F.B. Costa et al.
Biomedicine & Pharmacotherapy 102 (2018) 481–493
1. Introduction
(Diadema, SP, Brazil), respectively. Trypan blue dye, dimethyl sulfoxide
(
DMSO), ethanol, methanol and Triton X-100 were obtained from Vetec
Piperazine is a promising scaffold for drug development [1] due to
(Rio de Janeiro, RJ, Brazil), while May-Grünwald-Giemsa dye was
purchased from Merck (Darmstadt, HE, Germany). Hoechst 33342 dye
was acquired from Invitrogen (GrandIsland, NY, USA). Xylasine and
ketamine hydrochloride were obtained from Syntec (Cotia, SP, Brazil)
and König (Embu-Guaçu, SP. Brazil), respectively. Agar and an FITC
Annexin V Apoptosis Detection kit were purchased from BD Bioscience
(Franklin Lakes, NJ, USA). The mouse monoclonal anti-human cyto-
chrome c (6H2, sc-13561 PE) and anti-human tumor necrosis factor
receptor 1 (TNF-R1) (6A658, sc-73195 FITC) antibodies were acquired
from Santa Cruz Biotechnology (Dallas, TX, USA). RNeasy mini,
QuantiTect Reverse Transcription, Rotor-Gene SYBR Green PCR and
QuantiTect Reverse Transcription kits were purchased from Qiagen
(Hilden, Germany). The sunflower oil was obtained from Bunge
Alimentos (Gaspar, SC, Brazil).
its broad spectrum of biological activities [2]. It is present in a large
variety of commercially available anticancer drugs such as imatinib
mesylate, a tyrosine kinase inhibitor (TKI) that acts by inhibiting spe-
cific tyrosine kinases, such as BCR-ABL fusion oncoprotein in chronic
myeloid leukemia [3–5].
Chronic myeloid leukemia is a myeloproliferative disorder char-
acterized by the neoplastic transformation of hematopoietic stem cells
in the bone marrow and their accumulation in the bloodstream. Its
molecular hallmark is the Philadelphia chromosome (Ph), an aberrant
fusion gene originated by translocation between chromosomes 9 and 22
which results in a chimeric gene product BCR-ABL [6–8]. The mutations
of Ph and overexpression of BCR-ABL oncoprotein can promote re-
sistance to apoptosis induced by conventional chemotherapy, and make
chronic myeloid leukemia stem cells capable of escaping from imatinib
and other TKI agents [9–12]. Therefore, compounds which also pro-
mote other or additional cellular death mechanisms, such as regulated
necrosis, could be a new therapeutic option.
2.2. Cell cultures
Balb/c 3T3-A31 fibroblasts were kindly donated by Dr. Mari Cleide
Sogayar (Chemistry Institute, University of São Paulo, SP, Brazil); while
K562 chronic myelogenous leukemia cells were obtained from the Rio
de Janeiro Cell Bank (Rio de Janeiro, RJ, Brazil). The 3T3 and K562
cells were cultured in DMEM or RPMI-1640 medium, respectively,
supplemented with 10% (v/v) heat-inactivated FBS, 2 mM L-glutamine,
4.5 mM HEPES, 0.17 M sodium bicarbonate, 100 IU/mL penicillin and
In accordance with the Nomenclature Committee on Cell Death
NCCD), regulated cell death occurs as part of physiological programs
(
or can be activated once adaptive responses to perturbations of the
extracellular or intracellular microenvironment fail [13,14]. Regulated
necrosis, in turn, plays a major role in both physiological scenarios (e.g.
embryonic development) and pathological settings (e.g. ischemic dis-
orders); various types have been characterized, including necroptosis,
mitochondrial permeability transition (MPT)-dependent regulated ne-
crosis and parthanatos ([15–18]).
2
100 μg/mL streptomycin in a humidified atmosphere of 5% CO in air
at 37 °C.
Currently, studies show that necrosis is a regulated process invol-
ving a set of transduction pathways and degradative mechanisms
2.3. Animals
[
19–21]. In view of that, the term “necroptosis” can be defined as a
Female Swiss mice, weighing between 30 and 35 g, obtained from
the Bioterium at the Federal University of Goiás (Goiânia, GO, Brazil)
were used in this study. All efforts were realized to ensure the welfare of
mice. Parameters as loss of body weight, food/water consumption and
changes in activity and behavior of the animals were daily checked as
clinical conditions of animal suffering to determine when the animals
must be humanely sacrificed [26]. In addition, mice were acclimatized
for a week before beginning the experiments.
The animals were kept under constant environmental conditions
with a light-dark (12:12 h) cycle, controlled temperature (23 ± 2 °C),
water and food provided ad libitum. All procedures and protocols were
reviewed and approved by the Research Ethics Committee of the
Federal University of Goiás (UFG no. 137/2009). At the end of each
experiment, the mice were previously anesthetized with xylazine
(10 mg/kg) and ketamine hydrochloride (100 mg/kg) administered in-
traperitoneally and then euthanized by cervical dislocation [26].
receptor interacting protein kinase 3 (RIPK3)-dependent molecular
cascade promoting regulated necrosis [13]. Necrosis can be character-
ized by cell volume gain, organelle swelling, plasma membrane rupture
and loss of intracellular content, which can lead to inflammation
[
22,23]. The cellular signaling that triggers necrosis is complex and
requires different molecules working in concert. This process can be
initiated by death receptors such as the tumor necrosis factor (TNF)
receptor family, including TNF, Fas and TNF-related apoptosis-inducing
ligand (TRAIL) [24].
Considering this background, this study describes the synthesis of
the new piperazine-containing compound LQFM018 (2) [ethyl 4-((1-(4-
chlorophenyl)-1H-pyrazol-4-yl)methyl)piperazine-1-carboxylate],
a
closely related analogue of LASSBio579 (1), a compound obtained by
molecular simplification of atypical antipsychotic clozapine [25].
Moreover, the LQFM018 (2)-triggered cell death mechanisms in K562
cells as well as acute oral systemic toxicity and potential myelotoxicity
assessments of LQFM018 (2) were carried out.
2.4. General
2
. Materials and methods
NMR experiments were acquired at room temperature on a
BrukerAvance III 500 (11.75 T) spectrometer, using a 5 mm inverse-
detection probehead with z-gradient. To acquire 1H and C experi-
13
2.1. Chemicals
3
ments, samples containing 20 mg of LQFM018 (2) in CDCl and tetra-
An In Vitro Lactate Dehydrogenase Activity Assay kit, a Caspase-3
methylsilane (TMS) as internal standard were used. 1D and 2D pulse
sequences from the Bruker user library were used for all experiments.
Infrared (IR) spectra were obtained on a Nicolet-55a Magna spectro-
meter using KBr plates. Mass spectra (MS) were obtained with a
microTOF III (Brucker Daltonics Bremen, Germany). The sample pre-
paration for MS analysis consisted of diluting 1 μg of sample in 1 mL of
methanol. To perform the analysis in positive mode, 1 μl of formic acid
was added to the sample. The solution obtained was directly infused at
a flow rate of 3 μL/min into the ESI source. ESI(+) source conditions
were as follows: nebulizer nitrogen gas temperature and pressure of
2.0 bar and 200 °C, capillary voltage of −4,5 kV, transfer capillary
temperature of 200 °C; drying gas of 4 L min−1; end plate offset of
Colorimetric Assay kit, Dulbecco’s modified Eagle’s medium (DMEM),
Roswell Park Memorial Institute (RPMI)-1640 medium, gentamycin,
amphotericin B, propidium iodide (PI), RNAse, acetate 2,7-dichloro-
fluorescein (DCFH-DA), EDTA, bovine serum albumin (BSA), phos-
phatidylcholine, sodium taurodeoxycholate hydrate, rhodamine 123,
granulocyte-macrophage colony-stimulating factor (GM-CSF), 3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and
ethidium bromide were purchased from Sigma-Aldrich (St. Louis, MO,
USA). A CaspaTagTM Caspases-8 In Situ Assay kit was obtained from
TM
Millipore (Temecula, CA, USA). Fetal bovine serum (FBS) and acetic
acid were acquired from Gibco (Grand Island, NY, USA) and Cromoline
482