Biological evaluation of 3-benzylidenechromanones and their spiropyrazolines-based analogues

Article abstract of DOI:10.3390/molecules25071613

A series of 3-benzylidenechrmanones 1, 3, 5, 7, 9 and their spiropyrazoline analogues 2, 4, 6, 8, 10 were synthesized. X-ray analysis confirms that compounds 2 and 8 crystallize in a monoclinic system in P2₁/n space groups with one and three molecules in each asymmetric unit. The crystal lattice of the analyzed compounds is enhanced by hydrogen bonds. The primary aim of the study was to evaluate the anti-proliferative potential of 3-benzylidenechromanones and their spiropyrazoline analogues towards four cancer cell lines. Our results indicate that parent compounds 1 and 9 with a phenyl ring at C2 have lower cytotoxic activity against cancer cell lines than their spiropyrazolines analogues. Analysis of IC₅₀ values showed that the compounds 3 and 7 exhibited higher cytotoxic activity against cancer cells, being more active than the reference compound (4-chromanone or quercetin). The results of this study indicate that the incorporation of a pyrazoline ring into the 3-arylideneflavanone results in an improvement of the compounds’ activity and therefore it may be of use in the search of new anticancer agents. Further analysis allowed us to demonstrate the compounds to have a strong inhibitory effect on the cell cycle. For instance, compounds 2, 10 induced 60% of HL-60 cells to be arrested in G2/M phase. Using a DNA-cleavage protection assay we also demonstrated that tested compounds interact with DNA. All compounds at the concentrations corresponding to cytotoxic properties are not toxic towards red blood cells, and do not contribute to hemolysis of RBCs.

Full text of DOI:10.3390/molecules25071613

molecules
Article
Biological Evaluation of 3-Benzylidenechromanones
and Their Spiropyrazolines-Based Analogues
Angelika A. Adamus-Grabicka 1 , Magdalena Markowicz-Piasecka 2 , Marcin Cies´lak 3
,
Karolina Królewska-Golin´ska 3, Paweł Hikisz 4, Joachim Kusz 5, Magdalena Małecka 6 and
Elzbieta Budzisz 1,
*
1
Department of Cosmetic Raw Materials Chemistry, Faculty of Pharmacy, Medical University of Lodz,
Muszynskiego 1, 90-151 Lodz, Poland; angelika.adamus@umed.lodz.pl
2
3
4
Laboratory of Bioanalysis, Department of Pharmaceutical Chemistry, Drug Analysis and Radiopharmacy,
Medical University of Lodz, Muszynskiego 1, 90-151 Lodz, Poland; magdalena.markowicz@umed.lodz.pl
Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112,
90-363 Lodz, Poland; marcin@cbmm.lodz.pl (M.C.); kkrolews@cbmm.lodz.pl (K.K.-G.)
Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of Lodz,
Pomorska 141/143, 90-236 Lodz, Poland; pawel.hikisz@biol.uni.lodz.pl
Institute of Physics, University of Silesia, Uniwersytecka 4, 40-007 Katowice, Poland; joachim.kusz@us.edu.pl
Department of Physical Chemistry, Theoretical and Structural Chemistry Group, Faculty of Chemistry,
University of Lodz, Pomorska 163/165, 90-236 Lodz, Poland; magdalena.malecka@chemia.uni.lodz.pl
Correspondence: elzbieta.budzisz@umed.lodz.pl; Tel.: +48-42-272-55-95
5
6
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 8 February 2020; Accepted: 30 March 2020; Published: 1 April 2020
Abstract: A series of 3-benzylidenechrmanones
1,
3,
5,
7,
9
and their spiropyrazoline analogues
2, 4, 6, 8,
10 were synthesized. X-ray analysis confirms that compounds
2
and crystallize in a monoclinic system
8
in P21/n space groups with one and three molecules in each asymmetric unit. The crystal lattice of the
analyzed compounds is enhanced by hydrogen bonds. The primary aim of the study was to evaluate
the anti-proliferative potential of 3-benzylidenechromanones and their spiropyrazoline analogues
towards four cancer cell lines. Our results indicate that parent compounds
at C2 have lower cytotoxic activity against cancer cell lines than their spiropyrazolines analogues.
Analysis of IC50 values showed that the compounds and exhibited higher cytotoxic activity against
1 and 9 with a phenyl ring
3
7
cancer cells, being more active than the reference compound (4-chromanone or quercetin). The results
of this study indicate that the incorporation of a pyrazoline ring into the 3-arylideneflavanone results
in an improvement of the compounds’ activity and therefore it may be of use in the search of new
anticancer agents. Further analysis allowed us to demonstrate the compounds to have a strong
inhibitory effect on the cell cycle. For instance, compounds 2, 10 induced 60% of HL-60 cells to be
arrested in G2/M phase. Using a DNA-cleavage protection assay we also demonstrated that tested
compounds interact with DNA. All compounds at the concentrations corresponding to cytotoxic
properties are not toxic towards red blood cells, and do not contribute to hemolysis of RBCs.
Keywords: 3-benzylideneflavanone/3-benzylidenechromanone; spiropyrazolines; cytotoxicity;
lipophilicity; DNA interaction
1. Introduction
The chromanones and their flavanone derivatives comprise an interesting group of compounds
because they easily react with nucleophilic reagents, the most investigated of these reactions being
those which take place with hydrazine or its analogues, resulting in the production of a five-membered
heterocyclic ring. Pyrazolines are heterocyclic compounds having two adjacent atoms within the
ring. They have only one endocyclic double bond and are basic in nature. The dihydro derivative
Molecules 2020, 25, 1613; doi:10.3390/molecules25071613
www.mdpi.com/journal/molecules

Molecules 2020, 25, 1613
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of pyrazole is known as pyrazoline. Depending on the position of the double bond it can exist in
three separate forms:
1-pyrazoline (the double bond exists between the first and second nitrogen
atoms),
2-pyrazoline (the double bond occurs between the second nitrogen atom and the third carbon
atom) and ].
3-pyrazoline (the double bond occurs between the third and fourth carbon atom) [
As their chemical structure is based on the presence of electron-rich nitrogen heterocycles, they
display important biological activities [ ]. Pyrazoline derivatives widely occur in nature in the form
∆
∆
∆
1
2
of alkaloids, pigments and vitamins. Due to the anticancer (Figure 1A), antibacterial (Figure 1B),
antifungal (Figure 1C), anti-inflammatory (Figure 1D), and antidepressant (Figure 1E) properties of
spiropyrazole analogs, many studies have examined their biological activities [3–5]. Certain derivatives,
such as nitric oxide synthase (NOS) inhibitor (Figure 1F) and cannabinoid CB1 receptor antagonist
(Figure 1G), also demonstrate potent selective biological activity against certain receptors [
their biological activity, they have been used in the treatment of various diseases.
6,7]. Due to
B
C
A
D
E
F
G
Figure 1. Examples of pyrazoline analogs with biological activity: (
)–antifungal, ( )–anti-inflammatory, ( )–antidepressant, (
and (G)—cannabinoid CB1 receptor antagonist.
A
)–anticancer, (
B
)–antibacterial,
(
C
D
E
F)—nitric oxide synthase (NOS) inhibitor

Molecules 2020, 25, 1613
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Over the last thirty years, many methods have been developed for the synthesis of pyrazolines
and their analogues; however, while numerous such analogues have been described in the literature,
the 4,5-dihydro-1H-pyrazolines are the most often studied. Their best known method of synthesis
is based on the reaction of
α
,β-unsaturated ketones with hydrazines, which results in the formation
of a hydrazone, which is cyclized to form a 2-pyrazoline [
8]. Much attention has been focused
on the synthesis of 3-arylidenechromanones/flavanones, which are precursors in the reaction for
obtaining 3-spiropyrazolines; these were synthesized by reacting 3-benzylidenechromanones or
3-benzylidene-flavanones with diazomethane in anhydrous acetone [9]. According to the Toth [9]
the classical synthesis of 3-arylideneflavanones/chromanones with diazomethane allows only one
stereoisomer of spiro-1-pyrazolines to be obtained.
Following on from a previous paper [10], the present study broadens our studies of the reactivity
of 3-arylideneflavanones and 3-arylidenechromanone with diazomethane on a larger number of
compounds containing various benzylidene substituents. The formation of spiro- compounds as a
result of attack by a nucleophilic reagent on the carbon of the 3-arylideneflavanone most probably
depends on the degree of electropositivity of the carbon; this would govern reactions with the various
resonance structures of diazomethane [9]. It is possible that the resonance structure of diazomethane,
with a partial negative charge on the carbon atom, may be attacked in the reaction of the formation of
3-spiropyrazolines (Scheme 1).
R1
O
O
R
O
R
H
piperidine
+
OHC
0
150 C
R1
O
1,3,5,7,9,11,12
CH N
2
2
_
+
_
R
O
CH2
N
N
_
O
O
R1
or
N
H
O
R
N
Ph
_
_
+
CH2
N
N
_
2,4,6,8,10
Scheme 1. The route of synthesis of the examined compounds.
R1 = p-OCH3; : R = Ph, R1 = H; : R = H, R1 = H;
p-N(C2H5)2; 12: R = H, R1 = p-N(C2H5)2.
1
,
2
: R = Ph, R1 = p-OCH3;
3
,
4
: R = H,
5;
6
7;
8
9, 10: R = Ph, R1 = m-OCH3; 11: R = Ph, R1 =
The aim of this study is to compare the cytotoxic activities of a series of chromanones/flavanones
and spiropyrazoline analogues as substrates and products of their modification reactions with
diazomethane. The compounds were tested for biological activity on four cell lines: HL-60 (human
leukemia cell line), NALM-6 (human peripheral blood leukemia cell line), WM-115 (melanoma cell
line) and COLO-205 (human colon adenocarcinoma cells). It also examines the influence of various
substituents on the lipophilicity and cytotoxic effect of the compounds, as well as their interaction with
DNA and cell cycle arrested studies. Moreover, we searched for the less toxic compounds compared
to references compounds. Both the 3-benzylidene and spiro- analogues were digested of plasmid

Molecules 2020, 25, 1613
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DNA with BamHI restriction nuclease and the study of cell cycle phases using a flow cytometer has
been done.
Correct design and systematic examination of the structure-activity relationship of pyrazoline
compounds appears to be a good strategy for new drug design. The obtained compounds may be a
valuable basis for discovering safe potent drug candidates with lesser side effects.
2. Results and Discussion
Two structures, benzylideneflavanone and benzylidenechromanone substituted with diethylamine
group, have been examined previously in a study that compared the exacytotoxic activity of the two
compounds and checked whether they destroy erythrocyte membranes [10]. Encouraged by these
positive results, the present manuscript examines the relationship between the biological activity of the
tested compounds and their chemical structure: for this purpose ten 3-arylidenechromanone analogues
with a
their cytotoxicity, determined. All synthesized compounds were tested for their compatibility with
blood and whether they influenced the integrity of the erythrocyte membrane. Analogues 10,
∆
1-pyrazoline ring were synthesized and their physicochemical characteristics, together with
2
,
7,
11, and 12 exhibited the greatest cytotoxicity towards the tested cancer cell lines and thus were
taken for further study. The next step examined whether the compounds would inhibit digestion of
plasmid DNA in a similar way to a reference compound (daunorubicin); in addition, the compounds
were subjected to DNA interaction studies to identify the phase of the cell cycle they would block.
Finally, two analogues, 2 and 8, were chosen for further testing to describe their crystal structure and
characterize their intermolecular interactions in the crystal lattice.
2.1. Chemistry
The synthesis of the described compounds has been divided into two stages. In the first step the
compounds
or chroman-4-one and the appropriate aryl aldehyde. The exocyclic
synthesized in the piperidine medium; in a second step, these unsaturated ketones were reacted
with an ethereal solution of diazomethane in anhydrous acetone to produce spiro-1-pyrazolines
10 in a regioselective and stereospecific manner (Scheme 1). The structures of all compounds
1
,
3,
5
,
7
and
9
were prepared by a conventional reaction between 2-phenylchroman-4-one
α,β-unsaturated ketones were
2, 4,
6, 8,
were characterized using IR, 1H- and 13C-NMR, MS spectroscopy and elemental analysis. A detailed
description of the spectra of the tested compounds is provided in the Supporting Information with the
1H- and 13C-NMR spectra (see Figures S4–S13a). Part 3 (Materials and Methods) presents a detailed
description of the IR bands and signal shifts of the spectra.
2.2. X-ray Single-Crystal Structure of 2 and 8
The molecular structure of compounds
in a monoclinic system in P21/n; however, in
chroman skeleton in consists of a benzene and a pyran ring with a phenyl substituent at the C2 atom.
In addition, 4-(4-methoxyphenyl)-4,5-dihydro-3H-pyrazole is attached at the C3 position.
Molecule contains a chroman skeleton with a 4-phenyl-4,5-dihydro-3H-pyrazole at position
2
and
8 is shown in Figure 2. Molecules of 2 and 8 crystalize
8, an asymmetric unit has three molecules. The main
2
8
C3. The pyran ring and 4,5-dihydro-3H-pyrazole adopt an envelope conformation in both structures,
with puckering parameters and asymmetry parameters given in Table 1 [11,12].

Molecules 2020, 25, 1613
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O
O
OMe
N
N
(a)
(b)
H
O
O
H
N
N
(c)
(d)
(e)
Figure 2. The molecular structure of
) and ). Scheme. ( ) represents the overlay of three molecules in the asymmetric unit in the
compound 8 crystal structure.
2 (a) and 8 (c) with an atom numbering. The chemical structure of
2
(b
8
(d
e
The crystal packing of
of compound are linked by four intermolecular hydrogen bonds: C12-H12A . . . O1 (1/2
z), C2-H2 . . . O4 (1/2 x, 1/2 + y,1/2 z), C26-H26 . . . N2 (1/2 x, 1/2 + y, 1/2 z), C11-H11 . . . N1 (1/2
x, 1/2 + y, 1/2 − z), which are responsible for the formation of dimers (Figure 3).
2 and 8 is enhanced by a network of hydrogen bonds (Figure 2). The molecules
2
−x, 1/2 + y, 1/2
−
−
−
−
−
−
−

Molecules 2020, 25, 1613
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Table 1. Ring puckering parameters with asymmetry parameters and dihedral angle between chromanone
skeleton (A/B rings) and C ring.
Compound/Molecule
5-Membered Ring
6-Membered Ring
Asymmetry
θ (◦)
Φ (◦)
Conformation
Q (Å)
Parameter (◦)
2
0.211(2)
0.411(2)
-
108.9(3)
73.2(2)
∆Cs(C11) = 0.2(2)
∆Cs(C10) = 5.9(2)
E
E
51.2(2)
8/molecule 1
molecule 2
molecule 3
0.307(2)
0.469(2)
-
288.45(4)
83.8(3)
∆Cs(C11) = 0.4(2)
∆C2(C2-C3) = 9.5(2)
E
54.0(2)
distorted H
0.236(2)
0.431(2)
-
108.8(5)
78.6(3)
∆Cs(C41) = 0.2(2)
∆Cs(C32) = 10.5(2)
E
53.9(2)
distorted E
0.240(2)
0.414(2)
-
108.8(5)
74.7(3)
∆Cs(C61) = 0.1(2)
∆Cs(C62) = 7.4(2)
E
53.9(3)
distorted E
Q,
θ, Φ puckering parameters calculated according to Cremer & Pople [11], ∆Cs, ∆C2—asymmetry parameters
calculated due to Duax & Norton [12] E, B, S/B, T/B define conformations: envelope, boat, between screw-boat
& boat and between twist boat & boat. Molecule 1,2,3 (see Table 1) corresponds to three different molecules in
compound 8.
Figure 3. Partial molecular packing of 2 showing hydrogen-bonded dimers and chains.
Additionally, each molecule is linked by a C24-H24 . . . O4 (1 + x, y, z) hydrogen bond forming
chains propagating along a axis. The intermolecular hydrogen bonds in crystal structure 8 produce a
3-D net of hydrogen bonds (Figure 4). The geometric parameters for the hydrogen bonds are presented
in Table 2. Additionally, it is worth mentioning that compound
2 has higher values for all tested
cytotoxic activities compared to (Table 3); this is associated with its more extended spatial structure,
8
in which a bulky phenyl substituent is attached in the C2 position. A similar trend is observed for
benzylideneflavanone and benzylidenechromanone analogues [13]. However, taking into account the
structure
2 and 10, which are similar and differ only the position of methoxy group in phenyl fragment
cytotoxic activity is different. Therefore it could be concluded that in case of COLO-205 the position of
OCH3 group influences on the activity (higher when m-OCH3 group is attached). This trend is also
visible if we compare
2, 4 and 10 structures, for p-OCH3 substituent cytotoxic activity (COLO-205) is
lower that for m-OCH3 substituent.

Molecules 2020, 25, 1613
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Figure 4. Partial molecular packing of 8 showing the 3-D network of hydrogen bonds.
Table 2. Hydrogen bond geometry in (Å).
D-H
H . . . A
D . . . A
<D-H . . . A
2
C12-H12A . . . O1i
C2-H2 . . . O4ii
0.99
1.00
0.95
0.95
0.85
0.93
2.56
2.61
2.53
2.74
2.67
2.68
3.392(2)
3.388(2)
3.239(2)
3.416(2)
3.653(2)
3.497(2)
136
135
131
129
180
147
C24-H24 . . . O4iii
C26-H26 . . . N2i
C11-H11 . . . N1i
C11-H11 . . . O4intra
8
Molecule 1
C6-H6 . . . O1iv
0.95
0.95
1.00
2.59
2.60
2.45
3.304(2)
3.316(3)
2.863(2)
132
133
104
C14-H14 . . . N31
C11-H11 . . . O4intra
Molecule 2
C32-H32A . . . O34v
C36-H36 . . . O31iii
C48-H48A . . . N2vi
C44-H44 . . . O64
0.99
0.99
0.99
0.95
2.47
2.45
2.61
2.53
2.45
3.373(2)
3.361(2)
3.473(2)
3.418(2)
2.799(2)
152
161
146
157
100
C41-H41 . . . O34intra 1.00
Molecule 3
C66-H66 . . . O61iii
0.95
C71-H71 . . . O64intra 1.00
2.57
2.43
3.281(2)
2.783(2)
132
100
symmetry code: (i) 1/2
(vi): 3/2 − x, 1/2 + y, 1/2 − z.
−
x, 1/2 + y, 1/2
−
z; (ii): 1/2
−
x,
−
1/2 + y,1/2
−
z; (iii): 1 + x, y, z; (iv): 1 + x,y,z; (v):
−
1 + x, y, z;
2.3. Experimental Lipophilicity of the Synthesized Flavone Analogues
Lipophilicity is one of the most important physicochemical properties in pharmaceutical research.
It can be considered a key determinant of the pharmacokinetic properties of a drug. It is commonly
measured by the partition coefficient between water and n-octanol, being expressed as logP [14].
The coefficient logP shows the affinity of a molecule for a lipophilic environment [15
of the partition coefficient is valuable: it is frequently used in structure–activity relationship (SAR) and
quantitative structure–activity relationship (QSAR) studies [17 18]. The experimental lipophilicity of the
,16]. Knowledge
,
synthesized compounds, viz. benzylideneflavanones, benzylidenechromanones and spiropyrazolines,
was determined using RP-TLC. The logP results indicate that the tested compounds have lipophilic
character. The logP values ranged from 4.08 to 5.88 for all tested compounds except
2.35), indicating a possible relationship between lipophilicity and chemical structure. Compound
demonstrated some dependence in terms of spatial arrangement, and greater cytotoxicity than
8 (logP =
2
8

Molecules 2020, 25, 1613
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towards four cell lines (Table 3). This can be attributed to its more spatial structure, where a bulky
phenyl substituent is attached in the C2 position. A similar trend can be seen for benzylideneflavanone
analogues: all compounds with a phenyl ring in the C2 position have higher logP values. It was
also found that lipophilicity of a compound depends on its chemical structure: the presence of a
phenyl or methoxy substituent increases the logP value, and unsubstituted compounds have lower
lipophilicity (8). Compound 12 has a lower logP value (3.43) than 11 (5.69) (Figure 5). It is important
to examine the relationship between structure of the compound and its lipophilicity and cytotoxicity.
Compound 11, with a phenyl ring at C2, exhibits greater biological activity than the unsubstituted
benzylidenechromanone. The biological activity of the compounds increases with lipophilicity, but it
cannot exceed the maximum value. According to Lipinski’s rule and drug likeliness parameters,
compounds with higher lipophilicity, up to a maximum logP value of 5, may be good candidates for
drugs [19,20]. The compounds with logP greater than 3 present high lipophilicity and high potential
for bioaccumulation in cells.
2.4. Cytotoxicity toward Human Cancer Cells
Modern medicine is still struggling with the problem of fully effective chemotherapy. Therefore,
there is a strong need to develop and synthesize new chemotherapeutics characterized by adequate
effectiveness, selectivity and specificity of action against cancer cells. The need to identify new
compounds with effective anticancer activity, and high selectivity against cancer cells and low toxicity
to normal cells, demands the testing of a wide variety of candidates. Undoubtedly, the turning point
for the development of research on organometallic complexes was the discovery of cisplatin in the 20th
century, which is used to treat lung, ovarian, neck or head cancer, among others. However, its effective
biological anticancer activity is unfortunately associated with high systemic cytotoxicity, particularly
myelosuppression and nephro- and hepatotoxicity, which is a serious factor limiting its wide use.
Our present study evaluated the anticancer activity of a large group of 3-arylidenechromanone,
3-arylideneflavanone, spirochromanone and spiroflavanone analogues against four tumor lines.
The experiments serve as targeted basic research and are aimed at gaining new knowledge related to
the anticancer activity of selected 3-benzylidenechromanone/3-benzylideneflavanone analogues and
their pyrazolines.
The first stage of the study used the MTT viability test to determine the cytotoxicity of ten
compounds, i.e., 3-arylidenechromanone, 3-arylideneflavanone, spirochromanone and spiro- flavanone
analogues, against four human cancer cell lines: HL-60, NALM-6, WM-115 and COLO-205. In addition,
and 4-chromanone, quercetin, cisplatin and carboplatin were included in the tests as reference
compounds. The IC50 values for the entire series of tested compounds are given in Table 3.
Our studies indicate that the antiproliferative activity of the test compounds was determined by
their chemical structure. Compounds 2, 7 and 10 demonstrated the greatest antiproliferative activity
against cancer cells. As expected, they demonstrated dose-dependent inhibition of human cancer cell
line growth with a significant decrease in cell viability being associated with increasing concentrations.
The most active compound was 2, with IC50 < 10 µM against three cancer cell lines: HL-60, NALM-6
and WM-115; however, COLO 205 proliferation was slightly less inhibited (IC50 29.3 µmol/L).
Table 3. The lipophilicity values and cytotoxic activity of compounds against HL-60, NALM-6, WM-115
and COLO-205 cancer cell lines. The results are presented as IC50 values in the µM range.
Number of
Compound
IC50 (µM)
Chemical Structure
HL-60
NALM-6
WM-115
COLO-205
logP
1
53.38 ± 2.49
49.81 ± 2.35
58.13 ± 4.93
51.0 ± 2.3
4.79

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Table 3. Cont.
Number of
Compound
IC50 (µM)
Chemical Structure
HL-60
NALM-6
WM-115
COLO-205
logP
2
3.0 ± 0.3
6.8 ± 0.4
5.5 ± 0.4
29.3 ± 2.35
5.42
3
4
45.36 ± 4.77
74.05 ± 1.85
30.07 ± 3.15
72.60 ± 4.56
23.53 ± 1.71
75.27 ± 4.89
193.1 ± 2.9
26.5 ± 1.8
4.08
4.68
5
6
33.74 ± 3.87
>1000
27.02 ± 3.10
44.4 ± 6.3
47.41 ± 4.62
57.4 ± 5.8
77.1 ± 1.9
53.6 ± 1.6
4.90
5.88
7
8
34.78 ± 2.87
473.9 ± 29.0
6.83 ± 0.18
12.75 ± 1.83
415.0 ± 75.4
31 ± 1.4
>1000
4.23
2.35
281.6 ± 49.4
9
35.89 ± 2.96
15.7 ± 6.23
25.0 ± 3.3
16.89 ± 3.78
169.94 ± 3.81
487.1 ± 4.1
5.83
5.42
10
9.4 ± 0.4
49.2 ± 2.7
11
12
8.36 ± 0.63 *
9.08 ± 0.30 *
8.69 ± 0.40 *
673.7 ± 22.5
6.45 ± 0.69 *
-
5.69
3.43
2.19
11.76
±
1.97 *
18.09
±
3.14 *
-
4-Chromanone
**
676.7 ± 32.6
58.0 ±4.0
>1000
721.5 ± 6.4
Quercetin **
77.1 ± 7.8
177.5 ± 21.5
-
2.16

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Table 3. Cont.
Number of
Compound
IC50 (µM)
NALM-6 WM-115
Chemical Structure
HL-60
COLO-205
logP
Cisplatin **
0.8 ± 0.1
0.7 ± 0.3
0.7 ± 0.2
18.2 ± 4.3
61.45 ± 3.2
−2.21
−2.30
Carboplatin
**
4.3 ± 1.3
422.2 ± 50.2
354.6 ± 73.7
* See literature [10]; ** Reference compounds.
Compound
and WM-115 cell lines, with IC50 is equal to 6.83 and 12.75
that 10 also demonstrated noticeable cytotoxic effects against HL-60 and NALM-6 cells (IC50 about 9.4
and 25 mol/L respectively); however, WM-115 and COLO-205 were markedly more resistant. On the
7
demonstrated promising antiproliferative properties, especially towards NALM-6
µmol/L respectively. It is worth mentioning
µ
basis of these results, further studies were performed on the molecular mechanisms of the anticancer
activity of the complexes against the corresponding cell lines.
It is also worth mentioning that in further studies we also used two additional compounds:
(E)-3-(4-N,N-diethylaminobenzylidene)chroman-4-one (12) and (E)-3-(4-N,N-diethylaminobenzylidene)-
2-phenylchroman-4-one (11). Their synthesis, structure and promising antiproliferative properties
against cancer cells (HL-60, NALM-6 and WM-115) have been described previously [10]. Both
compounds showed high cytotoxic activity against the HL-60 cell line: 11.76
The IC50 values for NALM-6 cell line were similar: 8.69 12 and 9.08 11. Only for WM-115 cell
line the cytotoxicity values were not similar. The compound 11 exhibited high cytotoxic activity with an
IC50 value of 6.45 M, i.e., three times lower than for 12. Hence, both compounds appear to exhibit
µM 12 and 8.36 µM 11.
µM
µM
µ
selectivity for human leukemia cell lines.
Chromanone
Flavanone
8
8
6
4
2
0
6
4
2
0
*
(A)
Figure 5. Cont.

Molecules 2020, 25, 1613
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(B)
Figure 5. The effects of reference compounds, flavanone and chromanone (
A) and synthesized
compounds ( ) on the integrity of the erythrocyte membrane. The results are presented as the
B
percentage of hemolysis obtained from the interaction of studied compounds with 2% RBCs (red blood
cell) suspension, compared to the positive control Triton X-100 constituting 100% hemolysis. The results
are presented as mean ± SD; n = 4, * p < 0.05 vs. control; ** p < 0.01; *** p < 0.001.

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2.5. Red Blood Cell Lysis Assay
All synthesized compounds with potential biological activity have to be tested for blood
compatibility since xenobiotics will react with blood cells upon administration. In this case,
biocompatibility refers to the quantification of cellular and plasma components of the blood.
The measurement of the integrity of RBC membrane is considered a simple and reliable method
for estimating hemocompatibility [21,22]. The effects of the synthesized compounds, together with
those of the two reference compounds flavanone and chromanone, on RBC hemolysis are depicted
in Figure 6A,B. Neither flavanone nor chromanone was found to have any unfavourable effect on
the erythrocyte membrane over the whole concentration range. A statistically significant increase in
the rate of haemolysis was observed for most of the tested compounds at the higher concentrations.
For instance, compounds
3
and
4 contributed to a significant disintegration of the erythrocyte membrane
at concentrations above 25
µM, manifested by a higher percentage of hemolysis in comparison to
control. However, these values did not exceed 5%, which means that there is not a risk of pathological
hemolysis in in vivo conditions. Similar results were obtained for compounds 5, 6, 9, and 10.
In the case of compound
however, a significantly greater disintegration of RBC membrane was reported at concentrations of 250
and 500 M in the related compound . The differences in the tested concentrations of these compounds
7, no significant effects on RBC hemolysis were observed up to 35 µM;
µM
µ
8
stem from the fact that they affect the cellular growth (Table 3) at different concentration ranges.
2.6. Red Blood Cell Morphology
Studies on erythrocyte morphology complement the erythrotoxicity studies because they provide
information on potential immediate interactions of the tested compounds with the erythrocyte lipid
bilayer, and the possibility of creating pathological forms of erythrocytes, which are not able to carry
oxygen. The effects of the synthesized compounds, and the two reference compounds chromanone
and flavanone, on the morphology of erythrocytes is shown in Figure 6. Briefly, 2% RBC suspension
was incubated with 0.9% NaCl (control samples) or tested compounds at various concentrations
corresponding to their activity towards cancer cell lines. Dyscocytes were observed in control samples,
while echinocyte formation was observed in cultures treated with chromanone or flavanone over the
entire concentration range (Figure 6A).
(A)
Figure 6. Cont.

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(B)
Figure 6. Microscope images of erythrocytes treated with (
A
)—reference compounds, chromanone
and flavanone; ( )—compounds
B
1, 2, 3, 4, 5, 6, 7, 8, 9, 10. 2% erythrocyte suspension was treated at
37 ◦C for 60 min with indicated concentrations of compound. Representative phase-contrast images
are shown (magnification of 400 times).
Echinocytes are formed when the agents that preferentially accumulate in the outer layer of the
RBC membrane bilayer expand it, thus giving the cell an echinocytic shape [21]. In most samples, the
population of echinocytes exceeded 80% of the total erythrocytes observed in the microscopic field of
view. Incubation of erythrocytes with compound
anisocytes, while compound resulted mainly in echinocytosis. In addition, echinocytes accounted
for the major part of erythrocytes following treatment with compounds and at a concentration of
100 mol/L, and for compounds and , as well as and 10. Compound primarily led to echinocytosis,
1 led to the extensive formation of echinocytes and
2
3
4
µ
5
6
9
7
however single anisocytes could also be detected. In summary, in vitro exposure of RBCs to different
concentrations of examined compounds and reference molecules resulted mostly in the formation
of echinocytes. Nevertheless, the process of transformation of biconcave erythrocyte to echinocyte
occurs naturally in blood vessels, and results from the insertion of xenobiotics into the outer monolayer

Molecules 2020, 25, 1613
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of the erythrocyte membrane. As this is a reversible process [22] caused by a series of chemical and
physical factors including increased ion strength, alkaline pH and decreased ATP level, it may be
assumed that most of the analyzed compounds do not affect RBCs in a pathological manner at the
relevant concentrations.
2.7. Digestion of Plasmid DNA with BamHI Restriction Nuclease
Some of the test compounds showed significant toxicity toward cancer cells. For example
compounds 2 or 7 demonstrated IC50 values in the single micromolar range (Table 3). This cytotoxicity
was tested with regard to its effect on the cell cycle and the ability to modify DNA. In addition to the
compounds shown in Table 4, two highly-cytotoxic compounds (11 and 12) were also tested. Their
structure and biological activity were described in a previous article [10]. The chemical structures of
the test compounds suggests that they may intercalate with double-stranded nucleic acids. To test this
hypothesis, plasmid DNA was incubated with the test compounds and subsequently digested with
BamH1 endonuclease. As expected, daunorubicin (positive control) which is a strong DNA intercalator,
prevented linearization of DNA (Figure 7, lane B). Interestingly, chromanone/flavanone test compounds
also inhibited digestion of plasmid DNA (Figure 7, lanes C -I). This conclusion is supported by the
presence of the circular form of plasmid DNA after digestion with BamH1. These results suggest that
chromanone/flavanone analogues intercalate to genomic DNA.
Figure 7. Digestion of pcDNA3.1HisC (total length 5.5 kbp) with BamH1 endonuclease. M—marker
DNA; A—not digested plasmid DNA; B—plasmid DNA digested with BamH1 (DNA present in linear
form); C—plasmid DNA + daunorubicin + BamH1; D—plasmid DNA + quercetin + BamH1; E—plasmid
DNA + 2 + BamH1; F—plasmid DNA + 7 + BamH1, G—plasmid DNA +12 + BamH1, H—plasmid DNA
+ 11+ BamH1, I—plasmid DNA +10 + BamH1.
2.8. Cell Cycle Analysis
The next stage examined the effects of the selected compounds on cell cycle progression in HL-60
leukemia cells. The five chromanone/flavanone compounds and quercetin (reference) were tested at
concentrations of IC50 and 0.5
compounds is shown in Figure 8 and Table 4. As expected, in the presence of quercetin, about 50–60%
×
IC50. The cell cycle distribution of HL 60 cells after treatment with test
of cells were blocked in the G2/M phase (see Figure S2). Interestingly, the test compounds also induced
mitosis block in G2/M. Only
7 moderately inhibited cell cycle progression, arresting 20–26% of cells.
The effect of 10 11 and 12 was very pronounced and similar to quercetin. In the presence of these
2,
,
compounds, about 50–60% of cells were arrested in G2/M. These results confirm that test compounds
have a strong influence on the cell cycle and arrest the HL-60 cells in the G2/M phase.

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Table 4. The cell cycle distribution of HL 60 cells after treatment with test compounds. The results are given as mean +/
compounds (in µM) are given in the parenthesis and correspond to 1× IC50 and 0.5 × IC50 (µM). (n = 2) n-number of repetition (see Figure S3).
−
standars deviation. The concentrations of the
DMSO
Control
Quercetin Quercetin
HL 60
2 (3)
2 (1.5)
7 (35)
7 (18)
12 (12)
12 (6)
11 (4.5)
11 (9)
10 (9.4)
10 (4.7)
(58)
(29)
G1
G2
45.2
39.2
15.6
70.3 ± 2.2
20.5 ± 1.7
9.2 ± 3.9
11.6 ± 1.7
39.4 ± 2.5
49.0 ± 4.2
14.3 ± 2.4
25.0 ± 2.0
60.8 ± 0.4
21.3
±
±
±
10.9 23.5
±
21.7
50.4 ± 1.0
23.5 ± 0.9
26.1 ± 0.1
57.7 ± 7.2
21.5 ± 3.8
20.8 ± 3.4
19.5
±
12.3
19.3 ± 2.6
28.4 ± 8.8
52.4 ± 6.2
3.2 ± 2.8
29.1 ± 3.1
67.8 ± 0.3
6 ± 3.5
16.2
±
13.4 40.3
±
±
±
26.8
13.9
12.9
25.0
53.7
10.4
23 ± 2.3
28.0 ± 1.4
52.4
34.8
±
10.5
33.3 ± 1.1
50.5
30.2
G2/M
21.4 53.5
±
19.4
±
10.9
59.2 ± 6.9
±
12.2 29.4

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G1
S
G2/M
100%
80%
60%
40%
20%
0%
Figure 8. The effect of the test compounds and quercetin on the cell cycle in HL-60 cells. Cell
cycle distribution of HL-60 cells after treatment with the test compounds. The concentrations of the
compounds (in
µ
M) are given in the parenthesis and correspond to 1
×
IC50 and 0.5
×
IC50 (µM). 1%
DMSO was used as the vehicle control. Bars represent mean values from two independent experiments.
3. Materials and Methods
3.1. General Information
The compound pairs
Compounds and were synthesized according to Levai and Schag [23]. Compounds
synthesized according to Toth and Szollosy [9].
1
and
2
,
5
and
6
and
9
and 10 were synthesized according to Pijewska [
8].
3
7
4
and were
8
All compounds were purified by crystallization from methanol. All solvents (methanol, ethanol,
toluene) used in this work were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Polish
Chemical Reagents (Gliwice, Poland), and used without further purification. Melting points were
determined on a B-540 Melting Point apparatus (Büchi, Flawil, Switzerland) in capillary mode and
they were uncorrected. The infrared transmission spectra of the crystalline products were recorded at
the University of Lodz Faculty of Chemistry using a Nexus FT-IR spectrophotometer (Thermo Nicolet,
Waltham, MA, USA). The MS-ESI were measured at the University of Lodz Faculty of Chemistry on a
500–MS LC Ion Trap mass spectrometer (Varian, Palo Alto, CA, USA). Accurate mass measurements of
product ions were confirmed using a Waters (Manchester, UK) q-TOF Synapt quadrupole time-of-flight
hybrid mass spectrometer. Elemental analyses were performed at the Faculty of Chemistry (University
1
of Lodz) using a Vario Micro Cube analyzer by Elemental (Langenselbold, Germany). H- (600 MHz)
and 13C-NMR spectra (150 MHz) were recorded at the University of Lodz Faculty of Chemistry on an
Avance III 600 MHz instrument (Bruker, Billerica, MA, USA). The samples were dissolved in deuterated
DMSO and CDCl3. Chemical shifts are given in ppm, coupling constants in Hz. Chemical shifts are
referenced to the residual solvent signals, 2.50 ppm for 1H in DMSO-d6, 7.26 ppm for 1H in CDCl3 and
39.5 ppm for 13C in DMSO-d6, 77.0 ppm for 13C in CDCl3. Computational studies were used to predict
the properties and future use of potential drugs. Molinspiration Cheminformatics was used for the
calculation of important molecular properties, molecular processing, bioactivity and the high-quality
depiction of new molecules. Lipophilicity was determined by RP-TLC with small amounts of organic
solvents. The erythrotoxicity of the compounds was evaluated in Laboratory of Bioanalysis in the
Department of Pharmaceutical Chemistry, Drug Analysis and Radiopharmacy (Medical University of
Lodz). The erythrocyte morphology was evaluated using an Opta-Tech phase-contrast microscope
(Opta-Tech, Warsaw, Poland) with OptaView 7 software.

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Diazomethane was prepared for the synthesis spiropyrazolines analogues as follows: Briefly,
50% aqueous potassium hydroxide solution (30 mL) and 100 mL diethyl ether was placed in a
◦
500 mL flat-bottomed flask. The solution was cooled in an ice-salt mixture to 5 C and 0.2 M of
N-nitrosomethylurea (10.3 g) was added in portions. The mixture was stirred. The solution turned
yellow and was placed in a previously-cooled separatory funnel. The solution was separated and the
ether layer of the diazomethane was placed in a flask covered with pellets of potassium hydroxide.
The aqueous layer was placed into a flask with an aqueous solution of benzoic acid.
3.2. Synthesis of (E)-3-(4-methoxybenzylidene)-2-phenylchroman-4-one (1)
A mixture of 0.01 mol (2.243 g) 2-phenylchroman-4-one, 0.01 mol (1.362 g) 4-methoxy-benzaldehyde
and five drops of piperidine were heated in oil bath with mechanical stirring at 130 ◦C for five hours.
The progress of the reaction was tested by TLC (toluene:methanol 9:1). The mixture was left to cool
at room temperature and dissolved in methanol. After 24 h, the compound was precipitated and
then purified by crystallization from methanol. The compound
Yield: 41% m.p: 139.8–141.3 ◦C. MS (ESI+): m/z 343.3 C23H18O3 [M + H]+. IR (KBr)
(C-H aromat), 2945, 2910 (C-H aliph.), 1675 (C=O), 1602, 1578, 1506 (C=C), 1304 (C-O), 1172 (C-O),
1148 (C-O-C). 1H-NMR (DMSO-d6)
(ppm): 2.28 (3H, s, OCH3), 6.74 (1H, s, C2-H), 7.99 (1H, s, =CH),
7.04–7.81 (13H, m, C-H arom.) (Figure S4). 13C-NMR (CDCl3)
(ppm): 29.7 (OCH3), 77.9 (C2-H), 116.2,
1
was obtained as a white powder.
ν
(cm−1): 3030
δ
δ
118.6, 121.7, 126.0, 127.6, 127.7, 128.6, 132.5, 136.0, 138.1, 140.0 (CHarom, =CH), 122.3, 132.5, 158.6, 158.9
(Carom), 183.0 (C=O) (Figure S4a). Anal. Calc. For C23H18O3 (M = 342.38 g/mol) % C: 80.68; %H: 5.26.
Found %C: 80.60; % H: 5.25.
3.3. Synthesis of 50-(4-methoxyphenyl)-2-phenylo -40,50-dihydro-4H-spiro [chromano-3,30-pirazol]-4-one (2)
Briefly, 2.5 mmol (0.856 g) of compound
1 was dissolved in anhydrous acetone (5 mL) to give
a light yellow solution. The flask containing the solution was placed in the ice bath and an ethereal
solution of diazomethane (10 mmol) was added in excess. The mixture in the flask was left in the
freezer for 48 h. The yellow precipitate was filtered off and purified by column chromatography and
ν
crystallized from methanol. Yield: 34% m.p. 128–129 ◦C. IR (KBr) (cm−1): 3029 (C-H aromat.), 2958,
2837 (C-H aliph.), 1677 (C=O), 1605, 1580, 1547, 1512, 1472 (C=C, N=N), 1307 (C-N), 1240 (C-O), 1146
(C-O-C). 1H-NMR (DMSO-d6)
(ppm): 3.74 (3H, s, OCH3), 4.01 (1H, dd, JAB = 7.6 Hz, JBX = 6.9 Hz,
CH), 4.14 (1H, dd, JAB = 12.27 Hz, JBX = 7.15 Hz CH2), 4.23 (1H, dd, JAB = 12.27 Hz, JBX = 1.96 Hz
CH2), 5.86 (1H, s, C2-H), 6.87–7.68 (13H, m, C-H aromat) (Figure S5). 13C-NMR (CDCl3)
(ppm): 23.6
δ
δ
(OCH3), 55.2 (CH), 77.3 (CH), 79.5 (C2-H), 112.4, 113.9, 118.2, 119.2, 123.4, 127.3, 127.4, 128.5, 129.8,
138.9 (CHarom), 121.3, 135.7, 142.6, 152.8, 158.7, 159.6 (Carom), 186.3 (C=O) (Figure S5a). Anal. Calc. For
C24H20O3N2 (M = 384.43 g/mol) %C: 74.98; %H: 5.20; %N: 7.34. Found %C: 74.07; %H: 5.21; %N: 7.43.
MS (ESI+): m/z 385.4 C24H20O3N2 [M + H]+. HRMS (ESI+): m/z [M + H]+ calcd. for C24H21O3N2:
385.1552; found: 385.1548 (see Figure S5b).
3.4. Synthesis of (E)-3-(4-methoxybenzylidene)chroman-4-one (3)
A mechanically-stirred mixture of 0.01 mol (1.481 g) of chroman-4-one, 0.01 mol (1.362 g) of
p-methoxybenzaldehyde and five drops of piperidine were heated at 150 ◦C in an oil bath for four
hours. After cooling, the reaction mixture was left for 24 h at room temperature. The solidified product
was filtered and crystallized from methanol. Compound
Yield: 55% m.p: 133.8–134.85 ◦C. MS (ESI+): m/z 267.5 C17H14O3 [M + H]+. IR (KBr)
3000 (C-H aromat.), 2958, 2866 (C-H aliph.), 1665 (C=O), 1603, 1568, 1510, 1477,1463 (C=C), 1146
(C-O-C), 1112 (C-O). 1H-NMR (CDCl3)
(ppm): 1.58 (3H, s, OCH3), 3.87 (1H, s, =CH), 5.38 (2H, d,
JAB = 16.60 Hz C2-H), 6.96–8.03 (8H, m, C-H aromat.) (see Figure S6). 13C-NMR (CDCl3)
(ppm):
3
was obtained as a cream-colored powder.
ν
(cm−1): 3038,
δ
δ
55.5 (OCH3), 67.8 (CH2), 114.3, 117.9, 127.0, 127.9, 128.9 (CHarom, =CH), 121.9, 132.1, 135.7, 137.4,
160.1(Carom), 182.3 (C=O) (see Figure S6a). Anal. Calc. For C17H14O3 (M = 366.30 g/mol) %C: 76.67;
%H: 5.25. Found %C: 76.68; %H: 5.20.

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3.5. Synthesis of 40-[(4-methoxy)phenyl]-40,50-dihydro-4H-spiro[chroman-3,30-pirazol]-4-one (4)
Compound
3 (2.5 mmol) (0.666 g) was dissolved in anhydrous acetone (4.5 mL) to give a light
yellow solution. The flask with the solution was placed in the ice bath and an ethereal solution of
diazomethane (10 mmol) was added in excess. The mixture in the flask was left in the freezer. The
creamy precipitate was filtered off and purified by the column chromatography and crystallized from
ν
methanol. Yield: 56% m.p: 109–111.3 ◦C. IR (KBr) (cm−1): 3126, 3025 (C-H aromat.), 2968, 2934 (C-H
aliph.), 1684 (C=O), 1607, 1580, 1544, 1514, 1476, 1468 (C=C, N=N), 1311 (C-N), 1184 (C-O-C), 1115
(C-O). 1H-NMR (CDCl3)
(ppm): 1.58 (3H, s, OCH3), 3.74 (1H, dd, JAB = 8.4 Hz, JBX = 8.4 Hz CH2),
3.78 (3H, s, CH), 4.16 (1H, d, JAB = 12.6 Hz CH2), 4.55 (1H, d, JAB = 12 Hz CH2), 4.90 (1H, d, JAB
24 Hz CH2), 5.13 (1H, d, JAB = 13.39 Hz CH2), 6.82–7.68 (8H, m, C-H aromat) (see Figure S7). 13C-NMR
(CDCl3) (ppm): 41.9 (CH), 55.3 (OCH3), 69.5 (CH2), 85.8 (CH2), 98.0, 114.3, 118.2, 121.9, 128.0, 137.0
δ
=
δ
(CHarom),119.5, 128.8, 159.2, 161.7 (Carom), 186.2 (C=O) (see Figure S7a). Anal. Calc. for C18H16O3N2
(M = 308.34 g/mol) % C: 70.11; % H: 5.19; % N: 9.08. Found % C: 70.10; % H: 5.20; % N: 9.10. MS (ESI+):
m/z 309.4 C18H16O3N2 [M + H]+. HRMS (ESI+): m/z [M + H]+ calcd. for C18H17O3N2: 309.1239;
found: 309.1236 (see Figure S7b).
3.6. Synthesis of (E)-3-benzylidene-2-phenylchroman-4-one (5)
Compound
5 was synthesized by the same method as compound 1 [8]. Reagents: 0.01 mol (2.243 g)
2-phenylchroman-4-one, 0.01 mol (1.062 g) benzaldehyde and 5 drops of piperidine. Yield: 85% m.p:
103–105 ◦C. MS (ESI+): m/z 313.5 C22H16O2 [M + H]+. IR (KBr) (cm−1): 3068 (C-H aromat.), 2932
ν
(C-H aliph.), 1668 (C=O), 1604, 1577, 1506, 1492, 1472, 1461 (C=C), 1142 (C-O-C). 1H-NMR (DMSO-d6)
δ (ppm): 6.30 (1H, s, C2-H), 7.69 (1H, s, =CH), 6.99–7.81 (14H, m, CH aromat.) (Figure S8). 13C-NMR
(CDCl3)
δ (ppm): 77.3 (C2-H), 118.7, 121.8, 127.6, 128.7, 128.9, 129.8, 129.9, 136.1, 139.5 (CHarom, =CH),
122.2, 132.5, 134.1, 138.1, 158.9 (Carom), 182.7 (C=O) (Figure S8a). Anal. Calc. For C22H16O2 (M =
312.36 g/mol) %C: 84.59; %H: 5.16. Found %C: 84.55; %H: 5.18.
3.7. Synthesis of 20-phenylchromanone-30-spiro-3,4-phenyl-1-pyrazoline (6)
Compound
0.781 g) was dissolved in anhydrous acetone (6 mL) to give a clear solution. The next steps were
the same as for the synthesis of compound
. Yield: 78% m.p: 126.5–127 ◦C. MS (ESI+): m/z 355.3
C23H18O2N2 [M + H]+. IR (KBr)
(cm−1): 3061, 3032 (C-H aromat.), 1697 (C=O), 1602, 1579, 1493, 1473,
1460 (C=C, N=N), 1303 (C-N), 1146 (C-O-C). 1H-NMR (DMSO-d6)
(ppm): 4.07 (1H, dd, JAB=7.16 Hz,
JBX = 1.96 Hz CH), 4.20 (1H, dd, JAB = 11.96 Hz, JBX = 7.16 Hz CH2), 4.21 (1H, dd, JAB = 11.95 Hz, JBX
= 1.96 Hz CH2), 5.86 (1H, s, C2-H), 7.10–7.69 (14, m, C-H aromat.) (Figure S9). 13C-NMR (CDCl3)
6 was synthesized by the same method as compound 2 [8]. Compound 5 (2.5 mmol,
2
ν
δ
δ
(ppm): 55.2 (CH), 77.0 (CH), 79.5 (C2-H), 112.4, 113.9, 118.2, 121.3, 127.3, 128.0, 128.5, 129.8, 135.7, 138.9,
142.8 (CHarom), 100.1, 119.2, 138.9, 152.8, 159.6 (Carom), 185.3 (C=O) (see Figure S9a). Anal. Calc. For
C23H18O2N2 (M = 354.41 g/mol) %C: 77.95; %H: 5.11; %N: 7.90. Found %C: 78.80; %H: 5.20; %N: 7.70.
3.8. Synthesis of (E)-3-benzylidene-chroman-4-one (7)
Compound
were: 0.01 mol (1.481 g) chroman-4-one, 0.01 mol (1.062 g) benzaldehyde and five drops of piperidine.
Next steps were the same as for the synthesis of compound
. Yield: 85% m.p: 111–112 ◦C. MS (ESI+):
m/z 237.4 C18H16O3N2 [M + H]+. IR (KBr)
(cm−1): 3056, 3028 (C-H aromat.), 2904, 2854 (C-H aliph.),
1666 (C=O), 1600, 1572, 1461, 1454 (C=C), 1306 (C-O), 1144 (C-O-C). 1H-NMR (DMSO-d6)
(ppm): 5.43
7 was synthesized according to Levai and Schag [22]. Reagents used for the reaction
3
ν
δ
(1H, s, =CH), 7.59 (2H, d, JAB = 14.22 Hz C2-H), 7.05–7.90 (9H, m, C-H aromat) (see Figure S10). 13C-
NMR (CDCl3 δ (ppm): 67.6 (CH2), 108.2, 119.3, 121.9, 125.2, 128.6, 129.5, 129.9, 131.1, 134.5 (CHarom,
=CH), 137.5, 154.5, 155.9 (Carom), 182.3 (C=O) (see Figure S10a). Anal. Calc. for C16H12O2 (M =
236.26 g/mol) %C: 81.33; %H: 5.08. Found %C: 81.03; %H: 5.10.

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3.9. Synthesis of 40-phenyl-40,50-dihydro-4H-spiro[chromano-3,30-pirazol]-4-one (8)
Compound
8
was synthesized by the same method as compound
4
[9
]. Compound
7
(2.5 mmol,
0.590 g) was dissolved in anhydrous acetone (5 mL) to give a clear solution. Next step were the same
as for the synthesis of compound
. Yield: 92.5% m.p: 141–142 ◦C. MS (ESI+): m/z 279.4 C17H14O2N2
[M + H]+. IR (KBr)
(cm−1): 3064, 3031 (C-H, aromat.), 1677 (C=O), 1606, 1576, 1543, 1473, 1464 (C=C,
N=N), 1309 (C-N), 1148 (C-O-C). 1H-NMR (DMSO-d6)
(ppm): 4.15 (1H, dd, JAB = 12.01 Hz, JBX
6.98 Hz CH2), 4.46 (1H, dd, JAB = 7.58 Hz, JBX = 6.98 Hz CH), 4.94 (1H, d, JAB = 12.56 Hz CH), 5.12 (1H,
d, JAB = 12.56 CH), 7.09–7.68 (9, m, C-H aromat) (see Figure S11). 13C-NMR (CDCl3)
(ppm): 42.4
4
ν
δ
=
δ
(CH), 69.6 (CH2), 85.7 (CH2), 98.0, 118.2, 122.0, 128.0, 137.1, (CHarom), 119.5, 129.0, 161.8 (Carom), 186.1
(C=O) (see Figure S11a). Anal. Calc. for C17H14O2N2 (M = 278.31 g/mol) %C: 73.36; %H: 5.03; %N:
10.06. Found %C: 73.40; %H: 5.13; %N: 10.10.
3.10. Synthesis of (E)-3-(3-methoxybenzylidene)-2-phenylchroman-4-one (9)
Compound
(2.243 g) 2-phenylchroman-4-one, 0.01 mol (1.362 g) 3-methoxybenzaldehyde and five drops of
piperidine. Yield: 80% m.p.: 96.2–98.4 ◦C. MS (ESI+): m/z 343.5 C23H18O3 [M + H]+. IR (KBr) (cm−1):
9 was synthesized by the same method as compounds 1 and 5 [8]. Reagents: 0.01 mol
ν
3064 (C-H aromat.), 2958, 2829 (C-H aliph), 1663 (C=O), 1608, 1599, 1581 (C=C). 1176 (C-O), 1140
1
(C-O-C). H-NMR (DMSO-d6)
δ
(ppm): 2.28 (3H, s, OCH3), 6.74 (1H, s, C2-H), 7.99 (1H, s, =CH),
(ppm): 54.9 (OCH3), 77.2
7.04–7.81 (13H, m, C-H aromat) (see Figure S12). 13C-NMR (DMSO-d6)
δ
(C2-H), 115.1, 116.1, 118.8, 126.9, 127.4, 128.8, 129.0, 130.1, 134.6, 136.7, 138.9 (CHarom, =CH), 121.5,
122.1, 122.2, 131.8, 137.7, 158.3 (Carom), 181.3 (C=O) (see Figure S12a). Anal. Calc. For C23H18O3 (M =
342.39 g/mol) %C: 80.68; %H: 5.26. Found %C: 81.60; %H: 5.55.
3.11. Synthesis of 50-(3-methoxyphenyl)-2-phenyl-40,50-dihydro-4H-spiro[chromano-3,30-pyrazol]-4-one (10)
Compound 10 was synthesized by the same method as compounds
for the reaction were: 2.5 mmol (0.856 g) of compound dissolved in anhydrous acetone (6 mL) to
give a clear solution. Next steps were the same as for the synthesis of compound . Yield: 41.3% m.p.
126–128 ◦C. MS (ESI+): m/z 385.4 C24H20O3N2 [M + H]+. IR (KBr) (cm−1): 3027 (C-H aromat.), 2975,
2828 (C-H aliph.), 1677 (C=O), 1603, 1579, 1550, 1515, 1472 (C=C, N=N), 1307 (C-N), 1232 (C-O), 1146
(C-O-C). 1H-NMR (DMSO-d6)
(ppm): 2.13 (3H, s, OCH3), 2.31 (1H, dd, JAB = 3.1Hz, JBX = 6.9 Hz,
CH), 6.01 (1H, s, C2-H), 6.36 (1H, dd, JAB = 18Hz, JBX = 3Hz CH2), 6.37 (1H, dd, JAB = 18Hz, JBX
7.15 Hz CH2), 6.73–7.82 (13H, m, C-H aromat.) (Figure S13). 13C-NMR (DMSO-d6)
(ppm): 20.6 (CH),
2 and 6 [8]. Reagents used
9
2
ν
δ
=
δ
39.8 (OCH3), 81.1 (C2-H), 118.6, 121.8, 126.8, 127.8, 128.2, 128.6, 138.0 (CHarom) 102.2, 119.8, 134.7, 134.9,
136.1, 159.4 (Carom), 185.5 (C=O) (Figure S13a). Anal. Calc. For C24H20O3N2 (M = 384.43 g/mol) %C:
74.98; %H: 5.20; %N: 7.34. Found %C: 74.77; %H: 5.25; %N: 7.45.
3.12. Determination of Lipophilicity of Flavone Analogues Using a RP-TLC Method
The RP-TLC experiments were performed on TLC plates (5
×
10 cm) RP-18 F254S (Merck,
Darmstadt, Germany). The synthesized compounds were dissolved in N,N-dimethylformamide DMF
(2 mg/mL). DMF was obtained from Chempur (Piekary Slaskie, Poland) and used with high purity
(99.8%). The solutions of each compound in DMF were spotted on the plates, and observed under
UV light at
λ = 254 nm. A DMF-water solvent system was used as mobile phase. The composition of
the solvent system has changed from 50%:50% to 95%:5%. All experiments were performed at room
temperature. The logP parameter was calculated using the equation from the calibration curve.
3.13. Cells Cultures and Cytotoxicity Assay by MTT
Cytotoxicity was tested against human skin melanoma cells (WM-115, ECACC, Salisbury, UK),
two human leukemia cell lines, viz. promyelocytic leukemia (HL-60) and lymphoblastic leukemia
(NALM-6), and human colon adenocarcinoma cells (COLO-205). The cell lines COLO-205, WM-115 and

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HL-60 used in this work came from the ATCC American Type Culture Collection (Manassas, VA, USA),
whereas the NALM-6 cell line was purchased from the German Collection of Microorganisms and Cell
Cultures (Braunschweig, Germany). The leukaemia cells and colon adenocarcinoma were cultured in
RPMI 1640 medium (Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal bovine serum
(FBS; Invitrogen) and gentamicin (25 µg/mL; KRKA, Novo Mesto, Slovenia). For melanoma WM-115
cells, Dulbecco’s minimal essential medium (DMEM; Invitrogen) was used. All cell lines were cultured
at 37 ◦C in a humidified atmosphere of 5% CO2 in air.
For all experiments, the studied compounds were dissolved in DMSO (Sigma-Aldrich) and
were further diluted in culture medium to obtain <0.1% DMSO concentration. In each experiment,
controls with and without 0.1% DMSO were performed. The cytotoxicity of all compounds and of
the reference compounds 4-chromanone and 3-benzylideneflavanone was determined by the MTT
assay, i.e., 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma, St. Louis, MO, USA),
which measures cellular dehydrogenase activity [24]. Exponentially growing cells were seeded a day
before the experiment onto a 96-well microplates (Nunc, Roskilde, Denmark) at a density up to 6–8
×
103 cells/well (depending on the cell line). Subsequently, various concentrations of the studied
compounds freshly prepared in DMSO and diluted with complete culture medium were added. All
compounds were tested for their cytotoxicity at a final concentration of 10−7–10−3 M. After 46 h of
incubation with the studied compounds, the cells were treated with the MTT reagent and incubation
was continued for another two hours. MTT – formazan crystals were dissolved in 20% sodium dodecyl
sulphate (SDS, Sigma-Aldrich) and 50% DMF (Sigma-Aldrich) at pH 4.7; following this, absorbance
was read at 570 nm on a multifunctional Victor ELISA-plate reader (Perkin Elmer, Turku, Finland). The
IC50 values, i.e., the concentration of the test compound required to reduce the cell survival fraction to
50% of controls, were calculated from concentration-response curves and used as a measure of the
sensitivity of the cells to a given treatment. As a control, cultured cells were grown in the absence of
drugs. The data points represent the means of at least five to ten repeats ± standard deviation (S.D.).
3.14. Red Blood Cells Lysis Assay
The studies on biological material were approved by the Bioethics Committee of the Medical
University of Lodz, Poland (RNN/109/16/KE). The blood samples were obtained from healthy donors at
the Blood Donation Centre in Lodz. The procedure for plasma preparation for erythrotoxicity studies
was described previously [10]. The influence of synthesized compounds as well as two reference
compounds flavanone and chromanone on RBC membrane integrity was performed by lysis assay
which was conducted according to our previous protocol [10]. Briefly, 2% RBC suspension in 0.9%
◦
NaCl was incubated at 37 C for one hour with the tested compounds at various concentrations
corresponding to their activity towards cancer cell lines or 0.9% NaCl (control). The samples were
centrifuged at 3000 rpm for 10 min and the absorbance of the supernatant was measured at 550 nm.
The results are presented as percentage of haemolysis where a sample containing 10 µL of 2.0%
v/v Triton X-100 was regarded as a positive control contributing to 100% of haemolysis. A sample
containing saline solution represented spontaneous haemolysis of RBCs (control). The studies were
performed on four biological samples obtained from different blood donors (n = 4). The results are
presented as mean
W = 9.51%, n = 5.
±
standard deviation (SD). The coefficient of variability for the method was counted:
3.15. RBCs Morphology
A 2% erythrocyte suspension was incubated at 37 ◦C for one hour with various concentrations of
synthesized compounds. The morphology of the RBCs was examined by means of a phase contrast
Opta-Tech inverted microscope, at 400
image analysis.
×
magnification, equipped with software (OptaView 7) for

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3.16. Cell Cycle Analysis
One million HL-60 (subline CCL 240; cell type: promyeloblast) cells in 3 mL of RPMI 1640
supplemented with 20% FBS were seeded on 6-well plate. Following 20 h incubation with the test
compounds at the concentrations of 0.5
cells were centrifuged (6 min, 700 rpm, room temperature (RT)), washed with PBS (without Ca2+ and
Mg2+) and suspended in 1 mL of cold 70% ethanol in PBS. Fixed cells were stored in 20 ◦C for 2–3
days. Before analysis, cells were centrifuged (10 min, 1500 rpm, 4 ◦C) and washed twice with cold PBS.
After the second wash, the cells were suspended in 1mL of PBS containing 100 g of RNase A and
incubated in 37 ◦C for 10 min. Next, 5
L of solution of propidium iodide (1 mg/mL) was added to
×
IC50 and 1
×
IC50, or 1% DMSO as the vehicle control, the
−
µ
µ
each sample and incubated in dark for 30 min at RT. Stained cells were analyzed by flow cytometry
(FACS Calibur, BD Biosciences, San Jose, CA, USA). Data from 10,000 gated cells were collected using
CellQuestPro software (Ver 6.0, Becton Dickinson) and cell cycle analysis was performed with ModFit
LT software (Ver 3.2, Verity Software House, Topsham, ME, USA).
3.17. Digestion of Plasmid DNA with BamHI Restriction Nuclease
First, 0.5 µg of plasmid DNA (pcDNAHisC, total length 5.5 kbp) containing a unique BamHI
restriction site was dissolved in a 1x BamHI reaction buffer and incubated overnight at 37 ◦C with
the test compounds or daunorubicin, which was used as a positive control. The concentration of the
test compounds and daunorubicin in the samples was 10
all samples was 10%. In the next step, the reaction mixtures were digested with BamHI restriction
endonuclease (2 U/ L. Products of the reaction were
L) for 3 h at 37 ◦C. Total reaction volume was 10
µM. The final concentration of DMSO in
µ
µ
separated in the 1% agarose gel in TBE buffer. The gel was stained with ethidium bromide and DNA
fragments were visualized under a UV lamp (GBox, Syngene, Frederick, MD, USA).
3.18. X-ray Diffraction Experiment
X-ray data for compounds
Clara, CA, USA) at T = 100(2)K. The intensities were recorded with an Atlas detector with MoK
radiation ( = 0.71073 Å). For all data, multi-scan absorption was applied [25]. Both structures were
2 and 8 were collected on a SuperNova diffractometer (Agilent, Santa
α
λ
solved using direct methods and the least-square refinement on F2. All non-hydrogen atoms were
refined anisotropically. SHELXT [26] and SHELXL-2014/7 programs were used to solve and refine the
structures [27]. All non-hydrogen atoms were refined anisotropically. The positions of all H-atoms
were calculated based on known geometry; the respective C-H bond lengths for aromatic CH, methine
CH and methyl CH3 atoms are 0.95, 1.00 and 0.98 Å. The H atoms were included as riding contributions,
with isotropic thermal parameters set at 1.5 and 1.2 times the Ueq of the parent atom (both the methyl
and remaining atoms). To identify molecular geometries and hydrogen-bond patterns, PLATON [28
]
and MERCURY [29] were used. The experimental, refinement and crystallographic details are shown in
Table 5. Further crystallographic details for the structures reported in this paper may be obtained free
of charge on application to CCDC, 12 Union Road, Cambridge CG21, EZ, UK [fax: (44) 1223-336-033;
e-mail: deposit@ccdc.cam.ac.uk] on quoting the depository numbers: CCDC 1957835 for 2 and CCDC
1957892 for 8.

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Table 5. Crystallographic data for structures 2 and 8.
2
8
Chemical formula
Formula weight
Crystal system
Space group
Temperature (K)
a (Å)
C24H20N2O3 C17H14N2O2
384.42
Monoclinic
P21/n
100.0(1)
9.3885(2)
10.6241(2)
19.2202(5)
90
278.30
Monoclinic
P21/n
100.0(1)
6.7189(1)
23.6127(5)
25.0637(7)
90
b (Å)
c (Å)
α (◦)
β (◦)
90.768(2)
90
95.700(2)
90
γ (◦)
V (Å3)
1916.93(7)
4
3956.73(15)
12
Z
Radiationtype
Measuredreflections 15,186
Uniquereflections 3965
Mo Kα
Cu Kα
25,640
7120
Observedreflections 3578
5196
R[F2 > 2σ(F2)]
0.0334
0.0466
0.1228
1.00
7120
568
0
0.22
−0.25
wR(F2)
S
0.0874
1.02
2 3965
263
No. of reflections
No. of parameters
No. of restraints
0
(e Å−3
)
0.33
−0.20
∆ρ
max
∆ρmin (e Å−3
)
3.19. Statistical Analysis
Statistical analysis was conducted with a commercially-available package (GraphPad Prism 5,
GraphPad, San Diego, CA USA). All results are presented as means standard deviation (SD). Normal
±
distribution of continuous variables was confirmed with Shapiro-Wilk test, and homogeneity of variances
was checked using Levene’s test. The results were analyzed using one way ANOVA, and Dunnett’s post
hoc test. The results were considered significant at p-values lower than 0.05.
4. Discussion and Conclusions
This paper explores the biological activity of a series of ten compounds comprising various
3-benzylideneflavanones and 3-benzylidenechromanones, as well as their spiro- analogues with various
substituents at the C3 position. The tested compounds exhibited relatively high cytotoxicity against four
cancer cell lines: HL-60, NALM-6, WM-115 and COLO-205. We indicate that parent compounds
with a phenyl ring at C2 show lower cytotoxicity for the studied cancer cell lines than their spiropyrazolines
analogues. Regarding the compounds with a methoxy substituent, compound demonstrated lower
cytotoxic activity than . Similarly was found to display lower cytotoxic activity than . The most
1 and 9
1
3
5
7
cytotoxic compound was 2, which demonstrated an IC50 = 3.0 ± 0.3 µM against HL-60.
The presence of a fused pyrazoline ring may well play a key role in selective cytotoxicity.
The p-methoxy-substituted analogues of 3-arylideneflavanone exhibit high cytotoxicity against four
cancer cell lines [30,31]. The unsubstituted compound 6, with a phenyl ring bound to the pyrazoline
ring, exhibited more than 330-fold lower cytotoxicity against HL-60 than compound 2, bearing a
p-methoxy group on the phenyl ring.
Cytotoxicity was also strongly influenced by the position of any substituents in the phenyl ring of
the spiroflavanones or spirochromanones, especially against the HL-60 cell line. Both and 10 have
exhibited high activity
6
a methoxy group in the phenyl ring, and m-methoxy- 10 and p-methoxy-
6
towards the HL-60, NALM-6 and WM-115 cell lines.

Molecules 2020, 25, 1613
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One of the aims of this study is to evaluate the logP of the synthesized compounds. Unfortunately,
no significant relationship was observed between chemical structure and lipophilicity, nor between
cytotoxicity and the electron-attracting or electron-donating properties of the substituents. Some of
the analyzed complexes demonstrated promising anti-proliferative properties against specific human
cancer cell lines. It is worth noting that the ability to inhibit the proliferation of cancer cells was
determined by the structure of the studied compounds. Additional structure–activity relationship
(SAR) studies are needed to better understand the relationship between the structure of the compound
and its biological activity.
Our present findings indicate that the ability of the studied compounds to inhibit the growth of
cancer cells may result from cell cycle inhibition. The compounds were found to block the cell cycle of
several cancer cells in the G2/M phase. In addition, as indicated by their ability to prevent digestion of
plasmid DNA with BamHI restriction nuclease, the studied chromanone/flavanone analogues appear
to be capable of intercalating with genomic DNA of cancer cells, thus leading to cell damage and
ultimately apoptosis. It is noteworthy that commercially-used anticancer drugs, such as docetaxel,
doxorubicin, cisplatin or etoposide, are also known to arrest the cycle of cancer cells in the G2/M
phase [32–34]. Our findings correspond closely with those of previous reports on the cell cycle
distribution in HL-60 cells following treatment with cisplatin. Velma et al. [35] demonstrated that
cisplatin caused changes in the expression of genes involved in cell cycle regulation and programmed
cell death among HL-60 cells, with both cell cycle blockade and apoptotic pathway activation being
observed. It is worth noting that not only do our chromanone/flavanone analogues demonstrate similar
molecular anti-proliferative activity to cisplatin, but they also possess the ability to interact with DNA
and damage it [35]. Our results are in agreement with those of previous experiments carried out on
leukemia cells exposed to etoposide, another commonly-used chemotherapeutic drug. Etoposide has
also been shown to inhibit the cell cycle of HL-60 cells in the G2/M phase [35].
Our findings also indicate that the test compounds protected DNA against cleavage catalyzed by
BamHI endonuclease in a similar way to quercetin, which has also previously been shown to intercalate
with DNA [36]. This suggests that the test compounds interact with DNA, possibly by intercalation.
Furthermore, the test compounds arrested leukemia cells in the G2/M phase to an extent comparable
with quercetin, suggesting that they share a similar mechanism of action.
We intend to continue this line of research into chromanone/flavanone analogues towards better
understanding their influence on the mechanisms of cell cycle arrest of cancer cells. It is planned
to extend our research to include expression analysis of genes involved in cell cycle regulation
and apoptosis.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/25/7/1613/s1,
IR spectrum of compounds; the effect of the test compounds and quercetin on the cell cycle in HL-60 cells; the
cell cycle of the tested compounds; 1H NMR and 13C NMR spectrum spectrum of compounds are available in
supplementary materials.
Author Contributions: E.B. conceptualization, writing-review and edition, supervision; A.A.A.-G. methodology,
chemistry (synthesis and lipophilcity), formal analysis, writing—review and edition; J.K.—X-ray data measurements;
M.M. crystal structure solution & determination, molecular and crystal packing analysis, writing, review-edition of
crystallographic part; M.M.-P. investigation of erythrotoxicity, statistical analysis of RBCs lysis assay, writing—review
and edition; M.C. and K.K.-G. Digestion of Plasmid DNA with BamHI Restriction Nuclease and Cell cycle analysis,
writing-review, edition; P.H.—investigation; All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by Medical University of Lodz (grant No. 503/3-066-02/503-31-001 and grant
No. 502-03/3-066-02/502-34-118).
Acknowledgments: The authors would like to thank Joanna Sikora for her skillful assistance in biological
material collection.
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2020, 25, 1613
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References
1.
2.
3.
4.
5.
Marella, A.; Ali, R.; Alam, T.; Saha, R.; Tanwar, O.; Akhter, M.; Alam, M. Pyrazolines: A Biological Review.
Mini-Rev. Med. Chem. 2013, 13, 921–931. [CrossRef] [PubMed]
Yusuf, M.; Jain, P. Synthetic and biological studies of prrazolines and related heterocyclic compounds.
Arab. J. Chem. 2014, 7, 553–596. [CrossRef]
Amir, M.; Kumar, H.; Khan, S.A. Synthesis and pharmacological evaluation of pyrazoline derivatives as new
anti-inflammatory and analgesic agents. Bio-org. Med. Chem. Lett. 2008, 18, 918–922. [CrossRef] [PubMed]
Karthikeyan, M.S.; Holla, B.S.; Kumari, N.S. Synthesis and antimicrobial studies on novel chloro-fluorine
containing hydroxypyrazolines. Eur. J. Med. Chem. 2007, 42, 30–36. [CrossRef]
Prasad, Y.R.; Rao, A.L.; Prasoona, L.; Murali, K.; Kumar, P.R. Synthesis and antidepressant activity of some
1,3,5-triphenyl-2- pyrazolines and 3-(2”-hydroxynaphthalen-1”-yl)-1,5-diphenyl-2-pyrazolines. Bio-Org. Med.
Chem. Lett. 2005, 15, 5030–5034. [CrossRef]
6.
7.
Camacho, M.; Leon, J.; Entrena, A.; Velasco, G.; Carrion, M.; Escames, G.; Vivo, A.; Acuna-Castroviejo, D.;
Gallo, M.; Espinosa, A. 4,5-Dihydro-1H-pyrazole Derivatives with Inhibitory nNOS Activity in Rat Brain:
Synthesis and Structure−Activity Relationships. J. Med. Chem. 2004, 47, 5641–5650. [CrossRef]
Carrión, M.; Lopez, C.; Tapias, V.; Escames, G.; Acuña-Castroviejo, D.; Espinosa, A.; Gallo, M.; Entrena, A.
Pyrazoles and pyrazolines as neural and inducible nitric oxide synthase (nNOS and iNOS) potential inhibitors
(III). Eur. J. Med. Chem. 2008, 43, 2579–2591. [CrossRef]
8.
9.
Pijewska, L.; Kamiecki, J.; Perka-Karolczak, W. 3-Arylideneflavanones. Part 2 Reaction with diazomethane.
Pharmazie 1993, 48, 254–257.
Toth, G.; Szollosy, A.; L
évai, A.; Kotovych, G. Synthesis and stereochemistry of spiropyrazolines. J. Chem.
Soc. Perkin. Trans. II 1986, 12, 1895–1898. [CrossRef]
10. Adamus-Grabicka, A.; Markowicz-Piasecka, M.; Ponczek, M.; Kusz, J.; Malecka, M.; Krajewska, U.; Budzisz, E.
Interaction of Arylidenechromanone/Flavanone Derivatives with Biological Macromolecules Studied as
Human Serum Albumin Binding, Cytotoxic Effect, Biocompatibility Towards Red Blood Cells. Molecules
2018, 23, 3172. [CrossRef]
11. Cremer, D.; Pople, J.A. General definition of ring puckering coordinates. J. Am. Chem. Soc. 1975, 97,
1354–1358. [CrossRef]
12. Duax, W.L.; Norton, D.A. Atlas of Steroid Structure; Plenum Press: London, UK, 1975; pp. 3273–3274.
13. Suchojad, K.; Dołega, A.; Adamus-Grabicka, A.; Budzisz, E.; Małecka, M. Crystal structures of
(E)-3-(4-hydroxybenzylidene)chroman-4-one and (E)-3-(3-hydroxybenzylidene)-2-phenylchroman-4-one.
Acta Cryst. E. 2019, E75, 1907–1913. [CrossRef] [PubMed]
14. Pastuszko, A.; Majchrzak, K.; Czyz, M.; Kupcewicz, B.; Budzisz, E. The synthesis, lipophilicity and cytotoxic
effects of new ruthenium(II) arene complexes with chromone derivatives. J. Innorg. Biochem. 2016, 159,
133–141. [CrossRef] [PubMed]
15.
J
óz´wiak, K.; Szumiło, H.; Soczewin´ski, E. Lipofilowos´c´, metody wyznaczania i rola w działaniu biologicznym
substancji chemicznych. Wiad. Chem. 2001, 55, 1047–1074.
16. Gao, S.; Cao, C. A New Approach on Estimation of Solubility and n-Octanol/Water Partition Coefficient for
Organohalogen Compounds. Int. J. Mol. Sci. 2008, 6, 962–977.
17. Dearden, J.C. The History and Development of Quantitative Structure-Activity Relationships (QSARs).
IJQSPR 2016, 1, 1–44. [CrossRef]
18. Hoekman, D. Exploring QSAR Fundamentals and Applications in Chemistry and Biology, Volume 1.
Hydrophobic, Electronic and Steric Constants. J. Am. Chem. Soc. 1996, 118, 10678. [CrossRef]
19. Soskic, M.; Magnus, V. Binding of ring-substituted indole-3-acetic acids to human serum albumin.
Bioorg. Med. Chem. 2007, 15, 4595–5000. [CrossRef]
20. Lipinski, C.A.; Lombardo, F.; Dominy, B.W.; Feeney, P.J. Experimental and computational approaches to
estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv. Rev.
2001, 46, 3–26. [CrossRef]
21. Gallagher, P.G. Red Blood Cell Membrane Disorders. In Hematology, 7th ed.; Hoffman, R., Benz, E., Heslop, H.,
Weitz, J., Eds., Elsevier: Philadelphia, PA, USA, 2018.
22. Stasiuk, M.; Kijanka, G.; Kozubek, A. Changes in the shape of erythrocytes and the factors causing them.
Post. Biochem. 2009, 55, 425–433.

Molecules 2020, 25, 1613
25 of 25
23. Levai, A.; Schag, J.B. Synthesis of 3-benzylidenechroman-4-ones and -1-thiochroman-4-ones. Pharmazie 1979
34, 747–749.
,
24. Hansen, M.B.; Nielsen, S.E.; Berg, K. Re-examination and further development of a precise and rapid dye
method for measuring cell growth/cell kill. J. Immunol. Methods 1989, 119, 203–210. [CrossRef]
25. CrysAlis PRO; Agilent Technologies: Yarnton, Oxfordshire, UK, 2013.
26. Sheldrick, G.M. SHELXT—Integrated space-group and crystal-structure determination. Acta Crystallogr. A
2015, 71, 3–8. [CrossRef] [PubMed]
27. Sheldrick, G.M. Crystal structure refinement with SHELXL. Acta Cryst. C 2015, 71, 3–8. [CrossRef]
28. Spek, A.L. Structure validation in chemical crystallography. Acta Cryst. D. 2009, 65, 148–155. [CrossRef]
29. Macrae, C.F.; Bruno, I.J.; Chisholm, J.A.; Edgington, P.R.; McCabe, P.; Pidcock, E.; Rodriguez-Monge, L.;
Taylor, R.; Van de Streek, J.; Wood, P.A. New Features for the Visualization and Investigation of Crystal
Structures. J. Appl. Cryst. 2008, 41, 466–470. [CrossRef]
30. Kupcewicz, B.; Balcerowska-Czerniak, G.; Małecka, M.; Paneth, P.; Krajewska, U.; Rozalski, M.
Structure-cytotoxic activity relationship of 3-arylideneflavanone and chromanone (E, Z isomers) and
3-arylflavones. Bioorg. Med. Chem. Lett. 2012, 23, 4102–4106. [CrossRef]
31. Kupcewicz, B.; Małecka, M.; Zapadka, M.; Krajewska, U.; Rozalski, M.; Budzisz, E. Quantitative relationships
between structure and cytotoxic activity of flavonoid derivatives. An application of Hirshfeld surface derived
descriptors. Bioorg. Med. Chem. Lett. 2016, 26, 3336–3341. [CrossRef]
32. Velma, V.; Dasari, S.R.; Tchounwou, P.B. Low Doses of Cisplatin Induce Gene Alterations, Cell Cycle Arrest,
and Apoptosis in Human Promyelocytic Leukemia Cells. Biomark. Insights 2016, 24, 113–121. [CrossRef]
˙
33. Zuryn´, A.; Krajewski, A.; Szulc, D.; Litwiniec, .A.; Grzanka, A. Activity of cyclin B1 in HL-60 cells treated
with etoposide. Acta Histochem. 2016, 118, 537–543. [CrossRef]
34. Ghelli, A.; di Rora’, L.; Iacobucci, I.; Martinelli, G. The cell cycle checkpoint inhibitors in thetreatment of
leukemia. J. Hematol. Oncol. 2017, 10, 77. [CrossRef] [PubMed]
35. Dasari, S.R.; Velma, V.; Yedjou, C.G.; Tchounwou, P.B. Preclinical assessment of low doses of cisplatin in the
management of acute promyelocytic leukemia. Int. J. Cancer Res. Mol. Mech. 2015, 1, 1–6.
36. Srivastava, S.; Somasagara, R.; Hedge, M. Quercetin, a Natural Flavonoid Interacts with DNA, Arrests Cell
Cycle and Causes Tumor Regression by Activating Mitochondrial Pathway of Apoptosis. Sci. Rep. 2016, 6,
240–249. [CrossRef] [PubMed]
Sample Availability: Samples of the compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 are available from the author Angelika
A. Adamus-Grabicka.
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