The synthesis and anticancer activity of 2-styrylquinoline derivatives. A p53 independent mechanism of action

Article abstract of DOI:10.1016/j.ejmech.2019.05.061

A series of styrylquinolines was designed and synthesized based on the four main quinoline scaffolds including oxine, chloroxine and quinolines substituted with a hydroxyl group or chlorine atom at the C4 position. All of the compounds were tested for the

Full text of DOI:10.1016/j.ejmech.2019.05.061

Accepted Manuscript
The synthesis and anticancer activity of 2-styrylquinoline derivatives. A p53
independent mechanism of action
Anna Mrozek-Wilczkiewicz, Michał Kuczak, Katarzyna Malarz, Wioleta Cieślik,
Ewelina Spaczyńska, Robert Musiol
PII:
S0223-5234(19)30477-5
DOI:
https://doi.org/10.1016/j.ejmech.2019.05.061
EJMECH 11371
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 February 2019
Revised Date: 21 May 2019
Accepted Date: 21 May 2019
Please cite this article as: A. Mrozek-Wilczkiewicz, Michał. Kuczak, K. Malarz, W. Cieślik, E.
Spaczyńska, R. Musiol, The synthesis and anticancer activity of 2-styrylquinoline derivatives. A p53
independent mechanism of action, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.05.061.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
The synthesis and anticancer activity of 2-styrylquinoline derivatives. A p53
independent mechanism of action.
Anna Mrozek-Wilczkiewicza*, Michał Kuczakb, Katarzyna Malarza, Wioleta Cieślikb,
Ewelina Spaczyńskab and Robert Musiolb*
aA. Chełkowski Institute of Physics and Silesian Center for Education and Interdisciplinary Research,
University of Silesia, 75 Pułku Piechoty 1a, 41-500 Chorzów, Poland
bInstitute of Chemistry, University of Silesia, 75 Pułku Piechoty 1a, 41-500 Chorzów, Poland
*corresponding authors email: anna.mrozek-wilczkiewicz@us.edu.pl, robert.musiol@us.edu.pl
Keywords: 2-styrylquinoline derivatives, anticancer activity, p53 protein, reactive oxygen
species, apoptosis, cell cycle inhibition
ABSTRACT
A series of styrylquinolines was designed and synthesized based on the four main quinoline
scaffolds including oxine, chloroxine and quinolines substituted with a hydroxyl group or
chlorine atom at the C4 position. All of the compounds were tested for their anticancer
activity on wild-type colon cancer cells (HCT 116) and those with a p53 deletion. Analysis of
SAR revealed the importance of electron-withdrawing substituents in the styryl part and
chelating properties in the quinoline ring. The compounds that were more active were also
tested on a panel of four cancer cell lines with mutations in TP53 tumor suppressor gene. The
results suggest that styrylquinolines induce cell cycle arrest and activate a p53-independent
apoptosis. The apparent mechanism of action was studied for the most promising compounds,
which produced reactive oxygen species and changed the cellular redox balance.
1. Introduction
Styrylquinoline (vinyl-quinoline) derivatives (SQ) are a group of aromatic and
lipophilic compounds that have a broad spectrum of biological activity. They have been
reported as being antibacterial [1][2], antiparasitic [3] and antiviral agents [4]. Styrylquinoline
FZ-41 (Fig. 1.) entered clinical trials as an inhibitor of HIV integrase [5]. The antifungal

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activity of SQ has also been broadly described [6–9] and the WK14 derivative was
determined to be a selective inhibitor of the Cdr1p efflux pump [10]. More recently, their
potency in neurodegenerative diseases has also been reported [11]. Their anticancer activity
has been known for more than six decades since the works of Bahner [12,13] and Emmelot
[14] were published. Unfortunately, the simple 2- and 4-styrylquinolines that were tested
appeared to be rather non-selective and their cytotoxicity hampered their further development.
Later, some more promising data has been revealed with a study on SQ that had been
substituted with the carboxylic and hydroxyl groups in a quinoline moiety similar to FZ-41
[15]. Their lower lipophilicity correlated with a better solubility and selectivity. Several of
these compounds appeared to be active at a micromolar level in colon cancers (HCT 116) as
well as in neuroblastomas (SK-N-MC).
NH2
N
N
S
HO
N
Br
O
OH
OH
OH
N
OH
OH
N
FZ-41
WK14
1.
IC50 = 9.0 μg/mL (HepG2)
O
H3C
CH3
N
CH3
OH
N
H3C
CH3
N
CH3
HN
HN
N
OH
N
R
N
N
O
3.
H3C
CH3
O
R = 4-Cl IC50 = 0.77 μM (SK-N-MC)
CP-31398
IC50 = 18.63 μM (HCT 116)
2.
IC50 > 4.0 μg/mL (MCF-7)
4.
R = 2-F IC50 = 8.4 μM (HCT 116 p53-null)
5.
R = 2-OAc IC50 = 7.3 μM (HCT 116)
Fig. 1. Styrylquinoline as versatile scaffold for anticancer activity.
SQ that had been substituted with thiadiazole at the C4 position of quinoline (1 in Fig. 1.)
appeared to be active against liver cancer cell lines (HepG2) [16]. Moreover, it was found to
be an active inhibitor of EGFR kinase with a potency that was similar to sorafenib. Other SQ
that had been designed on 4-chloro or 4-phenylamino quinoline were described by Chang et

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al. [17]. These compounds (2 in Fig. 1.) were also characterized by a good submicromolar
activity level against breast cancer line (MCF-7). The authors postulated that the SQ block the
cell cycle at the S phase and induce apoptosis. These facts along with their larger, aromatic
structure may suggest a DNA intercalation. Unfortunately, their selectivity toward normal
cells was not tested. However, in another report concerning furanyl analogs, the active
compounds appeared to have a rather low selectivity [18]. A significant increase in their
activity level and selectivity has been observed for derivatives of 8-hydroxyquinoline. A
series of such derivatives that were halogenated in the styryl ring (3-5) had a superior activity
along with good selectivity indexes compared to the unsubstituted SQ [19]. Oxine (8-
hydroxyquinoline) is a known chelating agent and many compounds with this fragment exert
their biological activity through interactions with metal cations. Oxine itself is broadly used as
antibacterial, antifungal and antiparasitic agent in various applications. For these SQ, the
molecular mechanism of activity was established based on the strong correlation of the
activity with lipophilicity and the intercellular localization of the drug. Apparently, SQ
penetrates the mitochondrial membrane and causes leakage of the cytochrome c followed by
the activation of the caspase apoptosis pathway. A similar mechanism, which consists of
disrupting the mitochondrial functionality was proposed earlier for amine substituted SQ [20].
What is even more appealing is that the dihalogenated compounds (e.g. 4 in Fig. 1.) were
found to be considerably more active against colon cancer that had a deletion of TP53 tumor
suppressor gene. Mutations in the TP53 gene can be found in half of the cancers that are
diagnosed and are often considered to be a sign of drug resistance and a poor prognosis.
Typical cytostatic drugs are often less effective against cancer lines that have such mutations.
With this in mind, a high activity against p53-null cells combined with a good selectivity is
particularly appealing. Another compound, CP-31398, which is a quinazoline analog of SQ
(Fig. 1.), should also be mentioned in relation to p53. This styrylquinazoline is known to be a
reactivator of a misfolded mutant protein [21]. Although its mechanism of action is not clear,
it may stabilize the protein in the correct spatial (DNA-binding) form, thereby promoting its
proapoptotic activity. Although it was designed for mutants with a point mutation, it is also
active in the wild-type cells [22][23]. More recently, a similarity between CP-31398 and
kinase inhibitors has been exploited in the design of novel styrylquinazoline anticancer agents
[24].
Unfortunately, in some mutants, and particularly in cells that have more damage in the
genome (e.g. as a deletion), the reactivator appeared to be ineffective. Actually, compared to

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the wild-type and p53-null colon adenocarcinoma, the CP-31398 was found to be inactive in
concentrations that are cytotoxic to normal cells [19,24]. Thus, SQ that are equally potent
against the wild-type and mutant as well as p53-null cells are a promising class of compounds
that deserve further exploration. Unfortunately, there are some serious gaps in our knowledge
on SAR as only several compounds with a good activity level have been described to date.
Therefore, in our current work we plan to perform a more in-depth analysis of the structural
factors that determine the activity level. We designed a series of SQ from 8-hydroxyquinoline
and its 5,7-dichloroderivative as they are the most promising among those that have been
described in the literature [25][7]. Another scaffold that was reported is 4-substituted
quinoline, e.g. 4-hydroxy or 4-phenyl [17]. On the other hand we used various substituents in
the styryl part of the molecule in order to cover a relatively broad range of electronic effects.
2. Results and discussion
2.1. Chemistry
All of the compounds that were studied were synthesized from the commercially
available quinaldines A-D. The first step of this synthesis consisted of condensing the
appropriate aromatic aldehyde using the conventional or microwave heating methods
(MAOS)[26] according to Scheme 1. The conventional reactions were performed in a mixture
of boiling acetic anhydride and acetic acid. The result of such a selection of conditions is the
acylation of the hydroxyl groups of the reactants. Accordingly, the next step was the
hydrolysis of the resulting reaction of the acyl group(s). These groups were selectively
removed with pyridine/water or K2CO3/methanol mixtures as was previously described [19].

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Scheme 1. Synthesis of the compounds that were studied.
2.2. Biological studies
2.2.1. Antiproliferative assay
The anti-proliferative activity of the synthesized compounds was tested against the human
colon carcinoma cell lines, the wild type (HCT 116 p53+/+) and p53 negative (HCT 116 p53-/-)
with an MTS assay. Additionally, the compounds were also tested for their cytotoxicity
against normal cells – human fibroblasts (NHDF). The results related to the antiproliferative
activity assays are shown in Table 1.
Table 1. Anti-cancer activity of the compounds that were studied (ND – not determined).
IC50 [µM]
Group
No.
HCT 116
p53+/+
HCT 116
p53-/-
R1
R2
NHDF
2-OCH3
3-OCH3
4-OCH3
3,4-OCH3
3,5-OCH3
2-OEt
13.13 ± 1.98
9.70 ± 0.46
11.65 ± 0.73
16.84 ± 0.44
>25
13.44 ± 1.39
10.35 ± 0.84
11.16 ± 1.13
14.74 ± 0.56
>25
>25
>25
>25
> 25
ND
1a
2a
3a
4a
5a
20.26 ± 4.44
20.87 ± 0.84
5.40 ± 0.37
>25
19.76 ± 1.16
15.48 ± 1.42
4.82 ± 0.66
>25
>25
>25
> 25
ND
6a
A
8-OAc
4-OEt
7a
3-OAc
4-OAc
2-Cl
8a
9a
10.63 ± 0.41
14.47 ± 0.68
>25
>25
>25
>25
ND
10a
11a
12a
13a
8.86 ± 0.83
10.87 ± 1.71
9.38 ± 0.87
>25
3-Cl
2,3-Cl
2,6-Cl
>25

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2- NO2
2,4-NO2
4- OCH3
3,5-OCH3
4-OH
>25
ND
14a
15a
1b
4.60 ± 0.22
>25
4.54 ± 0.31
>25
>25
>25
ND
>25
19.28 ± 1.15
16.61 ± 1.59
13.53 ± 0.81
15.48 ± 1.84
18.61 ± 1.23
>25
>25
2b
13.53 ± 1.76
7.32 ± 1.01
12.36 ± 0.91
19.92 ± 0.50
>25
>25
3b
2-OAc
4b
6.92 ±1.72
>25
8-OH
4-OAc
5b
2-Ac-4Cl
2,6-Cl
>25
6b
ND
7b
2-Cl-6-F
2-NO2
>25
>25
ND
8b
11.72 ± 0.45
11.20 ± 0.47
13.85 ± 0.59
3.65 ± 0.22
3.83 ± 0.14
5.13 ± 0.27
1.20 ± 0.16
2.22 ± 0.27
5.93 ± 0.70
3.41 ± 0.36
3.70 ± 0.38
2.57 ± 0.37
4.93 ± 0.25
7.26 ± 0.36
20.90 ± 0.71
0.93 ± 0.12
1.39 ± 0.18
1.78 ± 0.53
2.71 ± 0.16
4.76 ± 0.47
4.22 ± 0.32
0.85 ± 0.11
1.74 ± 0.10
2.31 ± 0.30
0.28 ± 0.04
10.48 ± 2.14
>25
9b
2.61 ± 0.35
8.77 ± 1.44
11.46 ± 0.50
4.72 ± 0.26
4.39 ± 0.23
3.90 ± 0.16
2.40 ± 0.56
1.30 ± 0.13
6.06 ± 0.96
2.73 ± 0.37
2.90 ± 0.28
4.47 ± 1.00
4.49 ± 0.51
4.82 ± 0.52
12.15 ± 1.86
1.08 ± 0.11
1.47 ± 0.50
1.92 ± 0.19
2.61 ± 0.33
3.60 ± 0.70
4.52 ± 0.16
0.78 ± 0.13
1.01 ± 0.07
2.01 ± 0.53
0.27 ± 0.03
15.30 ± 1.84
2-OAc
>25
1c
2-OAc-5-Cl
2-OAc-3,5-Cl
2-OAc-3-Br-5-Cl
2-OAc-3,5-I
2-I
>25
2c
13.83 ± 0.46
13.36 ± 0.64
13.09 ± 0.75
15.39 ± 0.74
>25
3c
4c
5c
6c
2-Cl
7c
2,6-Cl
8c
9.55 ± 1.58
23.46 ± 1.44
>25
2-Cl-6-F
2-Br-6-F
2,5-F
9c
10c
11c
12c
13c
14c
15c
16c
17c
18c
19c
20c
21c
22c
23c
24c
20.38 ± 0.89
>25
2,6-F
5,7-Cl-8-OAc
2,6-F-3-Cl
2,6-F-4-Cl
2-CN
21.38 ± 2.21
>25
>25
3-CN
11.15 ± 2.53
20.00 ± 1.04
20.23 ± 1.27
22.16 ± 0.77
>25
4-CN
2-OAc-3-NO2
2-OAc-5-NO2
3-NO2-4-OAc
2-NO2
B
22.29 ± 1.01
11.09 ± 0.35
19.94 ± 1.98
14.59 ± 0.81
>25
3- NO2
4- NO2
2,4-NO2
2-OH
1d
2-OAc-3,5-Cl
2-OH-3-Br-5-Cl
2-Cl
16.99 ± 1.58
14.65 ± 0.99
12.57 ± 2.88
4.50 ± 0.27
>25
2d
3d
4.09 ± 0.28
3.28 ± 0.42
1.88 ± 0.85
5.27 ± 1.02
7.30 ± 1.02
3.29 ± 0.38
0.86 ± 0.07
8.51 ± 0.77
3.10 ± 0.04
3.63 ± 0.14
0.73 ± 0.12
2.77 ± 0.54
4.03 ± 0.34
2.58 ± 0.25
2.86 ± 1.00
4.00 ± 0.37
4.50 ± 1.10
1.85 ± 0.56
0.58 ± 0.08
5.28 ± 1.20
2.46 ± 0.34
3.28 ± 0.69
0.54 ± 0.17
1.03 ± 0.32
4d
2,3-Cl
5d
2-Cl-6-F
2-I
6d
>25
7d
5,7-Cl-8-OH
2-CN
>25
8d
3-CN
17.40 ± 0.85
7.97 ± 0.55
22.78 ± 0.97
>25
9d
4-CN
10d
11d
12d
13d
3-NO2-4-OAc
2-NO2
3-NO2
9.82 ± 1.10

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4-NO2
2,4-NO2
2-I
>25
>25
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
14d
15d
1e
6.48 ± 0.42
0.75 ± 0.30
>25
3.39 ± 0.35
0.76 ± 0.16
>25
4-OAc
4-OH
2-OAc
4-OAc
2-I
>25
>25
1f
>25
>25
2f
>25
>25
3f
2-Cl
>25
>25
4f
C
3-Cl
>25
>25
5f
2,3-Cl
2-CN
3- NO2
4-NO2
2,4-NO2
4-CN
2-NO2
3-NO2
4-NO2
2,4-NO2
-
>25
>25
6f
>25
>25
7f
>25
>25
8f
>25
>25
9f
>25
>25
10f
1g
>25
>25
>25
>25
2g
D
4-Cl
>25
>25
3g
>25
>25
4g
>25
>25
5g
18.63 ± 0.92
4.42 ± 0.70
0.34 ± 0.04
26.28 ± 1.41 12.26 ± 0.54
CP-31398
-
>25
5-Fluorouracil
Doxorubicin
4.69 ± 0.33
0.38 ± 0.03
-
0.14 ± 0.03
During the analysis of the structure-activity relationship, the effect of the quinoline
substituents as well as the phenyl ring on antitumor activity was observed. IC50 values below
1 are highlighted in red and those in the 1-10 range are bolded for better readability. All of the
compounds could be divided into four groups (A, B, C and D) according to their structural
scaffolds (Scheme 1). The biological activity results are consistent with this division. In
general, the styrylquinolines that were based on quinoline substituted at the C-4 position (C
and D groups) were inactive. Compounds that were based on 8-hydroxyquinoline or 8-
acyloxyquinoline (group A) were moderately active, while their analogs that were designed
on chloroquine (dichloro derivatives – group B) were the most active in this series. It seems
that the modulation of nitrogen pKa is not crucial factor. The quinolines that were unable to
chelate metal ions (unsubstituted at C-8 position) generally lacked any antitumor activity.
These results are in good agreement with our previous work [19]. The importance of the
hydroxy or acyloxy group at the C-8 position for antitumor activity suggest that metal
chelation is a relevant mechanism of this activity [27,28]. In most cases, a quinoline
substitution with an acyloxy group resulted in a better antitumor activity than a substitution
with a hydroxy group (compare: 14a/9b, 22c/13d, 16c/9d, 24c/15d, etc.). The exceptions to
this rule were very few and they were basically within the measurement error (compare:
21c/12d, 20c/11d and 15c/8d). This observation is an important clue for understanding the
possible mechanism of action. An obvious change in lipophilicity or solubility and an effect

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on pharmacodynamics is not the case in the conditions of in vitro tests that are performed on
cellular cultures. On the other hand, both 8-hydroxyquinoline and its acetylated derivative are
known for their ability to chelate metal cations. However, the 2-methyl derivative has higher
pKa of conjugate acid than its parent acetylated oxine. Moreover, in the presence of Cu2+
ions, the hydrolysis of the acetoxy group is more rapid and is the first order in attacking the
hydroxyl group [29]. This, in turn, may improve the redox strength of the resulting complex,
thereby allowing it to participate in the Fenton reaction and in the production of the hydroxyl
radical and other reactive oxygen species (ROS) [30]. Another clear trend in the substitution
pattern can be observed in the phenyl ring of the styryl group. The stronger effect of the
electron-withdrawing group; the higher the antitumor activity. Namely, it turns out that a
substitution of the phenyl ring at the 2-position affects the activity level of the individual
derivatives in the order that is shown in the Table 2 (entries 1 and 2). The derivatives with
cyano and nitro substituents showed the highest activity level. The most active were the ortho
derivatives followed by the meta and para derivatives, which were generally less active in
each group (Table 2, entry 3). The introduction of a second nitro group into the phenyl ring
increased the activity level even further and the 2,4-dinitro derivatives in group B were the
most active against both of the cancer cell lines that were tested. The most active compound
was 24c, which reached a 0.27 µM level on HCT 116 p53-/- cell line. At the same time, it was
favorably selective with the toxicity indexes that were higher than 55 (all of the selectivity
indexes are listed in Table S1 of the Supporting Information). As proof, the introduction of an
electron-donating group (acyloxy group) into the phenyl ring with the nitro substituent
reduced the activity level (compare: 22c vs. 18c, 19c, 20c and 13d vs. 11d). A striking
exception among the 8-acyloxyquinoline derivatives was 15a, which despite a 2,4-NO2
substitution appeared to be inactive in our tests. This is even more surprising compared to 14a
(2-NO2), which was the most active in this group of derivatives. When considering selectivity,
it should be noted that the styrylquinolines from the B group were less selective toward
normal fibroblasts. However, the majority of the active compounds that are presented in
Table 1 had a reasonable selectivity index within a range of 5-10. In general, the meta-
substituted compounds with the electron-withdrawing group were less selective (compare:
16c vs. 15c, 17c; 22c vs. 21c, 23c; 13d vs. 12d, 14d).

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Table 2. The substitution pattern of the anticancer activity of styrylquinolines.
Related anticancer activity
No.
1.
Structure
2-OH
<
<
<
2-Cl/I
2-Cl/I
<
<
<
2-CN
2-CN
<
<
<
2-NO2
2.
3.
2-OAc
2-NO2
4- NO2
4-CN
3- NO2
3-CN
2- NO2
2-CN
2,4-NO2
Based on the first cytotoxicity screen on the HCT 116 cell line (Table 1), we selected
a group of the most active compounds to be investigated further. In our previous studies, we
were focused on the activity toward cell lines with a mutation in the TP53 suppressor gene
[19][31]. The p53 protein is an attractive target for anticancer studies because of its common
mutations in malignant cells. As mentioned above more than half of all cancer cases have a
mutation in the TP53 gene, therefore, for our research we selected cell lines with a mutation
(U-251, PANC-1) or deletion (double-deletion) in TP53 (HCT 116 p53-/-) or with total
absence of TP53 (AsPC-1) for further examinations. These six cell lines represent colon
(HCT 116; p53+/+ and p53-/-) and pancreatic (PANC-1 and AsPC-1) cancers, which are some
of the most common types of tumors. Additionally, we selected a glioblastoma (U-251)
because of its aggressiveness, poor prognosis and difficulty in treatment. To determine the
toxicity toward normal cells, we selected normal human dermal fibroblasts (NHDF).
The glioblastoma line U-251 carry a point missense mutation that results in a change
of arginine into histidine at codon 273. The p53 protein for its regulatory function is important
for ROS-related activity. The TP53-induced glycolysis and apoptosis regulator (TIGAR) is
another regulatory protein that changes the cellular response to redox stress and damage. It
has often been found to be overexpressed in gliomas [32]. The PANC-1 cell line has also been
reported to have missense point mutations in 273 (Arg-His or Arg-Cys) [33]. However,
according to the source, different positions and types of mutations have also been reported for
these pancreatic cell lines [34,35]. Although p53 is typically considered to be the “guardian of
the genome”, its mutation does not cause a loss of function or increase in the subsequent
mutations. Because this protein is an important node between many pathways and also acts as
gene regulator, it is not possible to predict the behavior of mutated p53 in cancer cells [36,37].
Consequently, some mutations are of the gain-of-function type as was reported for this

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R273H in U-251 cells [38]. Through new interactions with other genes, the mutant p53 may
acquire an oncogenic function.
In order to determine the expression of TP53 in the cell lines that were tested, we
performed a qRT-PCR analysis (Fig. 2.). The highest TP53 expression was observed for HCT
116 p53+/+ and subsequently for PANC-1.
Fig. 2. mRNA expression of TP53 in a panel of cancer cell lines. The results are shown as
the mean ± SD of three independent measurements, each in triplicate.
As is presented in Table 3, all of the compounds that were tested were characterized
by a good activity level against all of the cancer cell lines as well as selectivity toward normal
cells. The IC50 parameter for cancer cells varied from 0.12 µM (compound 15d on U-251) to
11.65 µM (compound 14d on AsPC-1). In general, no significant differences were observed
between the HCT 116 wild type (p53+/+) and HCT 116 p53-/-. However, the cytotoxicity of the
compounds that were tested toward AsPC-1 had the lowest level, i.e. the compounds were
significantly less active. For the rest of the cell lines that were tested, major differences in
IC50 values were observed. The compounds that were investigated had rather similar levels of
activity, except for the AsPC-1 cell line for which they had a low specificity. The activity
level against fibroblasts was to determine the selectivity indexes (SI) of each cell line (Table
S1 in the Supporting Information).

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Table 3. Cytotoxicity of the selected compounds that were studied on a panel of cell
lines.
IC50 [µM]
Compound
R2
HCT 116 HCT 116
PANC-1
AsPC-1
U-251
NHDF
p53+/+
p53-/-
2.22 ±
0.27
1.30 ±
0.13
2.14 ±
0.28
6.07 ±
0.68
5.70 ±
1.09
>25
7c
17c
2-Cl
20.00 ±
1.04
1.78 ±
0.53
1.92 ±
0.19
1.82 ±
0.09
4.58 ±
0.74
3.67 ±
0.30
4-CN
10.91 ±
0.92
20.23 ±
1.27
2-AcO-3-
NO2
2.71 ±
0.16
2.61 ±
0.33
6.16 ±
0.54
5.11 ±
0.24
18c
22.29 ±
1.01
0.85 ±
0.11
0.78 ±
0.13
2.51 ±
0.09
4.97 ±
0.32
2.30 ±
0.25
21c
2-NO2
3-NO2
2,4-NO2
2-CN
2-NO2
4-NO2
2,4-NO2
-
11.09 ±
0.35
1.74 ±
0.10
1.01 ±
0.07
2.30 ±
0.09
6.34 ±
0.36
3.07 ±
0.25
22c
14.59 ±
0.81
0.28 ±
0.04
0.26 ±
0.03
0.57 ±
0.06
5.30 ±
0.14
0.35 ±
0.05
24c
0.86 ±
0.07
0.58 ±
0.08
3.39 ±
0.42
7.81 ±
0.84
3.08 ±
0.53
>25
>25
>25
>25
8d
0.73 ±
0.12
0.53 ±
0.17
0.89 ±
0.07
7.41 ±
0.78
2.04 ±
0.42
12d
11.65 ±
1.04
6.48 ±
0.42
3.39 ±
0.35
8.25 ±
1.20
8.78 ±
0.84
14d
0.75 ±
0.30
0.76 ±
0.16
0.49 ±
0.08
1.67 ±
0.17
0.12 ±
0.02
15d
18.63 ±
0.92
26.28 ±
1.41
18.77 ±
1.65
12.26 ±
0.54
>25
ND
CP-31398
Doxorubicin
0.34 ±
0.04
0.38 ±
0.03
0.730 ±
0.088
0.86 ±
0.13
0.05 ±
0.01
0.14 ±
0.03
-
2.2.2. Cell Cycle
The promising activity profiles against the cancer cell lines prompted us to run further
experiments. We hypothesized that the compounds that were tested could affect the inhibition
of the cell cycle, while the lack of any significant differences between the IC50 values for both
HCT 116 cell lines (p53+/+ and p53-/-) suggest a p53-independent mechanism of action.
However, some resistance was observed for the AsPC-1 cell line. Therefore, we decided to
explore the effect of the selected compounds, 12d and 24c, on the progression of the cell
cycle in the HCT 116 p53+/+ and U-251 cell lines that had been selected as the wild type and a
partially functional mutant of p53. The results of these experiments are presented in Fig. 3.
After incubation with the tested compounds, no significant differences were observed in the
G0/G1 phases for the controls and treated cells, but a negligible decreasing trend was

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noticeable. The reverse occurred for the S phase, which was increased; however, significant
growth was detected for HCT 116 p53+/+. These observations suggest that the compounds that
were tested inhibit the cell cycle in the S phase.
Fig. 3. Effect of the treatment with styrylquinolines (10xIC50) on the cell cycle in the
HCT 116 p53+/+ and U-251 cells. (A) Representative histograms from one of three
independent experiments showing the distribution of cells in the individual phases of the
cell cycle. (B) Table with the mean ± SD percentage of the cells in the individual phases
of the cell cycle from three independent experiments. (C) Diagram of the data with
statistical analysis using a one-way ANOVA with Bonferroni’s post-hoc test: *p < 0.05,
**p < 0.01, ***p < 0.001 compared to the respective control.
2.2.3. Annexin V Binding
The type of cell death was another factor in determining the potential mechanism of
action for SQ. The assay was based on measuring the FITC fluorescence, that was conjugated

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to Annexin V, in the apoptotic cells. Annexin V is a cellular protein that binds to
phosphatidylserine – an indicator of apoptosis. A damaged cell loses the integrity of the cell
membrane and phosphatidylserine is moved outside the cell, which results in the emission of a
green signal of the FITC and indicates apoptotic cell death. For this experiment, we selected
the same two cell lines – HCT 116 p53+/+ and U-251. Incubation with 12d and 24c enabled
the following conclusions to be drawn. While an apoptotic cells fraction was observed for
both derivatives, the strongest effect was detected for 24c for HCT 116 p53+/+ (63.10% of the
apoptotic; Fig. 4B). This increase in the apoptotic cells was also observed in the U-251 cells
but to a lesser extent.

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Fig. 4. Evaluation of the affect of the treatment with styrylquinoline analogs (20 µM) on
the induction of apoptosis in the HCT 116 p53+/+ and U-251 cells after 48h. (A)
Representative histograms from one of three independent experiments show the
percentage of live, early and late apoptotic cells. (B) Table shows the mean ± SD
percentage of live and total apoptotic cells from three independent experiments. (C)
Diagram of the data with a statistical analysis using a one-way ANOVA with
Bonferroni’s post-hoc test: *p < 0.05, **p < 0.01, ***p < 0.001 compared to the
respective control.

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2.2.4. ROS generation
As was mentioned above, chelating the metal cations that are redox active (such as
copper and iron) may lead to the formation of reactive oxygen species and cause oxidative
damage. In larger aromatic structures such as SQ, the same can be associated with DNA
intercalation, which results in minor, cumulative damage in a genome. This, in turn, leads to
an arrest of the cell cycle and apoptosis in proliferating cells. Our previous works [39][40][41]
promted us to examite the affect of the compounds that were tested on the generation of ROS.
For this purpose, we conducted a series of the experiments that involved the incubation of the
selected derivatives 12d and 24c with the HCT 116 p53+/+ and U-251 cell lines after which
the ROS level was measured using a fluorescent assay.
Fig. 5. Impact of the compounds that were tested (20 µM) on the generation of ROS in
the HCT 116 p53+/+ and U-251 cells. Data were normalized to control (untreated cells).
Results are presented as the mean ± SD of three independent measurements. Data were

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analyzed using the Student's t-test: *p < 0.05, **p < 0.01, ***p < 0.001 compared to the
control group.
The results presented in Fig. 5 indicated that ROS were generated by both of the
compounds that were tested on both cell lines. We performed kinetic measurements in the
time range of 1-24h, which indicated an increasing trend in all cases. The largest increase of
the ROS level was observed for HCT 116 p53+/+ (over 160%). The highest ROS concentration
was detected after 24h and was still increased. The level of ROS and their function of
signaling factors is closely correlated with the activity of p53 [42]. p53 has a downregulating
effect on MnSOD – one of the main proteins that constitutes an antioxidant defense of a cell.
The loss of the p53 function, which is caused by a mutation, may lead to higher antioxidative
potential in cancer cells [43,44]. The same can be observed in our results in which the
generation of ROS was higher in the wild-type cells.
2.2.5. Western Blot
After previous analyses, we had no doubt that the styrylquinoline derivatives that were
being examined affected the regulation of the cell cycle and the induction of apoptosis. In
order to confirm the inhibition of the cell cycle and apoptosis induction that were observed
and to investigate the mechanism of action of the compounds that were tested more deeply,
we performed a western blot analysis. Additionally, after the kinetic determination of the
intracellular ROS levels, we decided to examine the expression of the oxidative stress-related
proteins. A visualization of the protein bands with their corresponding densitometric analysis
is presented in Fig. 6.

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Fig. 6. Effect of the styrylquinoline analogs (20 µM) that were tested on the expression of
HIF-1α. PARP-1, p53, β-actin, cdc2, HO-1, p21 in the HCT 116 p53+/+ and U-251 cells.
(B) Densitometric analysis of the data that was obtained normalized to the reference
proteins are shown as the mean ± SD of four independent experiments.
A cytotoxicity analysis on the cell lines for various TP53 gene expressions indicated
that there was no correlation between the activity of the compounds that were tested and p53
activation. Indeed, in the western blot analysis, no effect of the tested 12d and 24c derivatives
on the activation of the p53 protein was observed. Although p53 was activated at a certain
level in U-251, there were no differences between the control cells and the cells after
treatment with the compounds that were investigated. The expression of this protein in U-251
is most likely related to a mutation at position 273 [38]. A similar level of p53 in this cancer
cell line was observed in a study of the menavolate pathway in glioblastomas [45]. Our results
clearly indicated a p53-independent mechanism of action of the styrylquinoline analogs and
confirmed our previous work on this group of compounds [19].

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One of the important enzymes that are associated with the apoptosis pathway is Poly
(ADP-rybose) polymerase-1 (PARP-1), which is the target of caspases. The main role of this
protein is to support the cells viability by its involvement in the repair of the DNA damage
that is triggered by stress factors. The degradation of PARP-1, which was observed as the
cleavage by caspases, promoted the destabilization of cells. Cleaved PARP is considered to be
a good indicator of apoptotic cells [46][47][48]. Clear cleavages were observed in both cell
lines after treatment with the 24c derivative. The most distinct cleavage was observed in HCT
116 (p53+/+) and cleavage for 12d was also detected for this cell line. Therefore, the result in
this case strongly confirms the data that was obtained from the annexin V binding assay.
The compounds that were tested also contributed to the arrest of the cell cycle in the S
phase in the case of HCT 116 (p53+/+), and to a lesser extent in U-251. A lower level of the
two proteins (cdc2 and p21), which are related to untreated cells, were observed in the
glioblastoma cell line. Cyclin-dependent kinase 2 (cdc2) participates in the transition of cells
from the G2 phase into the M phase [49]. On the other hand, p21WAF1/CIP1 regulates the
activity of cyclin and kinases during the progression of the cell cycle [50]. Downstream of
p21WAF1/CIP1, which inhibits the cell cycle in the S phase as well as significant increase
population of the apoptotic cells, was also observed in keranocytes after an anti-proliferative
factor was applied [51]. We suppose that in the U-251 cells, the derivatives that were tested
may cause arrest in both the S and G2/M phases. Confirmation of our thesis was the
downregulation of cdc2. In HCT 116 (p53+/+), however, there were no significant differences
between the cdc2 expression, which proved that the cell cycle had been inhibited in the earlier
G1 phase. Additionally, a p53-independent mechanism of G1 inhibition was observed in HCT
116 in which an increase of p21WAF1/CIP1 plays important role [52]. These results seem to be
opposite to ours due to the decrease in the level of p21WAF1/CIP1 and the arrest of the cell cycle
in other phases. However, the factors that are involved in the mechanism of action of a
specific drug depend on the cell line being tested, the dose of a drug, the time point being
tested and many other factors. Considering the dual role of the p21 protein [53], results that
were observed are not surprising although some more detailed research would be valuable to
explain these mechanisms.
In pathophysiological conditions, one of the crucial enzymes that are involved in
maintaining proper homeostasis is heme oxygenase-1 (HO-1). During normal states, the level
of HO-1 in cells is quite low. In response to numerous factors that lead to oxidative stress,
HO-1 is highly activated [54][55]. The significant increase of HO-1 after treatment with the
compounds that were tested in both cell lines is most likely the result of excessive

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intracellular generation of ROS. These results are in agreement with the literature data, which
suggest a correlation between increased ROS levels and the intensified activation of HO-1
[56]. The increasing production of ROS by mitochondria may also play an important role in
promoting the expression of hypoxia-inducible factor-1 (HIF-1). Although this transcription
factor is mostly connected with tumorogenesis, angiogenesis in solid tumors and a generally
poor prognosis for patients, its growth may be related to oxidative stress [57][58]. Liang et al.
[59] proved a time-dependent relationship between the growth of both the HIF-1 and ROS
levels. The results of our work, in addition to the expansion of the generation of ROS, showed
a high activation of HIF-1α after treatment with both SQ. Because the overexpression of this
protein is connected with an accumulation of mutations in the tumor suppressor genes such as
p53 [60], we detected a higher level of HIF-1 in U-251 than in HCT 116 (p53+/+).
Furthermore, in studies on the osteosarcoma cancer cell lines, apoptosis was partially
inhibited by upregulating the expression of HIF-1 [59]. In other words, HIF-1 is another
element of the cellular antioxidative potential along with MnSOD and the small molecular
redactors such as glutathione, which is predominantly upregulated in the p53-mutated cancer
cell lines. Moreover, the response of the wild-type p53 to ROS strongly depends on their
initial level. p53 may act as an anti- or pro-oxidative regulator to promote survival or
apoptosis respective to the severity of the damage [61]. On the other hand, p53 mutants
develop a higher antioxidative capacity in order to fulfill their metabolic demand on ROS,
which leads to a more fragile equilibrium that can easily be broken by drugs that increase the
ROS.
Based on all of the results that were obtained, we suggest that the anti-proliferative
effect of styrylquinolines is correlated with the overproduction of intracellular ROS. In our
research, the oxidative stress that was induced by the compounds that were tested clearly
activated the ROS-related pathways, thus leading to apoptosis. Apoptotic death via an
elevated level of ROS is the mechanism that has also been determined in other glioma cell
lines [62][63].
3. Conclusions
A series of styrylquinolines with diversified substituents were designed and
synthesized. Their activity against a panel of cancer cells was evaluated, which provided data
that was suitable for some SAR analyses. According to these results, the importance of the
hydroxyl group in position C8 of quinoline is confirmed. Apparently, it is its chelating
properties that extend the activity of SQ. This was also confirmed by a time-dependent

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generation of ROS in cells after their incubation with those active compounds. The same can
explain the high activity level of the acetylated derivatives, which can be more active in
generating the redox reactions with iron or copper ions. On the other hand, strong electron-
withdrawing substituents in the styryl part of a molecule are crucial for a high activity level.
In fully aromatic flat molecules such as styrylquinolines, these effects have some influence on
the electron density in the 8-hydroxyquinoline fragment, thus further increasing their
chelation properties. An investigation of the mechanism of action revealed that the active SQ
are also able to induce p53-independent apoptosis in cells that have a mutation in the TP53
gene or those that are p53-null (deletion). Thus, their activity is generally at a similar level
regardless of the p53 status of the cells, which is especially promising from the point of view
of further development.
4. Experimental
All of the reagents were purchased from Aldrich. A Kieselgel 60, 0.040-0.063 mm (Merck, Darmstadt,
Germany) was used for the column chromatography. TLC experiments were performed on alumina-
backed silica gel 40 F254 plates (Merck, Darmstadt, Germany). The plates were illuminated under UV
(254 nm) and evaluated in iodine vapor. The melting points were determined on an Optimelt MPA-
100 apparatus (SRS, Stanford CA). The purity of the final compounds was checked using HPLC.
Detection wavelengths of 210 and 250 nm were chosen for detection. The purity of individual
1
compounds was determined from the area peaks in the chromatogram of the sample solution. All H
and 13C NMR spectra were recorded on a Bruker Avance III 400 MHz FT-NMR spectrometer (400
1
MHz for H and 101/126 MHz for 13C, Bruker Comp., Karlsruhe, Germany). Chemical shifts are
reported in ppm (δ) using signal of the solvent (DMSO-d6, CD2Cl2-d2, CDCl3-d1) as the reference
against the internal standard Si(CH3)4. Easily exchangeable signals were omitted when they were
diffuse. Signals are designated as follows: s, singlet; br. s, broad singlet; d, doublet; dd, doublet of
doublets; ddd, doublet of doublet of doublets; t, triplet; tt, triplet of triplets; td, triplet of doublets; q,
quartet; m, multiplet. Mass spectra were measured using a LTQ Orbitrap Hybrid Mass Spectrometer
(Thermo Electron Corporation, USA) with direct injection into an APCI source (400°C) in the positive
mode.
4.1. Synthesis of compounds
General Method A: The appropriate quinoline derivative (2.5 mmol) in acetic anhydride with 80%
acetic acid was thoroughly mixed with one equivalent aldehyde and heated in an inert gas atmosphere
(N2) for 18-22 h at 130 °C. Then, the mixture was evaporated to dryness and a solid was crystallized
from ethanol, methanol or ethyl acetate.

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General Method B: The crude product from step A was transferred to a mixture of pyridine and
water at a ratio of 3:1 and further heated for 3 h at 100 °C. Then, the mixture was evaporated to
dryness and a solid was crystallized from ethanol, methanol or ethyl acetate.
General Method C: The appropriate styrylquinoline derivative (2.5 mmol) in methanol was
thoroughly mixed with 2.5 equivalent K2CO3 for 2 h at room temperature. Then, concentrated HCl
was added and the resulting precipitate was filtered and washed with H2O. The crude product was
recrystallized from ethanol, methanol or ethyl acetate.
General Method D: The appropriate quinoline derivative (2.5 mmol) and respective benzaldehyde
(7,5 mmol) in 1:3 ratio was heated in an oil-bath at 175-180 °C. After 30-45 min of heating, the
mixture was cooled to room temperature and washed with hexane (2x15mL) to remove excess
benzaldehyde. The residue was recrystallized from ethanol, methanol or ethyl acetate.
Physicochemical data with spectroscopic analyses for all compounds are given in Supporting
Information.
4.2. Biological studies
For biological experiments brief description is provided. For more details please refer to Biological
Studies in Supporting information.
4.2.1. Cell culture
The human colorectal carcinoma cell line HCT 116 with wild type of p53 (p53+/+) were obtained from
the ATCC. HCT 116 with deletion of TP53 gene (p53-/-) was kindly provided by prof. M. Rusin from
Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology in Gliwice, Poland. The
glioblastoma cell line U-251, two pancreatic cancer cell lines (PANC-1 and AsPC-1) were purchased
from Sigma-Aldrich (Germany) and normal human dermal fibroblast cell line (NHDF) from
PromoCell. Cells were grown as monolayer cultures in 75cm2 flasks in appropriate medium according
to typical procedures. Cells were cultured under standard conditions at 37° C in a humidified
atmosphere at 5% CO2. Additionally, all cell lines subjected to routine mycoplasma test to ensure that
there was no contamination.
4.2.2. Cytotoxicity studies
The exponentially growing cells were seeded into 96-well plates and cultured under standard
conditions at 37° C for 24 h. Then, medium was supplied by solutions with varying concentrations of
tested derivatives and further incubated for 72 h. After treatment, media solutions were replaced in
each well by 100 µL DMEM without phenol red and 20 µL of CellTiter 96® AQueous One Solution-
MTS (Promega), incubated for short time and measured. The results as medium for at least three
independent experiments, were estimated using GraphPad Prism 5 as IC50 values.

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4.2.3. Analysis of the level mRNA expression of TP53.
The total RNA was isolated from each cancer cell line according to the TRIzol procedure (Ambion).
Then reverse transcription was conducted with 5 µg of total RNA using a GoScript™ Reverse
Transcriptase kit (Promega) and Oligo(dT)23 Primers (Sigma). The Real-Time PCR in a 20 µL reaction
volume was performed with a CTX96 Touch™ Real-Time PCR Detection System (Biorad). All
primer pair sequences (see Supporting Information) were ordered in Sigma-Aldrich. The reaction was
performed and data was analyzed based on a comparison of the expression of the target gene to a
reference gene – ACTB (β-actin), using the 2–∆∆CT method. The experiments were repeated at least
three times.
4.2.4. Cell cycle assay
The cells were seeded in 3 cm Petri dishes at a density of 0.2·106 cells per dish and incubated under
standard conditions for 24 h. Afterwards, the medium was replaced by freshly prepared solutions of
the tested styrylquinoline derivatives in tenfold IC50 values. After 24h, assays were performed using a
Muse Cell-Cycle Kit (Millipore) in keeping with the manufacturer’s instructions. Samples were
incubated for 30 min at room temperature in the dark. After staining, the values of cellular
subpopulations in individual cell cycle phases were assessed. The experiments were repeated at least
three times.
4.2.5. Annexin V binding assay
The FITC Annexin V Apoptosis Detection KIT with 7-AAD (BioLegend) was performed according to
manufacturer’s instructions. The cancer cells were incubated on Petri dishes with medium solutions of
tested styrylquinoline analogues (20 µM) for 48 h. After then, the cells were detached and centrifuged
at 300 g for 5 min, then washed twice with cold PBS and next resuspended in 100 µL of Annexin V
Binding Buffer. Afterwards, the cells were incubated with 5 µL of FITC Annexin V and 5 µL 7-AAD
Viability Staining Solution for 15 min at room temperature in the dark. After staining, the events for
live, early and late apoptotic cells were counted using a Muse Cell Analyzer (Millipore). The
experiments were repeated at least three times.
4.2.6. Immunoblotting
The cells were incubated in solution of the tested compounds (20 µM) for 24 h. After then, the cells
were detached by trypsinization, collected in Eppendorf tubes and centrifuged at 2,000 rpm. Next, cell
pellets were suspended in 150 µL of complete RIPA Buffer (Thermo Scientific) containing a Halt
Protease Inhibitor Cocktail (Thermo Scientific) and Halt Phosphatase Inhibitor Cocktail (Thermo
Scientific) along with 0.5 M EDTA and lysed on ice for 20 min on a rocking plate. Subsequently,
obtained lysates were sonicated and centrifuged at 10,000 rpm for 10 min at 4° C and the supernatants

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were collected for further analysis. The protein concentration was measured using a Micro BCA™
Protein Assay Kit (Thermo Scientific) according to the manufacturer’s instruction. Equal amounts of
proteins (16 µg) were electrophoresed on SDS-Page gels and transferred onto nitrocellulose
membranes. The membranes were blocked in 5% non-fat milk prepared in TPBS (PBS containing
0.1% Tween-20 for 1 h). After blocking, the membranes were incubated with specific primary
monoclonal antibodies overnight at 4° C. The next day, membranes were washed in TPBS and
incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room
temperature. Finally the membranes were visualized using a ChemiDoc™ XRS+ System (BioRad).
The experiments were performed at least three-four times. The densitometric analysis was carried out
using ImageJ software (Wayne Rasband, National Institutes of Health, USA).
4.2.7. Time dependent measurement of ROS generation
The cells were incubated with the solutions of the tested compounds for 1, 3, 6, 9, 12 and 24 h. The
generation of ROS was assessed immediately according to CellROX® Green Reagent procedure. In
addition, the amount of cells in each well was determined using Hoechst 33342 (Molecular Probes™).
After time of incubation, the solutions of tested compounds in each well were changed for 100 µL of
CellROX Green Reagent (5 µM) and Hoechst 33342 (6 µM) and subsequently cells were incubated for
30 min at 37 ºC. The fluorescence was measured using a multi-plate reader (Synergy 4, Bio Tek) at
485 nm excitation and a 520 nm emission for CellROX Green Reagent and a 345 nm excitation laser
and a 485 nm emission filter for Hoechst 33342. The experiments were repeated three to four times.
ROS levels were expressed as the percentage of the control cells level.
4.2.8. Statistical analysis
The results are expressed as the mean ± standard deviation (SD) from at least three independent
experiments. Statistical analysis was performed using the one-way ANOVA with a Bonferroni post-
hoc test (in case of results from progression of cell cycle and Annexin V binding assays) or the two
tailed Student’s t-test (in case of results from ROS level measurement). A p-value of 0.05 or less was
considered to be statistically significant. GraphPad Prism 5 software (GraphPad Software, USA) was
used for the analysis.
Author contributions
AMW and RM created the research hypothesis; ES carried out the chemical syntheses
and designed the experiments, which were supervised by RM; AMW, MK and KM performed
the biological tests; AMW, MK, WC and RM wrote and edited the manuscript.

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Acknowledgements
This work was supported by the projects of the National Science Centre
2014/13/D/NZ7/00322 (A.M.W.) and 2016/23/N/NZ7/00351 (K.M.). Technical assistance of
Klaudia Krzykała is greatly acknowledged.
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