Synthesis of novel pyrazoline derivatives and the evaluation of death mechanisms involved in their antileukemic activity

Article abstract of DOI:10.1016/j.bmc.2018.12.012

Malignant neoplasms are one of the leading causes of death worldwide and hematologic malignancies, including acute leukemia (AL) is one of the most relevant cancer types. Current available chemotherapeutics are associated with high morbidity and mortality

Full text of DOI:10.1016/j.bmc.2018.12.012

Accepted Manuscript
Synthesis of novel pyrazoline derivatives and the evaluation of death mecha-
nisms involved in their antileukemic activity
Natália Marceli Stefanes, Jéssica Toigo, Mariana Franzoni Maioral, Amanda
Virtuoso Jacques, Louise Domeneghini Chiaradia-Delatorre, Daiane Mari
Perondi, Amanda Abdalla Biasi Ribeiro, Álisson Bigolin, Iris Mattos Santos
Pirath, Bruna Ficher Duarte, Ricardo José Nunes, Maria Cláudia Santos-Silva
PII:
S0968-0896(18)31537-2
https://doi.org/10.1016/j.bmc.2018.12.012
BMC 14661
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
31 August 2018
28 November 2018
6 December 2018
Please cite this article as: Stefanes, N.M., Toigo, J., Maioral, M.F., Jacques, A.V., Chiaradia-Delatorre, L.D.,
Perondi, D.M., Ribeiro, A.A.B., Bigolin, A., Pirath, I.M.S., Duarte, B.F., Nunes, R.J., Santos-Silva, M.C., Synthesis
of novel pyrazoline derivatives and the evaluation of death mechanisms involved in their antileukemic activity,
Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.12.012
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SYNTHESIS OF NOVEL PYRAZOLINE DERIVATIVES AND THE
EVALUATION OF DEATH MECHANISMS INVOLVED IN THEIR
ANTILEUKEMIC ACTIVITY
Natália Marceli Stefanes 1,2, Jéssica Toigo 3, Mariana Franzoni Maioral 1,2, Amanda
Virtuoso Jacques 1,2, Louise Domeneghini Chiaradia-Delatorre ³, Daiane Mari Perondi
1,2, Amanda Abdalla Biasi Ribeiro 1, Álisson Bigolin 1,2, Iris Mattos Santos Pirath 1,2
,
Bruna Ficher Duarte 1,2, Ricardo José Nunes 3, Maria Cláudia Santos-Silva 1,2
*
1 Experimental Oncology and Hemopathies Laboratory, Clinical Analysis Department, Federal University
of Santa Catarina, CEP: 88040-900, Florianópolis, SC, Brazil.
2 Post-Graduation Program in Pharmacy, Health Science Center, Federal University of Santa Catarina,
CEP: 88040-900, Florianópolis, SC, Brazil.
3 Structure and Activity Laboratory, Chemistry Department, Federal University of Santa Catarina, CEP:
88040-900, Florianópolis, SC, Brazil.
Natália Marceli Stefanes: nataliamarcelis@gmail.com
Jéssica Toigo: jessica.toigo@hotmail.com
Mariana Franzoni Maiora: marimaioral@gmail.com
Amanda Virtuoso Jacques: vjacquesamanda@gmail.com
Louise Domeneghini Chiaradia-Delatorre: louisedc@gmail.com
Daiane Mari Perondi: daia.perondi@gmail.com
Amanda Abdalla Biasi Ribeiro: amandibdallar@hotmail.com
Álisson Bigolin: ali.bigolin@gmail.com
Iris Mattos Santos Pirath: irismattossantos@gmail.com
Bruna Ficher Duarte: brufduarte@gmail.com
Ricardo José Nunes: nunesrjj@ufsc.br
Maria Cláudia Santos-Silva: maria.claudia.silva@ufsc.br
* Corresponding Autor:
Maria Cláudia Santos da Silva (maria.claudia.silva@ufsc.br)
Laboratório de Oncologia Experimental e Hemopatias (LOEH), Departamento de Análises
Clínicas, Centro de Ciências da Saúde - Universidade Federal de Santa Catarina 88040-
900 Florianópolis, SC, Brasil.
Funding:
This study was supported by grants and fellowships from CAPES (Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior) (Brazil) and Santos-Silva M.C. is
recipient of a Research Fellowship from CNPq (National Counsel of Technological and
Scientific Development) (Brazil).
Conflict of interest:
The authors declare no conflicts of interest regarding the publication of the present article.

Abstract
Malignant neoplasms are one of the leading causes of death worldwide and hematologic
malignancies, including acute leukemia (AL) is one of the most relevant cancer types.
Current available chemotherapeutics are associated with high morbidity and mortality
rates, therefore, the search for new molecules with antitumor activity, specific and
selective for neoplastic cells, became a great challenge for researchers in the oncology
field. As pyrazolines stand out in the literature for their great variety of biological
activities, the aim of this study was to synthesize and evaluate the antileukemic activity
of five new pyrazoline derivatives. All pyrazolines showed adequate physicochemical
properties for a good oral bioavailability. The two unpublished and most effective
pyrazoline derivatives have been selected for further experiments. These compounds are
highly selective for leukemic cells when compared to non-neoplastic cells and did not
cause lysis on human red blood cells. Additionally, selected pyrazolines induced cell
cycle arrest at G0/G1 phase and decreased cell proliferation marker KI67. Apoptotic cell
death induced by selected pyrazolines was confirmed by morphological analysis,
assessment of phosphatidylserine residue exposure and DNA fragmentation. Several
factors indicate that both intrinsic and extrinsic apoptosis occurred. These were: increased
FasR expression; the predominance of Bax in relation to Bcl-2; the loss of mitochondrial
membrane potential; AIF release; decreased expression of survivin (an antiapoptotic
protein); and the activation of caspase-3. The selected pyrazolines were also found to be
cytotoxic against neoplastic cells collected from the peripheral blood and bone marrow
of patients with different subtypes of acute leukemia.
Keywords: pyrazolines, acute leukemia, cytotoxicity, apoptosis.

1 Introduction
The isolation of natural compounds as potential chemotherapeutic drugs is limited by
their low-yield, as well as by the scarcity of their natural sources [1, 2]. In addition, some
natural compounds are highly cytotoxic to non-neoplastic cells; thus, although presenting
antitumor properties, their therapeutic use is limited [3]. For this reason, synthetic
heterocyclic compounds are of great importance due to their wide applicability in many
fields, including medicinal chemistry. Pyrazolines are important examples of biologically
active molecules with a five membered ring containing two atoms of nitrogen and a
double bond [4, 5]. Among the several biological properties of pyrazoline derivatives are
the antimicrobial, antioxidant, anti-inflammatory and antitumor activities [5], including
anticancer potential in cervix carcinoma (HeLa), and lung cancer (NCI-H522), among
other cell lines [6, 7]
.
Hematologic neoplasms are the most frequent cancer type in children, adolescents and
young adults, and also present a high incidence in patients over 60 years of age [8, 9]. Acute
leukemias (ALs) are a group of hematologic malignancies characterized by a blockage of
normal differentiation and by the exacerbated clonal proliferation of a single myeloid or
lymphoid progenitor [10]. Chemotherapy with cytotoxic and cytostatic drugs is the first
therapeutic option against AL; however, it has several limitations, such as severe adverse
effects and high relapse rates [6, 11]. In this perspective, since cancer is a major public
health problem, the search for novel molecules that may be used as prototypes for the
development of new drugs has motivated scientific researches in the oncology field [6, 12]
.
Thus, aiming to contribute to the discovery of safer and more effective compounds against
AL, this study developed a series of pyrazoline derivatives with cytotoxic effects on
leukemic cells that induce regulated cell death without significant cytotoxicity to non-
neoplastic cells.
2 Material and methods
2.1 Chemistry
2.1.1 Synthesis
Reagents used were purchased from Sigma-Aldrich® or Merck®. Chalcones were
obtained by aldol condensation of acetophenones (5.0 mmol) with aldehydes (5.0 mmol)
in ethanol (25 ml) and KOH (50% v/v), at room temperature and magnetic stirring for 24

h,[13] and neutralized with HCl (10%v/v). The precipitate was separated by vacuum
filtration and the product was recrystallized to give an average 80% yield of chalcones [3].
To the obtained chalcones (1.0 mml), acetic acid (20 mL) and hydrazine hydrate (0.2 mL,
3.0 mmol) was added, the mixture was refluxed under stirring for 6 h, and then poured in
ice bath and neutralized with NaHCO3(aq)[14]. The formed precipitate was filtered,
washed with cold water, and recrystallized in ethanol/EtOAc (80:20v/v) to give the
(3,4,5-trimethoxyphenyl-4,5-dihydro-1H-pyrazole-1-yl) ethanone derivatives. The new
1
13
five pyrazoline derivatives were characterized by the melting points (m.p.), H and C
nuclear magnetic resonance spectroscopy (NMR) and high-resolution mass spectra
(HRMS) and the physicochemical data on synthesized compounds are described in
Supp.Mat.1.
2.1.2 Physicochemical properties
Compounds were evaluated according to physicochemical parameters proposed by
Lipinski et al. (2001)[15] and Veber et al. (2002)[16]. Molecular weight (MW), cLog P
hydrogen bonding acceptors, hydrogen bonding donors, number of rotatable bonds and
polar surface area (PSA) were obtained from Osiris® (Acetylion Pharmaceuticals Ltd.,
available at: http://www.organic-chemistry.org).
2.2 Biological evaluation
All experiments with human samples were approved by the Research Ethics Committee
with Human Beings (CEPSH-UFSC nº746.486/2014) and all individuals signed the Free
and Informed Consent Form present in the approved research project.
2.2.1 Cells
Acute myeloid leukemia (AML) (K562) and T-cell acute lymphoblastic leukemia (T-
ALL) (Jurkat) cell lines were originally purchased from the American Type Culture
Collection (ATCC). Murine melanoma (B16-F10) and Burkitt lymphoma (BL) (Daudi)
cells were purchased from ATCC or the Rio de Janeiro Cell Bank (BCRJ). Peripheral
blood mononuclear cells (PBMC) and red blood cells (RBC) were isolated from six non-
smoking healthy volunteers by Ficoll-Hypaque as previously described[17]
.

2.2.2 Viability assays
Screening was performed in K562 and Jurkat cells (5x104cells/well) incubated with each
pyrazoline for 24h. Cell viability was assessed by the MTT assay[18] (Sigma-Aldrich®)
and optical density of the control groups (untreated cells) was considered as 100% of
viable cells. The most cytotoxic compounds were selected and incubated with K562,
Jurkat, B16-F10, Daudi and PBMC at different concentrations (1-100µM). The half-
maximal inhibitory concentrations (IC50) were calculated using GraphPad Prism 5
software and the selectivity index (SI) was calculated by dividing the IC50 obtained on
PBMC by the IC50 obtained on K562, Jurkat, Daudi and B16-F10 cells. A high selectivity
was considered as SI ≥ 5, a moderate selectivity as 2 ≤ IS < 5 and a low selectivity as IS
< 2[19]. Hemolysis test was performed on RBC using saline as a negative control (0%
lysis), distilled water as a positive control (100% lysis) and increasing concentrations of
selected pyrazolines. The hemolysis percentage (% hemolysis) was calculated as
previously described [20]
.
2.2.3 Cell cycle analysis
K562 and Jurkat cells were incubated with the selected pyrazolines at their respective 24
h IC50 (H3TM4: 32.0 µM and 30.08 µM on K562 and Jurkat cells respectively; H3TM6:
55.7 µM and 33.7 µM on K562 and Jurkat cells respectively) for 12 h. Cell cycle analysis
was assessed according to the kit protocol (PI/RNAse Solution Kit, Immunostep®).
Analysis was performed by flow cytometry (FACSCantoIITM, BD Immunocytometry
Systems®) and data were analyzed using Infinicyt software version 1.7 (Cytognos®).
2.2.4 Apoptosis assays
Apoptotic cell death was first evaluated on K562 and Jurkat cells by ethidium bromide
(EB) and acridine orange (AO) staining as previously described[17]. For the Annexin V
assay, K562 and Jurkat cells were incubated for 12 h with the selected pyrazolines (24h
IC50) and then labeled with Annexin V–FITC (BD Biosciences®) before flow cytometry
analysis. For the DNA fragmentation assay, DNA extraction of Jurkat cells was
performed using a commercial kit (QIAGEN®) and DNA samples were separated by
electrophoresis in 2% agarose gel embedded with EB (2µg/mL). All experiments were
performed with pyrazolines H3TM4 and H3TM6 at the 24 h CI50 (H3TM4: 32.0 µM
and 30.08 µM on K562 and Jurkat cells respectively; H3TM6: 55.7 µM and 33.7 µM on
K562 and Jurkat cells respectively).

2.2.5 Mitochondrial membrane potential (MMP) and protein expression
K562 and Jurkat cells were incubated for 12 h with the selected pyrazolines at the 24 h
CI50 (H3TM4: 32.0 µM and 30.08 µM on K562 and Jurkat cells respectively; H3TM6:
55.7 µM and 33.7 µM on K562 and Jurkat cells respectively).
For MMP evaluation, cells were incubated in a MitoView 633™ (Biotium®) solution
(diluted 1:10.000) and for protein expression, cells were first fixed and permeabilized
with BD Cytofix/Cytoperm (BD Biosciences®). Subsequently, cells were stained with
anti-Ki-67-FITC (BD Biosciences®), anti-Bax-PerCP (Santa Cruz Biotechnology®),
anti-Bcl-2-FITC (Invitrogen®), anti-survivin-PE (Santa Cruz Biotechnology®), anti-
AIF-FITC (Santa Cruz Biotechnology®), anti-FasR-PE (Santa Cruz Biotechnology®)
and anti-caspase-3-PE (BD Biosciences®) for flow cytometry acquisition.
2.2.6 Ex vivo assays
Two PB and four BM samples obtained from patients with AL were collected before the
first treatment at the diagnostic or at a relapse. MC were isolated as described above and
incubated with increasing concentrations of the selected pyrazolines for 24 h before cell
viability assessment.
2.3 Statistical analysis
Data are expressed as means ± SEM and each experiment has been repeated at least three
times independently. Statistical analysis was conducted in GraphPad Prism 5 software
using t test or ANOVA one-way and Bonferroni post-hoc test. p ≤ 0.05 was considered
as significant.
3 Results
3.1 Pyrazoline derivatives synthesis
Synthesis of the pyrazolines H3TM2, H3TM4, H3TM5, H3TM6 and H3TM7 was
conducted in two steps (Fig.1A). Theyields were considered good and found to be around
53 and 90%. Selected compounds H3TM4 and H3TM6 have not been previously
1
reported in the literature and led to regular yields (41–85%). The H NMR spectra
revealed a typical ABX system, in which each hydrogen is shown as a double doublet
with characteristic constants (J) and aromatic hydrogens at 6.5–7.5 ppm, according to the
13
substituent in the aromatic group. In the C NMR, methylene signals can be observed

between 20 and 45 ppm, a methoxy group between 55 and 60 ppm, and aromatic carbons
and the pyrazoline C=N signal at 100–150 ppm. The signal observed between 168 and
189 ppm, attributed to the carbonyl group, confirms the acetylation performed during the
synthesis.
Figure 1. Synthesis and screening of novel pyrazoline derivatives.
Figure 1. (A) Synthesis of precursor chalcones and pyrazolines (H3TM2, H3TM4-7). (i) ethanol, KOH
50% w/v, rt, 24h, HCl (10%); (ii) acetic acid, hydrazine hydrate, reflux 6h, NaHCO3(aq). (B) Screening of
five pyrazolines (100µM) on AML K562 and ALL Jurkat cells after 24h incubation. * p ≤0.05 when
compared to the control groups, one-way ANOVA followed by Bonferroni.
3.2 Pyrazoline derivatives have a good predicted oral bioavailability
All five pyrazolines synthesized have an MW lower than 500 g/mol, cLog P lower than
five and PSA lower than 140 angstroms (Table 1). They also have fewer than 10 hydrogen
acceptors, no hydrogen donors and flexible bonds, which is in agreement with the
physicochemical parameters considered ideal for a predicted good oral bioavailability, as
they have adequate solubility and permeability [15, 16]
.

Table 1. Physicochemical properties. Theoretical prediction of pyrazolines H3TM2, H3TM4-7
properties according to Lipinski et al. (2001) and Veber et al. (2002).
Properties/
Compounds
MW
cLogP
H --
acceptors
H –
donors
Rotatable
bonds
PSA
Violations
H3TM2
H3TM4
H3TM5
H3TM6
H3TM7
398.2
433.3
404.5
404.5
397.5
2.4
3.4
3.7
3.7
2.7
≤ 5
8
6
0
0
5
5
5
5
6
-
78.8
60.4
30.4
60.4
63.6
-
0
0
0
0
0
-
6
0
6
0
7
0
*Lipinski et
al. (2001)
≤
500
≤ 10
≤ 5
*Veber et
al. (2002)
-
-
≤ 12
≤ 12
≤ 10
≤ 140
-
3.3 Pyrazoline derivatives are cytotoxic against neoplastic cells
All five synthetic pyrazolines were cytotoxic to K562 and Jurkat cells at 100µM (Figure
1B). The most cytotoxic and unpublished compounds, H3TM4 and H3TM6 reduced the
cell viability of K562, Jurkat, Daudi and B16-F10 in a concentration- and time-dependent
manner (Supp.Fig.1); the IC50 values are shown in Table 2.
Table 2. IC50 values. IC50 for H3TM4 and H3TM6 on K562, Jurkat, Daudi, B16-F10 and non neoplastic
cells (PBMC).
H3TM4 IC50 (µM)
Cells/Time
24h
K562
Jurkat
Daudi
B16-F10
PBMC
32.0 ± 1.1
16.2 ± 0.3
15.1 ± 1.0
30.8 ± 0.6
17.2 ± 0.6
15.6 ± 0.5
49.8 ± 1.4
34.8 ± 1.2
33.0 ± 0.8
98.7 ± 1.6
≈ 339.8 ± 16.6
48h
72.7 ± 2.4 ≈ 287.1 ± 6.5
62.5 ± 1.3 ≈ 117.5 ± 2.4
72h
H3TM6 IC50 (µM)
Cells/Time
24h
K562
Jurkat
Daudi
B16-F10
72.3 ± 2.1
PBMC
55.7 ± 0.8
21.9 ± 0.4
15.5 ± 0.5
33.7 ± 1.2
27.7 ± 0.7
25.3 ± 0.6
46.2 ± 1.5
20.2 ± 0.8
5.1 ± 0.9
≈ 496.7 ± 23.2
48h
56.6 ± 1.2 ≈ 104.7 ± 2.9
23.3 ± 0.7
55.3 ± 1.6
72h
3.4 Selected pyrazolines H3TM4 and H3TM6 are highly selective for tumor cells
Table 2 shows that the selected pyrazolines did not reduce the viability of PBMC. In fact,
when compared to non-neoplastic cells, H3TM4 and H3TM6 were highly selective for
tumor cell lines, as demonstrated by the SI displayed in Table 3. Additionally, significant
hemolysis was observed only at concentrations 12 and 15 times higher than those found
in AL cells (Table 4).

Table 3. SI for H3TM4 and H3TM6. SI was calculated by the ratio IC50 PBMC/ IC50 Neoplastic cells , obtained
after 24h incubation.
H3TM4
≈ 10.6
≈ 11.0
≈ 6.8
H3TM6
≈ 14.7
≈ 8.9
Jurkat
K562
Daudi
≈ 10.7
≈ 6.9
B16-F10
≈ 3.4
Table 4. Hemolysis on RBC. Hemolysis percentage induced by H3TM4 and H3TM6 when compared
with the negative control (0% hemolysis).
% HEMOLYSIS
Concentration
1X IC50
H3TM4
0.9 ± 0.6
1.3 ± 1.0
1.6 ± 0.5
6.0 ± 5.7
12.9 ± 3.5*
15.8 ± 5.1*
H3TM6
1.9 ± 1.2
1.5 ± 1.2
1.6 ± 1.3
2.4 ± 1.8
3.2 ± 0.4
6.4 ± 1.5*
2X IC50
3X IC50
9X IC50
12X IC50
15X IC50
3.5 H3TM4 and H3TM6 induce cell cycle arrest, apoptosis and reduce cell proliferation
A significantly increased percentage of K562 cells at the G0/G1 phase was observed after
treatment with H3TM4 and H3TM6 (8.0% and 7.4%, respectively) (Fig.2A). In Jurkat
cells, a mild but non-significant increase was seen at the S phase. Treatment with H3TM4
and H3TM6 also reduced expression of the cell proliferation marker KI-67 in both cell
lines (Fig.2B). Morphological changes resulting from the cell death induced by H3TM4
and H3TM6 revealed apoptotic characteristics, such as membrane blebbing, chromatin
condensation and membrane integrity loss (Fig.2C). The percentage of Annexin-V
positive K562 cells increased by 25.0% and 18.8% after treatment with H3TM4 and
H3TM6, respectively, while an increase of 24.6% and 29.2% was observed in Jurkat cells
(Fig.2D). Finally, apoptosis, confirmed as the typical “ladder pattern”, was detected in
treated Jurkat cells (Fig.2E).

Figure 2. Cell cycle and apoptosis assays.
(A) Cell cycle analysis of K562 and Jurkat cells treated with H3TM4 and H3TM6 (24 h IC50), stained with
PI and evaluated by flow cytometry. (B) Ki67 expression (%) was determined by flow cytometry after
labeling with anti-Ki67. (C) Apoptotic K562 and Jurkat cells are indicated by the white arrows after AO/EB
staining. (D) Annexin-V positive K562 and Jurkat cells (%) were quantified by flow cytometry. (E) DNA
fragmentation assay on Jurkat cells. * p≤0.05 when compared to control groups, t-test or ANOVA one-way
followed by Bonferroni.

3.6 H3TM4 and H3TM6 induce intrinsic and extrinsic apoptosis on AL cells
Pyrazolines H3TM4 and H3TM6 significantly increased Bax expression in K562 (15.8%
and 16.8%) and Jurkat cells (101.4% and 20.5%) (Fig.3A), while the anti-apoptotic
protein Bcl-2 was significantly reduced only after treatment with H3TM4 (K562: 13.4%,
Jurkat: 12.9%) (Fig.3B). The percentage of K562 cells with an intact MMP was reduced
by 25.3% and 26.0% after treatment with H3TM4 and H3TM6, respectively (Figure 3C),
while Jurkat cells with an intact MMP reduced by 32.9% and 41.0%. The selected
pyrazolines also decreased survivin expression in both K562 (H3TM4: 21.2%, H3TM6:
22.2%) and Jurkat cells (H3TM4: 10.7%; H3TM6: 21.4%) (Figure 3D), and induced AIF
release in ALL cells (H3TM4: 33.2%; H3TM6: 6.0%) (Figure 3E). K562 cells treated
with H3TM4 and H3TM6 showed increased FasR expression (21.3% and 34.3%,
respectively), while only treatment with H3TM6 modulated FasR in Jurkat cells (30.3%)
(Figure 3F). Finally, treatment with H3TM4, but not H3TM6, significantly increased
active caspase-3 expression (20.0%) in K562 cells, whereas both compounds modulate
this protein in Jurkat cells (H3TM4: 128.3% and H3TM6: 6.6%) (Figure 3G).

Figure 3. Effect of selected pyrazolines in the apoptotic machinery.
(A-B) K562 and Jurkat cells incubated with H3TM4 and H3TM6 were labeled with anti-Bax-PerCP (A)
and anti-Bcl-2-FITC (B). (C) MMP % of K562 and Jurkat cells by MitoView. (D-G) K562 and Jurkat
labeled with anti-Survivin-PE (D), anti-AIF-FITC (E), anti-FasR-PE (F) and anti-caspase-3-PE (G). All
experiments were acquired by flow cytometry. * p≤0.05 when compared to the control groups using t-test.

3.7 Pyrazolines H3TM4 and H3TM6 are cytotoxic to AL patients’ cells
Pyrazolines H3TM4 and H3TM6 reduced the cell viability of all PB and BM samples
collected from AL patients in a concentration-dependent manner (Supp.Fig.2). Different
IC50 values were found for each case, as demonstrated in Table 5.
Table 5. Patients information and results. Clinical information about the seven AL patients and IC50
values calculated after MC incubation with H3TM4 and H3TM6.
Patient
Age
(years)
Sample
Diagnosis
Leuko
cytes/
mm³
Blast
cells
(%)
H3TM4
IC50
(µmol/l)
H3TM6
IC50
(µmol/l)
1
2
3
4
5
52
28
81
56
67
PB
PB
PB
PB
BM
AML
inv(16)
7090
9590
-
45
50
76
83
38
11.8 ± 1.3
37.1 ± 1.1
B-ALL
141.6 ± 2.7
71.3 ± 1.8
40.2 ± 1.9
31.0 ± 0.9
139.0 ±
3.6
Early T-cell
precursor ALL
46.0 ± 1.0
27.7 ± 0.7
16.2 ± 0.6
AML without
maturation
-
AML secondary to
MDS
5710
6
7
33
31
BM
BM
AML t(8;21)
8550
27
96
98.2 ± 2.8
39.9 ± 0,6
44.6 ± 1.2
29.0 ± 0.8
PML t(15;17) Bcr3+
FLT3 DIT+
11507
0
PML – Acute promielocytic leukemia
MDS – Myelodysplastic syndrome
4 Discussion
In this report, we have provided new information about the effects of a series of specific
and cytotoxic pyrazoline derivatives on malignant cells. The five compounds fit the oral
bioavailability parameters, which are important for the rational investigation of new drugs
[21]
as this may contribute to future structure-activity relationship (QSAR) studies.
Considering the chemical structures of these pyrazolines, the 3,4,5-trimethoxy
substitution on the A ring seems to be responsible for their cytotoxic activity. As already
stated in the literature, this group is essential for the antitumor activity of
chemotherapeutics like vincristine, used against ALL [3]. In addition, the presence or
absence of methoxyl groups (OCH3) linked to the A ring characterizes the lipophilicity
[22,
variation of the molecule and its presence is related to different biological activities
23]. The presence of electron density donor groups (OCH3) on the A ring also seems to be
relevant. At the B-ring, the presence of an electronegative (electron acceptor) group as
the bromine atom (H3TM4), may be responsible for the increased cytotoxic activity of

this compound, to the both cell lines. Furthermore, hydrophobic substituents as B-ring
(H3TM5 and H3TM6) also seems to be relevant for the cytotoxicity of the compounds,
especially the 2-naphthyl group (H3TM6) against Jurkat cells; the naphthyl group can
interact with other hydrophobic rings by interactions between the π orbitals, which may
favor their cytotoxic activity [24, 25]. On the other hand, an electron-donor group as a
dimethylamine (H3TM7) or a heteroatom ring as 3,4-methylenedioxy (H3TM2) in the
B ring caused reduction in cytotoxic activity, especially for K562 cells.
Pyrazolines H3TM4 and H3TM6, the two more cytotoxic compounds, were active
against T-ALL Jurkat, AML K562 and BL Daudi cells; the different IC50 values might be
explained by the heterogeneity of hematological malignancies [19]. The K562 cell line
originates from an AL with Bcr/abl oncogene, which is largely responsible for its
resistance to chemotherapy [26, 27], while BL affects mature B lymphoid cells and is also
a very aggressive disease [10, 28]. Considering that therapeutic protocols involve several
chemotherapy cycles, these results are very promising, especially when taking into
account that some cells are usually resistant to conventional chemotherapeutics in the first
24 h [26]. Similar studies evaluated the effect of B-ring substituted steroidal pyrazoline
derivatives and glycoside derivatives of ferrocenyl-pyrazolines on AL cells and found
IC50 values ranging from 10.6 μM to >200 μM [27, 29]. Interestingly, H3TM4 and H3TM6
were not effective against non-tumor cells, which suggests that these pyrazolines are
highly selective for hematological malignancies and have a high blood biocompatibility.
Since the non-selective toxicity of currently available chemotherapeutic agents is an
important factor influencing treatment failure [6, 11, 17], this was considered a very
promising result.
To date, few studies elucidating the anti-leukemic effect of pyrazolines have been
[30]
described in the literature. Bao et al.
reported that a pyrazoline derivative induced
[20]
G0/G1 arrest, while Santos-Bubniak et al.
reported cell cycle arrest at the S phase.
Cell cycle arrest at the S phase, as seen in Jurkat cells treated with H3TM4 and H3TM6,
suggests interference with DNA replication [31], whereas G0/G1 arrest, as seen in K562
cells, could be related to p53 activation. p53 acts in the first checkpoint, and is
fundamental to genome integrity, as it allows DNA repair mechanisms and the removal
of damaged cells through apoptosis [32, 33]. Similarly, as uncontrolled cell proliferation is
highlighted as one of the main features of oncogenesis, Ki67 expression is often used in
vitro and in clinics as a proliferation marker [34, 35]. H3TM4 and H3TM6 decreased KI67
expression in AL cells, suggesting that they might modulate neoplastic cell proliferation.

Cell death may occur through several regulated and non-regulated mechanisms [36]. We
have demonstrated that pyrazolines H3TM4 and H3TM6 induce apoptosis in AML and
ALL cells, which is in agreement with other studies found in the literature [20, 30]
.
The Bcl-2/Bax ratio after H3TM4 and H3TM6 treatment demonstrates Bax
predominance in relation to Bcl-2 in both K562 and Jurkat cells. According to the
literature, Bax and Bcl-2 modulation induces pores opening at the mitochondrial
membrane, resulting in MMP loss and the release of pro-apoptotic factors to the
mitochondria and the cytosol [37, 38]. Treatment with H3TM4 and H3TM6 increased AIF
expression in Jurkat but not K562 cells, suggesting that, in AML cells, other proteins
might be involved in cell death, such as cytochrome c, SMAC and EndoG. These results
are in agreement with two other studies that reported the activation of intrinsic apoptosis
by pyrazolines, as evidenced by an increase in Bax and AIF expression, decrease in Bcl-
[20]
2 expression
and MMP loss [20, 30]. Extrinsic apoptosis also seems to be involved in
cell death induced by H3TM4 and H3TM6 on AL cells, as evidenced by increased FasR
expression. FasR is one of the membrane receptors that triggers intracellular signaling
cascades, culminating in the activation of caspase-8 and effector caspases-3 and -7, which
results in apoptosis independent of the mitochondrial signaling pathway [39, 40]. Intrinsic
and extrinsic apoptosis led to caspase-3 activation in Jurkat cells, whereas only H3TM4
was able to increase active caspase-3 expression in K562 cells. The non-modulation of
caspase-3 by H3TM6 on K562 cells suggests that this pyrazoline might induce a caspase-
independent cell death with apoptotic characteristics; however, further studies are needed
to elucidate this question. Additionally, H3TM4 and H3TM6 decreased survivin
expression in K562 cells and H3TM6 did the same in Jurkat cells. Studies suggest that
survivin is related to radiation and chemotherapy resistance in clinics and this protein
inhibition may lead to a greater sensitivity of neoplastic cells to current cancer treatments
[41, 42]
.
Finally, H3TM4 and H3TM6 were evaluated in PB and MC cells collected from patients
with different AL subtypes. Different IC50 values have been found, which was expected
given that leukemias are heterogeneous diseases that react differently to treatment [10, 26,
27]. It is important to highlight that of all patients included in this study only Patient 7 had
relapsed when the cells were obtained. This patient was diagnosed with PML with a
t(15;17) Bcr3 positive and FLT3/DIT mutation. The presence of a DIT/FLT3 mutation in
patients with PML t(15;17) is associated with a very poor prognosis [10]. The results
obtained for H3TM4 and H3TM6 showed that they were both effective against these

resistant cells. Since not every PML patient responds well to conventional treatment, it is
very important to find new cytotoxic molecules against these AL subtypes. Pyrazolines
H3TM4 and H3TM6 were also cytotoxic against other aggressive AL subtypes such as
AML secondary to myelodysplastic syndrome (MDS), early T-cell precursor ALL and
AML without maturation [10]. It is evident that the small sample number does not allow
us to draw any definitive conclusion about the effect of pyrazolines on each AL subtype.
However, the results obtained so far are quite promising, as they show that H3TM4 and
H3TM6 are cytotoxic not only against neoplastic cell lines but also against cells collected
from patients with different AL subtypes. So far, no studies have been found reporting
the evaluation of pyrazoline in AL patient cells.
5 Conclusion
This study demonstrates that the previously unpublished pyrazolines H3TM4 and
H3TM6 are selective and cytotoxic to different hematological malignancies and that the
death mechanisms involve cell cycle arrest, the inhibition of cell proliferation and the
induction of intrinsic and extrinsic apoptosis. Altogether, the results suggest that these
novel compounds are promising candidates for the development of efficient new
prototypes against AL.
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HIGHLIGHTS
 H3TM4 and H3TM6 are highly cytotoxic and selective to several neoplastic cell
lines.
 H3TM4 and H3TM6 have physicochemical properties for a good oral
bioavailability.
 H3TM4 and H3TM6 induce cell cycle arrest and decrease cell proliferation.
 Cell death by H3TM4 and H3TM6 involves intrinsic and extrinsic apoptosis.
 H3TM4 and H3TM6 kill cancer cells from patients with different acute leukemia
subtypes.