Discovery of cytotoxic and pro-apoptotic compounds against leukemia cells: Tert-butyl-4-[(3-nitrophenoxy) methyl]-2,2-dimethyloxazolidine-3-carboxylate

Article abstract of DOI:10.1016/j.lfs.2011.09.012

Aims: We evaluated biological activity in leukemia cells lines of R and S enantiomers of tert-butyl 4-[(3-nitrophenoxy)-methyl]-2,2-dimethyloxazolidine-3- carboxylate (BNDC). Main methods: Cytotoxic activity was assessed by MTT assay. Flow cytometry assays were used to determined DNA fragmentation (Propidium Iodide-PI staining) and phosphatidylserine exposure (Annexin-V and PI staining). DNA condensation was evaluated by fluorescence microscopy using double-staining in leukemia cells (Hoechst and PI). Caspase activities were measured using Z-VAD-FMK, a non-selective caspase inhibitor, by flow cytometry and Z-DEVD-AMC, a selective caspase-3 substrate, by fluorescence spectrometry. Key findings: Both enantiomers displayed cytotoxic activity against leukemia cell lines (HL60, HL60.Bcl-2, HL60.Bcl-XL and Jurkat) with low toxicity against human peripheral blood mononuclear cell - PBMC based on IC₅₀ values. In HL60 cell lines, compounds induce exposure of phosphatidylserine and DNA fragmentation, which could be blocked by pretreatment of cells with Z-VAD-FMK. Confirming this observation, both enantiomers induced caspase-3 activation. Additional analysis revealed an increased percentage of apoptotic cells (defined as those with fragmented nuclei and condensed chromatin) after treatment with compounds. Significance: Taken together, the results indicate that BNDC compounds exhibited cytotoxic and pro-apoptotic activities and have a potential for developing a new class of anticancer drugs.

Full text of DOI:10.1016/j.lfs.2011.09.012

Life Sciences 89 (2011) 786–794
Contents lists available at SciVerse ScienceDirect
Life Sciences
journal homepage: www.elsevier.com/locate/lifescie
Discovery of cytotoxic and pro-apoptotic compounds against leukemia cells:
Tert-butyl-4-[(3-nitrophenoxy) methyl]-2,2-dimethyloxazolidine-3-carboxylate
Mauro C.X. Pinto a, Danielle F. Dias b, Helen L. Del Puerto a, Almir S. Martins a, Andréa Teixeira-Carvalho c,
Olindo A. Martins-Filho c, Bernard Badet d, Philippe Durand d, Ricardo J. Alves b, Elaine M. Souza-Fagundes a,
⁎
a
Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, 31270-901, Belo Horizonte-MG, Brasil
Faculdade de Farmácia, Universidade Federal de Minas Gerais, 31270-901, Belo Horizonte-MG, Brasil
Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz, 30190-002, Belo Horizonte-MG, Brasil
b
c
d
Institut de Chimie des Substances Naturelles-CNRS, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 May 2011
Accepted 27 August 2011
Aims: We evaluated biological activity in leukemia cells lines of R and S enantiomers of tert-butyl 4-[(3-
nitrophenoxy)-methyl]-2,2-dimethyloxazolidine-3-carboxylate (BNDC).
Main methods: Cytotoxic activity was assessed by MTT assay. Flow cytometry assays were used to deter-
mined DNA fragmentation (Propidium Iodide-PI staining) and phosphatidylserine exposure (Annexin-V
and PI staining). DNA condensation was evaluated by fluorescence microscopy using double-staining in
leukemia cells (Hoechst and PI). Caspase activities were measured using Z-VAD-FMK, a non-selective cas-
pase inhibitor, by flow cytometry and Z-DEVD-AMC, a selective caspase-3 substrate, by fluorescence spec-
trometry.
Keywords:
Apoptosis
Drug discovery
Hit compound
Scaffold
Key findings: Both enantiomers displayed cytotoxic activity against leukemia cell lines (HL60, HL60.Bcl-2,
HL60.Bcl-XL and Jurkat) with low toxicity against human peripheral blood mononuclear cell — PBMC
based on IC50 values. In HL60 cell lines, compounds induce exposure of phosphatidylserine and DNA frag-
mentation, which could be blocked by pretreatment of cells with Z-VAD-FMK. Confirming this observation,
both enantiomers induced caspase-3 activation. Additional analysis revealed an increased percentage of
apoptotic cells (defined as those with fragmented nuclei and condensed chromatin) after treatment
with compounds.
Anti-cancer
Significance: Taken together, the results indicate that BNDC compounds exhibited cytotoxic and pro-apoptotic
activities and have a potential for developing a new class of anticancer drugs.
© 2011 Elsevier Inc. All rights reserved.
Introduction
of apoptosis is another drug activity indicator in cancer cells (Hickman,
1992; Makin and Hickman, 2000; Li et al., 2006).
The investigation of the biological activities of novel synthetic
compounds is the first step in the discovery of new drugs (Keseru and
Makara, 2006; Braña and Sánchez-Migallón, 2006). The discovery
process begins with screening a large number of compounds selected
from chemical libraries to identify new hit compounds with biological
activity (Langer et al., 2009). New series of compounds structurally
related to these hit compounds are evaluated for their structure–activity
relationship to select a prototype or lead compound (Bleicher et al.,
2003). In the discovery of drugs for cancer treatment, in vitro cytotoxic-
ity assays are important for identifying new hit compounds. Once a
new compound is identified, it is necessary to investigate its biological
action mechanisms (Keseru and Makara, 2006), which include DNA
polymerase or topoisomerase inhibition, and DNA alkylation. Induction
Programmed cell death (apoptosis) is a natural process for remov-
ing unwanted cells, such as those with potentially harmful mutations,
aberrant substratum attachment, or alterations in cell-cycle control
(Fesik, 2005). The classical apoptosis are divided in two well de-
scribed pathways: extrinsic and intrinsic. In the extrinsic pathway,
endogenous factors like tumor necrosis factor and TRAIL binding on
‘death receptors’ such as TRAIL receptors and Tumor Necrosis Factor
Receptor 1 and trigger apoptosis by activating the initiator protease
caspase-8, which directly activates the effector protease caspase 3
(Hengartner, 2000). Intrinsic pathway is initiated by cellular stress
which leads the mitochondria to release cytochrome c, apoptotic pro-
tease activating factor-1 and AIF into the citosol (Hengartner, 2000).
This mitochondrial release of proteins is controlled by Bcl-2 family,
which is composed of anti-apoptotic (e.g. Bcl-2, Bcl-XL, MCL-1) and
pro-apoptotic (e.g. Bax, Bid, Bak) proteins, that regulate the mem-
brane potential of mitochondria and endoplasmic reticulum, being
crucial to transmit pro-apoptotic signals to cell death (Vander Heiden
⁎
Corresponding author. Tel./fax: +55 31 3409 2941.
E-mail address: elainefagundes@ufmg.br (E.M. Souza-Fagundes).
0024-3205/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2011.09.012

M.C.X. Pinto et al. / Life Sciences 89 (2011) 786–794
787
and Thompson, 1999; Janumyan et al., 2003). Once in the cytoplasm,
the procaspase-9, cytochrome c and apoptotic protease activating fac-
tor-1 form an apoptosome complex that activates caspase-9, which in
turn activates the caspase effectors. The active caspase effectors such
as caspases 3, 6 and 7 are responsible for most of the morphological
changes related to the phenomenon of apoptosis, such externaliza-
tion of phosphatidylserine (PS), chromatin condensation and DNA
fragmentation (Wang et al., 2005; Van Noorden, 2001; Ghavami
et al., 2009).
Over the past decade, substantial advances have been made in the
field of apoptosis-based therapeutics. Evasion has been identified as
one of the main processes associated with the pathophysiology of
cancer, which is thus associated with the aberrant regulation of regu-
lators and effectors of apoptotic death (Fleischer et al., 2006). Most of
the changes in proteins involved in cell death signaling are often re-
sistant to chemotherapy and are more difficult to treat using chemo-
therapeutic agents that act primarily by inducing apoptosis (Fesik,
2005). Therefore, the ability to understand and manipulate the ma-
chinery of cell death is a clear goal of medical research. Apoptosis-tar-
geting therapies are now advancing from preclinical/clinical trials to
actual application. This is a remarkable achievement, considering
that apoptotic cell death is a relatively recent discovery (Fischer and
Schulze-Osthoff, 2005; Jana and Paliwal, 2007).
Synthesis of (R)-tert-butyl 4-(3-(nitrophenoxy)methyl)-2,2-dimethyl-
oxazolidine-3-carboxylate (3)
Diethyl azodicarboxylate (0.26 mL, 1.65 mmol) was added to a mix-
ture of 3-nitrophenol (0.215 g, 1.54 mmol), alcohol 6 (R enantiomer)
(0.24 g, 1.04 mmol) and PPh3 (0.43 g, 1.65 mmol) in toluene (12 mL)
at room temperature under an argon atmosphere and the reaction mix-
ture was stirred for 7 h at 80 °C. The solution was cooled to room tem-
perature and washed with 1.0 M NaOH solution (20 mL) and then H2O
(3×20 mL). The organic phase was dried over MgSO4, filtered and
evaporated under reduced pressure. The residue was purified by flash
chromatography on silica gel (n-hexane/EtOAc 9:1) to give the desired
3 (0.29 g, 81%). Melting point: 59.6–60.1 °C. [α]D20 −70.3 (c 1.0, CHCl3).
HRMS (ESI+) calculated for C17H24N2O6Na (M+Na), 375.1532. Found:
375.1540. 1H NMR (500 MHz, DMSO-d6, 363 K) δ 1.45 (s, 9H, t-butyl);
1.48 (s, 3H, CH3); 1.55 (s, 3H, CH3); 3.96–4.24 (m, 5H H-4, H-5, H-6);
7.43–7.45 (d, 1H, J12–11=8.0 Hz, H-12).; 7.58 (t, 1H, J11–12=J11–10
=
8.0 Hz, H-11); 7.74 (bs, 1H, H-8); 7.80 (d, 1H, J10–11=8.0 Hz, H-10).
13C NMR (125 MHz, DMSO-d6, 363 K) δ 23.3 (CH3), 26.2 (CH3); 27.5
((CH3)3C); 55.1(C-4); 64.4 (C-5); 67.5 (C-6); 79.2 ((CH3)3C); 92.9 (C-
2); 108.9, (C-8); 115.2 (C-10); 121.3 (C-12); 130.1 (C-11); 148.6 (C-
9); 150.9 (C_O); 158.6 (C-7).
Using this background, we performed the screening of a series of
compounds from an in-house chemical library against four cancer
cell lines to identify new cytotoxic compounds that target the apopto-
tic machinery. Four chemically related compounds displayed cytotox-
icity against Jurkat and HL60 cells. The structures of compounds (1–4)
are shown in Fig. 1. To investigate drug resistance, we used the HL60
cell line ectopically expressing anti-apoptotic proteins that include
Bcl-2 and Bcl-XL since these proteins are often over-expressed in var-
ious human cancers, thereby conferring drug resistance (Brumatti
et al., 2003). The values of IC50 indicated that R and S enantiomers
of Tert-butyl-4-[(3-nitrophenoxy)methyl]-2,2-dimethyloxazolidine-
3-carboxylate — BNDC (compounds 3 and 4) were more active in
the HL60 cell line, with minor toxicity against normal human periph-
eral blood mononuclear cells — PBMC . These compounds induced
DNA fragmentation, phosphatidylserine exposure, morphological
changes and caspase-3 activation, suggesting that apoptosis was the
mechanism most likely to be involved. Thus, our data point to BNDC
as cytotoxic molecules that could provide a new scaffold for apoptosis
inducers useful in the development of a novel class of anti-cancer
agents.
Synthesis of (S)-tert-butyl 4-[(3-nitrophenoxy)methyl]-2,2-dimethyl-
oxazolidine-3-carboxylate (4)
Compound 4 (0.21 g, 73%) was prepared from the alcohol 6 (S en-
antiomer, 0.19 g, 0.82 mmol) under the same conditions and reagent
proportions as described above in preparing 3. Melting point: 59.2–
60 °C. [α]D20 73.3 (c 1.0, CHCl3). HRMS (ESI+) calculated for
C
17H24N2O6Na (M+Na), 375.1532. Found: 375.1534. 1H NMR and
13C NMR spectra of 4 were identical to those of 3, as expected.
Leukemia cell lines
HL60 cells (human promyelocytic leukemia HL-60 cells), HL60.Bcl-
2 and HL60.Bcl-XL (HL60 cells ectopically expressing Bcl-2 and Bcl-XL,
respectively) and human immortalized line of T lymphocyte (Jurkat
cells) were provided by Gustavo Amarante-Mendes, PhD (São Paulo
University, Brazil). All lineages were cultivated to log phase in RPMI
1640 Sigma-Aldrich (St. Louis, MO), supplemented with 100 U/mL
penicillin and 100 μg/mL streptomycin (GIBCO BRL, Grand Island,
NY), enriched with 2 mM of L-glutamine and 10% fetal bovine serum.
All cultures were maintained at 37 °C in a humidified incubator with
5% CO2.
Material and methods
Synthetic route
Compounds 1 and 2 were prepared as described elsewhere (Dias
et al., 2009). In the same vein, compounds 3 and 4 were prepared as
follows. Initially, the commercially available protected serine 5 (R or
S enantiomer) was reacted with LiBH4 in THF/CH3OH at −10 °C to
give the corresponding alcohol 6 (Garner and Park, 1987). Reaction
of 6 (R or S enantiomer) with 3-nitrophenol under Mitsunobu condi-
tions is described below.
PBMC
The PBMC were prepared using the protocol previously described
by Gazzinelli et al., 1983. Briefly, PBMC samples were obtained
through agreement with Minas Gerais Hematology and Hemother-
apy Center Foundation — HEMOMINAS (protocol no 105/2004)
from healthy adult volunteers of both sexes by centrifugation of
heparinized venous blood over Ficoll cushion (Sigma-Aldrich, St.
Louis, MO). PBMC were collected from the interphase after Ficoll
separation and washed three times in RPMI-1640 before further
processing (Gazzinelli et al., 1983). All cultures were carried out in
RPMI-1640 medium Sigma-Aldrich (St. Louis, MO), supplemented
with 5% (v/v) heat-inactivated, pooled AB (GIBCO/BRL, Grand Island,
NY) sera and 2 mM L-glutamine. An antibiotic/antimicotic solution
containing 1000 U/mL penicillin, 1000 μg/mL streptomycin and
25 μg/mL fungisone (GIBCO/BRL, Grand Island, NY) was added to
control fungal and bacterial contamination.
Fig. 1. Structures of compounds 1–4.

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M.C.X. Pinto et al. / Life Sciences 89 (2011) 786–794
Analysis of cell viability
1.43r (National Institute of Health, USA). The cell nuclei were counted
and classified as normal nuclei, condensed nuclei or fragmented nuclei.
Cells double-stained with HST and PI were considered late apoptotic or
necrotic, depending on the morphology of the nuclei. Apoptotic cells
were defined as HST+/PI-cells with condensed nuclei or fragmented
nuclei (Thuret, et al., 2003).
The leukemia cell lines and PBMC were cultured in 96 wells plate at
densities of 50,000 cells/well and 100,000 cells/well, respectively, in a
final volume of 200 μL/well. The plates were pre-incubated in a 5%
CO2/95% air-humidified atmosphere at 37 °C for 24 h to allow adapta-
tion of cells prior to the addition of the test compounds. All substances
were dissolved in dimethyl sulfoxide (DMSO) prior to dilution. The
half maximal inhibitory concentration (IC50) was determined over a
range of concentrations (10 nM–100 μM). All cell cultures were incu-
bated in a 5% CO2/95% air-humidified atmosphere at 37 °C for 48 h.
Cell viability was estimated by measuring the rate of mitochondrial re-
duction of yellow tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide; Sigma-Aldrich, St. Louis, MO) to in-
soluble purple formazan crystals (Mosmann, 1983). After incubation
with the test compounds, MTT solution (20 μL; 5 mg/mL) was added
to each well and incubated for 4 h. Afterward, the supernatant was re-
moved and 200 μL of 0.04 M HCl in isopropyl alcohol were added to
dissolve the formazan crystal. The optical densities (OD) were mea-
sured with a spectrophotometer at 590 nm. Results were normalized
with DMSO control (0.05%) and expressed as a percentage of cell via-
bility inhibition. Interactions of compounds and media were esti-
mated on the basis of the variations between the drug-containing
medium and drug-free medium to control for false-positive or false-
Evaluation of phosphatidylserine externalization
Apoptotic cells were detected using an Annexin-V FITC Apoptosis Kit
(Invitrogen Corporation, USA) and differentiated into viable, early, and
late apoptotic, as previously described (Martin, et al., 1995). Briefly, the
cells were treated with compounds (50 μM) and incubated for 24 h at
37 °C in an atmosphere of 5% CO2. The cells were washed with PBS and
then resuspended in Annexin-V Binding Buffer (10 mM HEPES/NaOH,
pH 7.4, 140 mM, NaCl, 2.5 mM CaCl2). They were double-stained with
an Annexin-V FITC and PI buffer and incubated at room temperature
for 15 min in the dark. Following incubation, the cells were analyzed in
FACScalibur flow cytometer (BD Biosciences) and the data analyzed by
FlowJo 7.5.5 (TreeStar Inc, CA).
Detection of caspase activity
Caspase activation was assessed by pre-treatment with
benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK),
a broad-spectrum caspase inhibitor. In assays of DNA fragmentation
inhibition, cells were incubated for 40 min with 40 μM Z-VAD-FMK
(Biomol, Research Laboratories, Plymouth Meeting, PA, USA) before
being treated with enantiomers at 50 μM. After 24 h of incubation,
the DNA content was evaluated as previously described. Control cul-
tures were treated with the appropriate amount of DMSO (0.05%).
Caspase-3 activation was determined by EnzChek® Caspase-3 Assay
Kit #1 (Molecular Probes, USA). Briefly, the cells were treated with a
lysis buffer and incubated on ice for 30 min. The lysed cells were cen-
trifuged at 5000 rpm for 5 min in a microcentrifuge and the superna-
tant collected. Microplate wells were filled with 50 μL of the
supernatant from each sample and 50 μL of substrate working solution
with 200 μM of Z-DEVD-AMC. The samples were incubated in the dark
at room temperature for 30 min. Afterward, the plate was analyzed in
a fluorescence spectrophotometer (Cary Eclipse, Varian, USA) with ex-
citation and emission wavelengths of 342 nm and 482 nm,
respectively.
negative results. The half maximal inhibitory concentration (IC50
)
values were obtained graphically from dose–effect curves using
Prism 5.0 (GraphPad Software Inc.). The cytotoxicity of cisplatin was
evaluated under the same experimental conditions for comparison.
DNA fragmentation assay
Cell cycle status and quantification of DNA fragmentation (hypo-
diploid DNA-content) were performed by propidium iodide (PI) stain-
ing according to Nicoletti et al. (1991). HL60 cells were treated with
the four compounds at the final concentration of 50 μM and incubated
in a 5% CO2/95% air-humidified atmosphere at 37 °C for 24 h. After-
ward, the cells were centrifuged, resuspended in a hypotonic fluoro-
chrome solution — HFS (50 μg/mL PI in 0.1% sodium citrate plus 0.1%
Triton X-100) and incubated at 4 °C for 4 h. The PI fluorescence of
20,000 individual nuclei was measured using a FACScalibur flow
cytometer (Becton Dickinson Immunocytometry Systems, San Jose,
Calif.). Data were analyzed using FlowJo software 7.5.5 (TreeStar Inc, CA).
Investigation of DNA condensation by Hoechst staining
RNA isolation, reverse transcription and quantitative real-time PCR
After incubation with the compounds (50 μM), nuclei were double-
stained with Hoechst 33342 (HST) and PI. The cells were examined by
fluorescence microscopy (Carl Zeiss, Germany) with two filters (DAPI
fluorescent filter, excitation 340–380 nm, barrier filter 430 nm and a
rhodamine filter, excitation 530–560 nm, barrier filter 580 nm). For
each experiment, three microscopic fields were photographed with an
AxioCam MRm ZEISS (Carl Zeiss, Germany) and results were averaged.
For each field, one photograph of HST and one of PI were taken for com-
parison. The images were analyzed and merged using Image J software
Total RNA was isolated from HL60 cells using 1 mL of Trizol® reagent
according to the protocol provided by the manufacturer (Invitrogen Life
Technologies, Carlsbad, CA, United States of America — USA). RNA sam-
ples were treated with Turbo DNA-free kit (Ambion Inc., Foster, CA,
USA). First-strand complementary DNA (cDNA) was synthesized from
2 μg total RNA using the Superscript first-strand synthesis system (Invi-
trogen Inc., Carlsbad, CA, USA). After denaturing the RNA template and
primers (25 pmol of each reverse oligonucleotide primer) at 70 °C for
10 min, 40 U reverse transcriptase was added in the presence of RT buffer
Table 1
Sequences of primers used in quantitative RT-PCR.
Primers
Nucleotide sequence
Size
Fragment size
80 bp
GenBank access number
NM_138765.3
BaxForw
BaxRev
Bcl2Forw
Bcl2Rev
BclXlForw
BclXlRev
GapdHForw
GapdHRev
5′-TCTACTTTGCCAGCAAACTGGTGC-3′
5′-TGTCCAGCCCATGATGGTTCTGAT-3′
5′-ATTTCCTGCATCTCATGCCAAGGG-3′
5′-TGTGCTTTGCATTCTTGGACGAGG-3′
5′-ATGACCAGACACTGACCATCCACT-3′
5′-ATGTAGTGGTTCTCCTGGTGGCAA-3′
5′-TTCCAGGACCAAGATCCCTCCAAA-3′
5′-ATGGTGGTGAAGACACCAGTGAAC-3′
24 nt
24 nt
24 nt
24 nt
24 nt
24 nt
24 nt
24 nt
90 bp
99 bp
86 bp
NM_000657
NM_001191
XM_001725661

M.C.X. Pinto et al. / Life Sciences 89 (2011) 786–794
789
Table 2
(50 mM KCl, 20 mM Tris–HCl, pH 8.4), 4 μL dNTP mix (250 μM each),
40 U RNase inhibitor and RNase-free water to complete the final volume.
The reaction mixture (50 μL) was incubated at 43 °C for 1 h, then stopped
at 4 °C and used immediately for PCR or kept at −80 °C until use.
Real-time PCR was carried out in an ABI Prism 7500 Sequence De-
tection System (Applied Biosystems, Foster City, CA, USA), using the
Power Sybr® Green Master Mix Kit (Invitrogen Life Technologies,
Carlsbad, CA, USA). Experiments were performed in two independent
experiments, and each sample run in duplicate in each experiment.
Samples were run on 96-well optical PCR plates in a final reaction vol-
ume of 25 μL. The PCR parameters were 1 cycle at 50 °C for 2 min, 1
cycle at 95 °C for 10 min, 40 cycles at 95 °C for 15 s and 60 °C for 1 min.
The primers used for PCR amplification of Bax, Bcl-2, Bcl-XL and
Gapdh, are listed in Table 1. Gapdh (Glyceraldehyde-3-Phosphate De-
hydrogenase) gene was used as a reference gene to normalize target
gene expression. Specific primers were designed using the sequences
obtained in GeneBank, through the Blast program (http://www.ncbi.
nlm.nih.gov/blast/blast.cgi). Subsequently, all sequences were designed
and analyzed using the Integrated DNA Technologies website program
(http://www.idtdna.com).
Concentrations of investigated compounds that induced 50% decrease (IC50) in malig-
nant and peripheral blood mononuclear cell survival.
Cell lines
1
2
3
4
Cisplatin
HL60
HL60.Bcl-2
HL60.Bcl-XL N100 78.21 6.62 N100
Jurkat
PBMC
N100 75.25 3.82 44.82 5.30
N100 N100 N100
12.22 2,13
25.66 0.52
12.20 5.55
13.61 2.30
1.69 0.44
5.77 3.98
4.91 3.00
2.24 0.66
N100 74.44 5.66 25.12 5.03
N100 43.31 7.26 71.61 14.94 60.46 11.24 31.87 14.54
Leukemia cells lines and PBMC were incubated with drugs in concentrations ranging
from 0.1 to 100 μM, at 37 °C in 5% CO2 atmosphere for 48 h. Viability was
determined by MTT assay. Each data represents mean SD from three (leukemia
cells) or seven (PBMC) independent experiments, performed in triplicate. Cisplatin
was used as positive control.
a
like) and HL60 cells (human promyelocytic leukemia cells), along
with two cell lines HL60.Bcl-2 and HL60.Bcl-XL (HL60 cells with ectopic
expression of Bcl-2 and Bcl-XL, respectively) known to be resistant to
anti-cancer drugs. Cytotoxic effects on normal cells were evaluated
using PBMC from healthy donors. The IC50 values for the reduction of
cell viability by these compounds using tumors and normal cells are
summarized in Table 2.
HL60 cells were more susceptible to treatment with compounds
than HL60.Bcl-2 and HL60.Bcl-XL, which ectopically express anti-
apoptotic proteins, but Jurkat cells were as susceptible as the
HL60 cells. Compound 1 was inactive in the tested concentration
for all cell lines. Compound 2 displayed cytotoxic effects on at
least one resistant lineage, while compound 4, as the most active
compound, was active against all the lineages. The compound 4 pre-
sented similar IC50 values against HL60, HL60.Bcl-XL and Jurkat cell
lines that were significantly smaller than those observed against
HL60.Bcl-2 (Student T-test, Pb0.05). Compound 3 displayed cyto-
toxic effects on Jurkat and HL60, but it was inactive in HL60.Bcl-2
and HL60.Bcl-XL. Similar profile was obtained for cisplatin, IC50
value against HL60 cells was three-times lower than those observed
against HL60.Bcl-2 and HL60 Bcl-XL cells. Cytotoxic activity differ-
ences between compounds 3 and 4 on Jurkat, HL60, HL60.Bcl-2
and HL60.Bcl-XL indicate an isomer-dependent effect (Student's t-
test, Pb0.05).
The PCR results were analyzed based on the ΔCT, which is the pri-
mary source of data variability. Relative gene expression was calculat-
ed by using the 2−ΔΔCT method, where CT is the threshold cycle.
Statistical analysis
All results were expressed as the mean SD of three independent
experiments carried out, at least, in triplicate. These data were analyzed
using Student's t-test for paired comparisons between compound and
DMSO control. One-way ANOVA test was used to analyze target gene ex-
pression. Results were considered statistically significant when Pb0.05.
Results
Synthetic route for preparation of BNDC
The compounds 1 and 2, R and S tert-butyl 4-((3-hydroxyphenoxy)
methyl)-2,2-dimethyloxazolidine-3-carboxylate — BHDC, were prepared
as described elsewhere (Dias, et al., 2009). Using a similar strategy, com-
pounds 3 and 4 (BNDC) were prepared, according to the synthetic route
shown in Scheme 1. Initially, the commercially available protected serine
5 (R or S enantiomer) was reacted with LiBH4 in THF/CH3OH at −10 °C to
give the corresponding alcohol 6 (Garner and Park, 1987; Meffre, et al.,
1994). Reaction of 6 (R or S enantiomer) with 3-nitrophenol under Mitsu-
nobu conditions at 80 °C produced 3 or 4, respectively (Pave, et al., 2003).
When considering the BNDC compounds, the IC50 values for tumor
cells were lower than those for normal PBMC. Compound 4 was ap-
proximately five times less cytotoxic to PBMC than to HL60 and Jurkat
cells, and for compound 3, the corresponding IC50 values were ap-
proximately two-times lower. Cisplatin was around fifty-times less
cytotoxic to PBMC than to leukemia cells (HL60 and Jurkat).
Cytotoxic activity of BNDC
Apoptotic nuclear changes induced by BNDC
To evaluate the antitumor potential of our compounds, we used two
wild-type leukemia cell lines, Jurkat cells (human T cell lymphoblast-
To investigate whether apoptosis was triggered by treatment with
the new compounds, DNA fragmentation and chromatin condensation
Scheme 1. Synthetic scheme for preparation of BHDC (compounds 1 and 2) and BNDC (compounds 3 and 4). Reagents and conditions: a) LiBH4, THF, MeOH, −10 °C; b) 3-benzoylphenol,
DEAD, PPH3, toluene, 80 °C; c) sodium methoxide/methanol, 0 °C; d) 3-nitrophenol, DEAD, PPH3, toluene, 80 °C.

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M.C.X. Pinto et al. / Life Sciences 89 (2011) 786–794
Table 3
in a marked increase in secondary necrosis in the apoptotic population
containing single or multiple clumps of rounded, bright and condensed
chromatin within a pale cytoplasm, different of control cell nuclei that
exhibited diffused and homogeneous staining of the chromatin. The re-
sults revealed that the morphological features characteristic of apoptosis
(chromatin condensation and nuclear fragmentation) was present in
HL60 cells following exposure to compounds 3 and 4 or to cisplatin.
DNA fragmentation induction by compounds 1–4 in leukemia cell lines.
Substances
HL60
Jurkat
Control
Cisplatin
1
2
3
4
6.70 3.41
92.22 2.48
16.99 2.37
17.90 3.44
71.78 17.78
65.97 15.72
5.37 2.37
47.64 11.00
5.27 2.69
3.30 1.17
7.02 4.44
11.98 6.18
⁎
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⁎
⁎
HL60 and Jurkat cells were incubated with 50 μM of each compound, at 37 °C in a 5%
CO2 atmosphere for 24 h. DNA content was assessed by flow cytometry analysis of
cells labeled with propidium iodide. Each data represents mean SD from 3 different
experiments.
Apoptotic membrane changes induced by BNDC
The discrimination between cell death by necrosis and apoptosis is
important for understanding the mechanism of action involved in the
cytotoxicity of new compounds. For this purpose the exposure of PS
was investigated. Under normal conditions, these phospholipids are
only present in the inner leaflet of the plasma membrane, but after
oxidative disordering it appears also in the outer side of the mem-
brane (Martin, et al., 1995).
Control cells presented 90.84 1.22% of cell without labeling, different
than found to compound 3 (70.71 8.52%) and compound 4 (72.16
5.20%) after 24 h of incubation (Student T-test, Pb0.05). Treatment of
cells with enantiomers 3 and 4 (50 μM) increased the proportion of
cells in late apoptosis (20.4% and 22.1% of Annexin-V⁄PI double-positive
cells, respectively), being similar effects observed with cisplatin treatment
under the same conditions, as shown in Fig. 3.
⁎
Pb0.05, Student T-test.
was evaluated after 24 h of incubation with 50 μM of compounds
(Nicoletti, et al., 1991; Nagata, 2000). Significant increases in DNA
fragmentation were detected after 24 h of treatment with compounds
3 and 4 (50 μM) in HL60 cells (Pb0.05, Student's t-test), but corre-
sponding increases were not observed in Jurkat cells (Table 3). Com-
pounds 1 and 2 were clearly less potent than compounds 3 and 4 in
HL60 cells.
Consistent with these results, morphological changes in cell nuclei
were observed in HL60 cells after treatment with BNDC compounds, as
shown in Fig. 2. The treatment of cells with enantiomers 3 and 4 resulted
Fig. 2. BNDC compounds induce morphological alterations in HL60 cells. Cells were incubated with compounds 3 and 4 (50 μM) or cisplatin for 24 h. The double staining cell with
Hoechst 33342 and propidium iodide distinguishes some typical features of apoptosis such as condensed chromatin (white arrows) and fragmented nuclei (yellow arrows). Overlap
of the two images allows the detection of double-labeled cells suggesting late apoptosis at this time.

M.C.X. Pinto et al. / Life Sciences 89 (2011) 786–794
791
Fig. 3. BNDC compounds induce phosphatidylserine exposure in HL60 cells. The cells were incubated with the enantiomers 3 and 4 at the concentration of 50 μM for 24 h. The cells
were stained with annexin V-FITC and propidium iodide. Analyses were performed by flow cytometry. Each data represents mean SD from three independent experiments
(*Pb0.05, T-test Student).
Caspase-dependent activity induced by BNDC
caspase-3-like activity 4.36- and 2.48-fold, when compared with con-
trol, respectively (Pb0.05, Student T-test). Cisplatin was included as a
positive control (Fig. 5). Our results demonstrated that cell death in-
duced by BNDC compounds involved activation of the classical path-
way of apoptosis.
Having demonstrated that the cytotoxic effects of enantiomers 3 and
4 on HL60 cells induced DNA fragmentation, chromatin condensation
and externalization of phosphatidylserine, we next investigated whether
the compounds induce a caspase-dependent apoptotic pathway. HL60
cells were pretreated with Z-VAD-FMK (40 μM) for 40 min and then
treated with BNDC compounds (50 μM) and incubated for 24 h, followed
by PI staining and flow cytometry analyses. As expected, the treatment
with the compounds 3 and 4 significantly increased the subdiploid
DNA content of HL60 cells, when compared with the DMSO control
(Student T-test, Pb0.05). However, cells pretreated with Z-VAD-FMK
and then treated with compounds 3 and 4 showed reduced levels of
subdiploid DNA content (Fig. 4), similar to the levels in the control, sug-
gesting that the compounds induced apoptosis by a caspase-dependent
mechanism.
As caspase-3 has been specifically implicated as the caspase re-
sponsible for DNA fragmentation and chromatin condensation, this
end point was examined in HL60 cells in response to treatment
with compounds 3 and 4. Caspase-3-like activity was measured by
monitoring fluorescence levels generated by proteolytic cleavage of
the Z-DEVD-AMC substrate. HL60 cells were treated with compounds
3 and 4 (50 μM) for 24 h. Exposure to compounds 3 and 4 increased
Changes in mRNA expression of Bcl-2 family induced by BNDC
BNDC enantiomers displayed cytotoxic activity and pro-apoptotic
effects against leukemia cell lines. In order to observe the effect of
BNDC in gene expression profile of Bcl-2 family, a Real-time PCR
was performed to the specific target genes (Bax, Bcl-2 and Bcl-XL)
in HL60 cells lines treated for 24 h with compound 4 in concentra-
tions of 6.25 μM, 12.5 μM, 25 μM and 50 μM, as shown in Fig. 6. Com-
pound 4 induced a concentration-dependent increase in mRNA
expression of Bax and Bcl-XL in HL60 cells lines. Bcl-2 expression of
mRNA decreased after treatment with 50 μM of compound 4. HL60
cells with DMSO vehicle were used as control.
Discussion
The present paper describes the discovery of a new class of com-
pounds with cytotoxic and pro-apoptotic activities. In drug discovery,

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M.C.X. Pinto et al. / Life Sciences 89 (2011) 786–794
100
80
60
40
20
Control
6.7 3.4
M1
100
101
102
103
104
100
100
80
60
40
20
100
80
60
40
20
Cisplatin
3
4
80
60
40
20
92.2 2.4*
(-) Z-VAD-FMK
71.7 17.7*
65.9 15.7*
M1
M1
M1
100
101
102
103
104
100
101
102
103
104 100
100
80
101
102
103
104
100
80
60
40
20
100
80
60
40
20
Cisplatin
3
4
9.0 3.5
10.2 2.7
10.9 2.0
M1
Z-VAD-FMK (40µM)
60
M1
M1
40
20
100
101
102
103
104
100
101
102
103
104 100
101
102
103
104
Propidium Iodide
Fig. 4. BNDC compounds induce caspase-dependent apoptosis in HL60 cells. HL60 cells were pretreated with 40 μM of Z-VAD-FMK for 40 min and incubated with compounds 3 and
4 for 24 h. The DNA content was assessed by staining with PI and flow cytometry analysis. Cells containing subdiploid DNA (fragmented DNA) is indicated as M1 in each histograms.
Cisplatin, positive control is demonstrated. Each data represents mean SD from three independent experiments (*Pb0.05, T-test Student).
low toxicity and high efficacy are the main criteria in selecting hit com-
pounds as candidates for drug development (Keseru and Makara, 2006;
Langer et al., 2009). To demonstrate that the new BNDC compounds are
useful hit compounds with anticancer potential, we evaluated their
cytotoxicity toward cancer and normal cell lineages. The BNDC enantio-
mers are the first representative compounds of a new class with high
potential, since the exchange of a hydroxyl group by the nitro group sig-
nificantly increases (up to five-fold) the cytotoxicity effectiveness of
these compounds. Furthermore an enantiomer-dependent effect was
observed in BNDC and BHDC compounds, which suggest site-specific ef-
fects of these compounds. In the present study a relevant finding is that
cytotoxicity effects of BNDC and BHDC against HL60 cells are greater
than for HL60. Bcl-2, as we know the stable expression of these anti-
apoptotic proteins in HL60 cells converted this apoptosis-sensitive cell
line into resistant cells against anticancer drugs (Brumatti et al.,
2003). These results strongly suggest that cytotoxicity effect induced
by the BHDC and BNDC compounds are involved with apoptosis. Inter-
estingly, the ectopic expression of Bcl-XL did not alter the cytotoxic
activity of compounds 2 and 4. According to these data, 50 μM of com-
pound 4 induces an increase in mRNA expression of Bcl-XL, but this
phenomenon does not seem to influence the cytotoxic effects in HL60
cells. On the other hand, this treatment presents downregulation of
Bcl-2 and upregulation of Bax mRNA expression, which suggest a differ-
ential activity through Bax/Bcl-2 pathways by BNDC. In normal cells,
Bcl-XL preferentially associates with Bak, while Bcl-2 associate with
Bax to trigger apoptosis, being a differential action in Bax/Blc-2 pathway
a good strategy to develop new anticancer drugs against cancer cells
with Bcl-XL overexpression (Willis et al., 2005; Park et al., 2004; Liu
et al., 2005).
Fig. 5. BNDC compounds induce caspase-3 activation in HL60 cells. The cells were incu-
bated with the enantiomers 3 and 4 in the concentration of 50 μM for 24 h. The fluores-
Based on these data, it was evaluated more closely the phenome-
non of apoptosis triggered by the cytotoxic effect of the compounds
against the two susceptible lineages, HL60 and Jurkat. With regard
to apoptotic nuclear morphology, the DNA condensation and
cence generated by exposure to substrate Z-DEVD-AMC was quantified in
a
fluorescence spectrophotometer. Each data represents mean SD from 3 different ex-
periments (*Pb0.05, T-test Student).

M.C.X. Pinto et al. / Life Sciences 89 (2011) 786–794
793
Fig. 6. Compound 4 induces change in mRNA expression of Bax, Bcl-2 and Bcl-XL in HL60 cells. HL60 were treated with different concentrations of compound 4 (50 μM, 25 μM,
12.5 μM and 6.25 μM) and incubated for 24 h. Data are plotted as mean SD of 2−ΔΔCT, which is directly proportional to the relative gene expression **Pb0.001, ***Pb0.0001
(One-way ANOVA test).
fragmentation, these events occurred in HL60 cells when treated with
BNDC and BHDC compounds, being the nitro compounds the most
potential apoptotic inductor. The Jurkat cells demonstrated to be re-
sistant to DNA fragmentation after 24 h of incubation with com-
pounds, being the differences between HL60 and Jurkat apoptosis
with apoptosis inducers also observed in other works (Reis et al.,
2010; Kizawa et al., 2006). Karpinich et al. (2002) demonstrated
that Jurkat cells lack expression of two critical pro-apoptotic proteins
responsible for induction of apoptosis by intrinsic pathway: Bax and
P53. The HL60 cells, also lack p53 protein, but normally express
Bax protein (Brumatti et al., 2003; Koeffler et al., 1986; Shimizu and
Pommier, 1997). Thus, the pro-apoptotic activity observed for these
compounds possible depend on Bax protein to trigger apoptosis,
which explains the greater resistance to DNA fragmentation in Jurkat
cells. Although no difference in DNA fragmentation was found after
treatment with both enantiomers 3 and 4, a relevant observation
was that the peak characteristic of cells in G0/G1 phase was preserved
in the treatments with both compounds, which was not observed in
the control of cisplatin. Cisplatin causes DNA damage when interact-
ing with DNA forming interstrand cross-link and protein–DNA
cross-link, which may lead to expression of p53 protein and G1
phase cell cycle arrest and apoptosis, in the early phase of this phe-
nomenon and is dose-dependent (Gonzalez et al., 2001). In our ex-
periments, cisplatin was used at higher concentration (50 μM) and
DNA measurement was performed after 24 h, where total DNA frag-
mentation is observed. In cells treated with BNDC, it is remarkable
that viable cells were the majority after incubation, which corrobo-
rates with G0/G1 peak found in the analysis of DNA content measure-
ment. The compounds seem to act in synthesis phase and/or G2/
Mitose, which is characteristic of substances that act during cell divi-
sion or causing DNA damage (Karpinich et al., 2002; Gonzalez et al.,
2001).
Furthermore, the DNA fragmentation triggered by compounds 3
and 4 was related to the activation of caspases, once the caspase in-
hibitor Z-VAD-FMK was able to block this effect, indicating that cas-
pase cascades were involved in the apoptotic cell death induced by
the new enantiomers. Consistent with these results, compounds 3
and 4 promoted caspase-3 activation. Caspase-3 is activated specifi-
cally during apoptosis (in the intrinsic and extrinsic pathways) and
is one of the most important markers of apoptotic activity. These en-
zymes normally remain in the cytoplasm of cells as pro-enzymes, are
particularly activated in the presence of a pro-apoptotic stimulus and
are responsible for the major cellular degradation found in the nucle-
us and in the cytoplasm (Thornberry, 1998; Wang et al., 2005). A pro-
apoptotic signal activates the initiator caspases, such as 8, 9 and 10,
which in turn, activate effector caspases such as caspases 3, 6 and 7
that bring about most of the morphological changes related to apo-
ptosis (Wang et al., 2005; Van Noorden, 2001). In addition, com-
pounds 3 and 4 showed more activity in induction of caspase-3
compared to cisplatin after 24 h of treatment. These results point to
different kinetics of apoptosis induction. At this time, cisplatin cell
control is in the end of the apoptosis process and the most part of
cells present total DNA fragmentation, which is associated with late-
phase of apoptosis. As observed by the caspase activity, there are dif-
ferences on activation of caspase-3, that is time-course and cell-type
dependent (Kolfschoten et al., 2002; Gonzalez et al., 2001). Consistent
with these data, analysis of phosphadidilserine exposure to measure
necrosis/apoptosis ratio, demonstrated that HL-60 cells treated with
cisplatin presents large number of cells in late apoptosis and necrosis
differently from BNDC, that can be related to a distinct mechanism of
action of cisplatin (Gonzalez et al., 2001). Likewise, caspase-3 activa-
tion also induces cleavage of lamin and nuclear mitotic apparatus
proteins that maintain the structural integrity of the nucleus (Ziegler
and Groscurth, 2004). Added to all of our findings, analysis of nuclear
morphology using Hoechst 33258 and PI staining in HL60 treated
with BNDC and cisplatin, corroborates with activation of caspase-de-
pendent apoptotic machinery.
Even though the new compounds 3 and 4 demonstrated pro-
apoptotic activity in our assays at 50 μM, there are screening pro-
grams designed to discover new hit compounds from assays at
higher concentrations. Keseru and Makara (2006) described that
hit compounds could be detected by high-throughput screening
(HTS) techniques, in which, single-point experiments are typically
performed with small molecules at concentrations of 1–50 μM with
a 30–50% activity cutoff to identify potential hit compounds. In
agreement with this report, Severson et al. (2008) screened 84,000
compounds against influenza virus at 114 μM and several high-activ-
ity compounds were revealed. The authors performed a structure–
activity relationship (SAR) analysis of compounds to identify the
most promising to lead compounds for further design and synthesis.
The evaluation of targeted analogs of various molecular scaffolds
should help to identify compounds exhibiting greatly improved po-
tency and selectivity, which can be subsequently, developed into
clinically useful therapeutic agents. In this context, the enantiomers
3 and 4 are interesting compounds that can be further modified by
simple chemical manipulation in order to improve activity (Keseru
and Makara, 2006; Bleicher et al., 2003; Li et al., 2006). Although
the exact mechanism(s) by which these compounds can activate
the apoptotic cascade in HL60 cells remains to be elucidated, our re-
sults provide a clear correlation between DNA fragmentation, mor-
phological alterations and caspase activation consistent with a pro-
apoptotic potential of the new enantiomers.
Conclusion
In conclusion, through this work we have identified new cytotoxic
agents with pro-apoptotic activity, preferentially toward tumor cell
division. Furthermore, our results suggest enantiomer-specific effects

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M.C.X. Pinto et al. / Life Sciences 89 (2011) 786–794
Kizawa Y, Furuya M, Saito K, Masuko T, Kusama T. Effects of dexamethasone and ami-
nophylline on survival of Jurkat and HL-60 cells. Biol Pharm Bull 2006;29(2):
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Koeffler HP, Miller C, Nicolson MA, Ranyard J, Bosselman RA. Increased expression of
p53 protein in human leukemia cells. Proc Natl Acad Sci 1986;83(11):4035–9.
Kolfschoten GM, Hulscher TM, Duyndam MCA, Pinedo HM, Boven E. Variation in the ki-
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and that relatively small changes in the structure of these molecules
could lead to new compounds with improved cytotoxic and pro-apo-
ptotic activity. Studies in this direction are already under way and will
be reported in due course.
Conflict of interest
Langer T, Hoffmann R, Bryant S, Lesur B. Hit finding: towards [‘]smarter’ approaches.
Curr Opin Pharmacol 2009;9(5):589–93.
There are no competing interests.
Li W, Lam MS, Birkeland A, Riffel A, Montana L, Sullivan ME, et al. Cell-based assays for
Acknowledgment
profiling activity and safety properties of cancer drugs.
Methods 2006;54(3):313–9.
J Pharmacol Toxicol
Liu JJ, Huang RW, Lin DJ, Peng J, Wu XY, Lin Q, et al. Expression of survivin and bax/bcl-2
in peroxisome proliferator activated receptor-Î3 ligands induces apoptosis on
human myeloid leukemia cells in vitro. Ann Oncol 2005;16:455–9.
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Martin SJ, Reutelingsperger CP, McGahon AJ, Rader JA, van Schie RC, LaFace DM, et al.
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Bcl-2 and Abl. J Exp Med 1995;182(5):1545–56.
This work was supported by grants of CAPES, CNPq and FAPEMIG
(Brazil). The authors thank the program for technological develop-
ment in tools for health — PDTIS-FIOCRUZ for use of its facilities.
ATC and OAMF thank CNPq for fellowships (PQ).
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