Synthetic 3-alkylpyridine alkaloid analogues as a new scaffold against leukemic cell lines: cytotoxic evaluation and mode of action

Article abstract of DOI:10.1007/s00044-019-02395-5

Leukemia is a malignant disease that originates in the bone marrow, local of blood cells production. Chemotherapy treatment is one of the alternatives to improve the survival of patients. In this context, the search for new compounds with potential cytotoxic action is justified. The present work evaluated the cytotoxic activity of 16 new synthetic analogs of 3-alkylpyridine alkaloids. The cytotoxic profiles were determined by MTT in vitro assay against THP-1 (acute monocytic leukemia), K562 (chronic myeloid leukemia) and PBMC (peripheral blood mononuclear) human cells. To investigate the mechanism of action, cell cycle analysis and alterations of TP53, p21, Bax, Bcl2, NOX-1, NOX-2, NOX-4 and p47-phox gene expressions were performed by qPCR, along with the measurement of reactive oxygen species (2’,7’-dichlorodihydrofluorescein diacetate and dihydroethidium). Compounds 4c, 5b, 5c and 6d were the most active and selective for THP-1 and compounds 7c and 11 were found to be more active for K562. All of this induced apoptosis in the tested strains. Concerning the investigation of the mechanism of action, it was observed that the pathway being activated is a p53-independent pathway. The data presented in this work indicate that 3-alkylpyridine alkaloid analogues are a potential class of compounds with cytotoxic action.

Full text of DOI:10.1007/s00044-019-02395-5

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02395-5
ORIGINAL RESEARCH
Synthetic 3-alkylpyridine alkaloid analogues as a new scaffold
against leukemic cell lines: cytotoxic evaluation and mode of action
1
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Fernanda Cristina Gontijo Evangelista
Aline de Freitas Lopes2 Silmara Nunes Andrade2
Camila de Souza Barbosa2 Julia Dias da Silva2 Alessandra Mirtes Marques Neves2
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Cristina de Mello Gomide Loures1 Larissa Froede Brito1 Lirlândia Pires de Sousa1 Karina Braga Gomes Borges1
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Gustavo Henrique Ribeiro Viana2 Fernando de Pilla Varotti2 Adriano de Paula Sabino1
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Received: 15 May 2019 / Accepted: 6 July 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
Leukemia is a malignant disease that originates in the bone marrow, local of blood cells production. Chemotherapy treatment
is one of the alternatives to improve the survival of patients. In this context, the search for new compounds with potential
cytotoxic action is justified. The present work evaluated the cytotoxic activity of 16 new synthetic analogs of 3-alkylpyridine
alkaloids. The cytotoxic profiles were determined by MTT in vitro assay against THP-1 (acute monocytic leukemia), K562
(chronic myeloid leukemia) and PBMC (peripheral blood mononuclear) human cells. To investigate the mechanism of
action, cell cycle analysis and alterations of TP53, p21, Bax, Bcl2, NOX-1, NOX-2, NOX-4 and p47-phox gene expressions
were performed by qPCR, along with the measurement of reactive oxygen species (2’,7’-dichlorodihydrofluorescein
diacetate and dihydroethidium). Compounds 4c, 5b, 5c and 6d were the most active and selective for THP-1 and compounds
7c and 11 were found to be more active for K562. All of this induced apoptosis in the tested strains. Concerning the
investigation of the mechanism of action, it was observed that the pathway being activated is a p53-independent pathway.
The data presented in this work indicate that 3-alkylpyridine alkaloid analogues are a potential class of compounds with
cytotoxic action.
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Keywords 3-alkylpyridine alkaloid analogues Leukemia Mode of action
Introduction
the granulocytic lineage and the decreased production of
normal blood cells (Fogliato et al. 2003; Puumala et al.
2013).
Acute Myeloid Leukemia (AML) is an aggressive malig-
nant disease of the hematopoietic system, characterized by
the uncontrolled proliferation of abnormal blastic cells of
AML has abrupt clinical onset and rapid evolution to
death if the patient is not treated. The aim of the treatment is
to eliminate or control the proliferation of leukemic clones,
and is based on the use of polychemotherapy and stem cell
transplantation (Estey and Döhner 2006; Queliane et al.
2011). The gold standard treatment includes the use of
cytarabine (anti-metabolic) and an anthracyclic compound
that causes the inhibition of topoisomerase II (daunorubicin,
idarubicin or mitoxantrone) (Gong et al. 2015). However,
most patients (58%) die due to a recurrence of AML (Estey
and Döhner 2006).
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-019-02395-5) contains supplementary
material, which is available to authorized users.
* Fernando de Pilla Varotti
varotti@ufsj.edu.br
* Adriano de Paula Sabino
adriansabin01@gmail.com
Chronic Myelogenous Leukemia (CML) is a chronic
myeloproliferative pathology arising from the clonal
expansion of transformed hematopoietic stem cells, and is
characterized by the presence of a specific cytogenetic
abnormality, the Philadelphia (Ph) chromosome (Heiblig
et al. 2014). This chromosome is a product of the t(9;22)
1
Departamento de Análises Clínicas e Toxicológicas, Faculdade de
Farmácia, Universidade Federal de Minas Gerais, Belo Horizonte,
Minas Gerais, Brazil
2
Núcleo de Pesquisa em Química Biológica, Universidade Federal
de São João Del Rei, Divinópolis, Minas Gerais, Brazil

Medicinal Chemistry Research
translocation (q34;q11) resulting in a hybrid BCR-ABL
gene, which encodes a chimeric BCR-ABL oncoprotein
with tyrosine kinase activity (Mughal and Goldman
2004).
allows the investigation of the associated biological impli-
cations. It also allows modifications in the molecular
architecture of analogues of natural products, which creates
opportunities to obtain new bioactive compounds (Netz and
Opatz 2015). In this context, synthetic analogs of alkaloids
of the 3-alkylpyridine (3-APA) type are good candidates for
a supposed anti-leukemic activity, since they have pro-
apoptotic activity, and anti-angiogenic, anti-proliferative
and cytotoxic actions against human tumoral lineages
(Wang et al. 2016a; Suzuki et al. 2017). These compounds
are characterized by the presence of at least one pyridine
ring and a diverse functionalized aliphatic chain attached to
the pyridine ring at the carbon of position 3 [15].
The disease that was previously considered incurable
now presents good results for remission and cure following
the transplantation of hematopoietic stem cells. However,
most patients are not able to receive this treatment, either
due to old age or due to the absence of a donor (Mughal and
Goldman 2004). The discovery that BCR-ABL is necessary
for the pathogenesis of CML and that its tyrosine kinase
activity is important in cell transformation has made it an
important target for therapeutic intervention (Harrington
et al. 2017). Thus, imatinib mesylate, which inhibits the
enzymatic activity of BCR-ABL, has been developed,
leading to the death of the leukemic cells by decreasing
their resistance to apoptosis (Mahalingam et al. 2009).
The alarming increase in the number of cases and cancer
deaths has boosted the search for new anti-tumor drugs.
Although radiotherapy and surgery continue to be employed
as first choice treatment strategies, recovery rates slightly
exceed 33% (Paubelle et al. 2017).
In view of these facts, this work aims to evaluate the
mechanism of the in vitro cytotoxic action of novel syn-
thetic molecules, 3-alkylpyridine alkaloids, against
myeloid-type human leukemic cell lines.
Materials and methods
Synthesis
Regarding CML, research has focused on the development
of new drugs capable of combating imatinib-resistant BCR-
ABL clones and, consequently, reducing the risk of resistance
(Ramachandran et al. 2016; Egan and Radich 2016).
In this way, chemical synthesis presents itself as an
important tool to obtain compounds on a larger scale and
The synthesis of the 3-APA analogs 4a-d, 5a-d, 6a-d, 7b-c,
11 and, 13 is depicted in Scheme 1. Characterization details
are presented in Supporting Information. The parent nucleus
of 3-alkylpyridine marine alkaloid (3-Pyridinepropanol 12)
was purchased from Sigma (cat. number: P71207).
Scheme 1 Synthesis of 3-Alkylpyridine Alkaloid Analogs 4a-d, 5a-d,
6a-d, 7b-c, 11, 13 and the parent nucleus 3-Pyridinepropanol 12.
Reagents, conditions, and yields: a (i) NaHSO4, DHP, DMSO, hexane,
40 °C, 16 h, 74%–89%; (ii) MsCl, Et3N, CH2Cl2, r.t., 10 h, 77%–87%;
(iii) 3-(pyrid-3-yl)propan-1-ol, NaOH/H2O, Bu4N+Br−, Et2O, r.t.,
72 h, 57%–73%; (iv) MeOH, HCl, r.t., 12 h, 71%–100%; (v) (COCl)2,
Et3N, DMSO, CH2Cl2, −60 °C, 25 min, 60−95%; (vi) MsCl, Et3N,
CH2Cl2, r.t. 10 h, 52%−83%; (vii) Bu4N+Br−, KSCN, THF, reflux,
55%−78%; (viii) HBr(aq), toluene, reflux, 69%; (ii) NaN3, DMSO, r.t.,
98%; (iii) MsCl, Et3N, CH2Cl2, 100%; (iv) NaOH, Bu4N+Br−, 63%
and b (COCl)2,Et3N, DMSO, CH2Cl2, −60 °C, 25 min, 57%

Medicinal Chemistry Research
Cell viability
Treatments were run for 48 h in both lines, with 1 × 106
cells/tube. After the treatments, the cells were washed 2
times with phosphate buffered saline (PBS) at 4 °C and
resuspended in 0.15 mL of annexin (1×) binding buffer.
Cells were labeled with 50 μL of the labeling mix con-
taining 2.5 μL annexin V-FITC and 2.5 μl IP (3 mM) and
incubated for 15 min at 4 °C in the dark. Here, 20,000
events were used for cell quantification on a flow cytometer
(LSR Fortessa BD biosciences). Non-stained controls were
used to evaluate the autofluorescence of cells and untreated
cell controls were labeled with annexin and propidium
iodide to evaluate the cell death of untreated cells. The
population of cells in each quadrant was determined using
the software FlowJo x10.0.7 (Zhang et al. 2013).
The anti-leukemic activity of the synthetic compounds was
evaluated by a colorimetric assay using MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide)
(Sigma, St. Louis, MO, USA) (Park et al. 1987b). Cytotoxic
activity was assessed in the human acute monocytic leuke-
mia cell line THP-1 (ATCC# TIB-202), human chronic
myelogenous leukemia cell line K562 (ATCC# CCL-243)
and Peripheral Blood Mononuclear Cells (PBMCs). PBMCs
were isolated from the peripheral blood obtained from 3
healthy individuals (The institutional Ethics Committee of
Federal University of Minas Gerais approved this study, and
informed consent was obtained from these control subjects)
by Ficoll gradient centrifugation (Ficoll-Paque Plus, GE
Healthcare Life Sciences) at room temperature. PBMC were
collected, washed and resuspended in RPMI-1640 medium
containing 10% of fetal bovine serum. THP-1, K562 and
PBMC cells were placed in 96-well plates (1 × 106 cells per
well, after cellular expansion and five maximum passages).
After incubation for 24 h at 37 °C in a humidified atmo-
sphere containing 5% CO2, the wells were washed with the
culture medium (RPMI-1640 + 20% inactivated fetal bovine
serum + 2 mmol/L L-glutamine) and incubated with Control
(Cytarabine—Meizler UCB Laboratories; Imatinib—EMS
S/A) and compound samples at concentrations ranging from
0.10 to 250.00 μM. After 48 h of incubation, the plates were
treated with MTT (5 mg/mL). All assays were performed in
triplicate. The optical density (OD) of the 96-well plate
contents was measured using a Spectramax plus 384
(Molecular Devices, Sunnyvale, CA, USA) spectro-
photometer microplate reader at 550 nm. The OD value of
formazan formed in untreated control cells was taken as
100% viability. Cytotoxicity was scored as the percent
reduction in absorbance versus untreated control cultures
(Park et al. 1987a). All experiments were performed in tri-
plicate. The results are expressed as the mean of the IC50
(the lethal drug concentration that reduced cell viability by
50%). The IC50 values were calculated using OriginPro
8.0 software (OriginLab Corporation, Northampton, MA,
USA). The selectivity index (SI) of the 3-APA analogs),
corresponding to the ratio between the cytotoxic activities of
each compound against the THP-1 and K562 cell lines and
the cytotoxicity against PBMCs, was calculated as follows:
SI = IC50 PBMC/IC50 tumor line.
Cell cycle analysis
The application of flow cytometry for the quantification of
DNA in cell suspensions, using PI (BD biosciences, cat #
556547, USA), allows the distribution of cells in the dif-
ferent phases of the cell cycle, G0/G1, S and G2/M, to be
evaluated at any given moment; the fluorescence intensity
of the IP is proportional to the amount of DNA at
certain times.
For the cell cycle evaluation, 1 × 106 cells/tube were
incubated with 1 mL of 70% ethanol (1 × PBS) at 4 °C for
30 min. The sample was then centrifuged (5 min at 300 × g)
and resuspended in 0.2 ml PSSI solution (1.4 μl 1% Triton
X-100, 20 μl RNAse (20 mg), 60 μl IP 2 mg/mL, 10 mL qsp
of 1 × PBS) and incubated at 4 °C for 20 min. The obtained
suspension was then evaluated on the flow cytometer (LSR
Fortessa BD biosciences) using an excitation wavelength of
488 nm; the filter used for the emission reading was 585/
42 nm, relative to the excitation wavelengths and IP emis-
sion, with 20,000 events being evaluated in each analysis
(M AWTAR KRISHAN (From the Sidney Farber Cancer
Center and Harvard Medical School, Boston, Massachusetts
02115 In mammalian cell cycle studies 1975).
Gene expression analysis—Comparative real-time
PCR
Total RNA was extracted with Trizol LS reagent (Invitro-
gen, EUA) according to the manufacturer’s instructions.
RNA was quantified by the Nanovue plus spectro-
photometer (GE Healthcare Life Sciences) and the quality
was assessed by gel electrophoresis.
Complementary DNA (cDNA) was synthesized from
1.5 µg of RNA in a total volume of 20 µL using the High-
Capacity cDNA Reverse Transcription Kit, respectively
(Life Technologies®, Carlsbad, California, USA).
Analysis of apoptosis/necrosis by the Annexin V/
propidium iodide method
To evaluate cells in apoptosis, the Annexin V-FITC
Apoptosis Detection kit (BD biosciences, cat # 556547,
USA) was used following the manufacturer’s instructions
strictly by the flow cytometry method.
Real-time PCR assays of the tumor suppressor protein
p53 (TP53), cyclin-dependent kinase inhibitor 1A (p21),

Medicinal Chemistry Research
BCL2 homologous antagonist/killer (BAK), B-cell lym-
phoma 2 (Bcl-2), NADPH oxidase 1 (NOX-1), NADPH
oxidase 2 (NOX-2), NADPH oxidase 4 (NOX-4), neutrophil
cytosolic factor 1 (p47-phox) and human glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) were carried out in
duplicate in a 10 μL volume using SYBR Green (Thermo
Fisher Scientific Inc., Asheville NC), and the following
primers: TP53 (5′-TGCAGCTGTGGGTTGATTCC-3′)-(5-
AAACACGCACCTCAAAGCTGTTC-3′); p21 (5′-TGAG
CCGCGACTGTGATG-3′)-(5′- GTCTCGGTGACAAAGT
CGAAGTT-3′); BAK (5′-TCTGGCCCTACAC-3′)-(5′-
ACAAACTGGCCCAACAGAAC-3′); Bcl-2 (5′-TCCGCA
TCAGGAAGGCTAGA-3′)-(5′-AGGACCAGGCCTCCA
AGCT-3′); NOX-1 (5′-GCCTGTGCCCGAGCGT CTGC-
3′)-(5′-ACCAATGCCGTGAATCCCTAAGC-3′); NOX-2
(5′-GGAGTT TCAAGATGCGTGGAAACTA-3′)-(5′-GC
CAGACTCAGAGTTGGAGATGCT-3′); NOX-4 (5′-GT
CATAAGTCATCCCTCAGA-3′)-(5′-TCAGCTGAAAGA
CTCTTTAT-3′); p47-phox (5′-TTGAGAAGCGCTTCG
TACCC-3′)-(5′-CGTCAAACCACTTG GGAGCT-3′); and
GAPDH (5′-GGTCGGAGTCAACGGATTTG-3′)-(5′-AT
GAGCCCCA GCCTTCTCC AT-3′). Relative TP53, p21,
BAK, Bcl-2, NOX-1, NOX-2, NOX-4 and p47-phox mRNA
expression levels were calculated from normalized ΔCT
(cycle threshold) relative to the housekeeping gene
(GAPDH). For the detection of changes in gene expression,
normalized ΔCT values for each sample were used. The
obtained values were converted to a linear scale (2-ΔΔCT)
and reported as fold-change in expression (arbitrary
units).
Western blotting
THP-1 and K562 cells were washed with PBS and whole-
cell extracts were prepared as previously described (Vago
et al. 2012). Protein amounts were quantified with the
Bradford assay reagent from Bio-Rad (Bio-Rad, Redmond,
WA, USA). Extracts were separated by electrophoresis on
denaturing 10% polyacrylamide-SDS gels and electro-
transferred to nitrocellulose membranes, as described pre-
viously (Lima et al. 2017). Membranes were blocked for 2 h
at room temperature with PBS containing 5% (w/v) nonfat
dry milk and 0.1% Tween-20. After, the membranes were
washed three times with PBS containing 0.1% Tween-20
and then incubated with the specific primary antibody anti-
PARP (Cell Signaling Technology #9542) and normalized
to anti-β-actin (Sigma #A5316) using a dilution of 1:1000 in
PBS containing 5% (w/v) BSA and 0.1% Tween-20. After
washing, membranes were incubated with appropriated
horseradish peroxidase-conjugated secondary antibody
(1:3000). Immunoreactive bands were visualized using the
ECL detection system, as described by the manufacturer (GE
Healthcare, Piscataway, NJ, USA). The values of cleaved
PARP (89 kDa fragment) were quantified by using a den-
sitometric analysis software (ImageJ, Image Processing and
Analysis in Java; National Institutes of Health, Bethesda,
MD). Changes in protein levels were estimated, and results
are expressed as cleaved PARP in arbitrary units, and nor-
malized to the values of b-actin in the same sample
Statistical analysis
The real-time PCR assay was performed on a StepOne
Real-Time PCR System (Applied Biosystems®) thermo-
cycler under the following conditions: 95 °C for 10 min, 40
cycles of 15 s at 95 °C and 60 °C for 1 min. At the end of
the process the stage of the dissociation curve is increased
(95 °C for 15 s).
Cell viability results were expressed as the mean standard
deviation (Shapiro-Wilk normality test) and each experi-
ment was performed in triplicate and repeated at least three
times. The IC50 values were calculated using the OriginPro
8.0 program (OriginLab Corporation, Northampton, MA,
USA). The results of the gene expression were analyzed
using the program “Sigma Stat” version 2.03 (Systat Soft-
ware I 2013). For the data that presented normal distribution
was used analysis of variance (ANOVA) followed by
Tukey multiple comparison test. For those who did not
present normal distribution, it was the KrusKal-Wallis non-
parametric test followed by the Holm-Sidak multiple com-
parison test. A significant value of p < 0.05 was considered.
measurement of reactive oxygen species (ROS)
2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) and
dihydroethidium (DHE) fluorescent probes were used to
measure the intracellular generation of hydrogen peroxide
(H2O2) and superoxide anions, respectively. Briefly, 1 × 106
THP-1 or K562 cells were plated in 6-well plates and
allowed to attach overnight. Cells were treated or not with
3-APA analogs and then reacted with 5 μM of H2DCF-DA
or 2 μM of DHE for 30 min at 37 °C. Cells were collected
and fluorescence was analyzed by flow cytometry. The first
flow cytometric analysis of cell suspension was performed
in the absence of the selective fluorescence probe for
reactive oxygen species to verify the basal fluorescence of
blank samples. The analyses of data acquired by the flow
cytometer were collected for 10,000 events.
Results and discussion
Cell viability assay
In order to evaluate the in vitro cytotoxic activity of the
compounds, MTT assays were performed on the human cell
lines THP-1, K562 (tumor lines) and PBMC (non-tumoral),

Medicinal Chemistry Research
Table 1 In vitro cytotoxic
activity of new 3-APA analogs
against THP-1 and K562
cell lines
IC50 (μM) SD*
SI
Compound
THP1
K562
PBMC
THP1
K562
4a
59.17 5.08
20.36 2.82
33.53 2.02
ND
88.78 6.23
28.12 2.44
49.78 5.20
92.33 8.97
ND
126.06 4.64
42.85 3.57
136.34 5.10
225.14 10.55
ND
2.13
2.10
4.06
ND
1.42
1.52
2.73
2.43
ND
4b
4c
4d
5a
ND
ND
5b
22.11 3.33
27.19 2.46
ND
140.55 4.33
74.09 8.36
ND
192.37 6.54
215.93 6.50
246.86 10.62
77.10 4.84
148.61 5.76
72.21 4.73
194.63 4.59
156.78 10.41
75.47 5.10
81.37 9.69
48.51 5.61
240.90 8.05
180.72 10.45
159.34 7.87
8.70
7.94
ND
1.37
2.91
ND
5c
5d
6a
59.71 2.61
48.26 4.52
85.2 5.88
37.99 6.88
182.82 16.87
20.25 2.82
39.41 3.77
22.06 2.95
244.87 6.45
40.75 4.45
ND
91.62 6.69
118.68 13.70
136.53 14.61
194.25 6.90
ND
1.29
3.07
0.84
5.12
0.85
3.72
2.06
2.19
0.98
4.43
ND
0.84
1.25
0.53
1.01
1.35
2.01
3.75
3.15
1.10
2.21
4.60
6b
6c
6d
13
7b
37.71 0.61
21.66 2.47
15.37 2.34
217.83 7.10
81.53 4.87
34.58 4.2
7c
11
12
Cytarabine
Imatinib
*Values presented as mean standard deviation. ND Not determined. SI selectivity index
using cytarabine and imatinib as standard cytotoxic com-
pounds. Table 1 shows the cytotoxicity and selectivity test
data in 48 h.
effects of marine alkaloids against leukemia cell lines,
especially the 3-APA alkaloid (Mioso et al. 2017).
According to the observed results, it was possible to
establish a relationship between the IC50 values and the
chemical structure of the compounds (Table 1 and Fig. 1). It
is noted that with a radical change of the protecting group,
such as carboxylic acid (compound 4c), to a hydroxyl group
(compounds 5b and 5c) or to a thiocyanate group (com-
pounds 7c and 11), the biological effect increased sig-
nificantly (p < 0.05), mainly correlating compound 4c with
the change to thiocyanate. In relation to the molecules that
have a subtle difference in the chemical structure, specifi-
cally the amount of carbon in the alkyl chain, it was
observed that the compounds with ten carbon atoms in the
alkyl chain, compounds 5b and 11, had the lowest values of
IC50 for the two tumoral strains. In addition, compound 5b
exhibited the highest SI of the series, 8.70 for the THP-1
lineage. Compounds with a carbonyl group on the radical
were also evaluated, and compound 6d, which has five
carbons in the alkyl chain, showed a significant (p < 0.05)
increase in cytotoxic activity and SI.
It was observed that for the THP-1 line, in 48 h the
compounds 4c, 5b, 5c and 6d, whose IC50 values were
33.53, 22.11, 27.19, and 37.99 μM, respectively, were
comparable and more selective than the standard used,
cytarabine (40.75 μM), a compound which has already been
used as a chemotherapeutic agent (p < 0.05). In the K562
line, the most effective and selective compounds were 15
(21.66 μM) and 16 (15.37 μM) (p < 0.05), compared to
imatinib (34.58 μM). Thus, these compounds demonstrated
a better cytotoxic potential compared to the cellular models
studied (Table 1). The parent nucleus of 3-alkylpyridine
marine alkaloid (3-Pyridinepropanol) showed no cytotoxic
activity in the leukemic strains (THP-1 and K562) and
PBMC (Table 1).
In an earlier study, our research group evaluated the
in vitro cytotoxic potential of different 3-APA analogs in
human colorectal cancer and cervical cancer cell lines and
found very promising results (Gonçalves et al. 2014). Dif-
ferent classes of alkaloids have been described in the lit-
erature with similar in vitro cytotoxic effects in several cell
lines (Gomathi and Gothandam 2016; Yao et al. 2016).
However, few studies have demonstrated the cytotoxic
Thus, compounds 4c, 5b, 5c, 6d, 7c and 11 were shown
to be strongly cytotoxic and selective for the leukemic
strains and were therefore selected for evaluation of the
mechanism of action.

Medicinal Chemistry Research
Fig. 1 Synthesis of 3-Alkylpyridine Alkaloid Analogs 4a-d, 5a-d, 6a-d, 7b-c, 11, 13 and the parent nucleus 3-Pyridinepropanol 12
Cell death induced by test compounds in leukemic
cells
Apoptosis pathway involves the activation of a family of
cysteinyl-aspartate proteases (caspases). Once activated,
caspases are able to cleave PARP (Pavithra et al. 2018).
DNA damage promotes an increase of PARP activity
(Chaitanya et al. 2010). However, caspases may inactivate
PARP with apoptosis induction, impairing the ability of
PARP to repair damaged DNA (Zhang et al. 2017).
Thus, to further delineate the mechanism by which
compounds induced apoptosis in THP-1 and K562 cells,
Western blotting assay was performed, revealing remark-
ably increased the PARP cleavage (Fig. 2). Several studies
agree with our results, which demonstrated that the increase
in PARP cleavage is linked to the induction of apoptosis
through the caspase-dependent pathway (Pavithra et al.
2018).
In order to characterize the cell death induced by the
compounds, tests of Annexin-V and Propidium Iodide were
performed by flow cytometry, which allowed the quantifi-
cation of cells in the different stages of cell death.
After analysis of the cells by flow cytometry, a graph of
the number of cells found with each type of labeling was
obtained. Each of the quadrants obtained represents a dif-
ferent cell stage. The upper left quadrant (Q1) represents
cells in necrosis, the upper right quadrant (Q2) denotes cells
in late apoptosis, the lower right quadrant (Q3) represents
cells in apoptosis, and the lower left quadrant (Q4) indicates
viable cells. Apoptosis was the type of major cell death
induced by all alkaloids (compounds 4c, 5b, 5c, 6d, 7c and
11) within 48 h (Fig. 1S). Clearly, approximately 50% of
the cells were killed by apoptosis (treatment dose used was
IC50). The expressed results were considered statistically
significant (p < 0.05) when compared with the viable cell
control group (without treatment).
The results confirm that the synthetic 3-APA alkaloids
induced the cells to undergo apoptosis and are therefore
promising for the study of new chemotherapeutics. Other
authors have also reported that different marine alkaloids
induced leukemic cell apoptosis (Wang et al. 2016b; Pala-
nisamy et al. 2017) and other tumor types (Dyshlovoy et al.
2017; Habli et al. 2017).
Effect of synthetic marine alkaloids on the cell cycle
Molecular studies of carcinogenesis and apoptosis have
shown that cell cycle regulation plays a role in the trans-
formation of tumor cells and the development of resistance
to chemotherapeutic agents (Schwartz and Shah 2005).
These observations led to the investigation of the effect of
synthetic molecules on the cell cycle in leukemic cells
(Florean et al. 2016).
Similarly, in order to elucidate a possible mechanism of
action of the synthetic 3-APA alkaloids in the cell cycle, the
effect of the same after 48 h of incubation with the IC50
concentration in different phases of the cell cycle was
evaluated in this study.
The Annexin V/PI assay revealed that the compounds
induced apoptosis in the leukemic cells, which was further
confirmed by the increased expression of Poly(ADP-ribose)
polymerase (PARP) cleavage (Fig. 2).
In comparison to the control group, THP-1 cells treated
with the compounds 5c and 6d induced cell cycle arrest in

Medicinal Chemistry Research
Fig. 2 Effect of compounds 4c, 5b, 5c, 6d, 7c and 11 against K562 and
THP-1 cells. THP-1 and K562 line were transferred to a 96-well plate
at a density of 50,000 cells per well. Subsequently, cells were either
left untreated, or treated with compounds 4c, 5b, 5c, 6d, 7c and 11 for
48 h. a and f—PARP were assessed by western blot analysis. Blots
were normalized with β-actin and are representative of three inde-
pendent experiments using pooled cells from at least four wells; b, c, e
and f—Densitometry data for PARP are represented graphically. *p <
0.05; **p < 0.01; and ***p < 0.001 when compared to the positive
control group
Fig. 4 Effect of 3-APA alkaloids analogues on the cell cycle against
K562 cells. K562 cells were incubated with the alkaloids (Compounds
7c and 11) and with imatinib, used as a positive control. The SubG0,
G0/G1, S and G2/ M phases were determined by DNA content ana-
lysis after labeling with IP and analyzed in flow cytometry. The per-
centage of cells in each phase was quantified by the FlowJo
x10.0.7 software
Fig. 3 Effect of 3-APA alkaloids analogues on the cell cycle against
THP1 cells. THP1 cells were incubated with the alkaloids (Com-
pounds 4c, 5b, 5c and 6d) and with cytarabine, used as a positive
control. The SubG0, G0/G1, S and G2 / M phases were determined by
DNA content analysis after labeling with IP and analyzed in flow
cytometry. The percentage of cells in each phase was quantified by the
FlowJo x10.0.7 software
the S phase (Fig. 3) and compounds 4c, 5b, 5c and 6d
induced block also G2/M phase. The K562 line treated with
molecules 7c and 11 showed a similar profile to the positive
control (imatinib) with a predominance in the S phase and
G0/G1 (Fig. 4). The SubG0 phase corresponds to cells in
apoptosis. The positive control, cytarabine, induced cell
cycle arrest in the S phase, as expected, since the
mechanism of action of this drug is anti-metabolic in that it
exerts its effects mainly by blocking the S phase of the cell
cycle.
The CKIs (cyclin inhibitory kinases) act by blocking
CDKs and inducing cell cycle arrest. The p21, p27 and p57
proteins are important CKIs that act to inhibit cell pro-
liferation (Wiman and Zhivotovsky 2017). From this, CKIs
may be related to cell cycle arrest in G0/G1 or S seen in cell
lines after incubation with the test molecules.

Medicinal Chemistry Research
In THP-1 cells, proliferation blockade caused an increase
in the proportion of cells in the S and G2/M phases of the
cell cycle and a corresponding decrease in G0/G1 phase
cells (Fig. 3). The K562 strain also had a block in the G0/
G1 phase, but there was no decrease in the S phase (Fig. 4).
In general, this means that progression to the S-phase of the
cycle in these cell lines is prevented by the treatments.
of the apoptosis signal, the anti-apoptotic proteins involved
are inhibited; however, if the inhibition signal does not
persist, they are expressed.
The compounds also induced a strong decrease in p21
expression. The p21 protein is a kinase-dependent cyclin
inhibitory protein, which participates in cell cycle regula-
tion, and acts by inducing cell cycle arrest (Schwartz and
Shah 2005; Kumar et al. 2015; Wiman and Zhivotovsky
2017). However, the p21 protein may also act in an anti-
apoptotic manner by inhibiting caspase-3 cleavage and
inhibiting the stress pathways JNK1/SAPK and MAPK
ASK1/MEKK5 (Kumar et al. 2015). Additionally, a high
expression of the p21 protein in the cytoplasm could lead
the cells to become resistant to apoptosis (Shen et al. 2007).
Our results demonstrate that compounds 4c, 6d, 7c and 11
induced a reduction or non-alteration of p21 expression at
48 h, possibly thus facilitating the activation of caspase-3
and consequently apoptosis (Figs. 5, 6) Compounds 5b and
5c, however, showed a statistically significant increase in
the expression of p21 with the IC50 concentration in 48 h
(Figs. 5, 6).
In general, a dose-response relationship was noted
between the expressions at the same treatment time, in
which some discrepancies in the expression at the IC50
concentration were noted (Figs. 5, 6).
These data suggest that the compounds induce a p53-
independent pathway to activate apoptosis, showing that
these complexes possibly interfere with DNA replication,
probably by interacting directly with DNA. These in vitro
results suggest that the compounds are a potential prototype
of a DNA-damaging anticancer agent against AML and
CML. Other studies suggest that alkaloid derivatives have a
cytotoxic effect on cancer cells regardless of p53 status
(Benton and Ravandi 2017).
Most of the conventional anticancer drugs induce cell
death, with the main focus of action being the induction of
cellular stress and DNA lesions (Mahalingam et al. 2009).
Thus, to date, the data presented in this work indicate that 3-
alkylpyridine alkaloid analogues are a potential class of
compounds with cytotoxic action in vitro, showing DNA
damage through the activation of apoptosis via the p53-
independent pathway. Thus, the investigation of the
induction of apoptosis, for example via Reactive oxygen
species (ROS), becomes interesting to explain the
mechanism of action of the test molecules. Facing this
scenario, the screening of new compounds becomes an
extremely complex and challenging but indispensable task.
Evaluation of gene expression by qPCR
In order to determine which signaling pathway was acti-
vated in apoptosis in the leukemic cells treated with the
selected compounds, gene expression analysis of genes
involved in this pathway was performed by a quantitative
PCR assay. In Figs. 5, 6, the relative expressions of each
target gene relative to the control cells (considered as 1.0)
are shown, using GAPDH as the normalizing gene.
A common feature in many tumor cells is the presence of
the TP53 gene. However, in many leukemia cell lines, the
lack of expression of the TP53 gene is identified (Benton
and Ravandi 2017). The mechanism responsible for the
silencing of this gene in myeloid tumors remains unknown.
The p53 protein is an important cell transcription factor
involved in the induction of apoptosis (activation of the Bax
gene, whose protein inhibits the anti-apoptotic action of the
Bcl-2 protein) and also in the activation of the p21 protein,
which results in the G1 phase of the cell cycle being stopped
(Dyshlovoy et al. 2017).
The cellular response to DNA damage involves the
transcription of the TP53 gene, which is responsible for the
induction of the G1/S checkpoint of mitosis and pro-
apoptotic molecules. However, THP-1 and K562 cell lines
show a deficiency in TP53 expression (Figs. 5, 6) (Hu et al.
2013). In leukemia models, the detection of mutations and
chromosomal deletions at this locus is associated with
partial or total resistance to treatment both in vitro and
in vivo (Yao et al. 2016; Benton and Ravandi 2017). In this
sense, treatment should be focused on p53-independent
pathways.
Thus, the absence of TP53 expression observed 48 h
after exposure to treatments was expected. There was also
only a slight or no change in the expression of all other
target genes (p21, Bak and Bcl2) after 48 h (Figs. 5, 6).
For Bak levels, no significant difference was observed in
relation to the control cells in any of the treatments (Figs. 5,
6). Bak, a key pro-apoptotic signaling regulator, showed
elevated levels related to tumor suppression in an apoptotic
p53-independent pathway. However, in some studies, the
low expression of Bak was able to induce apoptosis by
activating caspase 3 in gastric cancer cells in vitro in a p53-
independent pathway (Kubo et al. 2015).
Analysis of ROS
ROS can induce proliferation, apoptosis, and gene expres-
sion changes (Zhang et al. 2017). Cytotoxic drugs used in
leukemia therapy has as mechanism of action the alteration
In addition, these alkaloids either decreased or did not
alter Bcl-2 expression, which suggests that upon initiation

Medicinal Chemistry Research
Fig. 5 Effects of the compounds 4c(I), 5b(II), 5c(III) and 6d (IV) on
the expression of target genes against THP-1 cell line. a Relative
quantification of the TP53 gene, b p21, c Bak and (#) Bcl2. Cells (1 ×
106) were incubated with compound 4c for 48 h. Each bar represents
the mean standard deviation (n = 3) of relative quantification. The
data were expressed in relation to the control of cells without treatment
considered as 1. Value of *p < 0.05 was considered statistically sig-
nificant when compared to the control without treatment

Medicinal Chemistry Research
Fig. 6 Effecst of the compounds 7c(I) and 11(II) on the expression of
target genes against K562 cell line. a Relative quantification of the
TP53 gene, b p21, c Bak and d Bcl2. Cells (1 × 106) were incubated
with the compound 7c for 48 h. Each bar represents the mean
standard deviation (n = 3) of relative quantification. The data were
expressed in relation to the control of cells without treatment con-
sidered as 1. Value of *p < 0.05 was considered statistically significant
when compared to the control without treatment
of the DNA replication process of the malignant cells
(Antoszewska-smith et al. 2017). Oxidants compounds are
used for the treatment of hematological malignancies,
despite their negative effects (Chen et al. 2017). An increase
in ROS can induce cell death, lipid peroxidation, protein
oxidation and mutation, increased mitochondrial stress and
apoptosis, and activation of a G2/M phase cell cycle
checkpoint (Chen et al. 2017).
Pro-oxidant treatment and malignant cells can cause
depletion of antioxidant defenses, inducing increased pro-
duction of ROS (Trachootham et al. 2009). However, if
oxidative species are duplicated by the treatment action,
malignant cells may become sensitized and prolong treat-
ment or activate the apoptosis pathway (Al. HDH et 2008).
ROS can induce damage to the genetic material and
oxidize proteins and lipids, leading to cell death (Liu et al.
2016). Various ROS-generating drugs have proven to be
effective in cancer, due to its action of provoking an
increase in ROS levels, which causes damage to cellular
components and the activation of cellular signaling path-
ways (Chen et al. 2017).
cells lines, there was a significant increase in the DHE
marker in relation to the negative control (no-treatment). In
the case of H2DCFDA, there was a significant increase in
the level of ROS in THP-1 and K562, compared to the
negative control, while for molecule 7c, there was an
increase, but it was not significant (Fig. 2S). All analogous
molecules of alkaloids of the 3-alkylpyridine showed levels
of ROS which were similar to or even greater than the
positive control (phorbol myristate acetate) and the tradi-
tional drug (cytarabine and imatinib).
Main ROS are generated by the NOX family of NADPH
oxidases (NOX) which are regulatory molecules cell growth
and migration (Liu et al. 2016). The three NOX homo-
logues, namely NOX1, NOX2 and NOX4, have been
identified in mammalian cells (Chen et al. 2017) and the
p47phox is a cytosolic regulatory subunit of NOX (Antos-
zewska-smith et al. 2017).
Each NADPH oxidase seems to have a particular pattern
of expression, and most cells express different members of
this family. The importance of these enzymes in cancer is
beginning to be recognized and, interestingly, it has recently
been shown that NADPH oxidase inhibitors are efficient at
preventing the growth of cancer. This suggests that NADPH
oxidases could be exploited as therapeutic targets in the
cancer treatment setting.
Many antitumor agents, such as vinblastine, cisplatin,
mitomycin C, doxorubicin, camptothecin and neocarzinos-
tatin, exhibit antitumor activity through the activation of
ROS-dependent apoptotic cell death, suggesting the poten-
tial use of ROS as an cytotoxic agent (Chen et al. 2017).
Thus, an anticancer strategy called “oxidation therapy” was
developed by the induction of cytotoxic oxidative stress for
the treatment of cancer.
Therefore, the genetic expression of NOX1, NOX2,
NOX4 and p47phox is shown in Figs. 3S, 4S.
In the THP-1 line, there was a significant increase in the
calibration of NOX-1 (5b, 5c and cytarabine), NOX-2 (5b,
5c, 6d and cytarabine), NOX-4 (4c, 5b, 6d and cytarabine)
and p47phox (4c, 5b, 5c, 6d and cytarabine) (Fig. 3S). In the
K562 line, a significant increase in gene expression was
The basal fluorescence intensity emitted by H2DCFDA
and DHE cell lines treated with the compounds was
increased when compared to the control (Fig. 2S). In both

Medicinal Chemistry Research
observed for the NOX-1 (7c, 11 and imatinib), NOX-2 (11
and imatinib), NOX-4 (7c and 11) and p47phox (7c, 11 and
imatinib) genes (Fig. 4S).
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In this way, the increase in oxidative stress in cells
produce oxidized lipids, proteins, and damaged DNA,
which would consequently lead to cell death. Thus, the
analogous molecules of alkaloids of the 3-alkylpyridine are
in some way inducing the production of ROS, thereby
causing DNA damage, which contributes to the cytotoxic
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Our study shows that the selected compounds induced
apoptosis and it was observed that the pathway being acti-
vated is independent of p53. Overall, the data presented in this
work indicate that 3-alkylpyridine alkaloid analogues are a
potential class of compounds with cytotoxic action in vitro.
Acknowledgements The authors are grateful to Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq), Fundação de
Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Coor-
denação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
and Pro-Reitoria de Pesquisa (PRPq) da Universidade Federal de
Minas Gerais, for sponsoring this study.
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Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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