Induction of intrinsic apoptosis pathway in colon cancer HCT-116 cells by novel 2-substituted-5,6,7,8-tetrahydronaphthalene derivatives

Article abstract of DOI:10.1016/j.ejmech.2014.03.021

2-Acetyl tetralin (1) reacted with N,N-dimethylformamide dimethylacetal (DMF-DMA) to afford the enaminone 3. The reaction of 3 with piperidine and morpholine afforded the trans enaminone 5a,b, respectively. Compound 3 was treated with primary aromatic amines to give secondary enaminones 6a-e. The enaminone 3 reacted with acetylglycine and hippuric acid to yield pyranones 10a, b, respectively. The reaction of enaminone 3 with 1,4-benzoquinone and 1,4-naphthoquinone gave benzofuranyl tetralin derivatives 14a,b, respectively. Also, when 3 reacted with 5-amino-3-phenyl-1H-pyrazole 15a and 5-amino-1,2,3-triazole 15b, it afforded the new pyrazolo[1,5-a]pyrimidine 17a and 1,2,3-triazolo[1,5-a]pyrimidine 17b, respectively. While the reaction of 3 with pyrimidines 18a, b resulted in the formation of pyrido[2,3-d]pyrimidine derivatives 20a, b, respectively. Investigations of the cytotoxic effect of those compounds against different human cell lines indicated that some compounds showed high selective cytotoxicity against colon cancer HCT-116 cells. Some of these compounds led to DNA damaging and fragmentation that was associated with the induction of apoptosis via mitochondrial pathway. This pathway is initiated by the impairment of mitochondrial transmembrane potential (Δψ _m) and in response to that the mitochondria released cytochrome c increased, that in turn activated caspase-9 and caspase-3 and induced apoptosis. Compounds 17b and 20b were promising anti-cancer agents that induced intrinsic apoptosis pathway in colon cancer cells.

Full text of DOI:10.1016/j.ejmech.2014.03.021

European Journal of Medicinal Chemistry 77 (2014) 323e333
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Induction of intrinsic apoptosis pathway in colon cancer HCT-116 cells
by novel 2-substituted-5,6,7,8-tetrahydronaphthalene derivatives
,
b
,
b,c
Amira M. Gamal-Eldeen a , Nehal A. Hamdy , Hatem A. Abdel-Aziz
,
*
*
Enas A. El-Hussieny d, Issa M.I. Fakhr b
a Cancer Biology Laboratory, Center of Excellence for Advanced Sciences, National Research Center, Dokki, 12622 Cairo, Egypt
b Applied Organic Chemistry Department, National Research Centre, Dokki, Cairo, Egypt
c Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia
d Zoology Department, Faculty of Science, Ain Shams University, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
2-Acetyl tetralin (1) reacted with N,N-dimethylformamide dimethylacetal (DMF-DMA) to afford
the enaminone 3. The reaction of 3 with piperidine and morpholine afforded the trans enaminone
5a,b, respectively. Compound 3 was treated with primary aromatic amines to give secondary enaminones
6aee. The enaminone 3 reacted with acetylglycine and hippuric acid to yield pyranones 10a, b,
respectively. The reaction of enaminone 3 with 1,4-benzoquinone and 1,4-naphthoquinone gave ben-
zofuranyl tetralin derivatives 14a,b, respectively. Also, when 3 reacted with 5-amino-3-phenyl-1H-pyr-
azole 15a and 5-amino-1,2,3-triazole 15b, it afforded the new pyrazolo[1,5-a]pyrimidine 17a and 1,2,3-
triazolo[1,5-a]pyrimidine 17b, respectively. While the reaction of 3 with pyrimidines 18a, b resulted in
the formation of pyrido[2,3-d]pyrimidine derivatives 20a, b, respectively. Investigations of the cytotoxic
effect of those compounds against different human cell lines indicated that some compounds showed
high selective cytotoxicity against colon cancer HCT-116 cells. Some of these compounds led to DNA
damaging and fragmentation that was associated with the induction of apoptosis via mitochondrial
Received 26 October 2010
Received in revised form
7 March 2014
Accepted 9 March 2014
Available online 12 March 2014
Keywords:
2-Acetyl tetralin
Enaminone
Pyranone
Pyrazolo[1,5-a]pyrimidine
1,2,3-Triazolo[1,5-a]pyrimidine
Pyrido[2,3-d]pyrimidine
Mitochondrial membrane potential
Apoptosis
pathway. This pathway is initiated by the impairment of mitochondrial transmembrane potential (Djm
)
and in response to that the mitochondria released cytochrome c increased, that in turn activated caspase-
9 and caspase-3 and induced apoptosis. Compounds 17b and 20b were promising anti-cancer agents that
induced intrinsic apoptosis pathway in colon cancer cells.
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
as Fas receptor) and tumor necrosis factor receptor [5], followed by
the activation of caspase-8, which activates the downstream
Apoptosis, programmed cell death, plays an important role in
developmental processes by eliminating unwanted cells so as to
maintain homeostasis in healthy tissue [1]. Perturbations in its
regulation contribute to numerous pathological conditions,
including cancer and autoimmune and degenerative diseases [2,3].
On the other hand, a wide variety of chemotherapeutic agents have
been shown to cause the death of cancer cells by inducing apoptosis
[4]. Two principal signal pathways have been established for the
induction of apoptotic cell death, the extrinsic pathway (the death
receptor pathway), which is triggered following the activation of
cell-surface-expressed death receptors such as CD95 (also known
effector caspases ꢀ3 and ꢀ7, and then ꢀ6. The intrinsic apoptotic
pathway (the mitochondrion-mediated pathway) is initiated in
response to a variety of stress signals [6], and a complex interplay of
Bcl-2 proteins relays this signal to the mitochondrial outer mem-
brane (OM) to initiate Bak and Bax activation, oligomerisation and
OM damage. Breaching the mitochondrial OM releases apoptogenic
factors, including cytochrome c and Smac, which activate a group of
aspartate-specific proteases (caspases) [7]. Caspases, in turn, cleave
several hundred cellular proteins to coordinate the destruction of
the cell [8,9]. By contrast, the extrinsic apoptotic pathway can
activate caspases without the participation of mitochondria [5].
In certain types of cells, effector caspase activation requires
amplification of death-inducing signaling complex signals by
engagement of the cell-intrinsic pathway. A critical step in the cell-
intrinsic pathway is the activation of Bax, leading to dissipation of
mitochondrial transmembrane potential (Djm) and cytochrome c
* Corresponding authors.
E-mail
addresses:
aeldeen7@yahoo.com
(A.M.
Gamal-Eldeen),
drnehalhamdy63@hotmail.com (N.A. Hamdy).
http://dx.doi.org/10.1016/j.ejmech.2014.03.021
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

324
A.M. Gamal-Eldeen et al. / European Journal of Medicinal Chemistry 77 (2014) 323e333
release into the cytosol [10]. The compounds that induce apoptosis
may be implemented by the cell-intrinsic pathway, which regularly
begins with the disruption of Djm and the release of apoptogenic
factors such as cytochrome c from the intermembrane space into
the cytosol [11]. These factors activate caspase-9, which in turn
activates the executioner caspase-3. In this pathway, mitochon-
drion is the center of cell death control, and the mitochondrial
membrane is the primary site of action by proapoptotic and anti-
apoptotic factors [10].
The biological activities for pyrazolo[1,5-a]pyrimidines have
stimulated chemists to develop the chemistry of this class of
compounds [12], however, enaminones have been reported as
useful precursors for the synthesis of pyrazolo[1,5-a]pyrimidines
[13,14]. Enaminones are versatile reagents and their utility in
heterocyclic synthesis has received a considerable attention [15].
On the other hand, a great deal of interest has been focused on
the synthesis of tetralin derivatives due to their biological po-
tentialities [16]. Moreover, pyridopyrimidine derivatives were
reported as adenosine kinase inhibitors and potent anticancer
drugs [17]. Similarly, the importance of pyran-2-one derivatives
as building blocks in the field of synthetic and medicinal chem-
istry has been well established as a consequence of their inter-
esting structural features and diverse pharmacological properties
[18]. In addition, many benzofuran derivatives are reported as
interesting pharmaceutical compounds [19e21]. They have
potent pesticidal and insecticidal [22], antitumor [23], anthel-
mintic [24], nematocidal [24], antifungal [25], and anti-
inflammatory [26] activities, and its nucleus is incorporated in
various natural products [27].
elemental analysis and spectral data, however, the E-configuration
of the latter enaminone 3 have been reported [28,30].
The reactivity of the enaminone 3 towards some nitrogen nu-
cleophiles was investigated. Thus compound 3 was reacted with
some secondary amines namely piperidine 4a and morpholine 4b
in refluxing ethanol to yield the corresponding tertiary amines 5a, b
respectively. The IR spectra of the latter products showed carbonyl
absorption bands at the region 1637, 1657 cmꢀ1 respectively. Their
1H NMR spectra are free of signals characteristic for the dimethyl-
amine protons, in addition the value of the coupling constant
(J ¼ 12.2 Hz) for the olefinic protons indicates that the enaminones
5a, b exist in the trans configuration.
The enaminone 3 reacted with primary aromatic amines namely
aniline, 4-chloroaniline, 4-toludine, 4-anisidine and 4-nitroaniline
to yield the corresponding secondary amines 6aee, respectively,
through nucleophilic addition substitution reaction as a singlet
product in each case (as examined by TLC). The stereochemistry of
each compound (6aee) was assigned, based on the 1H NMR spec-
troscopy in DMSO-d6. They were found to exist in the E/Z geometric
conjugation forms. 1H NMR spectra revealed two sets of doublet
each belonging to the NH group of the cis and trans conformers
were observed between
d 9.78e10.50 ppm and d 11.95e12.20 ppm
for a total of one proton. The up-field lines of eNH protons were
assigned to cis conformer and downfield lines of the protons of the
same group to trans conformer [31].The formation of the Z-form
was proven by the coupling constant of the hydrogen atoms
attached on the double bond (J ¼ 8.0 Hz) and also by a distinct
splitting of the hydrogen atom on the NH group (J ¼ 12.5 Hz) fixed
via an intramolecular hydrogen bond [32]. The E-form was
confirmed by the coupling constant of the hydrogen atoms
attached on the double bond (J ¼ 12.5 Hz). In general, tertiary
enaminones tend to adopt the E form, which are sterically less
hindered while secondary enaminones exist predominantly, and in
many cases completely, in the Z form in polar solvents. This allows
for intramolecular hydrogen bonding. This observation is also in
accordance with the results in the literature [33e35].
All the previous findings and in continuation of our interest in
the synthesis of a wide variety of heterocyclic systems for biological
screening [28,29], we report here the potentiality of E-3-(N,N-
dimethylamino)-1-(5,6,7,8-tetrahydronaphthalen-2-yl)prop-2-en-
1-one (3) in the synthesis of the title compounds. We also evaluated
their influence on the cancer cell death and of both of the extrinsic
and intrinsic pathways of apoptosis.
Reacting compound 3 with acetylglycine (7a) or hippuric acid
(7b) in acetic anhydride afforded the 3-pyranyl tetrahydronaph-
thalene derivatives 10a, b, respectively. These are assumed to be
formed via initial cyclization of acetylglycine and hippuric acid into
the oxazolones 8a, b which then added to the activated double
bond system of a molecule of compound 3 yielding compounds 9a,
b followed by further rearrangement to give 10a, b, as shown in
2. Results and discussion
Treatment of 2-acetyl tetralin (1) with N,N-dimethylformamide
dimethylacetal (DMF-DMA) in refluxing dry toluene, afforded E-3-
(N,N-dimethylamino)-1-(5,6,7,8-tetrahydronaphthalen-2-yl)prop-
2-en-1-one (3) [28] (Scheme 1) and its structure was confirmed by
Scheme 1. Synthesis of enaminones 5a, b and 6a-e.

A.M. Gamal-Eldeen et al. / European Journal of Medicinal Chemistry 77 (2014) 323e333
325
Scheme 2. This is similar to the well known Kepe pyranone syn-
thesis [36a] that has been adopted by Elnagdi et al. [36b]. The
structures of latter compounds were confirmed on the basis of the
elemental analysis and spectral data. For example, their IR spectra
revealed absorption bands at 3287 and 3391 cmꢀ1, respectively, due
to NH function in addition to two absorption bands in the regions
1704e1705 cmꢀ1 and 1677e1658 cmꢀ1, respectively due to two
carbonyl function. Their 1H NMR spectra showed the protons of
pyran moiety in addition to D2O exchangeable broad singlet due to
NH function.
phenyl-1H-pyrazole (15a) or 3-amino-1,2,4-triazole (15b) in
refluxing ethanol, in the presence of a catalytic amount of piperi-
dine furnished in each case a single product identified as pyrazolo
[1,5-a]pyrimidine 17a and 1,2,3-triazolo[1,5-a]pyrimidine 17b de-
rivatives, respectively (Scheme 3). The structures of the latter
compounds were confirmed by elemental analysis and spectral
data. For example, the IR spectrum of 17a, b revealed no bands due
to amino or carbonyl functions. Moreover, the 1H NMR spectrum for
17a revealed a singlet signal due to pyrazole proton at
addition to doublet signals at 8.12, 8.56 ppm due to pyrimidine
protons. Also the 1H NMR spectrum for 17b revealed doublet sig-
nals at 8.23, 8.86 ppm due to pyrimidine protons, in addition to a
singlet signal due to the triazole proton at 8.73 ppm. Although the
d 5.31 ppm in
d
Compound 3 reacted with 1,4-benzoquinone (11a) and 1,4-
naphthoquinone (11b) yielding the benzofuran 14a and naph-
thofuran 14b, respectively. It is believed that compound 3 firstly
added to the quinone to yield the intermediate phenolic adduct
that cyclized via loss of dimethylamine to yield final isolable
products. Similar reaction sequence has been proposed to account
for the formation of benzofuran and naphthofuran from reaction of
enaminones with quinones [37]. Structures of 14a and 14b were
supported by spectral data. For example, their IR spectra showed
broad band of hydroxyl group at 3330e3241 cmꢀ1 and it also
d
d
endo cyclic nitrogen in compounds 15a, b is the most nucleophilic
site [38,39], it is sterically hindered site. Formation of compounds
17a, b are therefore assumed to take place via an initial Michael
addition of the exo-cyclic amino group in compounds 15a, b to the
activated double bond in compound 3 to give the acyclic non-
isolable intermediate 16a, b which undergo cyclization via loss of
both dimethylamine and water molecules to produce the final
isolable products 17a, b as depicted in Scheme 3.
revealed the carbonyl absorption band at 1619e1626 cmꢀ1
respectively. Their 1H NMR spectrum showed a characteristic
downfield signal at 8.54 and 8.69 ppm of C1 proton of furan
,
Enaminone
3 condensed readily with 6-aminopyrimidin-
d
2,4(1H,3H)-dione (18a) or 6-amino-2-thioxo-2,3-dihydropyrimidin-
4(1H)-one (18b) in boiling acetic acid to give pyrido[2,3-d]pyrimidine
derivatives 20a, b not the isomeric structures 21a, b (Scheme 3). The
1H NMR spectra of the resulting products 20a, b displayed two
doublet signals recognizable as arising from two CH of pyridine in the
moiety and revealed D2O exchangeable broad singlet at
10.21 ppm due to hydroxyl group, respectively.
d 9.40 and
The reaction of enaminone 3 with some heterocyclic amines as
potential precursors for fused heterocyclic systems was also
investigated. Thus when compound 3 was treated with 5-amino-3-
regions
d 7.89e8.29 and 7.86e8.26 ppm and two NH groups of
Scheme 2. Synthesis of pyranones 10a, b, benzofuran 14a and naphthofuran 14b.

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A.M. Gamal-Eldeen et al. / European Journal of Medicinal Chemistry 77 (2014) 323e333
Scheme 3. Synthesis of pyrazolo[1,5-a]pyrimidine 17a, 1,2,3-triazolo[1,5-a]pyrimidine 17b and pyrido[2,3-d]pyrimidines 20a, b.
pyramidie in the region
d
8.92e11.68 and 8.99e11.93 ppm, respec-
is produced through oxidative phosphorylation by the mitochon-
drial respiratory chain (RC) that localized in the inner membrane of
the mitochondria and includes cytochrome c as an electron carrier
[41] The electron transport generates an electrochemical proton
gradient across the inner membrane, measured as Djm, which
drives ATP synthesis by the ATP synthase [41]. Since loss of Djm
induces release of apoptogenic factors into cytoplasm and decrease
of ATP generation, leading to cell death, in the present study, we
have demonstrated that the preferential target of some compounds
(6e, 10a, 17b, and 20b) was the mitochondria and through inducing
of mitochondrial dysfunctions. The mitochondrial dysfunction was
assessed by measuring Djm in HCT-116 cells. Interestingly, the
ability to lower the Djm was observed to be dominant with 17b
(P < 0.001) and 20b (P < 0.001), which were more effective than the
positive control; CCCP (Fig. 2A). Moreover, the results indicated 5b,
6b and 14a did not impair Djm, which may suggest that these
compounds induced apoptosis through death receptors pathway
rather than mitochondrial mediated pathway.
tively. The formation of compounds 20a, b can be explained on the
basis of an initial Michael type addition of the amino group in ami-
nopyrimidines 18a, b to the double bond in enaminone 3 to afford the
non-isolable intermediates 19a, b, which underwent cyclization and
aromatization via loss of both dimethylamine and water molecules
producing the final isolable pyridopyrimidine derivatives 20a, b and
not 21a, b on the basis of the spectral data of the isolated products,
which in agreement with that recently reported [40].
Screening of the cytotoxic effect of the synthesized compounds
against variable human cancer cell line (Hep-G2, HeLa, HCT-116 and
MCF-7), using MTT assay, revealed that most of the tested com-
pounds showed low cytotoxic effect against Hep-G2, HeLa, and
MCF-7 cells as indicated by their high IC50 values (Table 1). On the
other hand, some compounds showed high selective cytotoxicity
against HCT-116 cells as indicated by their low IC50 values (<10 mg/
ml) in the following order 20b < 17b < 6e < 5b < 6b < 10a < 14a
(Table 1). 20b showed the highest potential cytotoxicity against
HCT-116 cells as effective as that of paclitaxel as concluded from
their relatively close IC50 values. According to these findings, we
selected HCT-116 cell line to explore the cell death mechanism due
to the treatment with the promising cytotoxic compounds: 5b, 6b,
6e, 10a, 14a, 17b, and 20b.
Table 1
The cytotoxicity effect of different synthesized compounds of Schemes 1e3 as
measured by MTT assay, after 48 h of incubation. The results are expressed as IC50
;
mg/ml.
Compounds
(IC50; mg/ml)
To detect the type of cell death, the IC50 of each compound in
HCT-116 cells were examined and the apoptosis and necrosis per-
centages were recorded using acridine orange/ethidium bromide
staining and analysis under fluorescence microscope. As shown in
Fig. 1A, all of the tested compounds led to an apoptosis-dependant
cell death 77e96% of the total number of dead cell population,
while the percentage of necrotic cells were only 0e23% of the total
number of dead cell population. Comparing the untreated cells
with paclitaxel-treated HCT-116 cells indicated that the later
significantly induced the DNA fragmentation up to 44% (P < 0.001),
as shown in Fig. 1B. Similarly the treatment of HCT-116 cells with
different compounds resulted in a high, to variable extents, DNA
fragmentation levels that were significantly different from control
(P < 0.01e0.001), in the following order: 20b > 17b > 10a <
5b < 6b < 6e < 14a.
Hep-G2
HeLa
HCT-116
MCF-7
3
5a
5b
6a
6b
6c
6d
6e
10a
10b
14a
14b
17a
17b
20a
20b
Paclitaxel
22.3
39.1
18.2
19.9
30.3
42.7
31.8
27.4
45.3
29.3
39.2
31.7
37.6
15.9
39.9
27.0
0.59
33.4
35.6
41.2
30.6
19.9
28.7
46.1
48.4
30.4
38.3
41.6
40.5
36.2
21.8
29.1
25.3
0.47
18.9
35.4
7.2
41.6
7.9
42.1
31.8
6.8
8.4
47.7
9.5
36.2
26.7
2.1
18.6
0.49
0.38
29.3
41.1
38.6
32.2
28.7
43.6
43.1
28.5
20.9
27.5
40.3
48.7
19.9
48.2
24.4
18.2
0.61
Mitochondria are subcellular organelles that are essential in the
regulation of cellular bioenergetics as a major source of ATP, which

A.M. Gamal-Eldeen et al. / European Journal of Medicinal Chemistry 77 (2014) 323e333
327
Fig. 1. A. The type of cell death was investigated in HCT-116 cells after the treatment
with different compounds, using acridine orange/ethidium bromide staining to
compare between the percentage of necrotic cells (black segment) and the apoptotic
cells (gray segment). B. The effect of different compounds (gray bars) and paclitaxel
(white bars) on the percentage of DNA fragmentation in colon cancer HCT-116 cells.
The fragmented DNA was determined with the diphenylamine reaction. Values are
means of three measurements (Mean ꢁ SD), where (*) represent the P < 0.05 in
comparison with control (black bars).
Fig. 2. A. The effect of different compounds (gray bars) on mitochondrial membrane
potential (Djm) of colon cancer HCT-116 cells. CCCP was the positive control (white
bars). Values are expressed as percentage of zero time readings and represent mean of
three readings ꢁ S.D. B. Dot blot analysis of the cytosolic content of cytochrome C in
the compounds-treated HCT-116 cells. Data are expressed as the mean percentage of
color intensity of dots of three replicates, where (*) represent the P < 0.05 in com-
parison with control (black bars).
Besides respiration, mitochondria also play important roles in
the regulation of apoptosis. The mitochondrial pathway of
apoptosis is initiated by the proapoptotic Bcl-2 family proteins,
such as Bax and Bak, which form pores and induce mitochondrial
outer membrane permeabilization [41]. This leads to a release of
cytochrome c, loss of Djm, and activation of various caspases. These
caspases cleave specific substrates within the cell to produce
changes associated with apoptosis (Djm is important for ATP pro-
duction and mitochondrial protein transport) [42,43]. On the other
hand, disruption of Djm is also implicated in various apoptotic

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A.M. Gamal-Eldeen et al. / European Journal of Medicinal Chemistry 77 (2014) 323e333
phenomena [44] and mitochondrial dysfunction and dysregulation
of apoptosis are implicated in many diseases such as cancer.
To investigate the effect of Djm loss in the release of apopto-
genic factors into cytoplasm we investigated the alteration in cy-
tochrome C and caspases release after the treatment with 6e, 10a,
17b, and 20b. Cytochrome c, a component of the mitochondrial
electron transfer chain that is present in the intermembrane space,
is released into the cytosol during the early phases of apoptosis
[45]. Therefore, we assayed the accumulation of mitochondrial
cytochrome c release into the cytosol by immunoblotting of the cell
lysate of compounds-treated HCT-116 cells. As shown in Fig. 2B,
cytosol from untreated cells contained low cytochrome c content.
In contrast, cytochrome c accumulated significantly in the cytosol
of the cells after treated with 6e and 10b (P < 0.01) and highly
accumulated after treated with 17b, and 20b (P < 0.001).
In many apoptotic systems, release of cytochrome c into the
cytosol results in the activation of the executioner caspases of
apoptosis. To determine whether caspases activation is involved
in the compounds associated apoptosis, caspases were investi-
gated in HCT-116 cells. The analysis revealed that there was a
dramatic increase in the total caspases level after the treatment
with 6e and 10a (P < 0.05), 17b and 20b (P < 0.01), as shown in
Fig. 3. On the other hand, the investigation of the individual cas-
pases indicated that the treatment of HCT-116 cells with 17b and
20b led to a significant increase (P < 0.01) in caspase-3 and -9
activities; whereas 17b led to a significant enhancement (P < 0.01)
in caspase-3 but not caspase-9, in addition to 10a which led to
significant enhancement (P < 0.01) in caspase-9 but not caspase-
3, as shown in Fig. 3.
3. Conclusion
The important characteristic in anychemotherapeutic drugs is its
ability to induce Djm collapse and cytochrome c release, thereby
induce the activation of the downstream caspases and apoptosis
[46]. In the present work, some of the synthetic compounds showed
high selective cytotoxicityagainst colon cancer HCT-116 cells among
all the tested human cancer cell lines. The treatment of cells HCT-116
with 6e, 10a, 17b, and 20b resulted in DNA damaging and frag-
mentation that was associated with the induction of apoptosis via
mitochondrial pathway. This pathway is initiated by 6e,10a,17b, and
20b treatment through the impairment of Djm and in response to
that besides the DNA damage and the mitochondria released cyto-
chrome c that may in turn activated caspase-9 through auto-
proteolysis. The treatment with 17b and 20b resulted in activation
of caspase-9 proteolyses activated caspase-3, which activates other
downstream caspases causing the apoptotic phenotype. Com-
pounds 17b and 20b are promising anti-cancer agents that induced
intrinsic apoptosis pathway in colon cancer cells. These findings
required further in vitro and in vivo investigations for confirmation.
4. Experimental
4.1. Chemistry
Melting points were measured with a Gallenkamp apparatus and
are uncorrected. IR spectra were recorded on Shimadzu FT-IR 8101
PC infrared spectrophotometer. The 1H NMR spectra were recorded
on a Varian Mercury VX-300 NMR spectrometer. 1H NMR spectra
were run at 300 MHz and 13C NMR spectra were run at 75.46 MHz in
deuterated dimethylsulfoxide (DMSO-d6). Chemical shifts are
In the present work the mechanistic evaluation of the cytotoxic
compounds against HCT-116 cells revealed that 6e,10a,17b and 20b
are the most promising compounds as intrinsic apoptosis inducer
agents. This activity may be due to the presence of the nitrophenyl
group in 6e, acetamide group in 10a, triazolopyrimidine ring in 17b
and the pyridopyrimidine ring in 20b. These active ring systems
represent the characteristic difference of each compound from its
corresponding family of compounds in Schemes 1e3 that may be
responsible for their mechanistic role in apoptosis process.
quoted in
d (ppm) and were related to that of the solvents. Mass
spectra were measured on a GCMS-QP1000 EX spectrometer at 70
e.V. Elemental analyses was carried out at the Microanalytical center
of Cairo University. 1-(5,6,7,8-Tetrahydronaphthalen-2-yl)ethanone
(1) [47] was prepared by the reported method and 5-amino-3-
phenyl-1H-pyrazole (15a), 3-amino-1,2,4-triazole (17b), Glycine
derivatives 7a-7b and pyrimidine derivatives 18ae18b were used as
obtained commercially.
4.1.1. E-3-(N,N-dimethylamino)-1-(5,6,7,8-tetrahydronaphthalen-
2-yl) prop-2-en-1-one (3) [28]
To a mixture of 1-(5,6,7,8-tetrahydronaphthalen-2-yl)ethanone
(1) [47] (1.74 g, 10 mmol) in dry toluene (50 mL),
dimethylformamide-dimethylacetal (DMF-DMA) (1.34 g, 10 mmol)
was added and the mixture was refluxed for 5 h. The solvent was
evaporated and the residual reddish brown viscous liquid was taken
in ether. The resulting yellow crystals were collected by filtration,
washed thoroughly with ether, dried and finally recrystallized from
EtOH to afford compound 3 [28] as yellow crystals in 58% yield, mp
70e72 ꢂC; IR (KBr) (
(ppm) 1.71 (m, 4H, aliphatic 2CH2 of tetrahydronaphthalene), 2.72
n
, cmꢀ1): 1634 (C]O); 1H NMR (DMSO-d6):
d
(m, 4H, aliphatic 2CH2 of tetrahydronaphthalene), 2.87 (s, 3H, CH3),
3.10 (s, 3H, CH3), 5.76 (d,1H, J ¼ 12.2 Hz, eCOeCH ¼ ), 7.05e7.58 (m,
3H AreH), 7.65 (d, 1H, J ¼ 12.2 Hz, ]CHeNe); 13C NMR (DMSO-d6):
d
(ppm) 20.12, 20.13 (2CH3), 22.48, 22.60, 28.70, 28.75 (4CH2), 90.93
(COeCH ¼ ), 124.25,127.67,128.44,136.17, 137.52,139.62 (Aromatic-
C), 153.63 (]CHeNe), 185.70 (C]O); MS m/z (%): 229 (Mþ, 92.4),
212 (99.0),159 (43.5), 98 (100), 70 (74.5). Analysis (Calc/found %): for
C15H19NO (229.32): C, 78.56/78.24; H, 8.35/8.30; N, 6.11/6.28.
4.1.2. General procedure reaction of the enaminone 3 with
piperidine and morpholine
A mixture of E-3-(N,N-dimethylamino)-1-(5,6,7,8-tetrahydro-
naphthalen-2-yl)prop-2-en-1-one (3) (1.15 g, 5 mmol) and the
Fig. 3. The effect of different compounds on the level of total caspases (black bars),
caspase-3 (white bars) and caspase-9 (gray bars) in the HCT-116 cells. Data are
expressed as fold of control untreated cells activity, where (*) represent the P < 0.05 in
comparison with control.

A.M. Gamal-Eldeen et al. / European Journal of Medicinal Chemistry 77 (2014) 323e333
329
appropriate secondary amine piperidine 4a or morpholine 4b
(3 mL) in ethanol (30 mL) was refluxed for 2 h, then left to cool. The
solid so formed, in each case, was collected by filtration and
recrystallized from ethanol to afford 5a and 5b, respectively.
(DMSO-d6): d (ppm) 1.74 (m, 4H, aliphatic 2CH2 of tetrahy-
dronaphthalene), 2.25 (s, 3H, CH3), 2.76 (m, 4H, aliphatic 2CH2
of tetrahydronaphthalene), 6.08 and 6.44 (d, 1H, J ¼ 8.0 and
12.5 Hz, eCOeCH], cis and trans conformers), 7.13e7.95 (m, 8H,
AreH þ ]CHeNe, cis and trans conformers), 9.98 and 11.99 (d, 1H,
J ¼ 12.5 Hz, NH, D2O exchangeable, cis and trans conformers); MS
m/z (%): 292 (Mþ þ 1, 25.3), 291 (Mþ, 92.6), 290 (100.0), 160 (60.5),
91 (45.8). Analysis (Calc/found %): for C20H21NO (291.40): C, 82.44/
82.17; H, 7.26/7.40; N, 4.81/4.92.
4.1.2.1. E-3-(piperidin-1-yl)-1-(5,6,7,8-tetrahydronaphthalen-2-yl)
prop-2-en-1-one (5a). Yield (64%); mp 134e136 ꢂC; IR (KBr) (
n,
cmꢀ1): 1637 (C]O); 1H NMR (DMSO-d6):
d (ppm) 1.56 (m, 6H, 3CH2
of piperidine), 1.71 (m, 4H, aliphatic 2CH2 of tetrahydronaph-
thalene), 2.73 (m, 4H, aliphatic 2CH2 of tetrahydronaphthalene),
3.39 (m, 4H, (CH2)2 ¼ Ne of piperidine), 5.94 (d, 1H, J ¼ 12.2 Hz, e
COeCH ¼ ), 7.07e7.58 (m, 3H AreH), 7.60 (d, 1H, J ¼ 12.2 Hz, ]CHe
Ne); MS m/z (%): 269 (Mþ, 100%), 252 (96.2), 186 (65.8), 159 (80.2),
138 (73.3), 110 (98.6), 84 (99.8). Analysis (Calc/found %): for
4 . 1. 3 . 4 . 3 - [ ( 4 - M e t h o x y p h e n y l ) a m i n o ] - 1 - ( 5 , 6 , 7 , 8 -
tetrahydronaphthalen-2-yl) prop-2-en-1-one (6d). Yield (70%); mp
122e124 ꢂC, the ratio of cis/trans: 69/31; IR (KBr) ( , cmꢀ1): 3385
n
(NH), 1617 (C]O); 1H NMR (DMSO-d6):
d (ppm) 1.74 (m, 4H,
C
18H23NO (269.39): C, 80.26/80.08; H, 8.61/8.74; N, 5.20/5.03.
aliphatic 2CH2 of tetrahydronaphthalene), 2.75 (m, 4H, aliphatic
2CH2 of tetrahydronaphthalene), 3.76 (s, 3H, CH3), 6.00 and 6.34 (d,
1H, J ¼ 8.0 and 12.5 Hz, eCOeCH], cis and trans conformers), 6.92e
7.24 (m, 8H, AreH þ ]CHeNe, cis and trans conformers), 9.78 and
12.05 (d, 1H, J ¼ 12.5 Hz, NH, D2O exchangeable, cis and trans
conformers); MS m/z (%): 308 (Mþ þ 1, 15.8), 307 (Mþ, 5.3), 306
(100.0),159 (96.8), 85 (99.8). Analysis (Calc/found %): for C20H21NO2
(307.40): C, 78.15/77.96; H, 6.89/7.83; N, 4.56/4.44.
4.1.2.2. E-3-morpholino-1-(5,6,7,8-tetrahydronaphthalen-2-yl)prop-
2-en-1-one (5b). Yield (65%); mp 115e117 ꢂC; IR (KBr) ( , cmꢀ1):
1657 (C]O); 1H NMR (DMSO-d6):
(ppm) 1.72 (m, 4H, aliphatic
2CH2 of tetrahydronaphthalene), 2.74 (m, 4H, aliphatic 2CH2 of
tetrahydronaphthalene), 3.00 (m, 4H, (CH2)2 ¼ Ne of morpholine),
3.92 (m, 4H, eCH2eOeCH2e of morpholine), 5.70 (d, 1H,
J ¼ 12.2 Hz, eCOeCH ¼ ), 7.05e7.58 (m, 3H, AreH), 7.7 (d, 1H,
J ¼ 12.2 Hz, ]CHeNe); MS m/z (%): 271 (Mþ, 23.3), 174 (32.7), 159
(100), 131 (58.5), 91 (48.6). Analysis (Calc/found %): for C17H21NO2
(271.36): C, 75.25/75.25; H, 7.80/7.80; N, 5.16/5.16.
n
d
4.1.3.5. 3-[(4-Nitrophenyl)amino]-1-(5,6,7,8-tetrahydronaphthalen-
2-yl)prop-2-en-1-one (6e). Yield (78%); mp 195e197 ꢂC, the ratio of
cis/trans: 41/59; IR (KBr) (
NMR (DMSO-d6): (ppm) 1.76 (m, 4H, aliphatic 2CH2 of tetrahy-
n
, cmꢀ1): 3442 (NH), 1645 (C]O); 1H
d
4.1.3. General procedure for the reaction of enaminone 3 with
primary aromatic amines
dronaphthalene), 2.77 (m, 4H, aliphatic 2CH2 of tetrahydronaph-
thalene), 5.75 and 6.61 (d, 1H, J ¼ 8.0 and 12.5 Hz, eCOeCH], cis
and trans conformers), 6.92e7.58 (m, 8H, AreH and ]CHeNe, cis
and trans conformers), 10.50 and 12.20 (d, 1H, J ¼ 12.0 Hz, NH, D2O
exchangeable, cis and trans conformers); 13C NMR (DMSO-d6):
To a mixture of enaminone derivative 3 (1.15 g, 5 mmol) in acetic
acid (20 mL), the appropriate primary aromatic amine namely
(aniline, 4-chloroaniline, 4-toludine, 4-anisidine and 4-
nitroaniline) (5 mmol) was added and the reaction mixture was
stirred for 3 h. The precipitated product was collected by filtration,
washed with ethanol and dried. Crystallization from ethanol
afforded the corresponding derivatives 6aee, respectively.
d
(ppm) 22.48, 22.51, 28.75, 28.85 (4CH2), 96.38 and 101.70 (eCOe
CH ¼ , cis and trans conformers), 124.50, 125.79, 128.09, 129.07,
135.48, 136.14, 140.84, 141.35, 141.62, 141.70, 141.80, 143.25 (Aro-
matic-C), 146.18, 147.240 (]CHeNe, cis and trans conformers),
187.500, 190,320 (C]O, cis and trans conformers); MS m/z (%): 323
(Mþ þ 1, 34.6), 322 (Mþ, 100.0), 159 (93.7), 91 (76.4). Analysis (Calc/
found %): for C19H18N2O3 (322.37): C, 70.79/70.58; H, 5.63/5.54; N,
8.69/8.61.
4.1.3.1. 3-Phenylamino-1-(5,6,7,8-tetrahydronaphthalen-2-yl)prop-
2-en-1-one (6a). Yield (71%); mp 160e162 ꢂC, the ratio of cis/trans:
60/40; IR (KBr) (
d6): (ppm) 1.75 (m, 4H, aliphatic 2CH2 of tetrahydronaphthalene),
n
, cmꢀ1): 3226 (NH), 1660 (C]O); 1H NMR (DMSO-
d
2.76 (m, 4H, aliphatic 2CH2 of tetrahydronaphthalene), 6.07 and
6.41 (d, 1H, J ¼ 8.0 and 12.5 Hz, eCOeCH ¼ , cis and trans con-
formers), 7.12e7.96 (m, 9H, AreH þ ]CHeNe, cis and trans con-
formers), 9.90 and 11.99 (d, 1H, J ¼ 12.0 Hz, NH, D2O exchangeable,
cis and trans conformers); MS m/z (%): 277 (Mþ, 23.3), 276 (100),
159 (85.0), 146 (42.2), 91 (55.4). Analysis (Calc/found %): for
4.1.4. General procedure for the reaction of enaminone 3 with
glycine derivatives 7a and 7b
A solution of the enaminone 3 (2.85 g, 10 mmol), acetylglycine
(7a) or hippuric acid (7b) (1.7 g, 10 mmol) in acetic anhydride
(30 mL) was refluxed for 3 h. The solid product obtained upon
cooling was filtered off and recrystallized from DMF/H2O to afford
the pyran derivative 10a and 10b, respectively.
C
19H19NO (277.37): C, 82.28/82.15; H, 6.90/6.83; N, 5.05/4.88.
4 . 1 . 3 . 2 . 3 - [ ( 4 - C h l o r o p h e n y l ) a m i n o ] - 1 - ( 5 , 6 , 7 , 8 -
4.1.4.1. N-[2-Oxo-6-(5,6,7,8-tetrahydronaphthalen-2-yl)-2H-pyran-
tetrahydronaphthalen-2-yl)prop-2-en-1-one (6b). Yield (75%); mp
3-yl]acetamide (10a). Yield (58%); mp 200e202 ꢂC; IR (KBr) (
n,
142e144 ꢂC, the ratio of cis/trans: 57/43; IR (KBr) (
n
, cmꢀ1): 3350
cmꢀ1): 3287 (NH),1704,1677 (2C]O); 1H NMR (DMSO-d6):
d (ppm)
(NH), 1634 (C]O); 1H NMR (DMSO-d6):
d
(ppm) 1.75 (m, 4H,
1.76 (m, 4H, aliphatic 2CH2 of tetrahydronaphthalene), 2.33 (s, 3H,
CH3), 2.77 (m, 4H, aliphatic 2CH2 of tetrahydronaphthalene), 7.0e
7.12 (m, 3H, AreH), 7.50 (d, 1H, J ¼ 8.0 Hz, pyran), 8.20 (d, 1H,
J ¼ 8.0 Hz, pyran), 9.36 (s, 1H, NH, D2O exchangeable); 13C NMR
aliphatic 2CH2 of tetrahydronaphthalene), 2.77 (m, 4H, aliphatic
2CH2 of tetrahydronaphthalene), 6.09 and 6.43 (d, 1H, J ¼ 8.0 and
12.5 Hz, eCOeCH ¼ , cis and trans conformers), 7.14e7.90 (m, 8H,
AreH þ ]CHeNe), 10.0 and 11.95 (d, 1H, J ¼ 12.5 Hz, NH, D2O
exchangeable, cis and trans conformers); MS m/z (%): 312 (Mþþ1,
73.8), 311 (Mþ, 100), 201 (84.7), 159 (37.8), 91 (23.3). Analysis (Calc/
found %): for C19H18ClNO (311.81): C, 73.19/73.00; H, 11.37/11.23; N,
4.49/4.62.
(DMSO-d6):
d (ppm) 20.34 (CH3), 22.41, 22.47, 28.57, 28.63 (4CH2),
121.630, 123.71, 124.53, 124.84, 128.15, 128.63, 137.94, 139.03,
153.04, 158.15 (Aromatic-C), 164.89, 168.49 (2C]O); MS m/z (%):
384 (Mþ þ 1, 24.1), 283 (Mþ, 100.0), 159 (56.7), 91 (35.7). Analysis
(Calc/found %): for C17H17NO3 (283.33): C, 72.07/72.32; H, 6.05/6.14;
N, 4.94/5.11.
4.1.3.3. 3-[(4-Tolyl)amino]-1-(5,6,7,8-tetrahydronaphthalen-2-yl)
prop-2-en-1-one (6c). Yield (68%); mp 134e136 ꢂC, the ratio of cis/
4.1.4.2. N-[2-Oxo-6-(5,6,7,8-tetrahydronaphthalen-2-yl)-2H-pyran-
3-yl]benzamide (10b). Yield (70%); mp 218e220 ꢂC; IR (KBr) (
n,
trans: 62/38; IR (KBr) (
n
, cmꢀ1): 3258 (NH), 1628 (C]O); 1H NMR

330
A.M. Gamal-Eldeen et al. / European Journal of Medicinal Chemistry 77 (2014) 323e333
cmꢀ1): 3391 (NH),1705,1658 (2C]O); 1H NMR (DMSO-d6):
d
(ppm)
155.39, 155.73, 168.41 (Aromatic-C); MS m/z (%): 250 (Mþ, 100), 249
(93.36), 222 (18.73). Analysis (Calc/found %): for C15H14N4 (250.31):
C, 71.98/71.80; H, 5.64/5.68; N, 22.38/22.42.
1.75 (m, 4H, aliphatic 2CH2 of tetrahydronaphthalene), 2.77 (m, 4H,
aliphatic 2CH2 of tetrahydronaphthalene), 7.08e7.54 (m, 8H, Are
H), 7.93 (d, 1H, J ¼ 7.8 Hz, pyran), 8.20 (d, 1H, J ¼ 7.8 Hz, pyran), 9.55
(s, 1H, NH, D2O exchangeable); MS m/z (%): 346 (Mþ þ 1, 30.6), 345
(Mþ, 62.3), 212 (46.2), 105 (100.0), 77 (68.4). Analysis (Calc/found
%): for C22H19NO3 (345.40): C, 76.50/76.53; H, 5.54/5.77; N, 4.06/
3.92.
4.1.7. General procedure for the reaction of enaminone 3 with
pyrimidine derivatives (18a, b)
A mixture of enaminone 3 (0.57 g, 2 mmol) and the appropriate
pyrimidine derivative 18a or 18b (10 mmol) in glacial acetic acid
(25 mL) was refluxed for 6 h, the solid product was filtered off,
washed with ethanol, dried and finally recrystallized from DMF/
H2O to afford the corresponding fused compounds 20a and 20b,
respectively.
4.1.5. General procedure for the reaction of enaminone 3 with p-
benzoquinone and 1,4-naphthoquinone
To a stirred solution of the enaminone 3 (2.29 g, 10 mmol) in
glacial acetic acid (50 mL), p-benzoquinone or 1,4-naphthoquinone
(10 mmol) was added. Stirring was continued overnight at room
temperature. The solid product was filtered off and recrystallized
from EtOH/DMF to afford the corresponding derivatives 14a and
14b, respectively.
4.1.7.1. 5-(5,6,7,8-Tetrahydronaphthalen-2-yl)pyrido[2,3-d]pyrimi-
dine-2,4(1H,3H)-dione (20a). Yield (64%); mp 251e253 ꢂC; IR (KBr)
(n
, cmꢀ1): 3171, 3050 (2NH), 1705, 1675 (2C]O), 1605 (C]N); 1H
NMR (DMSO-d6):
d (ppm) 1.73 (m, 4H, aliphatic 2CH2 of tetrahy-
dronaphthalene), 2.74 (m, 4H, aliphatic 2CH2 of tetrahydronaph-
thalene), 7.14e7.79 (m, 3H, AreH), 7.89 (d, 1H, J ¼ 4.5 Hz, pyridine),
8.29 (d, 1H, J ¼ 4.5 Hz, pyridine), 8.92 (br. s, 1H, NH, D2O
exchangeable), 11.68 (br, s, 1H, NH, D2O exchangeable); MS m/z (%):
294 (Mþ þ 1, 34.8), 293 (Mþ, 100.0), 131 (34.8), 77 (87.3). Analysis
(Calc/found %): for C17H15N3O2 (293.33): C, 69.61/69.43; H, 5.15/
5.32; N, 14.33/14.25.
4 . 1. 5 . 1. ( 5 - H y d r o x y - 1 - b e n z o f u r a n - 3 - y l ) ( 5 , 6 , 7 , 8 -
tetrahydronaphthalen-2-yl)methanone (14a). Yield (70%); mp 206e
208 ꢂC; IR (KBr) ( , cmꢀ1): 3330 (OH), 1619 (C]O); 1H NMR (DMSO-
n
d6): d (ppm) 1.76 (m, 4H, aliphatic 2CH2 of tetrahydronaphthalene),
2.80 (m, 4H, aliphatic 2CH2 of tetrahydronaphthalene), 6.84e7.57
(m, 6H, AreH), 8.54 (s, 1H, furan), 9.40 (s, 1H, OH, D2O exchange-
able); MS m/z (%): 293 (Mþ þ 1, 43.3), 292 (Mþ, 100.0), 161 (80.4),
105 (50.1), 77 (39.5). Analysis (Calc/found %): for C19H16O3 (292.34):
C, 78.06/77.89; H, 5.52/5.48.
4.1.7.2. 5-(5,6,7,8-Tetrahydronaphthalen-2-yl)-2-thioxo-2,3-
dihydropyrido[2,3-d]pyrimidin-4(1H)-one (20b). Yield (68%); mp
288e290 ꢂC; IR (KBr) ( , cmꢀ1): 3385, 3064 (2NH), 1679 (C]O),
n
4.1.5.2. (5-Hydroxynaphtho[1,2-b]furan-3-yl)(5,6,7,8-tetrahydro-
1607 (C]N); 1H NMR (DMSO-d6):
d (ppm) 1.74 (m, 4H, aliphatic
naphthalen-2-yl)methanone (14b). Yield (55%); mp 268e270 ꢂC; IR
2CH2 of tetrahydronaphthalene), 2.75 (m, 4H, aliphatic 2CH2 of
tetrahydronaphthalene), 7.17e7.84 (m, 3H, AreH), 7.86 (d, 1H,
J ¼ 4.5 Hz, pyridine), 8.26 (d, 1H, J ¼ 4.5 Hz, pyridine), 8.99 (br. s, 1H,
(KBr) (
(ppm) 1.77 (m, 4H, aliphatic 2CH2 of tetrahydronaphthalene), 2.81
n
, cmꢀ1): 3241 (OH), 1626 (C]O); 1H NMR (DMSO-d6):
d
(m, 4H, aliphatic 2CH2 of tetrahydronaphthalene), 7.26e8.25 (m,
8H, AreH), 8.69 (s, 1H, furan), 10.21 (s, 1H, OH, D2O exchangeable);
MS m/z (%): 343 (Mþ þ 1, 25.7), 342 (Mþ, 100.0), 211 (71.6), 131
(80.8), 77 (78.2). Analysis (Calc/found %): for C23H18O3 (342.40): C,
80.68/80.54; H, 5.30/5.22.
NH, D2O exchangeable), 11.93 (br, s, 1H, NH, D2O exchangeable); 13
C
NMR (DMSO-d6): d (ppm) 22.39, 22.49, 28.63, 28.75 (4CH2), 109.96,
116.23, 124.27, 127.73, 129.21, 133.75, 136.73, 136.98, 139.79, 151.24,
159.34, (Aromatic-C), 160.88 (C]O), 175.81 (C]S). MS m/z (%): 310
(Mþ þ 1, 14.5), 309 (Mþ, 58.9), 308 (100.0), 128 (14.7). Analysis
(Calc/found %): for C17H15N3OS (309.39): C, 66.00/59.84; H, 4.89/
5.03; N, 13.58/13.44; S, 10.36/10.14.
4.1.6. General procedure for the reaction of enaminone 3 with
heterocyclic amines 15a, b
A mixture of enaminone 3 (0.57 g, 2 mmol) and the appropriate
amine 15a or 15b (10 mmol) in ethanol (25 mL), in the presence of
catalytic amount of piperidine, was refluxed for 6 h. the reaction
mixture left to cool, the solid product was filtered off, washed with
ethanol, dried and finally recrystallized from DMF/H2O to afford the
corresponding derivatives 17a and 17b, respectively.
4.2. Cell culture
A panel of human cell lines was used in investigating the anti-
cancer activity including: Hepatocellular carcinoma (Hep-G2),
cervical carcinoma (HeLa), colon carcinoma (HCT-116), histiocytic
lymphoma and breast adenocarcinoma (MCF-7) (ATCC, VA, USA).
HCT-116 cells were grown in Mc Coy’s medium, while all cells were
routinely cultured in DMEM (Dulbeco’s Modified Eagle’s Medium)
at 37 ꢂC in humidified air containing 5% CO2. All media were sup-
plemented with 10% fetal bovine serum (FBS), 100 units/ml peni-
4.1.6.1. 2-Phenyl-7-(5,6,7,8-tetrahydronaphthalen-2-yl)pyrazolo[1,5-
a] pyrimidine (17a). Yield (63%); mp 180e182 ꢂC; IR (KBr) (
n,
cmꢀ1): 1606 (C]N); 1H NMR (DMSO-d6):
d (ppm) 1.76 (m, 4H,
aliphatic 2CH2 of tetrahydronaphthalene), 2.77 (m, 4H, aliphatic
2CH2 of tetrahydronaphthalene), 5.31 (s, 1H, pyrazole), 7.17e8.03
(m, 8H, AreH), 8.12 (d, 1H, J ¼ 4.5 Hz, pyrimidine), 8.56 (d, 1H,
J ¼ 4.5 Hz, pyrimidine); MS m/z (%): 325 (Mþ, 24.6), 324 (100.0), 128
(15.3), 77 (66.8). Analysis (Calc/found %): for C22H19N3 (325.42): C,
81.20/81.40; H, 5.89/5.97; N, 12.91/13.12.
cillin G sodium, 2 mM L-glutamine 100 units/ml, streptomycin
sulfate, and 250 ng/ml amphotericin B. Monolayer cells were har-
vested by trypsin/EDTA treatment. Cell culture material was ob-
tained from Cambrex BioScience (Copenhagen, Denmark), and all
chemicals were from Sigma (USA). The tested compounds were
diluted before assays and then tested for endotoxin using
PyrogentÒ Ultra gel clot assay that indicated endotoxin free of the
samples. All experiments were repeated four times, unless
mentioned, and the data was represented as (mean ꢁ S.D.).
4.1.6.2. 7-(5,6,7,8-Tetrahydronaphthalen-2-yl)[1,2,4]triazolo[1,5-a]
pyrimidine (17b). Yield (60%); mp 130e132 ꢂC; IR (KBr) ( , cmꢀ1):
n
1608 (C]N); 1H NMR (DMSO-d6):
d (ppm) 1.76 (m, 4H, aliphatic
2CH2 of tetrahydronaphthalene), 2.77 (m, 4H, aliphatic 2CH2 of
tetrahydronaphthalene), 7.06e7.84 (m, 3H, AreH), 8.23 (d, 1H,
J ¼ 4.5 Hz, pyrimidine), 8.73 (s, 1H, triazole), 8.86 (d, 1H, J ¼ 4.5 Hz,
4.3. Cytotoxicity assay
The cytotoxic influence of the compounds on the growth of
different human cancer cell lines was estimated by the 3-(4,5-
pyrimidine); 13C NMR (DMSO-d6):
d
(ppm) 22.31, 22.41, 28.74, 28.80
(4CH2), 109.07, 126.54, 129.10, 129.88, 137.05, 141.12, 147.35, 154.77,
dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide

A.M. Gamal-Eldeen et al. / European Journal of Medicinal Chemistry 77 (2014) 323e333
331
(MTT) assay [48]. Cells (5 ꢃ 104 cells/well) were incubated for 48 h
with various concentrations of the compounds at 37 ꢂC in a FBS-free
medium, before submitted to MTT assay. The absorbance was
measured with microplate reader (FluoStarOptima, BMG) at
570 nm. The relative cell viability was determined by the amount of
MTT converted to the insoluble formazan salt. The data are
expressed as the mean percentage of viable cells as compared to
untreated cells. The relative cell viability was expressed as the
mean percentage of viable cells as compared to the respective
untreated cells (control). The half maximal growth inhibitory con-
centration (IC50) value was calculated from the line equation of the
doseedependent curve of each compound. The results were
compared with the cytotoxic activity of paclitaxel, a known anti-
cancer drug.
see a relatively high ratio of 590/527 nm. Fluorescence values were
monitored in plates at zero time and after 2h by microplate fluo-
rescence reader (FluoStarOptima, BMG). Values are expressed as
percent of zero time reading. Carbonyl cyanide m-chlor-
ophenylhydrazone (CCCP, 10
mM) was used as a positive control.
4.7. Preparation of cell lysates
After harvesting of Compounds-treated and untreated HCT-116
cells, they were washed and centrifuged for 10 min at 1000ꢃ g.
The cell pellet was lysed in 0.5 mL of ice-cold lysis buffer (50 mM
TriseHCl, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 20 mM NaF,
100 mM Na3VO4, 0.5% NP40, 1% Triton X-100, 1 mM phenyl-
methylsulfonyl fluoride, 10 mg/ml aprotinin, and 10 mg/ml leu-
peptin (pH 7.4). The lysates were passed through a 21-gauge needle
to break up cell aggregates. After centrifugation at 14,000ꢃ g for
15 min at 4 ꢂC, the supernatants (total cell lysates) were submitted
to measure cytochrome C and caspases. The cellular total protein
content of the lysates was measured by bicinchoninic acid (BCA)
assay [57] and bovine serum albumin (BSA), as a standard.
4.4. Apoptosis and necrosis staining
The type of cell death was investigated in compounds-treated
and untreated cells using acridine orange/ethidium bromide
staining [49,50]. In brief, a mixture of 100
mg/ml acridine orange
and 100 g/ml ethidium bromide was prepared in PBS. The cell
m
uptake of the stain was monitored under a fluorescence micro-
scope, and the apoptotic, necrotic, and viable cells were counted.
The early apoptotic cells had yellow chromatin in nuclei that were
highly condensed or fragmented. Apoptotic cells also exhibited
membrane blebbing. The late apoptotic cells had orange chromatin
with nuclei that were highly condensed and fragmented. The
necrotic cells had bright orange chromatin in round nuclei. Only
cells with yellow, condensed, or fragmented nuclei were counted as
apoptotic cells in a blinded, nonbiased manner.
4.8. Immunoblotting of cytochrome C
Cytochrome C was assessed in cell lysate of from compounds
-treated and -untreated HCT-116 cells. Briefly, 20
mg of isolated
soluble proteins were applied at dot blotting set using nitrocellu-
lose membranes. The change in cytochrome c protein was detected
by dot immunoblotting using Cytochrome C Releasing Apoptosis
Assay kit (#K257, BioVision, CA, USA). After being washed, bound
antibody was detected using rabbit anti-goat antibody linked to
horseradish peroxidase (Dako), and bound complexes were
detected using O-phenylenediamine dihydrochloride (OPD)
(Amersham-Pharmacia). The percentage of the enhanced color
intensity was shown as fold induction after normalization of dot
intensity with actin control. The dot photographing and analysis
was performed using gel documentation system (Biometra, Goet-
tingen, Germany).
4.5. DNA fragmentation
DNA fragmentation was essentially assayed as reported previ-
ously [51]. Briefly, compounds-treated and untreated cells pellet
was re-suspended in 250 ml 10 mM Tris, 1 mM EDTA, pH 8.0 (TE-
buffer), and incubated with an additional volume lysis buffer (5 mM
Tris, 20 mM EDTA, pH 8.0, 0.5% Triton X-100) for 30 min at 48 ꢂC.
After lysis, the intact chromatin (pellet) was separated from DNA
fragments (supernatant) by centrifugation. Pellets were re-
suspended in TE-buffer and samples were precipitated by 0% tri-
chloroacetic acid at 48 ꢂC. The sample pellets were added to 5%
trichloroacetic acid and boiled. DNA contents were quantified using
the diphenylamine reagent [52,53]. The percentage of DNA frag-
mented was calculated as the ratio of the DNA content in the su-
pernatant to the amount in the pellet [54].
4.9. Evaluation of caspases activity
The cell lysates of the compounds-treated and untreated HCT-
116 cells were submitted to different kits to measure the level of
total caspases, caspase-3, and caspase-9, according to the manu-
facturer instructions. Red Multi-Caspase Staining Kit (# PK-
CA577-K190), PromoKine, Heidelberg, Germany was used for
analysis of total caspases in a black microtiter plate with fluo-
rescence plate reader at Ex. ¼ 540 nm and Em. ¼ 570 nm. The
assay utilizes the caspase family inhibitor VAD-FMK conjugated to
sulfo-rhodamine (Red-VAD-FMK) as the fluorescent in situ
marker. Caspase-3 Colorimetric Detection Kit (# 907e013),
Stressgen biotechnologies, Canada, was used to measure caspase-
3 at 405 nm. One unit of Caspase-3 activity is defined as the
amount of enzyme needed to convert one picomole of substrate
per minute at 30 ꢂC (U/ml). Caspase-9 Colorimetric Assay Kit (#
PK-CA577-K119), PromoKine Heidelberg, Germany, was used to
evaluated Caspase-9 activity. The method is based on spectro-
photometric detection of the chromophore p-nitroanilide (pNA)
after cleavage from the labeled substrate LEHD-pNA. The pNA light
emission can be quantified at 405 nm. In the assay, a constant
4.6. Mitochondrial transmembrane potential (Djm
)
5,59,6,69-Tetrachloro-1,19,3,39-tetraethybenzimidazol carbo-
cyanine iodide (JC-1, Molecular Probes) is a lipophilic, cationic dye
that enters mitochondria in proportion to the membrane potential
and forms J-aggregates at the high intramitochondrial concentra-
tions induced by higher Djm values [55]. Measurement of mito-
chondrial transmembrane potential (Djm) was carried out by JC-1
[55,56]. In 96 well plates, cells were seeded and then preloaded
with 10 m
g/ml JC-1 dissolved in HBSS for 30 min, 37 ꢂC. The JC-1
loaded cells were incubated with and without compounds at
37 ꢂC for 2 h period. JC-1 exist as a monomer (emission 527 nm) at
low Djm but forms J-aggregates (emission 590 nm) at high Djm
,
which can be assessed by JC-1 by monitoring fluorescence emission
ratios at (emission 590:527 nm). The ratio of red (590 nm) to green
(527 nm) gives an index of the Djm: the higher the Djm the greater
proportion of JC-1 aggregates in the mitochondria, the greater the
intensity of the red light signal and so with active mitochondria we
substrate concentration (200 mM final concentration) was added
to the assay reaction. A comparison of the fluorescence or absor-
bance readings of the treated and untreated-cells allows deter-
mination of the fold increase in the total caspases, caspase-3, and
caspase-9 activities.

332
A.M. Gamal-Eldeen et al. / European Journal of Medicinal Chemistry 77 (2014) 323e333
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Advanced Sciences and Applied Organic Chemistry Department,
National Research Center, Dokki, Cairo, Egypt.
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