-(4-Methylbenzylidene)hydrazinyl)-N-phenylacetamido)
cyclohexane-1-carboxamide 6d
Yellowish brown solid, m.p. 185 ꢀC, yield 95%, IR (KBr, cm−1) 2935.13,
2860.88 (NH2), 1742.37, 1660.41 (2 × C O), 1HNMR (CDCl3)
1.06–1.65 (m, 10H, 5CH2, cyclohexyl), 2.43 (s, 3H, CH3), 4.66 (s, 2H,
CO─CH2), 6.61 (br.s, 2H, NH2), 6.91–7.25 (m, 5H, Har.), 7.28 (d, 2H,
J = 7.9, Har.), 7.78 (d, 2H, J = 7.9, Har.), 8.70 (s, 1H, CH─C6H5); 13CNMR
(CDCl3) 21.60 (CH3), 21.68, 22.84, 34.56 (3 × CH2, cyclohexyl),
52.32 (CO─CH2), 63.52 (Cq), 127.84, 127.94, 128.76, 129.37,
131.08 (5 × CHar.), 136.87, 141.40, 141.93 (3 Car.), 148.74
(CH─C6H5), 162.00 (CO─CH2), 192.02 (CO─NH2). (EI) m/z (%):
392.50 ([M]+, 15%), C23H28 N4O2: C,70.38; H,7.19; N, 14.27
Found: C, 70.41; H, 7.25; N, 14.33.
General procedures for the synthesis of 1-(2-(2-(un)substituted-
benzylidene)hydrazinyl)-N-phenylacetamido)cyclohexane-
1-carboxamides 6a-h
To a solution of 5 (10.3 mmol) in ethanol (5 mL), appropriate aldehyde
(15 mmol) was added and the mixture was refluxed while stirring for

ABD-ALLAH ET AL.
5
45%), C20H25N5O2: C,65.37; H,6.86; N, 19.06; Found: C, 65.52; H,
6.59; N, 20.21.
1-(2-(2-((E)-3-(3-(Dimethylamino)phenyl)allylidene)hydrazinyl)-N-
phenylacetamido) cyclohexane-1-carboxamide 6e
Reddish solid, m.p. 235 ꢀC, yield 89%, IR (KBr, cm−1) 3460, 3446
(NH2), 1672, 1666 (2 × C O), 1HNMR (CDCl3) 1.32–1.62 (m, 10H,
5CH2, cyclohexyl), 3.06 (s, 6H, 2CH3), 4.56 (s, 2H, CO─CH2), 6.53 (d,
2H, J = 7.9, Har.), 6.56 (d, 2H, J = 7.9, Har.), 6.57 (s, 1H, CH CH─C6H5),
6.68 (br.s, 2H, NH2), 6.71 (s, 1H, CH CH─C6H5), 6.90 (s, 1H, N CH),
7.18–7.40 (m, 5H, Har.); 13CNMR (CDCl3) 22.84, 23.10, 25.14 (3 × CH2,
cyclohexyl), 40.09 (2CH3), 51.59 (CO─CH2), 60.07 (Cq), 111.76,
116.13, 121.72, 123.73, 128.48 (5 × CHar.), 129.74, 129.46, 130.31
(N CH─CH, N CH─CH, CH─C6H5), 142.67, 144.08, 152.41 (3Car.),
162.51 (CO─CH2), 193.78 (CO─NH2); (EI) m/z (%): 477.26 ([M]+, 40%),
General procedures for the synthesis of 1-(2-(2-(1-(un)substituted
phenyl)ethylidene) hydrazinyl)-N-phenylacetamido)cyclohexane-
1-carboxamides 7a-e
To a solution of 5 (3.04 g, 0.0105 mol) in absolute ethanol (10 mL),
appropriate acetophenone (0.0105 mol) was added. The reaction mix-
ture was refluxed with stirring for 5 hr, and then the obtained solid
was filtered off and recrystallized from ethanol to afford 7a-e.
1-(N-Phenyl-2-(2-(1-phenylethylidene)hydrazinyl)acetamido)
cyclohexane-1-carboxamide 7a
C26H33 N5O2: C,69.77; H,7.43; N, 15.65 Found: C, 69.52; H,
White solid, m.p. 105 ꢀC yield 85%, IR (KBr, cm−1) 3467, 3421 (NH2),
1747, 1630 (2 × C O), 1HNMR (CDCl3) 1.82–2.12 (m, 10H, 5CH2,
cyclohexyl), 2.28 (s, 3H, CH3), 3.35 (s, 2H, CO─CH2), 6.57 (br.s, 2H,
NH2), 7.39–7.50 (m, 5H, Har.), 7.91–7.93 (m, 5H, Har.), 13CNMR
(CDCl3) 17.1 (CH3), 21.3, 22.5, 23.4 (3 × CH2, cyclohexyl), 52.2
(CO─CH2), 62.6 (Cq), 113.3, 127.4, 128.1, 128.5, 129.4, 130.1
(6 × CHar.), 137.5, 139.2 (2Car.), 166.76 (CO─CH2), 168.7 (C N),
180.2 (CO─NH2). (EI) m/z (%): 392.50 ([M]+, 10%), C23H28 N4O2:
C,70.38; H,7.19; N, 14. 27 Found: C, 70.35; H, 7.25; N, 14.31.
7.21; N, 15.95.
1-(2-(2-(4-Methoxybenzylidene)hydrazinyl)-N-phenylacetamido)
cyclohexane-1-carboxamide 6f
Yellow viscous oil, yield 85%, IR (KBr, cm−1) 3446, 3421(NH2), 1687,
1670 (2 × C O), 1HNMR (CDCl3) 1.52–2.19 (m, 10H, 5CH2,
cyclohexyl), 3.86 (s, 3H, OCH3), 4.64 (s, 2H, CO─CH2), 6.95 (br.s, 2H,
NH2), 7.04 (d, 2H, J = 7.4, Har.), 7.17 (d, 2H, J = 7.4, Har.), 7.74–7.76
(m, 5H, Har.), 8.49 (s, 1H, N CH); 13CNMR (CDCl3) 22.84, 24.52,
34.55 (3 × CH2, cyclohexyl), 52.44 (CO─CH2), 55.60 (OCH3), 63.48
(Cq), 114.24, 126.44, 128.72, 129.39, 132.00 (5 × CHar.), 136.91,
151.98, 161.96 (3Car.), 166.21 (CO─CH2), 190.82 (CO─NH2), (EI) m/z
(%): 408.50 ([M]+, 25%), C23H28 N4O3: C,67.63; H,6.91; N, 13.72
Found: C, 67.71; H, 7.02; N, 13.80.
1-(2-(2-(1-(4-Methoxyphenyl)ethylidene)hydrazinyl)-N-
phenylacetamido)cyclohexane-1-carboxamide 7b
Yellowish brown, m.p. 212 ꢀC yield 85%, IR (KBr, cm−1) 3460, 3430
(NH2), 1771, 1650 (2 × C O), 1HNMR (CDCl3) 1.07–2.25 (m, 10H,
5CH2, cyclohexyl), 2.28 (s, 3H, CH3), 3.33 (s, 2H, CO─CH2), 3.78
(s, 3H, OCH3), 6.90 (br.s, 2H, NH2), 7.46 (d, 2H, J = 8.4, Har.), 7.71
(d, 2H, J = 8.4, Har.), 7.87–7.95 (m, 5H, Har.); 13CNMR (CDCl3) 14.86
(CH3), 21.12, 22.45, 24.00 (3 × CH2, cyclohexyl), 52.31 (CO─CH2),
55.63 (OCH3), 62.84 (Cq), 113.66, 128.14, 129.80, 130.21, 131.31
(5 × CHar.), 136.42, 141.52, 142.51 (3Car.), 166.86 (CO─CH2), 168.04
(C N), 180.22 (CO─NH2). (EI) m/z (%): 422.53 ([M]+, 35%), C24H30
N4O3: C,68.22; H,7.16; N, 13.26 Found: C, 68.26; H, 7.21; N, 13.27.
1-(N-Phenyl-2-(2-(3,4,5-trimethoxybenzylidene)hydrazinyl)
acetamido)cyclohexane-1-carboxamide 6g
Yellowish brown solid, m.p. 185 ꢀC, yield 90%, IR (KBr, cm−1) 3446,
3419 (NH2), 1672, 1666 (2 × C O), 1HNMR (CDCl3) 1.21–1.94 (m,
10H, 5CH2, cyclohexyl), 3.69 (s, 6H, 2OCH3), 3.93 (s, 3H, OCH3), 4.65
(s, 2H, CO─CH2), 7.03 (br.s, 2H, NH2), 7.16–7.44 (m, 7H, Har.), 8.52
(s, 1H, N CH); 13CNMR (CDCl3) 22.82, 24.47, 34.48 (3 × CH2,
cyclohexyl), 52.76 (CO─CH2), 56.31 ((2OCH3), 58.27 (OCH3), 63.54
(Cq), 104.97, 106.17, 128.76, 129.19 (4 × CHar.), 136.80, 140.55,
152.09, 153.48, 153.50 (5Car.), 141.53 (N CH), 166.38 (CO─CH2),
180.31 (CO─NH2). (EI) m/z (%): 468.55 ([M]+, 32%), C25H32 N4O5:
C,64.09; H,6.88; N, 11.96 Found: C, 64.12; H, 6.93; N, 12.22.
1-(2-(2-(1-(4-Aminophenyl)ethylidene)hydrazinyl)-N-
phenylacetamido)cyclohexane-1-carboxamide 7c
Reddish solid, m.p. 200 ꢀC yield 95%, IR (KBr, cm−1) 3440, 3423
(NH2), 1670, 1633 (2 × C O), 1HNMR (CDCl3) 1.79–2.12 (m, 10H,
5CH2, cyclohexyl), 2.38 (s, 3H, CH3), 3.47 (s, 2H, CO─CH2), 4.46 (br.s,
2H, NH2─C6H5), 6.63 (br.s, 2H, NH2), 6.57 (d, 2H, J = 8.4, Har.), 6.70
(d, 2H, J = 8.4, Har.), 7.64–7.70 (m, 5H, Har.); 13CNMR (CDCl3) 18.10
(CH3), 20.81, 24.22, 30.11 (3 × CH2, cyclohexyl), 52.23 (CO─CH2),
63.21 (Cq), 115.22, 127.81, 128.54, 130.25, 131.34 (5 × CHar.),
132.33, 138.22, 148.27 (3Car.), 166.12 (CO─CH2), 169.00 (C N),
180.22 (CO─NH2); (EI) m/z (%): 407.52 ([M]+, 28%), C23H29N5O2:
C,67.79; H,7.17; N, 17.19 Found: C, 67.83; H, 7.21; N, 17.22.
1-(2-(2-((1H-Pyrrol-2-yl)methylene)hydrazinyl)-N-phenylacetamido)
cyclohexane-1-carboxamide 6h
White solid, m.p. 110 ꢀC, yield 85%, IR (KBr, cm−1) 3446, 3423 (NH2),
1747, 1633 (2 × C O), 1HNMR (CDCl3) 1.00–2.03 (m, 10H, 5CH2,
cyclohexyl), 4.41 (s, 2H, CO─CH2), 6.30–6.69 (m, 5H, 3CH-pyrrole,
NH2), 7.19–7.45 (m, 5H, Har.), 8.32 (s, 1H, N CH); 13CNMR (CDCl3)
22.76, 25.22, 32.29 (3 × CH2, cyclohexyl), 54.45 (CO─CH2), 62.90
(Cq), 110.59, 116.13, 123.59, 127.17, 128.67, 129.42 (3CH-pyrrole,
3 × CHar.), 132.31 (C─NH─pyrrole), 136.80 (N CH), 144.42 (Car.),
166.84 (CO─CH2), 178.79 (CO─NH2). (EI) m/z (%): 367.45 ([M]+,
1-(2-(2-(1-(2-Hydroxyphenyl)ethylidene)hydrazinyl)-N-
phenylacetamido)cyclohexane-1-carboxamide 7d
White solid, m.p. 155 ꢀC yield 90%, IR (KBr, cm−1) 3440, 3420 (NH2),
1752, 1639 (2 × C O), 1HNMR (CDCl3) 1.26–1.65 (m, 10H, 5CH2,

6
ABD-ALLAH ET AL.
cyclohexyl), 2.68 (s, 3H, CH3), 4.42 (s, 2H, CO─CH2), 6.71 (br.s, 2H,
NH2), 6.80–7.4 (m, 9H, Har.), 10.31 (br.s, 1H, OH), 13CNMR (CDCl3)
17.2 (CH3), 21.22, 25.31, 30.52 (3 × CH2, cyclohexyl), 52.11
(CO─CH2), 63.1 (Cq), 117.1, 127.2, 127.54, 128.5, 128.9, 130.1,
131.7 (7 × CHar.), 137.8, 138.2 (2Car.), 162.4 (C─OH), 166.3
(CO─CH2), 169.3 (C N), 180.5 (CO─NH2); (EI) m/z (%): 408.50 ([M]+,
13%), C23H28 N4O3: C,67.63; H,6.91; N, 13.72 Found: C, 67.61; H,
7.02; N, 13.95.
180.41 (CO─NH2); (EI) m/z (%): 360.4 ([M]+, 29%), C19H28N4O3:
C,63.31; H,7.83; N, 15.54 Found: C, 63.36; H, 7.89; N, 15.61.
2.2 | Biological evaluation
2.2.1 | Cell lines and cell culture
Human HepG2 liver, colorectal, MCF-7 breast, and A549 lung cell
lines were obtained from the Egyptian Company for Production of
Vaccines, Sera and Drugs (VACSERA), Cairo, Egypt. HepG2 and A549
cells were maintained in RPMI-1640 medium (Biowest) while MCF-7
and HCT-116 cells were cultured in DMEM-F12 (Gibco). Media were
supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin
(Sigma) and 10% (v/v) fetal bovine serum (FBS; Gibco). Cells were cul-
tured and kept at 37 ꢀC in a 5% CO2 humidified incubator.
1-(2-(2-(1-(4-Butylphenyl)ethylidene)hydrazinyl)-N-phenylacetamido)
cyclohexane-1-carboxamide 7e
White solid, m.p. 140 ꢀC yield 95%, IR (KBr, cm−1) 3442, 3432 (NH2),
1747, 1633 (2 × C O), 1HNMR (CDCl3) 0.91 (s, 6H, 2CH3), 1.48 (s,
1H, CH─(CH3)2), 1.60–2.06 (m, 10H, 5CH2, cyclohexyl), 1.83 (s, 3H,
CH3), 2.59 (s, 2H, CH2─CH─(CH3)2), 4.41 (s, 2H, CO─CH2), 7.11 (br.s,
2H, NH2), 7.13 (s, 4H, Har.), 7.45 (s, 5H, Har.); 13CNMR (CDCl3) 14.21
(CH3─CH─C N)), 22.38 (CH─(CH3)2), 22.77, 24.44, 30.23 (3 × CH2,
cyclohexyl), 34.82 (CH─(CH3)2), 45.25 (CH2─C6H5), 54.51 (CO─CH2),
62.24 (Cq), 127.07, 128.31, 128.64, 129.21, 129.29 (5 × CHar.),
130.06, 137.03, 142.21 (3Car.), 166.25 (CO─CH2), 168.67 (C N),
180.23 (CO─NH2). (EI) m/z (%): 448.61 ([M]+, 20%), C27H36N4O2:
C,72.29; H,8.09; N, 12.49 Found: C, 72.22; H, 8.13; N, 12.52.
2.2.2 | In vitro cytotoxicity assay
The target compounds under investigation were evaluated by colori-
metric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide) assay to examine their cytotoxic potential against HepG2, HCT-
116, MCF7, and A549 human carcinoma cell lines. The assay is
depending on the ability of mitochondrial dehydrogenases present in
viable cells to metabolize yellow soluble MTT salt into water insoluble
purple formazan crystals. The intensity of solubilized blue formazan
product is directly proportional to the number of metabolically active
cells. Briefly, cells were seeded at 1 × 104 cells/well in a 96-well flat
bottom plate and allowed to adhere overnight. The tested compounds
were added at a range of concentrations (100, 50, 25, and 12.5 μM) in
triplicate. Negative control cells were treated with 0.5% DMSO while
doxorubicin treated cells worked as positive control. After 48 hr, the
medium was removed and then exchanged with 50 μL MTT solution
(0.5 mg/mL; Sigma) for 4 hr at 37 ꢀC. The reaction is stopped and the
formed crystals were dissolved by adding 200 μL/well of 10% sodium
dodecylsulfate in deionized water and incubated overnight at 37 ꢀC.
The absorbance was then measured at 595 nm with a reference
wavelength of 620 nm. The inhibition percent of viable cells was cal-
culated according to the literature equation (Mosmann, 1983). The
IC50 was determined using Graph-Pad PRISM version-6 for Windows
software (La Jolla, California).
General procedure for the synthesis of 1-(2-(2-acetyl or
butyrylhydrazinyl)-N-phenyl acetamido)cyclohexane-1-carboxamides
8a and 8b
To a solution of 5 (0.2 g, 0.001 mol) in glacial acetic acid (3 mL), appro-
priate anhydride (0.002 mol) was added and the mixture was refluxed
under stirring for 2 hr. The separated product was filtered off and rec-
rystallized from ethanol to afford 8a,b.
1-(2-(2-Acetylhydrazinyl)-N-phenylacetamido)cyclohexane-
1-carboxamide 8a
White solid, m.p. 90 ꢀC yield 90%, IR (KBr, cm−1) 2935.13, 2860.88
(NH2), 1742.37, 1660.41 (2 × C O), 1HNMR (CDCl3) 1.01–1.84 (m,
10H, 5CH2, cyclohexyl), 1.87 (s, 3H, CH3), 4.47 (s, 2H, CO─CH2), 7.14
(s, 2H, NH2), 7.15–7.45 (m, 5H, Har.); 13CNMR (CDCl3) 20.61 (CH3),
22.63, 24.30, 34.19 (3 × CH2, cyclohexyl), 54.25 (CO─CH2), 63.16
(Cq), 128.75, 129.45, 129.99 (3 × CHar.), 136.87 (Car.), 166.46
(CO─CH2), 168.28 (CO─CH3), 180.23 (CO─NH2); (EI) m/z (%): 332.40
([M]+, 27%), C17H24 N4O3: C,61.43; H,7.28; N, 16.86 Found: C,
61.46; H, 7.31; N, 16.92.
1-(2-(2-Butyrylhydrazinyl)-N-phenylacetamido)cyclohexane-
1-carboxamide 8b
2.2.3 | Caspase enzyme assay
White solid, m.p. 160 ꢀC yield 85%, IR (KBr, cm−1) 2935.13, 2860.88
(NH2), 1742.37, 1660.41 (2 × C O), 1HNMR (CDCl3) 0.99 (t, 3H,
J = 10, CH3─CH2), 1.17–1.61 (m, 10H, 5CH2, cyclohexyl), 1.84 (s, 2H,
CH2─CH3), 2.31 (s, 2H, CO─CH2─CH2), 4.47 (s, 2H, CO─CH2─NH),
7.13 (s, 2H, NH2), 7.15–7.45 (m, 5H, Har.); 13CNMR (CDCl3) 9.50
(CH3), 20.64, 22.63, 24.31, 27.14 (3 × CH2, cyclohexyl, CH2─CH3),
34.19 (CH2─CH2─CH3), 54.37 (CO─CH2), 63.16 (Cq), 128.73, 129.44,
130.00 (3 × CHar.), 136.90 (Car.), 166.69 (CO─CH2), 173.63 (CO─NH),
Effect of compounds 6a and 8a on caspases-3, -8, and -9 in A549 cell
line was evaluated at their cytotoxic IC50 concentrations against A549
cells using Invitrogen (California) Caspase 3 Human ELISA® kit
(KHO1091), Caspase 8 Human ELISA kit (BMS2024), and Caspase
9 Human ELISA kit (BMS2025) measured on ROBONIK P2000 (India)
spectrophotometer at 450 nm against untreated control cells (nega-
tive control), and doxorubicin (positive control) applying the standard
protocols of the manufacturer.

ABD-ALLAH ET AL.
7
2.2.4 | Cell cycle analysis
2.3.2 | Buffer solution preparation
In vitro DNA-flow cytometry
Equal volume of 50 mM Hepes (2.98 g/250 mL) and 100 mM KNO3
(2.525 g/250 mL) were mixed and the pH was adjusted to 7.2 using
1N NaOH. The prepared buffer was used as solvent and diluent.
A549 cells (2 × 105/well) were treated with compounds 6a and 8a at
their IC50 concentrations for 24 hr. After treatment, cells were washed
twice with ice-cold phosphate buffer saline (PBS), collected by centrifu-
gation, and fixed in ice-cold 70% (v/v) ethanol at 4 ꢀC for 30 min,
washed with PBS at 37 ꢀC for 30 min. Cells were collected by centrifuga-
tion at 2000 rpm for 5 min and stained with propidium iodide (PI) buffer;
samples were mixed gently and incubated at room temperature in the
dark for 20 min. The DNA content was analyzed using BD FACS CALI-
BER flow cytometer. All experiments were done in duplicates.
2.3.3 | Standard drug 250 μM (6a) solution
preparation
A 250 μM solution of 6a was prepared through weighing 0.0236 g in
250-mL measuring flask, dissolved and diluted to volume using the
prepared buffer.
In vitro measurement of apoptosis using annexin-V-FITC apoptosis
A549 cells (4 × 106/well) untreated and treated with compounds 6a and
8a at their IC50 for 24 hr were prepared for apoptosis assay using
Annexin V-FITC/ PI (fluorescein isothiocyanate /propidium iodide) Apo-
ptosis Detection Kit [ab#139418] (Bio Vision Research Products, Linda
Vista Avenue, California) according to the manufacturer's instruction.
Cells were washed with ice-cold PBS at least three times, resuspended in
PBS and stained with 5 μL Annexin V-FITC and 5 μL PI binding buffer for
15 min at room temperature in the dark, and then analyzed using BD
FACS CALIBER flow cytometer. All experiments were done in duplicates.
2.3.4 | Standard zinc(II) metal preparation
A 250 μM solution of ZnSO4.7H2O was prepared by accurately
weighing 0.0179 g in 250-mL measuring flask, dissolved and diluted
to volume using the prepared buffer.
2.3.5 | Instruments
Double beam UV visible spectrophotometer (Shimadzu, Kyoto, Japan)
model 1650 PC, connected to HP 7540 compatible computer and HP
laser 1018 inkjet printer, the bundle software was a UV probe spec-
troscopy software version 2.21. The spectra band width was 2 nm
and wavelength scanning speed was 2,800 nm min−1. The data points
for the zero-order spectra were then collected at every 0.1 nm.
A Jenway 3505 pH-meter (Jenway, UK), equipped with combined
glass electrode was used for pH adjustment.
2.2.5 | Cytotoxic in MCF-10 human breast normal
cell line
Compounds 6a and 8a were evaluated on MCF-10 (ATCC® CRL-
10318™) human breast normal cell line for their cytotoxic effect using
the same MTT assay protocol for human HepG2 liver, HCT-116 colo-
rectal, MCF-7 breast, and A549 lung cell lines.
2.3.6 | Continuous variation method
A series of buffer solutions of 250 μM Zn(II) and 250 μM of the 6a
ligand were prepared in buffer solution pH 7.2 such that the sum of
metal and ligand concentration was constant (250 μM). The mole frac-
tion of 6a was varied between 0.1 and 1.0. A series of buffer solutions
of compound 6a alone at the same concentration was also prepared.
For calculating of the maximum absorbance, the absorbance of 250 μM
6a alone was subtracted from the absorbance of 250 μM 6a with
250 μM ZnSO4 while the normalized 6a absorbance for each mole frac-
tion at 242 nm was then calculated by subtracting the absorbance of
6a at the appropriate concentration from the corresponding 6a/Zn2+
mixed sample and dividing by the corrected maximum absorbance value
(Likussar & Boltz, 1971). The normalized absorbance was then plotted
versus mole fraction. The peak of the plot gives the stoichiometry of
the interaction (based on the mole fraction) and was used to calculate
the formation constant.
2.2.6 | Effects of compounds 6a and 8a on cell cycle
regulatory molecules
The cell cycle arrest induced by compounds 6a and 8a was further sub-
stantiated through assessing the expression levels of p53, Bax, and Bcl-2
regulatory molecules in A549 cells; using Human p53 ELISA kit
(RAB0500 Sigma), Merck KGaA, Germany, Human Bax ELISA Kit (EIA-
4487), DRGR, Germany, and Invitrogen Zymed® Bcl-2 human ELISA Kit
(BMS244–3), respectively, measured on ROBONIK P2000 spectropho-
tometer at 450 nm against untreated control cell (negative control) and
doxorubicin (positive control). Analysis was confirmed with three differ-
ent sets of extracts. All experiments were done in triplicates.
2.3 | Evaluation of Zn chelation for compound 6a
2.3.1 | Reagents and standard solutions
2.4 | Computational studies
All the reagents used were of analytical grade; Hepes buffer free acid
(C8H18N2O4S) (TM media, India), KNO3 (ACS reagent ≥99.0%, Sigma-
Aldrich), ZnSO4.7H2O (ACS reagent ≥99.0%, Merck) and double dis-
tilled water.
In order to predict the interactions and binding modes of 6a and 8a
synthesized inhibitors in the caspase-3 active site, as well as evalua-
tion of their relative binding affinities, Discovery Studio 2016 client

8
ABD-ALLAH ET AL.
was used to perform the molecular docking studies. The crystal struc-
ture of caspase-3 protein with small molecule was downloaded
from the Protein Data Bank (PDB code 3H0E). Regarding protein
preparation, the small molecule involved in 3H0E protein was
extracted, hydrogen atoms were added and crystal water mole-
cules were deleted using Prepare Protein Structure module. To
enhance the accuracy of docking study, the co-crystallized inhibi-
tor was considered as the reference molecule during performing
the docking.
3
| RESULTS AND DISCUSSION
3.1 | Chemistry
The procedures for synthesis of the target compounds 6a-h, 7a-e, and
8a-b are outlined in Schemes 1 and 2. The starting compounds 1–4
were synthesized via adopting the reported methods in literature
(Abd-Allah et al., 2017). Hydrazinolysis of 4 yielded the corresponding
key intermediate hydrazide 5 (Scheme 1). Compounds 6a-h and 7a-e
SCHEME 1 Synthesis of compounds
1–5 pok
SCHEME 2 Synthesis of
compounds 6a-h, 7a-e, and 8a-b

ABD-ALLAH ET AL.
9
TABLE 1 Antitumor activity for the synthesized compounds in MTT assay
MCF-7
IC50 (μM)a
HepG2
IC50 (μM)a
A549
IC50 (μM)a
HCT-116
IC50 (μM)a
MCF-10
IC50 (μM)a
Compound no.
6a
18.43 0.20
>100
6.673 0.066
>100
3.03 0.18
38.22 0.18
11.76 1.15
17.38 0.43
87.31 2.42
27.92 1.09
84.07 2.32
40.83 1.24
66.05 1.21
44.11 1.56
20.71 0.51
60.83 0.96
50.45 2.41
5.21 0.72
22.51
24.88 0.0.08
>100
320.21 21.39
6b
ND
6c
74.24 0.09
10.19 0.25
>100
>100
>100
ND
6d
7.044 0.167
>100
12.52 0.09
>100
ND
6e
ND
6f
34.30 0.12
>100
57.76 0.714
>100
84.69 0.25
58.00 0.19
50.88 0.36
56.19 0.06
27.42 0.14
78.94 0.19
65.90 0.11
78.41 0.07
21.81 0.14
>100
ND
6g
ND
6h
>100
52.87 0.08
86.89 0.26
39.62 0.08
82.26 0.12
38.86 0.03
>100
ND
7a
36.35 0.35
19.82 0.17
>100
ND
7b
ND
7c
ND
7d
>100
ND
7e
>100
ND
8a
8.82 0.22
>100
7.073 0.099
54.05 0.0.18
3.52 0.23
131.30 4.38
ND
8b
Doxorubicin
4.21 0.16
3.01 1.21
2.78 0.25
ND
Note: ND, not determined.
aResults are the mean of three separate experiments and represented as value SD.
were obtained through coupling of compound 5 with different alde-
hydes and/or ketones, respectively. Finally compounds 8a and 8b were
synthesized by reacting of compound 5 with appropriate anhydrides.
TABLE 2 Effect of compounds 6a and 8a on caspases-3, -8,
and -9
Caspase-3 Caspase-8 Caspase-9
Compound no.
IC50 μMa (ng/mL)
(ng/mL)
(ng/mL)
23.52
28.52
38.55
1.11
Reagents and conditions: (i) Cyclohexanone, KCN, glacial acetic
acid, 24 hr, r.t (ii) H2SO4, 48 hr, r.t (iii) ClCOCH2Cl, CHCl3, 24 hr, r.t
(iv) NH2-NH2, ethanol, reflux, 8 hr.
6a
3.03
7.07
3.01
–
0.50
0.58
0.70
0.05
1.50
8a
2.10
Reagents and conditions: (i) Appropriate aldehydes, ethanol, reflux,
5 hr. (ii) Appropriate acetophenones, ethanol, reflux, 5 hr. (iii) Appropriate
anhydrides, glacial acetic acid, reflux, 2 hr.
Doxorubicin
Negative control
3.19
0.16
aDetermination of the levels of caspases was performed in A549 cells
treated with the tested compounds at their IC50 Concentrations.
3.2 | Biological evaluation
The cyclohexane carboxamides derivatives 6a-h, 7a-e, and 8a-b
also screened against HepG2 cell line. The results showed that the
tested congeners were weaker than DOX. Moreover, the 1-(2-(2-
benzylidenehydrazineyl)-N-phenylacetamido)cyclohexane-1-carboxamide
derivative 6a that the containing unsubstituted benzylidenehydrazineyl,
1-(2-(2-(4-methylbenzylidene)hydrazineyl)-N phenylacetamido)cyclohex-
ane-1-carboxamide 6d that containing P-methyl benzylidene)hydrazineyl,
and 1-(2-(2-acetylhydrazineyl)-N phenylacetamido)cyclohexane-
1-carboxamide 8a that containing acetyl moiety were the most
active congeners against HepG2 cell line (IC50: 6.67, 7.04 μM and
7.07 μM vs, 3.52 μM, respectively). The activity of the tested com-
pounds against HepG2 cells had the following descending order:
(6a > 6d > 8a > 7d > 7b > 6 h > 6f > 7c > 7a).
3.2.1 | Antiproliferation assay
The antiproliferative activity for the target compounds 6a-h, 7a-e, and
8a-b was assessed against four human cancer cell lines MCF-7
(breast), HepG2 (liver), A549 (lung), and HCT-116 (colon) using MTT
assay. Doxorubicin was used as a positive control in this work. Most of
the compounds exhibited anticancer activity against the four human
tumor cell lines compared with doxorubicin. The antiproliferative activ-
ity of the synthesized target compounds is listed in Table 1. Concerning
MCF-7 cell line, it was clear that all of the tested cyclohexane carbox-
amides derivatives 6a-h, 7a-e, and 8a-b showed moderate activity. On
the other hand, the 1-(2-(2-acetylhydrazineyl)-N-phenylacetamido)
cyclohexane-1-carboxamide 8a that containing acetylhydrazineyl
showed nearly half the potency of the reference drug DOX (IC50
8.82 μM vs. 4.21 μM, respectively). The activity of the tested com-
pounds against MCF-7 cell line had the following descending order:
(8a > 6d > 6a > 7b > 6f > 7a > 6c).
Regarding the activity of cyclohexane carboxamides 6a-h, 7a-e,
and 8a-b against resistant A549 human lung cell line, our results revealed
that the 1-(2-(2-benzylidenehydrazineyl)-N-phenylacetamido)cyclohex-
ane-1-carboxamide derivative 6a that the containing unsubstituted
benzylidenehydrazineyl induced the highest degree of cytotoxicity

10
ABD-ALLAH ET AL.
(Table 1). It was equipotent to reference drug DOX (IC50 3.03 μM
vs. 3.01 μM, respectively). On the other hand, the 1-(2-(2-
acetylhydrazineyl)-N-phenylacetamido)cyclohexane-1-carboxamide
8a that containing acetyl moiety showed half potency of DOX (IC50
5.21 μM vs. 3.01 μM, respectively). The antiproliferative activity of
the tested cyclohexane carboxamides against resistant A549 human lung
cell line had the following descending order: (6a > 8a > 6c > 6d > 7c >
8b > 6f > 6b > 7b > 7e > 7d > 7a > 6g > 6e).
of the tested compounds against HCT-116 cell line had the follow-
ing descending order: (6d > 8a > 6a > 7b > 6 h > 7a > 6g > 7d >
7e > 7c > 6f). Conclusively, most of the tested compounds produced
potent cytotoxic properties against A549 human lung cell line.
3.2.2 | Caspase enzyme assay
The apoptotic execution pathway is a family of aspartate-directed cys-
teine proteases termed caspases. Caspases function as either
upstream initiator or downstream effector proteases which cleave and
activate each other to create amplifiable cascades of proteolysis. Acti-
vation of the most proximal (upstream) caspases arises through pro-
tein interactions which inspire clustering of the inactive zymogens.
The activated initiator caspase subsequently induces the proteolytic
About HCT-116 cell line, the cyclohexane carboxamide derivatives
6a-h, 7a-e, and 8a-b exhibited moderate to weak activity compared
with standard reference drug DOX. Although, the 1-(2-(2-(4-
methylbenzylidene)hydrazineyl)-N phenylacetamido)cyclohexane-
1-carboxamide 6d that containing P-methyl benzylidene)hydrazineyl
induced the highest activity against the HCT-116 cell line. The activity
FIGURE 4 DNA flow cytomertic analysis of compounds 6a, 8a, and doxorubicin on A549 cells for 24 hr at their IC50 (μM)

ABD-ALLAH ET AL.
11
FIGURE 5 Representative dot plots of A549 cells treated with compounds 6a and 8a and doxorubicin at their IC50 (μM) for 24 hr analyzed by
flow cytometry after double staining of the cells with annexin-V FITC and PI
processing of effector caspases, such as caspase-3, -6, and -7. Here,
we reported the growth inhibitory effect of compounds 6a and 8a at
their IC50 concentration in comparison with the untreated control cells
(negative control) and doxorubicin (positive control) on the lung carci-
noma cell line A549 cells and the results are illustrated in Table 2.
Compounds resulted in variable degree of cell viability reduction
comparing with untreated control cells. Therefore, we attempted to
examine whether this viability reduction triggered by these com-
pounds are indeed due to apoptotic induction utilizing the Invitrogen
active caspase-3 Human ELISA® kit (KHO1091). Indeed, these com-
pounds appeared to induce caspase-3 activation as compared to the
untreated control cells, as drug-treated cells exhibited more active
caspase-3 expression. Furthermore, caspase-3 activation in this set-
ting was attributed to the upstream caspase-8 (suggests their
involvement in the extrinsic apoptotic pathway) and caspase-9 (sug-
gests their involvement in the intrinsic apoptotic pathway) activation
which is confirmed by utilizing the Invitrogen Caspase 8/FLICE
Human ELISA Kit (BMS2024) and the caspase-9 human ELISA Kit
(BMS2025), respectively. So, here, we showed a typical apoptotic
induction pathway induced by these compounds and involving activa-
tion of the effector caspase-3 and the upstream initiator caspase-8
and -9 in the lung carcinoma cell line A549 cells.
3.2.3 | Cytotoxic effect against normal cell line
Compounds 6a and 8a with high activity for caspase enzymes were
evaluated against normal breast cancer MCF-10 for their safety. Their
attained IC50 values were 320.21 and 131.3 μM, respectively, indicating

12
ABD-ALLAH ET AL.
TABLE 4 Continuous variation method for the estimation of
stoichiometry of Zn(II) and 6a complex
Zn(II)
Volume (cm3)
6a
Mole fraction
L/(Zn2+ + L)
Absorbances at
Volume (cm3)
λ 242 nm
0
1
2
3
4
5
6
7
8
9
10
9
1
0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
1
8
0.13
0.23
0.59
0.80
0.95
0.59
0.32
0
7
6
5
4
3
2
1
concentrations, compared with untreated control cells. The increasing
of Annexin-V positive cells percentage more than that of untreated
controls indicates an apoptosis induction as shown in Figures 5 and 6.
FIGURE 6 (a) Statistical cell cycle analysis of compounds 6a, 8a,
and doxorubicin on A549 cells for 24 hr at their IC50 (μM).
(b) Statistical analysis of Annexin V-FITC staining of compounds 6a,
8a, and doxorubicin on A549 cells for 24 hr at their IC50 (μM)
Effects of compounds 6a and 8a on cell cycle regulatory molecules in
A549 cell line
The significant cell cycle arrest induced by compounds 6a and 8a at
their IC50 concentrations against A549 cell line was further substanti-
ated through assessing the expression levels of three 12 control. The
results of their effect on p53, Bax, and Bcl-2 regulatory molecules in
A549 cells are listed in Table 3.
TABLE 3 Effect of compounds 6a and 8a on P53, Bax, Bcl-2, and
Bax/Bcl-2 ratio in A549 cell line
Compound no.
6a
P53 (ng/mL)
Bax (ng/mL)
Bcl-2 (ng/mL)
0.84
0.33
5.88
8a
0.88
1.44
0.11
0.29
0.39
0.04
7.42
4.98
10.28
It is observed that, level of p53 in A549 cells was increased in com-
pounds 6a and 8a by 7.6- and 8-folds, respectively, more than the
untreated control. The p53 (proapoptotic gene triggers both extrinsic
and intrinsic apoptotic pathways) [38], so the increase in p53 levels
forced by compounds 6a and 8a indicates their involvement in both
apoptotic pathways. On the other hand, Bax levels were significantly
high in cells treated with compounds 6a and 8a by eightfold and seven-
folds, respectively, as compared with the untreated cells. Since Bax is
one of the key proapoptotic proteins regulating the intrinsic pathway,
the increase in its levels supports the suggested mechanism by which
these compounds act as apoptotic inducers. Further estimation of the
intrinsic proapoptotic properties of compounds 6a and 8a was done
through testing their effect on Bcl-2 levels which is an anti-apoptic
member of the Bcl-2 family proteins, the key regulators of the intrinsic
pathway. The level of Bcl-2 in compounds 6a and 8a treated cells was
examined relative to the untreated control. The Bax/Bcl-2 ratio is con-
sidered as an indicator of the therapeutic response to chemotherapy.
Compounds 6a and 8a showed high increase in Bax/Bcl-2 ratio by 18.6
and 13, respectively, as compared with the untreated control, and
0.71-fold as compared with doxorubicin, which inspires further study.
Doxorubicin
Negative control
aDetermination of the levels of caspases was performed in A549 cells
treated with the tested compounds at their IC50 Concentrations.
safe profile and excellent selectivity toward the tested A549 cell line.
Compounds 6a and 8a were >100 and 25 times more cytotoxic to the
A549 cancer cell line as compared with the MCF-10 human breast nor-
mal cell line. This very high selectivity index may indicate a selective
effect of compounds 6a and 8a on some upregulated signaling path-
ways in A549 cells and suggests further study.
3.2.4 | In vitro cell cycle analysis
In vitro DNA-flow cytometry
Compounds 6a and 8a were selected to evaluate their cytotoxic effect
on cell cycle of A549 cells using flow cytometry assay. The selected
compounds were analyzed at their IC50 concentrations against A549
cell line, where they induce apoptosis at pre G1 phase and cell cycle
arrest at G2/M phase, as illustrated in Figures 4 and 6.
3.3 | Evaluation of compound 6a as a Zn chelator
In vitro Annexin V-FITC staining
Apoptosis rate in A549 cells was assessed by flow cytometry, using
Annexin V-FITC/PI double staining assay after treatment of A549
cells with compounds 6a, 8a, and doxorubicin at their IC50
Compound 6a revealed a remarkable complex formation with Zn ions
as shown in Figure 7a. The formation constant of the complex was
determined by Job's method.

ABD-ALLAH ET AL.
13
FIGURE 7 (a) The UV spectrum of (6a-Zn + 2) chelate and the normalized absorbance versus mole fraction of 6a. (b) The stoichiometry was
determined to be 2:3 (6a: Zn + 2)
FIGURE 8 2D representation of the binding mode of compounds 6a, 8a, and the co-crystallized ligand at the active site of caspase-3 (PDB
code: 3H0E)

14
ABD-ALLAH ET AL.
+ n
M+ n + X6a = ðM−6aÞ
The C N group of compound 6a acts as a hydrogen bond accep-
tor forming H-bond and u-u stacking with His 121; its phenyl group
showed a hydrophobic interaction with Met 61. In addition, its cyclo-
hexane moiety showed interaction with some crucial amino acid resi-
dues (Ser 205, Tyr 204, and Phe 256).
+ n
ðM6ax
Þ
x
À
Á
Kf =
M+ n ð6aÞ
Regarding compound 8a, one carbonyl group formed three hydro-
gen bonds with Arg 207, Arg 64, and Gln 161, and another one
formed hydrogen bond with Arg 207. Additionally, NH─ formed
hydrogen bond with Ser 205, the second NH─ formed hydrogen bond
with Cys 163 and phenyl group formed u-u with Phe 256.
where Kf is the formation constant of the formed complex.
The normalized absorbance resulting from continuous variation
method was plotted against mole fraction of the ligand. A triangular shaped
curve was obtained. The ratio of the stoichiometry was determined from
the mole fraction at the point of intersection formed by the extrapolation
of the legs of the triangle. The results are shown in Table 4.
The normalized absorbances were plotted against mole fraction as
shown in Figure 7b.
4
| CONCLUSION
The formation constant of the complex formed was calculated
from the following equation:
In the present work, the newly synthesized candidates 6a-h, 7a-e, and
8a-b displayed in vitro broad spectrum antitumor activities against the
four tested cancer cell lines. Compared to doxorubicin treatment, com-
pounds 6a and 8a induced caspases-3, -8, and -9 activities and G2/M
growth arrest in A549 carcinoma cell line. The expression levels of p53,
Bax and the Bax/Bcl-2 ratio were all higher than those in doxorubicin-
treated cells. Moreover, compounds 6a and 8a appeared to exhibit higher
selectivity against MCF-10 human breast normal cell line. Molecular
modeling studies showed the binding modes and the interactions of the
prepared inhibitors into caspase-3 active site. In addition, the most potent
compound 6a formed a complex with Zn(II) which was verified using UV
spectroscopy. All these findings unveiled that these compounds may pos-
sibly act as apoptotic inducers and caspase activators.
A=AexCm
Kf =
X
ðCm −A=AexCmÞðCL −A=AexCmÞ
where Kf is formation constant, A is observed absorbance (0.95),
Aex is extrapolated absorbance (1.1), CL is concentration of 6a
ligand (250 μM), Cm is concentration of the metal zinc (250 μM),
and X = 0.66, because 2:3 complex.
0:95=1:1 × 250
Kf =
= 0:944
0:66
ð250−0:95=1:1 × 250Þ250ð1−0:95=1:1Þ
Kf was found to be 0.944 using Zn2+
.
Gibbs free energy changes (ΔG) at 25 ꢀC was also calculated
CONFLICT OF INTEREST
according to the following equation (Ghiamati & Jalaeian, 2018):
The authors declare no conflict of interest.
ΔG = −2:303RTlog Kf
ORCID
where R is gas constant (8.314 J/ꢀ/mol), T is absolute temperature
(ꢀC + 273). Using the above equation, ΔG was found to be −142.81.
The high free energy (ΔG) indicated the stability of formed chelate
(Esmaielzadeh & Mashhadiagha, 2017).
Walaa Hamada Abd-Allah https://orcid.org/0000-0003-2659-2764
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How to cite this article: Abd-Allah WH, Salman A, Sabry
Saad S. Anticancer activity of newly synthesized 1,1-
disubstituted cyclohexane-1-carboxamides: in vitro caspases
mediated apoptosis activators in human cancer cell lines and
their molecular modeling. Drug Dev Res. 2019;1–15. https://
doi.org/10.1002/ddr.21573