Novel Chrysin-de-allyl PAC-1 hybrid analogues as anticancer compounds: Design, synthesis, and biological evaluation

Article abstract of DOI:10.3390/molecules25133063

New chrysin-De-allyl-Pac-1 hybrid analogues, tethered with variable heterocyclic systems (4a-4o), were rationally designed and synthesized. The target compounds were screened for in vitro antiproliferative efficacy in the triple-negative breast cancer (TNBC) cell line, MDA-MB-231, and normal human mammary epithelial cells (HMECs). Two compounds, 4g and 4i, had the highest efficacy and selectivity towards MDA-MB-231 cells, and thus, were further evaluated by mechanistic experiments. The results indicated that both compounds 4g and 4i induced apoptosis by (1) inducing cell cycle arrest at the G2 phase in MDA-MB-231 cells, and (2) activating the intrinsic apoptotic pathways in a concentration-dependent manner. Physicochemical characterizations of these compounds suggested that they can be further optimized as potential anticancer compounds for TNBC cells. Overall, our results suggest that 4g and 4i could be suitable leads for developing novel compounds to treat TNBC.

Full text of DOI:10.3390/molecules25133063

molecules
Article
Novel Chrysin-De-Allyl PAC-1 Hybrid Analogues as
Anticancer Compounds: Design, Synthesis,
and Biological Evaluation
Buthina A. Al-Oudat 1,†, Hariteja Ramapuram 2,†, Saloni Malla 2, Suaad A. Audat 3
,
Noor Hussein 2, Jenna M. Len 2, Shikha Kumari 4, Mel F. Bedi 5 , Charles R. Ashby, Jr. 6 and
Amit K. Tiwari 2,
*
1
Department of Medicinal Chemistry and Pharmacognosy, Faculty of Pharmacy, Jordan University of Science
and Technology, P.O. Box 3030, Irbid 22110, Jordan; baoudat@just.edu.jo
Department of Pharmacology and Experimental Therapeutics, College of Pharmacy & Pharmaceutical
Sciences, University of Toledo, Toledo, OH 43614, USA; hzr0030@tigermail.auburn.edu (H.R.);
saloni.malla@rockets.utoledo.edu (S.M.); noor.hussein@rockets.utoledo.edu (N.H.);
jenna.len@rockets.utoledo.edu (J.M.L.)
2
3
4
5
6
Department of Chemistry, College of Science and Arts, Jordan University of Science and Technology,
P.O. Box 3030, Irbid 22110, Jordan; saaudat@just.edu.jo
Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center,
Omaha, NE 68198, USA; fnu.shikha@unmc.edu
Department of Medicinal and Biological Chemistry, College of Pharmacy & Pharmaceutical Sciences,
University of Toledo, Toledo, OH 43614, USA; Fernand.Bedi@utoledo.edu
Department of Pharmaceutical Sciences, College of Pharmacy & Pharmaceutical Sciences,
St. John’s University, Queens, NY 11439, USA; cnsratdoc@optonline.net
*
†
Correspondence: Amit.Tiwari@UToledo.edu; Tel.: +1-419-383-1913; Fax: +1-419-383-1909
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Qiao-Hong Chen
Received: 2 June 2020; Accepted: 30 June 2020; Published: 4 July 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: New chrysin-De-allyl-Pac-1 hybrid analogues, tethered with variable heterocyclic systems
4a 4o), were rationally designed and synthesized. The target compounds were screened for in vitro
(
–
antiproliferative efficacy in the triple-negative breast cancer (TNBC) cell line, MDA-MB-231, and
normal human mammary epithelial cells (HMECs). Two compounds, 4g and 4i, had the highest
efficacy and selectivity towards MDA-MB-231 cells, and thus, were further evaluated by mechanistic
experiments. The results indicated that both compounds 4g and 4i induced apoptosis by (1)
inducing cell cycle arrest at the G2 phase in MDA-MB-231 cells, and (2) activating the intrinsic
apoptotic pathways in a concentration-dependent manner. Physicochemical characterizations of
these compounds suggested that they can be further optimized as potential anticancer compounds
for TNBC cells. Overall, our results suggest that 4g and 4i could be suitable leads for developing
novel compounds to treat TNBC.
Keywords: triple-negative breast cancer; cytotoxicity; chrysin analogues; flavonoid; anticancer
compounds
1. Introduction
Breast cancer is the second leading cause of death among all cancers affecting women [1].
Triple-negative breast cancer (TNBC) lacks the expression of hormone receptors (estrogen (ER) or
progesterone (PR)) and/or human epidermal growth factor receptor 2 (HER2), which are more amenable
to targeted therapy [2]. TNBC often presents as a high-grade invasive ductal carcinoma (IDC) in
Molecules 2020, 25, 3063; doi:10.3390/molecules25133063
www.mdpi.com/journal/molecules

Molecules 2020, 25, 3063
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patients, accounting for one-fourth of all breast cancer deaths. Consequently, there is an urgent
need for the development of compounds that are efficacious and safe for the treatment of breast
cancer. Flavonoids are ubiquitous naturally occurring polyphenolic compounds that are commonly
found in fruits and vegetables [
compounds, including flavanones, anthocyanidins, flavonols, flavanols, isoflavones, dihydroflavonols,
and flavones [ ]. Chrysin (5, 7-dihydroxyflavone, Figure 1) is a natural flavone found in many
plant extracts, such as the blue passionflower, as well as in honey and propolis [ ]. Chrysin has
been reported to have properties, such as antioxidant [ ], antihypertensive [10], antibacterial [11],
anti-inflammatory [12], antiviral [13], antiallergic [14], antidiabetic [15], anxiolytic [16], and anticancer
3]. Flavonoids comprise several classes of low molecular weight
4–6
7,8
9
efficacy [17
,18]. The activation of apoptosis plays a major role in producing the anticancer efficacy of
chrysin [19
–21]. Mechanistically, apoptosis involves a cascade of initiator and effector caspases [22].
Among these caspases, caspase-3 and caspase-7 are downstream executioner caspases that play
an essential role in inducing apoptosis by cleaving a variety of cellular substrates [23]. Therefore,
caspase-dependent apoptosis pathways represent targets for the development of efficacious anticancer
drugs. Recently, a number of studies have been done to augment the pharmacological activity
of chrysin by producing synthetic analogues [24
chrysin-based compounds have in vitro efficacy in breast cancer cells [30
procaspase-activating compound 1 (PAC-1), induces apoptosis in different types of cancer cells by
activating caspase-3 and/or caspase-7 [33 36]. Previously, we reported that molecular hybridization
–
29]. Moreover, there are studies indicating that
–32]. The compound, de-allyl
–
between chrysin and de-allyl PAC-1 can be used to produce novel hybrid molecules with cytotoxic
efficacy [29]. Molecular hybridization is a process that comprises the amalgamation of two or more
pharmacophoric moieties of different bioactive molecules into a single molecular framework [37,38].
Figure 1. Chemical structures of chrysin, de-allyl PAC-1, and a representative designed hybrid
molecule 4a.
In this study, new chrysin-de-allyl PAC-1 hybrid analogues, substituted with variable aromatic
heterocyclic cores, were synthesized and evaluated to identify a more potent bioactive hybrid against
breast cancer cells (Figure 1). The in silico parameters of the synthesized compounds were calculated to
predict their pharmacokinetic profile and drug-likeness using SwissSimilarity ADME, a web tool [39].
Subsequently, the target compounds were screened for antiproliferative efficacy in the human breast
cancer cell line MDA-MB-231, using the MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium

Molecules 2020, 25, 3063
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Bromide) colorimetric assay. Finally, we determined the effects of the most potent compounds on the
cell cycle.
2. Results and Discussions
2.1. Chemistry
The target compounds (4a–4o) were prepared according to Scheme 1, starting from the
commercially available chrysin, using previously published synthetic procedures [29]. Briefly, chrysin
was added to methyl 2-bromoacetate at a low temperature in the presence of K2CO3, yielding the
desired alkylation product
2
. The conversion of ester
2
into hydrazide
3
was accomplished using 80%
1
hydrazine hydrate and a few drops of hydrochloric acid at low temperature. H and 13C NMR data
confirmed the formation of hydrazide
3 as a pure single product. Target compounds (4a–4o) were
obtained by adding hydrazide to an appropriate aldehyde in the presence of a catalytic amount of
3
hydrochloric acid at room temperature. All analogues gave adequate analytical and spectroscopic data,
1
which were in full accordance with their structures. H and 13C NMR spectra showed that compounds
4a
–
4o existed as geometrical isomers (E/Z isomers). The E:Z ratio for each compound was determined
1
from H NMR spectra utilizing the integration of the neat methylene group peaks that appeared
as two separated singlets for each isomer. Target compounds were analyzed using SwissSimilarity
(Swiss Institute of Bioinformatics, Lausanne, Switzerland). Compounds 4a–4j and 4n–4o were the
most structurally similar to apigenin, a flavonoid similar to chrysin. The physicochemical properties of
the compounds (4a–4o) are shown in Table 1.
Scheme 1. Reagents and conditions: (
a
) methyl 2-bromoacetate, K2CO3, DMF, 0 ◦C, 3 h; (
) different substituted aldehydes, CH3OH, HCl (cat), rt, 1 h.
b) hydrazine
hydrate 80%, EtOH, 0 ◦C, HCl (cat.), 1 h; (
c
Table 1. Physiochemical properties of the compounds 4a–4o collected from Swiss ADME.
Solubility
Pharmacokinetics
Drug-Likeness
Medicinal Chemistry
Lipophilicity
Log P
Compound
MW
GI
BBB
Permeant
Lipinski
Violations
Lead-likeness
Violations
PAINS
Alerts
ESOL Class
Absorption
Moderately
soluble
Moderately
soluble
Moderately
soluble
Moderately
soluble
Moderately
soluble
Moderately
soluble
4a
4b
4c
4d
4e
4f
430.41
414.41
444.44
458.46
430.41
444.44
High
High
High
High
High
High
No
No
No
No
No
No
0
0
0
0
0
0
2
2
3
3
2
3
1
0
0
0
1
0
3.14
3.38
3.62
3.95
3.07
3.62

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Table 1. Cont.
Solubility
Pharmacokinetics
Drug-Likeness
Medicinal Chemistry
Lipophilicity
Log P
Compound
MW
GI
BBB
Permeant
Lipinski
Violations
Lead-likeness
Violations
PAINS
Alerts
ESOL Class
Absorption
Moderately
soluble
Moderately
soluble
Moderately
soluble
Moderately
soluble
Moderately
soluble
Moderately
soluble
Moderately
soluble
Poorly
4g
4h
4i
446.41
474.46
462.41
462.41
404.37
415.4
Low
High
Low
Low
High
High
High
Low
Low
No
No
No
No
No
No
No
No
No
0
0
0
0
0
0
0
0
1
2
3
2
2
2
2
2
2
3
2
0
3
2
0
0
0
1
1
2.71
3.65
2.38
2.43
2.81
2.66
2.72
4.04
2.52
4j
4k
4l
4m
4n
4o
415.4
480.47
475.41
soluble
Moderately
soluble
ESOL Class: Estimated Solubility Class [40]. GI Absorption: Gastrointestinal Absorption. BBB Permeant: Blood
Brain Barrier Permeant. Lipinski Violations examines orally active compounds to determine ranges for high
probability to be an oral drug. Lead-likeness Violations examines compounds’ likeliness to become a lead based on
a rule-based method [41]. PAINS Alerts: compounds that give false positives where the compound nonspecifically
binds to numerous biological targets, instead of one desired target [42]. Log P: partition coefficient measuring
lipophilicity [41].
2.2. 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide-Based Cytotoxicity Assay
The synthesized chrysin derivatives were evaluated for cytotoxic efficacy in the human breast
cancer cell line, MDA-MB-231, using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide
(MTT) colorimetric assay and doxorubicin was used as a reference anticancer drug. The IC50 values
(concentration of compound in µM required to reduce 50% of cell viability) are shown in Table 2.
Table 2. The effects of chrysin derivatives (4a–4o) on the survival of the MDA-MB-231 cancer cell line.
Compound
Structure
IC50 (µM)
4a
4b
4c
6.8 ± 2.76
9.4 ± 3.38
23.3 ± 6.98
100
4d
4e
54.8 ± 5.7 a

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Table 2. Cont.
Compound
Structure
IC50 (µM)
11.9 ± 3.5 a
4f
4g
4h
4i
5.98 ± 2.25
100
9.40 ± 1.45
4j
100
4k
66.65 ± 11.10
4l
100
100
4m
4n
100
4o
7.90 ± 1.82
0.2 ± 0.1
Doxorubicin
NA
Cell survival was determined by MTT assay as described in materials and methods. The IC50 values are represented
as mean ± SD of two independent experiments performed in triplicate. The data was taken from [29].
a

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As shown in Table 2, the structure–activity relationship (SAR) results indicated that the cytotoxic
efficacy of the synthesized compounds was affected by the modifications on the benzene ring (ring
D) of the parent compound 4a. Compound 4a, possessing a hydroxyl group at C-2 of ring D, had
cytotoxic efficacy, with an IC50 value of 6.8 µM. To determine the importance of the hydroxyl group at
the C-2 position, compound 4b was synthesized, and its efficacy was similar to compound 4a. The
methylation of the hydroxyl group in 4a yielded compound 4c, which had a lower efficacy than 4a and
4b, whereas ethylation of the hydroxyl group, which yielded compound 4d, produced a significant
decrease in cytotoxic efficacy, compared to compound 4a. Previously, we reported that the shifting
of the hydroxyl group from C-2 to C-4 (4e) produced a significant reduction in the cytotoxic efficacy
(IC50 > 50
was ~5 times more efficacious than 4e [29]. The addition of a second hydroxyl group at the C-4
position in 4a resulted in compound 4g (IC50 = 5.98 M), which had cytotoxic efficacy similar to
µM). However, methylation of the hydroxyl group in 4e yielded compound 4f, which
µ
compound 4a. Interestingly, the methylation of the two hydroxyl groups in 4g, yielding compound
4h, significantly decreased the cytotoxic efficacy. The addition of a third hydroxyl group to 4g at
position 3 of ring D, yielding compound 4i, did not significantly alter the antiproliferative efficacy
of the compound, whereas adding a third hydroxyl group at position 6 of ring D yielded a totally
inactive compound, 4j. Next, we determined the effect of ring D on the cytotoxic efficacy of the
synthesized compounds. Therefore, compounds 4k
with aromatic heterocyclic moieties. Compounds 4k
–
4m were synthesized, where ring D was replaced
4l, and 4m, containing furan, 3-pyridine, and
,
2-pyridine moieties, respectively, did not have significant cytotoxic efficacy. Furthermore, adding
another fused benzene ring to ring D, while keeping the C-2 hydroxyl group (2-hydroxynaphthalene),
yielded the inactive compound, 4n. The addition of a nitro group at the C5-position of ring D in 4a
yielded compound 4o, which had an efficacy similar to compound 4a (IC50 = 7.9 µM).
Next, the cytotoxicity and selectivity of the most active compounds, 4g and 4i, were determined
in a panel of cell lines, including the normal cell lines, HMECs, and cancer cell lines, BT-20, U-251, and
HCT116, as shown in Figure 2 and Table 3. Compound 4g had antiproliferative efficacy in the BT-20,
U-251, and HCT116, with IC50 values of 5.32, 7.64, and 2.68, respectively. In contrast, compound 4i had
IC50 values ranging from 10–25 µM. The results indicated that although compound 4g is more potent
than compound 4i in the three cancer cell lines, compound 4i is more selective for the cancer cells than
the normal cells.
A
Figure 2. Cont.

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B
Figure 2. The efficacy and selectivity of 4g and 4i in MDA-MB-231, BT-20, U-251, and HCT116, and
compared to the normal cell line, HMEC ( ) the viability curves for cancer cells (MDA-MB-231,
BT-20, U-251, HCT116), and the normal cell line, HMEC; ( ) the IC50 values of 4g and 4i for
A
B
these cell lines are also compared to the normal cell line. Cell survival was determined using
the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The IC50 values are
represented as the means
**** p < 0.0001 vs. control group.
±
SD of three independent experiments performed in triplicate with * p < 0.05,
Table 3. The effects of the chrysin-like compounds (4a–4o) on the survival of cancer cell lines (breast,
glioblastoma, and colon) and a normal non-cancerous cell line (normal human mammary epithelial cells).
Compound
Breast Cancer
BT-20
Brain Cancer
U-251
Colon Cancer
HCT116
Normal Cells
HMEC
4a
4g
4i
4.41 ± 0.99
5.32 ± 0.72
10.43 ± 0.20
7.25 ± 1.82
8.63 ± 4.86
7.64 ± 0.11
25.36 ± 7.53
11.27 ± 2.84
2.88 ± 0.09
2.68 ± 0.15
20.09 ± 0.13
3.85 ± 0.34
7.12 ± 4.55
8.00 ± 1.33
>100
7.17 ± 4.10
4o
Cell survival was determined by MTT assay as described in the materials and methods. The IC50 values (µM) are
represented as the mean
±
SEM of three independent experiments performed in triplicate. The compounds were
screened on breast (MDA-MB-231, BT-20), brain (U-251), and colon (HCT116) cancer cell lines and normal human
mammary epithelial cells (HMECs).
2.3. 4g and 4i Induce Apoptosis and G2 Cell Cycle Arrest in a Triple-Negative Breast Cancer Cell Line
Apoptosis, a type of programmed cell death, is one of the major mechanisms by which
chemotherapeutic drugs produce their therapeutic efficacy [43]. Morphologically, apoptosis is
characterized by cellular shrinkage, which is accompanied with nuclear chromatin condensation and
fragmentation followed by blebbing of the plasma membrane. This leads to the formation of small
apoptotic bodies that have an intact cellular membrane and unaltered organelle integrity. These bodies
are then released in the extracellular environment and removed by the process of phagocytosis [44,45].
Apoptosis can occur by two pathways: The extrinsic pathway and intrinsic pathway. In either pathway,
when the cell is exposed to certain extrinsic or intrinsic stimuli, the integrity of the inner mitochondrial
membrane of the cell is compromised, resulting in the loss of the mitochondrial membrane potential,
and causing the release of several apoptotic factors, including cytochrome c [46,47]. Numerous
studies indicate that during apoptosis, phosphatidylserine (PS), in the cytoplasmic side of the plasma
membrane, is translocated to the extracellular cell surface [48]. The flipped anionic PS binds to the
Ca2+-dependent phospholipid-binding protein, annexin V [49]. We discovered and reported several
potent apoptosis-inducing compounds [50–57]. In this study, the result of our morphological studies in
MDA-MB-231 cells after incubation with our lead compounds, 4g and 4i, indicated that apoptosis was

Molecules 2020, 25, 3063
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occurring (Figure 3A). At a concentration of 20
µM, both compounds decreased the number of adherent
MDA-MB-231 cells and induced cellular shrinkage. The cells were rounded and loosely attached,
and apoptotic bodies were present. Similarly, the incubation of MDA-MB-231 cells with compounds 4g
or 4i for 24 h produced a significant loss of the mitochondrial membrane potential. For compound 4g
the population of cells undergoing apoptosis increased from 14.30% at 0 M to 26.56% and 58.05% at 5
and 10 M, respectively (p value < 0.0001; Figure 3B,C). Compound 4i also produced a significant shift
in the apoptotic cell population in quadrant II, from 14.78% at 0 M to 32.25% and 42.56% at 5 and
10 M, respectively (p value < 0.0001, Figure 3B,C). Cell cycle analysis indicated that compounds 4g and
,
µ
µ
µ
µ
4i produced a significant disruption in the cell cycle of MDA-MB-231 cells (Figure 3D). Data obtained
using flow cytometry indicated that, when incubated with vehicle alone, MDA-MB-231 cells had a
normal cell cycle (5.43%, 83.4%, 4.63%, and 4.46% in the subG1, G1, S, and G2 phases, respectively).
However, incubation of MDA-MB-231 cells with compound 4g resulted in a significant shift towards
the G2 phase (59.12% and 54.13% for 5 and 10 µM, respectively (p value < 0.0001, Figure 3E).
0 hr
24 hrs
48 hrs
72 hrs
A
Control
4g(5μM)
4g(20μM)
4i(5μM)
4i(20μM)
Figure 3. Cont.

Molecules 2020, 25, 3063
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B
4g(µM)
5
0
10
105
105
103
105
103
Q1
Q2
Q1
Q2
Q1
Q2
85.12%
14.30%
39.90%
70.09%
58.05%
26.56%
103
101
101
101
Q3
Q3
Q3
Q4
Q4
Q4
-101
-101
-101
0.58%
0.00%
2.05%
0.00%
3.36%
0.00%
101 102 103 104 105
101 102 103 104 105
101 102 103 104 105
4i(µM)
5
0
10
Q1
53.08%
Q2
42.56%
Q1
63.94%
Q2
32.25%
105
103
105
103
101
Q1
83.16%
Q2
14.78%
105
103
101
101
Q3
Q3
Q3
Q4
Q4
Q4
-101
-101 4.36%
0.00%
-101
3.81%
0.00%
2.06%
0.00%
101 102 103 104 105
101 102 103 104 105
101 102 103 104 105
Alexa Fluor 488 Dye Fluorescence
C
Figure 3. Cont.

Molecules 2020, 25, 3063
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4g(µM)
5
D
0
10
G2:59.12%
85
64
43
21
0
85
64
43
21
0
85
G1:28.43%
G1:24.68%
G1:83.40%
SubG1:
0.39%
SubG1:
0.82%
SubG1:
5.43%
64
43
21
0
G2:54.13%
S:4.63%
G2:4.46%
S:17.67%
S:12.35%
101
102
103
104
105
101
102
103
104
105
101
102
103
104
105
4i(µM)
0
5
10
G2:43.44%
85
85
64
43
21
0
85
64
43
21
0
G1:34.95%
G1:33.41%
G1:83.40%
SubG1:
G2:48.15%
SubG1:
1.56%
SubG1:
1.33%
64
43
21
0
5.43%
S:4.63%
G2:4.46%
S:19.88%
S:16.76%
101
102
103
104
105
101
102
103
104
105
101
102
103
104
105
FL2-H (PI)
E
Figure 3. Effects of 4g and 4i on cellular morphology, mitochondrial membrane potential, and cell cycle
) Morphological observations of MDA-MB-231 cells incubated with 0, 5, and 20 M concentrations
of 4g and 4i at different time intervals of 0, 24, 48, and 72 h, respectively; ( ) The MDA-MB-231 cells in
complete medium were incubated with 4g and 4i at 0, 5, or 10 M for 24 h. Cells were then incubated
(
A
µ
B
µ
with the reagents of the MitoTracker Red and Alexa Fluor 488 annexin V kits for flow cytometry.
Representative results of MDA-MB-231 cells from two independent experiments, each performed in
triplicate, are shown; (C) Histograms quantitatively summarize the results following incubation with
4g and 4i, respectively; (
shown. The MDA-MB-231 cells were incubated with different concentrations (0, 5, and 10
and 4i for 24 h and were subjected to cell cycle analysis by flow cytometry of PI (X axis)/cell counts
(Y axis); ( ) A histogram quantitatively summarizing the change in % of cells in each phase of the
cell cycle due to incubation with 4g and 4i. The data represents means SEM of three independent
experiments performed in triplicate with **** p < 0.0001 vs. control group.
D) The induction of cell cycle arrest in MDA-MB-231 cells by 4g and 4i is
µM) of 4g
E
±

Molecules 2020, 25, 3063
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2.4. Compounds 4g and 4i Activate Apoptosis by Activating the Intrinsic Apoptotic Pathway
Apoptosis can be induced by the activation of two major pathways: The intrinsic and extrinsic
apoptotic pathways [22]. The activation of the intrinsic apoptotic pathway induces the activation of
proapoptotic proteins, such as apoptosis regulator Bak (Bcl-2 homologous antagonist/killer) and Bax
(Bcl-2-associated X protein) [58]. The activated the Bax and Bak proteins subsequently permeabilize
the mitochondrial outer membrane by forming pores on its outer surface [58–60]. Consequently,
cytochrome c (Cyt C) is released into the cytosol, where it combines with the adaptor protein (Apaf-1)
to form an apoptosome [22]. The initiator caspases (i.e., caspase-2, caspase-8, caspase-9, or caspase-10)
are activated and recruited to large protein complexes, resulting in the cleavage of the executioner
caspases, caspase-3 or caspase-7 [61].
Since MDA-MB-231 cells incubated with compounds 4g and 4i had a decrease in the mitochondrial
membrane potential, which is an early event of intrinsic apoptosis, i.e., altered permeability of the
inner mitochondrial membrane, we conducted experiments to determine if these compounds altered
the expression of key apoptotic proteins, including cytochrome c, in MDA-MB-231 cells using Western
blotting analysis. Our results indicated that compounds 4g at 5 µM, and compound 4i at 10 µM
produced a significant increase in the expression of cytochrome c, compared to cells incubated with
vehicle (Figure 4A,B). This may be due to an increase in the expression of Bak following incubation with
4g and 4i (Figure 4A,B), compared to cells incubated in the absence of lead compounds. In addition,
both compounds (4g at 5
caspase 9, in MDA-MB-231 cells, compared to cells incubated with vehicle (Figure 4A,B). These events
activated caspase 7 in breast cancer cells incubated with both concentrations of 4g and 10 M of 4i
µM and 4i at 10 µM) produced significant cleavage of the initiator caspase,
µ
,
compared to cells incubated with vehicle (Figure 4A,B). In contrast, there was no significant change
in the mammalian target of rapamycin (mTOR) expression, indicating that cell death induced by 4g
and 4i in MDA-MB-231 cells is not due to autophagy (Figure 4A,B). Thus, our results suggest that
cytochrome c release induces intracellular initiator caspase activation, followed by the activation of
executioner caspases, thus activating apoptotic cell death machinery through intrinsic the pathway.
A
Figure 4. Cont.

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B
Figure 4. The effect on apoptosis induction at different concentrations of 4g and 4i in MDA-MB-231
cells. ( ) Western blots for the proteins caspase 3, caspase 7, cleaved caspase 7, caspase 9, cleaved
caspase 9, Bak, cyt C and mTOR following incubation with 4g and 4i at 0, 5, or 10 M. The values of the
proteins were normalized to -actin levels. ( ) Histograms summarizing the levels of each protein are
also shown. All the data are presented as the means SEM of three independent studies with * p < 0.05,
A
µ
β
B
±
** p < 0.01, *** p < 0.001 vs. control group. Cleaved is abbreviated as Clvd.
3. Materials and Method
3.1. Chemistry
All chemicals and solvents were procured from commercial sources (reagent grade) and were used
without further purification. The reaction progress was monitored by thin layer chromatography (TLC)
1
using precoated TLC plates of silica gel 60 F254. H-NMR and 13C-NMR spectra were recorded using a
400 MHz Bruker Avance Ultrashield spectrometer. The spectra were obtained in ppm using automatic
calibration to the residual proton peak of the solvent, dimethyl-sulphoxide (DMSO-d6). The 1H NMR
data are presented as follows: Chemical shift (δ ppm), multiplicity (s = singlet, d = doublet, t = triplet,
q = quartet, m = multiplet), coupling constants (Hz), and integration. The 13C NMR analyses were
reported in terms of the chemical shift. The 1H and 13C NMR spectra for all compounds are included
in the supporting information (Figure S1). HRMS data were acquired using a Thermo QExactive
Plus mass spectrometer equipped with an electrospray ionization source (Thermo Fisher Scientific,
Greensboro, NC, USA).
3.1.1. Synthesis of methyl 2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetate (2)
The title compound was synthesized as previously described [62].
3.1.2. Synthesis of 2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetohydrazide (3)
The title compound was synthesized according to our previously published procedure [29].
3.1.3. General Procedure for the Synthesis of N’-arylidene-2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-
7-yl)oxy)acetohydrazide (4a–4o)
To a stirred suspension of hydrazide
3 (1.0 g, 3.0 mmol) in anhydrous methanol (60 mL),
the appropriate aldehyde (3.0 mmol) was added, along with a few drops of concentrated hydrochloric
acid. After one hour, the reaction was completed and the formed precipitate was separated by filtration,
washed with methanol, and dried in air to give pure compounds in good yields.

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(E,Z)-2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)-N’-(2-hydroxybenzylidene)acetohydrazide (4a
)
1
The product was obtained as a pale-yellow powder. Yield (64%). H NMR (DMSO-d6) (E:Z = 1:1):
δ
12.84–12.81 (m, 2H), 11.84 (s, 1H), 11.62 (s, 1H), 10.99 (s, 1H), 10.05 (s, 1H), 8.57 (s, 1H), 8.33 (s, 1H),
8.12–8.10 (d, J = 8 Hz, 4H), 7.77–7.55 (m, 8H), 7.31–7.24 (m, 2H), 7.07–6.85 (m, 8H), 6.51–6.42 (m, 2H),
5.31 (s, 2H), 4.87 (s, 2H). 13C NMR (DMSO-d6):
δ
182.11, 182.06, 167.94, 164.42, 163.65, 163.61, 163.48,
163.37, 161.17, 161.07, 157.36, 157.22, 156.45, 148.27, 141.63, 132.18, 132.12, 131.59, 131.29, 130.57, 130.54,
129.18, 129.15, 129.11, 126.56, 126.47, 120.01, 119.40, 118.65, 116.39, 116.14, 105.44, 105.39, 105.31, 105.09,
98.81, 98.72, 93.62, 93.46, 66.57, 65.36. HRMS (ESI, m/z): calculated for C24H19N2O6 [M + H]+ 431.1237;
found 431.1238.
(E,Z)-N’-Benzylidene-2-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetohydrazide (4b)
The synthesis and full characterization of the title compound have been previously reported [29].
(E,Z)-2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)-N’-(2-methoxybenzylidene)acetohydrazide (4c
)
1
The product was obtained as a yellow powder. Yield (55%). H NMR (DMSO-d6) (E:Z = 1:0.5):
δ
12.84–12.80 (m, 1.5H), 11.64 (s, 1.5H), 8.68 (s, 0.5H), 8.37 (s, 1H), 8.11–8.09 (d, J = 8 Hz, 3H), 7.91–7.89
(d, J = 8 Hz, 1H), 7.82–7.80 (d, J = 8 Hz, 0.5H), 7.63–7.55 (m, 4.5H), 7.44–7.4 (m, 1.5H), 7.12–7.0 (m, 4.5H),
6.88–6.84 (m, 1.5H), 6.5–6.42 (m, 1.5H), 5.32 (s, 2H), 4.82 (s, 1H), 3.86 (s, 4.5H). 13C NMR (DMSO-d6):
δ
182.09, 182.03, 168.09, 164.43, 163.71, 163.60, 163.48, 163.22, 161.13, 161.05, 157.80, 157.64, 157.21,
143.56, 139.73, 132.15, 132.10, 131.72, 131.48, 130.55, 129.09, 126.45, 125.67, 125.53, 121.94, 120.72, 120.66,
111.84, 111.77, 105.42, 105.31, 105.06, 98.77, 98.70, 93.62, 93.45, 66.65, 65.36, 55.68, 54.87. HRMS (ESI,
m/z): calculated for C25H21N2O6 [M + H]+ 445.1394; found 445.1392.
(E,Z)-N’-(2-Ethoxybenzylidene)-2-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetohydrazide (4d
)
1
The product was obtained as a light-brown powder. Yield (80%). H NMR (DMSO-d6) (E:Z = 1:0.5):
δ
12.84–12.80 (m, 1.5H), 11.68–11.62 (m, 1.5H), 8.66 (s, 0.5H), 8.40 (s, 1H), 8.11–8.10 (d, J = 4 Hz, 3H),
7.90–7.80 (m, 1.5H), 7.60–7.57 (m, 4.5H), 7.41–7.37 (m, 1.5H), 7.10–6.84 (m, 6H), 6.5–6.42 (m, 1.5H), 5.31
(s, 2H), 4.82 (s, 1H), 4.14–4.09 (q, J = 8 Hz, 3H), 1.39–1.35 (t, J = 8 Hz, 4.5H).13C NMR (DMSO-d6):
δ
182.08, 182.03, 168.03, 164.42, 163.77, 163.57, 163.46, 163.23, 161.14, 161.04, 157.20, 157.13, 156.98,
143.46, 140.01, 132.10, 131.67, 131.42, 130.54, 129.09, 126.45, 125.76, 125.58, 122.14, 122.09, 120.61, 112.75,
105.41, 105.29, 105.05, 98.71, 93.63, 93.45, 66.62, 65.36, 63.80,14.63. HRMS (ESI, m/z): calculated for
C26H23N2O6 [M + H]+ 459.1550; found 459.1548.
(E,Z)-2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)-N’-(4-hydroxybenzylidene)acetohydrazide (3e
)
The synthesis and full characterization of the title compound were previously published [29].
(E,Z)-2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)-N’-(4-methoxybenzylidene)acetohydrazide (3f
)
The synthesis and full characterization of the title compound were previously published [29].
(E,Z)-N’-(2,4-Dihydroxybenzylidene)-2-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-
yl)oxy)acetohydrazide (4g)
1
The product was obtained as an off-white powder. Yield (85%). H NMR (DMSO-d6) (E:Z = 1:0.8):
δ
12.84–12.80 (m, 1.8H), 11.67 (s, 1H), 11.43 (s, 0.8H), 11.15 (s, 1H), 9.98 (s, 1.8H), 9.83 (s, 0.8H), 8.42
(s, 1H), 8.20 (s, 0.8H), 8.12–8.10 (d, J = 8 Hz, 3.6H), 7.63–7.53 (m, 6.2H), 7.33–7.31 (d, J = 8 Hz, 1H),
7.08–7.06 (d, J = 8 Hz, 1.8H), 6.89 (d, J = 2 Hz, 1H), 6.8 (d, J = 2.4 Hz, 0.8H), 6.5 (d, J = 2 Hz, 1H), 6.4 (d,
J = 2 Hz, 0.8H), 6.36–6.30 (m, 3.6H), 5.26 (s, 1.6H), 4.83 (s, 2H). 13C NMR (DMSO-d6):
δ182.10, 182.05,
167.42, 164.44, 163.68, 163.64, 163.50, 162.86, 161.14, 161.04, 160.86, 160.53, 159.35, 158.07, 157.23, 149.28,
142.68, 132.18, 132.12, 131.10, 130.55, 129.15, 129.12, 128.27, 126.47, 111.49, 110.37, 107.84, 107.77, 105.45,

Molecules 2020, 25, 3063
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105.37, 105.32, 105.07, 102.60, 102.37, 98.81, 98.72, 93.63, 93.45, 66.59, 65.33. HRMS (ESI, m/z): calculated
for C24H19N2O7 [M + H]+ 447.1186; found 447.1187.
(E,Z)-N’-(2,4-Dimethoxybenzylidene)-2-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-
yl)oxy)acetohydrazide (4h)
1
The product was obtained as a yellow powder. Yield (96%). H NMR (DMSO-d6) (E:Z = 1:0.5):
δ
12.81 (s, 1.5H), 11.49 (s, 1.5H), 8.58 (s, 0.5H), 8.27 (s, 1H), 8.11–8.10 (m, 3H), 7.84–7.57 (m, 6H), 7.05 (s,
1.5H), 6.88–6.84 (m, 1.5H), 6.63–6.41 (m, 4.5H), 5.29 (s, 2H), 4.79 (s, 1H), 3.86–3.82 (m, 9H). 13C NMR
(DMSO-d6): 182.04, 167.79, 164.43, 163.69, 163.55, 163.44, 162.91, 162.55, 162.33, 161.09, 161.02, 159.19,
δ
159.02, 157.18, 143.62, 139.82, 132.10, 130.53, 129.09, 126.78, 126.43, 114.76, 106.41, 105.38, 105.28, 105.02,
98.68, 98.24, 98.07, 93.59, 93.42, 66.66, 65.35, 55.74, 55.40.HRMS (ESI, m/z): calculated for C26H23N2O7
[M + H]+ 475.1499; found 475.1499.
(E,Z)-2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)-N’-(2,3,4-
trihydroxybenzylidene)acetohydrazide (4i)
1
The product was obtained as a yellow powder. Yield (85%). H NMR (DMSO-d6) (E:Z = 1:0.5):
δ
12.84–12.80 (m, 1.5H), 11.71 (s, 1H), 11.47 (s, 0.5H), 11.18 (s, 1H), 9.57–9.51 (m, 1.5H), 9.37 (s, 0.5H),
8.54–8.50 (m, 1.5H), 8.38 (s, 1H), 8.18–8.10 (m, 3.5H), 7.61–7.59 (m, 4.5H), 7.07–7.0 (m, 2H), 6.90–6.78 (m,
2.5H), 6.51–6.38 (m, 3H), 5.26 (s, 1H), 4.85 (s, 2H). 13C NMR (DMSO-d6):
182.12, 182.07, 167.34, 164.42,
δ
163.68, 163.54, 162.93, 161.16, 161.07, 157.25, 150.45, 148.91, 148.42, 147.47, 146.65, 144.18, 132.76, 132.71,
132.21, 132.16, 130.56, 129.18, 126.49, 121.11, 118.36, 112.11, 110.70, 107.84, 107.73, 105.46, 105.40, 105.34,
105.10, 98.83, 98.75, 93.65, 93.48, 66.59, 65.32. HRMS (ESI, m/z): calculated for C24H19N2O8 [M + H]+
463.1134; found 463.1133.
(E,Z)-2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)-N’-(2,4,6-
trihydroxybenzylidene)acetohydrazide (4j)
1
The product was obtained as a brown powder. Yield (76%). H NMR (DMSO-d6) (E:Z = 1:0.2):
δ
12.83–12.79 (m, 1.2H), 11.65 (s, 1H), 11.48 (s, 0.2H), 10.99 (s, 2H), 10.32 (s, 0.4H), 9.85 (s, 1.2H), 8.72 (s,
1H), 8.41 (s, 0.2H), 8.09–8.08 (m, 2.4H), 7.59–7.58 (m, 3.6H), 7.04 (m, 1.2H), 6.88–6.84 (m, 1.2H), 6.5–6.42
(m, 1.2H), 5.86–5.83 (m, 2.4H), 5.2 (s, 0.4H), 4.82 (s, 2H). 13C NMR (DMSO-d6):
182.08, 166.56, 164.28,
δ
163.61, 163.51, 162.55, 161.74, 161.46, 161.12, 161.04, 159.73, 159.24, 157.20, 147.32, 144.33, 132.15, 130.54,
129.13, 126.45, 105.43, 105.37, 105.11, 98.85, 98.79, 94.37, 93.61, 93.50, 66.58, 65.26. HRMS (ESI, m/z):
calculated for C24H19N2O8 [M + H]+ 463.1134; found 463.1134.
(E,Z)-N’-(Furan-2-ylmethylene)-2-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetohydrazide(4k
)
1
The product was obtained as a yellow powder. Yield (87%). H NMR (DMSO-d6) (E:Z = 1:0.5):
δ
12.83–12.79 (m, 1.5H), 11.63–11.57 (m, 1.5H), 8.23 (s, 0.5H), 8.11–8.09 (d, J = 8 Hz, 3H), 7.92 (s, 1H),
7.85 (s, 1.5H), 7.63–7.55 (m, 4.5H), 7.06–7.05 (m, 1.5H), 6.95–6.83 (m, 3H), 6.64 (m, 1.5H), 6.49–6.39 (m,
1.5H), 5.24 (s, 2H), 4.83 (s, 1H). 13C NMR (DMSO-d6):
δ182.03, 167.97, 164.34, 163.66, 163.59, 163.46,
163.35, 161.13, 161.02, 157.21, 149.12, 148.94, 145.34, 145.10, 137.86, 134.20, 132.11, 130.54, 129.09, 126.45,
113.95, 113.78, 112.16, 105.42, 105.29, 105.08, 98.70, 93.60, 93.38, 66.65, 65.14.HRMS (ESI, m/z): calculated
for C22H17N2O6 [M + H]+ 405.1081; found 405.1077.
(E,Z)-2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)-N’-(pyridin-3-ylmethylene)acetohydrazide (4l
)
1
The product was obtained as a yellow powder. Yield (86%). H NMR (DMSO-d6) (E:Z = 1:0.5):
δ
12.82(bs, 1.5H), 11.98 (bs, 1.5H), 8.43 (s, 0.5H), 8.30–8.28 (d, J = 8 Hz, 3H), 8.13–7.96 (m, 7H), 7.62–7.55
(m, 4.5H), 7.07–7.06 (m, 1.5H), 6.88 (s, 1.5H), 6.5–6.45 (m, 1.5H), 5.39 (s, 2H), 4.9 (s, 1H). 13C NMR
(DMSO-d6):
δ 182.12, 168.50, 164.42, 163.67, 163.55, 161.08, 157.27, 150.51, 148.52, 145.40, 141.22, 133.75,
132.20, 130.59, 129.96, 129.16, 126.51, 123.94, 105.36, 105.12, 98.76, 93.55, 66.61, 65.40. HRMS (ESI, m/z):
calculated for C23H18N3O5 [M + H]+ 416.1241; found 416.1240.

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(E,Z)-2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)-N’-(pyridin-2-
ylmethylene)acetohydrazide (4m)
1
The product was obtained as a brown powder. Yield (52%). H NMR (DMSO-d6) (E:Z = 1:0.5):
δ
12.84–12.81 (m, 1.5H), 11.89–11.87 (m, 1.5H), 8.61 (m, 1.5H), 8.35 (s, 0.5H), 8.11–8.03 (m, 5H), 7.94–7.87
(m, 2H), 7.62–7.55 (m, 4.5H), 7.44–7.41 (m, 1.5H), 7.07–7.06 (m, 1.5H), 6.88 (m, 1.5H), 6.51–6.45 (m, 1.5H),
5.37 (s, 2H), 4.88 (s, 1H). 13C NMR (DMSO-d6):
δ
182.02, 168.45, 164.34, 163.75, 163.65, 163.59, 163.48,
161.13, 161.05, 157.21, 152.93, 152.78, 149.51, 149.44, 148.26, 144.45, 136.86, 136.75, 132.09, 130.53, 129.07,
126.44, 124.52, 124.34, 119.98, 119.90, 105.42, 105.30, 105.09, 98.70, 93.63, 93.48, 66.61, 65.30. HRMS (ESI,
m/z): calculated for C23H18N3O5 [M + H]+ 416.1241; found 416.1238.
(E,Z)-2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)-N’-((2-hydroxynaphthalen-1-
yl)methylene)acetohydrazide (4n)
1
The product was obtained as a yellow powder. Yield (54%). H NMR (DMSO-d6) (E:Z = 1:0.5):
δ
12.86–12.81 (m, 1.5H), 12.46 (s, 1H), 11.91 (s, 1H), 11.68 (s, 0.5H), 10.77 (s, 0.5H), 9.40 (s, 1H), 8.89 (s,
0.5H), 8.77–8.75 (d, J = 8 Hz, 0.5H), 8.30–8.28 (d, J = 8 Hz, 1H), 8.12–8.09 (m, 3H), 7.94–7.84 (m, 3H),
7.63–7.58 (m, 6H), 7.42–7.36 (m, 1.5H), 7.24–7.21 (m, 1.5H), 7.08–7.06 (m, 1.5H), 6.94–6.88 (m, 1.5H),
6.55–6.46 (m, 1.5H), 5.38 (s, 1H), 4.94 (s, 2H). 13C NMR (DMSO-d6):
δ182.09, 182.03, 167.52, 164.38,
163.64, 163.54, 163.49, 163.17, 161.17, 161.06, 157.95, 157.23, 156.88, 147.37, 143.29, 132.91, 132.52, 132.16,
132.10, 131.59, 131.25, 130.53, 129.13, 128.89, 128.68, 128.13, 127.80, 127.75, 126.46, 123.54, 123.40, 120.97,
118.75, 118.13, 110.08, 108.45, 105.45, 105.33, 105.10, 98.87, 98.73, 93.68, 93.46, 66.72, 65.59.HRMS (ESI,
m/z): calculated for C28H21N2O6 [M + H]+ 481.1394; found 481.1394.
(E,Z)-2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)-N’-((2-hydroxy-5-
nitrobenzylidene)acetohydrazide (4o)
1
The product was obtained as a yellow powder. Yield (91%). H NMR (DMSO-d6) (E:Z = 1:0.75):
δ
12.85–12.80 (m, 1.75H), 11.97 (bs, 2H), 11.78 (s, 1.5H), 8.64–8.54 (m, 2.75H), 8.33 (s, 1H), 8.15–8.10 (m,
5.25H), 7.64–7.55 (m, 5.25H), 7.08–7.04 (m, 3.25H), 6.90–6.86 (m, 1.75H), 6.52–6.44 (m, 1.75H), 5.39 (s,
2H), 4.89 (s, 1.5H). 13C NMR (DMSO-d6):
182.08, 182.03, 168.28, 164.42, 163.71, 163.61, 163.46, 162.04,
δ
161.14, 161.04, 157.20, 144.30, 139.91, 138.65, 132.16, 132.10, 130.55, 129.12, 129.08, 126.73, 126.45, 123.37,
121.68, 120.98, 119.95, 117.10, 116.72, 105.43, 105.37, 105.30, 105.08, 98.78, 98.73, 93.62, 93.46, 66.53, 65.42.
HRMS (ESI, m/z): calculated for C24H18N3O8 [M + H]+ 476.1088; found 476.1089.
3.2. Biological Studies
3.2.1. Cell Lines and Cell Culture
A panel of cancer cell lines, including breast (MDA-MB-231, BT20), brain (U251), and colon
(HCT116), as well as a normal cell line (human mammary epithelial cells: HMECs), were grown as
adherent monolayers in flasks with Dulbecco’s Modified Eagle Medium (DMEM), supplemented with
10% fetal bovine serum (FBS) and 1% penicillin and streptomycin in a humidified incubator with 5%
CO2 at 37 ◦C.
3.2.2. MTT Assay
The (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay was used to
determine the cytotoxicity of the 15 chrysin derivatives in the above-mentioned cell lines. Briefly,
cells were harvested with 0.05% trypsin, 2.21 mM ethylenediaminetetraacetic acid (EDTA), 1
Corning (Corning, NY, USA), and suspended at a final density of 5
103 cells/well. Cells were seeded
(180 L/well) into 96-well plates. Initially, four different concentrations of each compound were added
to find the compounds with the greatest antiproliferative efficacy in the MDA-MB-231 cell line (0, 1,
10, and 100 M). Subsequently, eight different concentrations (0.1, 0.3, 1, 3, 10, 30, and 100 M) of
each compound were added to the remaining cell lines mentioned above. After 68 h of incubation,
×
from
×
µ
µ
µ

Molecules 2020, 25, 3063
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20
This allowed viable cells to biotransform the yellow-colored MTT into dark-blue formazan crystals.
Subsequently, the medium was discarded, and 150 L of DMSO were added to each well to dissolve
the formazan crystals. The absorbance was determined at 590 nm using a DTX 880 multimode detector
(Beckman Coulter life sciences, IN, USA). The IC50 SD concentrations were calculated from three
µL of the MTT solution (4 mg/mL) were added to each well, and the plates were incubated for 4 h.
µ
±
experiments performed in triplicate/duplicate. The IC50 values were calculated from the cell survival
percentages obtained for each compound tested at different concentrations. Similarly, the cytotoxicity
of the test compounds was compared to the normal cell line (HMEC).
3.2.3. Cell Cycle, Apoptosis, and Mitochondrial Membrane Potential Analysis
MDA-MB-231 cells were plated into 6-well plates at 2.5
with 0, 5, or 10 M of compounds 4g or 4i and incubated for 12 h. Next, the cells were trypsinized with
0.05% trypsin, 2.21 mM EDTA, 1 , washed, counted, and resuspended in 1 mL of ice-cold PBS. The cells
then were stained with propidium iodide (PI) dye and incubated for at least 15 min. The distribution
of the cells in each cell cycle phase for the different concentrations was measured using a BD Accuri
×
105 cells/well. The cells were incubated
µ
×
™
C6 flow cytometer from BD Biosciences (Becton-Dickinson, San Jose, CA, USA) and analyzed using
FCS Express 5 plus De Novo software (Glendale, CA, USA).
MitoTracker Red and Alexa Fluor 488 annexin V kits for flow cytometry (Molecular Probes Inc.,
Invitrogen, Eugene, OR) were used to measure the mitochondrial membrane potential and apoptosis
in MDA-MB-231 cells. Briefly, cells were seeded into 6-well plates and incubated with 0, 5, or 10
of compounds 4g and 4i for 12 h. The cells were then lysed using 0.05% trypsin, 2.21 mM EDTA,
, counted, and 4 L of 10 M of the MitoTracker Red working solution were added to 1 mL of the
harvested cells. The cells were incubated at 37 ◦C with 5% CO2 for 30 min. The cells were washed once
with PBS and resuspended in 100 L of the annexin binding buffer. The cell suspensions were incubated
with 5 L of Alexa Fluor 488 annexin V for 15 min. This was followed by the addition of 400 L of the
µM
1×
µ
µ
µ
µ
µ
annexin-binding buffer. Finally, flow cytometry was used to detect the fluorescence of stained cells at
the following excitation/emission maxima: Alexa Fluor® 488 annexin V: 499/521 nm; MitoTracker®
Red: 579/599 nm with the BD Accuri™ C6 flow cytometer from BD Biosciences (Becton-Dickinson, San
Jose, CA, USA) and analyzed using FCS express 5 plus De Novo software (Glendale, CA, USA).
3.2.4. Protein Expression Analysis Using Western Blot
To measure the expression of Bak, cytochrome c, caspase-7, caspase-9, and mTOR, Western blotting
was performed by lysing MDA-MB-231 cells using a lysis buffer (50 mM Tris–HCl, 150 mM NaCl,
1 mM EDTA, 0.5% NP-40, 1% Triton, 0.1% SDS) containing a protease inhibitor cocktail that consisted
of Aprotinin, Bestatin, E-64, Leupeptin, and Pepstatin A (Sigma-Aldrich Life Science, St. Louis, MO,
USA). The bicinchoninic acid (BCA) quantification assay was used to determine the protein levels
in the cell extracts (G-BIOSCIENCES, St. Louis, MO, USA). The extracted proteins were loaded
onto a 10–20% tris-glycine gel. After separation, the proteins were transferred from the gel onto
a polyvinylidene difluoride (PVDF) membrane. The membranes were blocked using 5% milk in
Tris-buffered saline Tween 20 for 30 min and incubated overnight with primary antibodies against
Bak (1:1000), cytochrome C (1:1000), caspase-3 (1:1000), caspase 7 (1:1000), caspase 9 (1:1000), mTOR
(1:1000), or B-actin (1:2000) in 5% BSA (bovine serum albumin) at 4 ◦C. The next day, membranes were
washed and incubated with horseradish peroxidase-labelled (HRP) anti-rabbit secondary antibody
(1:5000 dilutions). The membrane was incubated with the antibody for an additional 1 h. Subsequently,
the membranes were washed and developed by Clarity Western ECL substrate (Bio-Rad; Hercules, CA,
USA). Protein was detected using a ChemiDoc Imaging System (Bio-Rad). Densitometry analyses of
the blots for the detected protein were quantified using the ImageJ software. Data was calculated as
ratios of protein/β-actin.

Molecules 2020, 25, 3063
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4. Conclusions
In conclusion, a series of novel chrysin derivatives were designed, synthesized, and characterized.
Upon screening these compounds for their antiproliferative efficacy in the TNBC cell line, MDA-MB-231,
and normal breast HMEC cells, two compounds, 4g and 4i, had the highest efficacy and selectivity
towards MDA-MB-231 cells. Upon investigating the mechanism by which these compounds produce
cytotoxicity, it was determined that 4g and 4i cause the death of MDA-MB-231 cells by inducing
apoptosis, producing cell cycle arrest at the G2 phase, and activating the intrinsic apoptotic pathway.
Physicochemical characterizations of these compounds suggested that they can be further optimized
as potential anticancer compounds for TNBC cells. The compounds were determined to have some
solubility issues that need to be overcome in the future design of additional compounds. Overall, our
results suggest that 4g and 4i could be suitable leads for developing novel compounds to treat TNBC.
Supplementary Materials: The following are available online, Figure S1: Chemical characterization of compounds
4a–4o.
Author Contributions: Conceptualization, B.A.A.-O., S.A.A. and A.K.T.; methodology, B.A.A.-O., S.A.A., H.R.,
S.M., N.H. and J.M.L.; software, S.M. and N.H.; validation, S.M. and N.H.; formal analysis, B.A.A.-O.; investigation,
H.R., S.M., N.H., J.M.L.; resources, B.A.A.-O. and A.K.T.; data curation, B.A.A.-O., H.R., S.M., N.H. and J.M.L.;
writing—original draft preparation, B.A.A. and S.M.; writing—review and editing, B.A.A.-O., S.A.A., S.M., S.K.,
M.F.B., C.R.A.J. and A.K.T.; visualization, A.K.T.; supervision, A.K.T.; project administration, A.K.T.; funding
acquisition, B.A.A.-O., and A.K.T. All authors have read and agreed to the published version of the manuscript.
Funding: This work was funded by the Deanship of Scientific Research at Jordan University of Science and
Technology (grant # 20190360 to BAA). This manuscript has been supported, in part, by University of Toledo
startup grants (F110760 to AKT) and Susan G. Komen Breast Cancer Foundation (CCR18548498 to AKT).
Acknowledgments: The authors acknowledge the financial support given by the Deanship of Research at the
Jordan University of Science and Technology (grant # 20190360 to BAA). The high-resolution mass spectrometry
data were acquired from the Triad Mass Spectrometry Laboratory at the University of North Carolina at Greensboro,
Greensboro, NC, USA. This manuscript has been supported, in part, by the University of Toledo startup grants
(F110760 to AKT) and Susan G. Komen Breast Cancer Foundation (CCR18548498 to AKT).
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 4a, 4g, 4i and 4o are available from the authors on request.
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2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).