In vitro apoptotic activity of 2,2-diphenyl-1,3,2-oxazaborolidin-5-ones in L5178Y cells

Article abstract of DOI:10.1016/j.lfs.2006.11.034

Compounds containing B-N bonds have shown interesting biological activity. One class of such molecules is the 2,2-diphenyl-1,3,2-oxazaborolidin-5-ones (3a-j), which contain a B-N bond, have an α-amino acid moiety in the heterocycle, and have an exocyclic

Full text of DOI:10.1016/j.lfs.2006.11.034

Life Sciences 80 (2007) 1007–1013
www.elsevier.com/locate/lifescie
In vitro apoptotic activity of 2,2-diphenyl-1,3,2-oxazaborolidin-5-ones in
L5178Y cells
Benjamín Velasco a,c, José G. Trujillo-Ferrara a, Luis H. Fabila Castillo b,
René Miranda c, Luvia E. Sánchez-Torres b,
⁎
a
Departamento de Bioquímica y Sección de Estudios de Posgrado e Investigación, Escuela Superior de Medicina-IPN, México D.F., C.P. 11340, México
b
Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas-IPN, México D.F., C.P. 11340, México
c
Departamento de Ciencias Químicas, Facultad de Estudios Superiores Cuautitlán-UNAM, Cuautitlán Izcalli, Estado de México, C.P. 54740, México
Received 15 August 2006; accepted 21 November 2006
Abstract
Compounds containing B–N bonds have shown interesting biological activity. One class of such molecules is the 2,2-diphenyl-1,3,2-
oxazaborolidin-5-ones (3a–j), which contain a B–N bond, have an α-amino acid moiety in the heterocycle, and have an exocyclic moiety related
to an amino acid. The purpose of this work was to determine the inhibitory effects of 3a–j on the proliferation of murine L5178Y lymphoma cells.
A new five-membered heterocyclic nucleus with apoptotic activity was found. The target products showed potent cytotoxicity in the L5178Y cell
line. Among them, 3a exhibited the highest antineoplastic activity in L5178Y cells with an IC50 value of 22.5 0.2 μM.
© 2006 Elsevier Inc. All rights reserved.
Keywords: Apoptosis; Diphenyloxazaborolidinones; Cytotoxicity; Antineoplastic; L5178Y cell
Introduction
Cell growth is regulated by the balance between cell
proliferation and apoptosis. Deregulated cell proliferation and
suppressed cell death together provide the underlying platform
for neoplastic progression (Evan and Vousden, 2001). It is well
known that the regulating mechanisms of apoptosis are
extremely complex, but it is also known that the apoptotic
process varies, depending on whether the cells involved are
tumoral or healthy (Evan and Vousden, 2001; Sanmartín et al.,
2005). Apoptosis results from activation of the genes of the cell,
which in turn activate preprogrammed biochemical pathways
leading to cell death (Fischer, 1994; Wright et al., 1994;
Jacobson et al., 1997). These pathways are all closely related to
the mitochondria alteration that induces changes in electron
transport and loss of mitochondrial transmembrane potential
(Ming-Jie et al., 2004; Shakibaei et al., 2005). After such an
alteration, there is a redistribution of phosphatidylserine in the
extracellular membrane which is an early and common
phenomenon in the process of cell apoptosis (Yuan et al.,
2006). Apoptotic cells are also characterized by cell shrinkage,
skeletal disruption, membrane blebbing, chromatin condensa-
tion and ordered cleavage of DNA (Bursh et al., 1992). Another
important event is the G0/G1 phase arrest, which is a crucial
Cell growth and division are highly regulated, although a
notable exception is provided by the cancer cell, which arises as
a variant that has lost the usual proliferation control pathways.
Consequently, there is growing interest in the search for anti-
cancer substances with high efficacy, low toxicity, and
minimum side effects (Wang et al., 2005). In particular, certain
compounds containing B–N bonds possess broad biological
activity including insecticidal, fungicidal, herbicidal, antibacte-
rial and calcium channel blockers (Morin, 1994; Dembitsky and
Srebnik, 2003; Jabbour et al., 2004; Dobrydneva et al., 2006)
and antineoplastic activities. Recently, the proteosome inhibitor
PS341, an α-amido-boronic acid, was approved as an effective
antineoplastic agent (Adams et al., 1999; Teicher et al., 1999).
Several aminoboron compounds have been used in boron
neutron capture therapy (BNCT) as well as in chemotherapeutic
forms of cancer treatment (Soloway et al., 1998).
⁎
Corresponding author. Tel./fax: +52 55 57296300x62489.
E-mail address: luviasanchez@hotmail.com (L.E. Sánchez-Torres).
0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2006.11.034

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B. Velasco et al. / Life Sciences 80 (2007) 1007–1013
via the previously reported methods (Chremos et al., 1961;
Flückiger et al., 1984; Farfán et al., 1993; Trujillo et al., 1998).
The compounds (3a–j) were assayed using several different
apoptotic indicator models, such as the inhibitory effect on cell
growth measured by the MTT [3-(4,5-dimethylthiazo-2-yl)-2,5-
diphenyl-tetrazolium bromide] assay, phosphatidylserine mem-
brane externalization measured by Annexin V, and DNA
distribution (sub-G0/G1 peak) measured by PI staining, the
last two using flow cytometry. We employed DMD with
positive control (Kosower et al., 1969).
Fig. 1. General structure of 2,2-diphenyl-1,3,2-oxazaborolidin-5-ones 3a–j.
DNA damage checkpoint and acts as an important safeguard for
genomic stability (Chen et al., 2004; Deng et al., 2004).
Apoptotic pathways might be significantly altered in cancer
cells with respect to untransformed cells, and these differences
may represent a therapeutic window that can be explored for the
development of new antineoplastic drugs. There are reports in
the literature of the cytoprotector effects of glycine and its
nitrogen derivatives, evaluated in different models of damage
related to the accumulation of free radicals (Tripathi et al., 2000;
Yan-Jun et al., 2000; Ascher et al., 2001; Jacob et al., 2003;
Tanonaka et al., 2002). Substantial data supports the existence
of a glycine receptor in the membrane of the cell, whose
activation reduces the release of reactive oxygen species (ROS),
thus protecting the cell from damage by the latter (Yan-Jun
et al., 2000; Jacob et al., 2003). This, in turn, maintains the
mitochondrial integrity. Recent data indicated that, when either
the amino or the carboxylic hydroxyl group in glycine is
modified the cytoprotective effect is maintained; whereas, if
both groups are modified, the cytoprotective effect is lost. In the
latter case, specifically in glioma C6 cells, such compounds
induce cell death by apoptosis, as demonstrated by in vitro
assays (Trejo-Solís et al., 2005).
With the aim further investigating glycine-related com-
pounds, we studied the apoptotic activity of a set of ten amino
acid derivatives, 3a–j. Two important elements were taken into
account when devising these experiments: 1) the B–N bond
contained in these compounds has shown biological activity and
2) the glycine fragment has activity as an apoptotic inducer
when both the amino and carboxylic hydroxyl groups have been
modified. Thus, a family of 2,2-diphenyl-1,3,2-oxazaborolidin-
5-ones (DPOXB) was generated in which both functional
groups were modified, forming five member heterocyclic
compounds (Fig. 1). These compounds, which all contain a
B–N bond, were obtained via the reaction of diphenylborinic
acid with the corresponding α-amino acid (Fig. 2) rather than
Materials and methods
Synthesis of 2,2-diphenyl-1,3,2-oxazaborolidin-5-ones (3a–j)
In a typical procedure, 0.5 g (6.6 mmol) of glycine was
dissolved in 3 mL H2O and the pH was adjusted to 8 with
5% NaOH. Diphenylborinic acid was prepared from 1.6 g of
2-aminoethyldiphenylborinate this was dissolved in a mini-
mum amount of ethanol, hydrolyzed by addition of 1 M hydro-
chloric acid with agitation, and the gummy, water-insoluble
diphenylborinic acid is extracted with ether. The ethereal solution
was added in to the glycine solution. The mixture was refluxed for
4 h, then the solvent was slowly evaporated and the white product
was filtered and washed with cold water and hexane to obtain 1.5 g
(6.26 mmol) of 3a. The same methodology was employed to obtain
compounds 3b–j, using the appropriate α-amino acid. All the
compounds obtained by this new methodology were characterized
1
by the following methods: the H-, and 13C-NMR spectra were
determined on a JEOL GSX-270 instrument in DMSO-d6 using
TMS as an internal reference, the 11B-NMR spectra were obtained
on a JEOL FX90Q spectrometer in DMSO-d6 using BF3.OEt2 as
an external reference. The IR spectra were measured on a
MSERIES spectrophotometer using KBr. The physical and
spectroscopic constants were found to be equivalent to those in
the literature from the derivatives generated by the method
previously employed. (Farfán et al., 1993; Trujillo et al., 1998).
Cell line and culture maintenance
For the experiments, 2×106 L5178Y tumor cells (ATCC)
were implanted into the peritoneal cavity of 8- to 10-week-old
BALB/C mice. On day 12 after tumor implantation, the mice
Fig. 2. Synthesis of 2,2-Diphenyl-1,3,2-oxazaborolidin-5-ones amino acid derivatives.

B. Velasco et al. / Life Sciences 80 (2007) 1007–1013
1009
Table 1
Production of 2,2-diphenyl-1,3,2-oxazaborolidin-5-ones 3a–3ja
Table 2
Cytotoxicity assay against murine L5178Y lymphoma cells
DPOXB α-aa
employed
R
m. p. °C % Yield
DPOXB
IC50 (μM)a L5178Y
3a
3b
3c
3d
3e
3f
3g
3h
3i
22.5 0.2
42.7 1.8
30.9 1.1
47.8 1.4
102.7 1.9
N200
38.6 0.6
48.7 0.4
75.1 2.5
44.5 0.9
3a
3b
Gly
R=H
241–243 94
221–223 93
Ile
3c
Arg
244–246 89
3j
3d
3e
3f
Leu
Asp
His
171–174 90
273–275 65
275–277 92
a
Viable L5178Y cells were determined using the MTT assay, as described
previously. The cells were incubated with 3a–j (6.25–100 μM) for 24 h. Control
cells were incubated with DMSO. The results are presented as the mean SD of
data from at least three independent experiments.
Determination of cytotoxicity in murine L5178Y lymphoma
cells using the MTT assay
3g
3h
3i
Val
Ser
Pro
243–245 80
259–260 93
268–269 98
Antiproliferative in vitro screening was performed on the
murine L5178Y lymphoma cells. Cytotoxicity was assessed by
the MTT assay. The L5178Y cells were suspended at a final
concentration of 5×104 /mL, seeded in 96-well microtiter plates
and treated with concentrations ranging from 6.25 to 100 μM of
one of the DPOXB 3a–j dissolved in DMSO. The final
concentration of solvent was less than 0.1% in cell culture
medium. Incubation was carried out at 37 °C for 24 h after
exposure to one of the 3a–j compounds, the cells were incubated
with MTT (0.5 mg/mL) for 4 h. MTT is reduced by the
mitochondrial dehydrogenases of viable cells to a purple formazan
product. The MTT/formazan product was dissolved in 100 μL of
DMSO and the corresponding absorbance, at 560 nm, was
measured with a microplate reader. The concentrations required in
order to inhibit cell growth by 50% (IC50) were calculated.
3j
Orn
213–215 90
aAll the products were characterized by their spectroscopical data (1H NMR, 13
NMR, 11B NMR, EIMS, IR).
C
were euthanized and the tumors were excised under sterile
conditions and washed three times with PBS 1X. The cells were
maintained in an RPMI 1640 medium supplemented with 10%
fetal calf serum, 2 mM L-glutamine, 100 IU/mL penicillin,
100 μg/mL streptomycin and 1% non-essential amino acids at
37 °C under a 5% CO2 atmosphere with 95% humidity.
Flow cytometry analysis for DNA distribution
Cell suspensions (1×106) were pelleted, resuspended in
500 μL of phosphate-buffered saline (PBS) and fixed for at least
Table 3
Apoptosis induced by DPOXB 3a–j in L5178Y cells
DPOXB
Percentage of cells Annexin V-FITC/PI+
⁎
3a
3b
3c
3d
3e
3f
3g
3h
17.7 1.7
7.1 2.1
10.8 1.9
8.3 2.6
6.9 1.2
9.0 2.3
12.3 1.1
9.4 1.7
6.4 2.1
10.9 0.8
38.7 1.8
6.1 1.3
3i
3j
DMD
DMSO Control
Fig. 3. Effect of compound 3a on L5178Y viability. The MTT assay was
performed at 24 h, as described in the Materials and methods section. Data are
represented as the mean SD, n=3.
Cells were treated with 100 μM of DPOXB 3a–j for 24 h. Annexin V-FITC/PI
double stained of cells was analyzed by flow cytometry. The percentages of cells
⁎
were calculated by CELL Quest Pro software. (mean S.D., n=3) p≤0.05.

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B. Velasco et al. / Life Sciences 80 (2007) 1007–1013
Statistical analysis
All the values shown are expressed as the mean SD of three
independent determinations. Thestatistical significance of the data
was assessed using the Kruskal–Wallis test, and p values≤0.05
were considered significant.
Results
2,2-diphenyl-1,3,2-oxazaborolidin-5-ones (3a–j)
The glycine derivatives were prepared using a new
methodology as described in Materials and methods (Table 1).
The glycine derivatives with substituents R in the fourth
positions were prepared by reacting commercially available α-
amino acids and diphenylborinic acid under alkaline conditions.
The overall synthetic method is outlined in Fig. 2.
Inhibition of cell growth
The cytotoxic effect of the 2,2-diphenyl-1,3,2-oxazaborolidin-
5-ones was first evaluated in the murine L5178Y lymphoma cells.
Treatment with 6.25–100 μM of each of the compounds 3a–j
caused a concentration-dependent decrease in cell viability. These
results demonstrated that 3a–j exhibited cytotoxic effects on the
L5178Y cell line tested. The best effect was achieved for
compound 3a with IC50 =22.5 0.2 μM (Fig. 3), while compounds
3b, 3c, 3d, 3g, 3h, 3i and 3j showed from good to moderate IC50
values. Compound 3f showed a markedly lower activity
IC50 N200 μM (Table 2) in relation to the other compounds.
Phosphatidylserine externalization
L5178Y cells were exposed to compounds 3a–j at 100 μM
and the pro-apoptotic effect was analyzed in order to evaluate
externalization of phosphatidylserine, which is an early event in
the apoptotic process. Cells were double stained with Annexin
V-FITC and PI and measured by flow cytometry (Table 3). 3a
Fig. 4. Effect of 3a on the cellular volume. FSC vs SSC dot plot of L5178Y cells
incubated for 24 h: A) untreated and B) treated with 3a 100 μM. One
representative experiment out of three is shown. The cells indicated in the circle
are those with reduced cellular volume.
24 h at −20 °C in 70% ethanol. Fixed cells were washed twice
with PBS, stained with 300 μL of 20 μg/mL PI (Sigma), and
incubated for at least 30 min at room temperature. The stained
cells were analyzed on a FACScalibur flow cytometer (Becton
Dickinson, San Jose, CA) for relative DNA distribution. We
employed DMD with positive control (100 μM).
Table 4
Effect of 2,2-diphenyl-1,3,2-oxazaborolidin-5-ones on the cell cycle in L5178Y
cells at 24 h
DPOXB
% G0/G1
% S
%G2/M
% Sub G0/G1
⁎
3a
3b
3c
3d
3e
3f
3g
3h
39.85
49.38
52.54
49.05
59.87
55.60
49.34
49.26
60.41
56.31
23.43
61.86
31.33
31.70
31.93
32.03
30.13
32.52
34.46
31.58
31.83
30.99
27.24
30.19
7.54
8.01
8.29
10.93
6.01
5.75
9.10
9.88
4.13
7.12
7.30
3.60
21.27 4.2
10.92 2.9
7.24 2.4
7.99 2.6
3.99 1.3
6.12 1.1
7.10 2.2
9.28 1.4
3.60 1.2
5.58 1.3
42.03 2.3
4.35 2.1
Flow cytometry analysis of apoptosis
Apoptosis was quantified by measuring phosphatidylserine
externalization (considered an early marker of apoptosis), using
a kit from CALTAG (Laboratories Burlingame, USA) and
according to the manufacturer's instructions. Briefly, the cells
were treated with 100 μM of one of the DPOXB 3a–j and
stained with Annexin-V-FITC. We employed DMD with
positive control. Apoptosis was determined in a FACScalibur
flow cytometer. At least 10,000 events were acquired and
analyzed using CELL Quest Pro software.
3i
3j
DMD
DMSO Control
Cells were incubated with 100 μM DFB for 24 h, then fixed and stained with PI as
described. The stained cells were analyzed by flow cytometry. The percentages of
cells were calculated by CELL Quest Pro software. (mean S.D., n=3). Results from
⁎
one representative experiment representing 10,000 events are shown. p≤0.05.

B. Velasco et al. / Life Sciences 80 (2007) 1007–1013
1011
turned out to be the most effective molecule, inducing 17.7
1.7% apoptotic cells after 24 h of treatment at 100 μM (Fig. 5).
The cell shrinkage characteristic of apoptotic cells was also
evident in the FSC vs SSC dot plots generated by 3a. This cell
shrinkage was observed as a reduction in the FSC, as seen in
Fig. 4A and B.
DNA distribution
The induction of apoptosis was also analyzed in PI-stained
cells by flow cytometry. The appearance of the sub-G0/G1 phase
indicates that the compound tested induced apoptosis in the
cells assayed. This technique detects DNA fragmentation,
which represents a very late stage of apoptosis. L5178Y cells
were treated with 100 μM DPOXB 3a-j for 24 h, then stained
with PI and analyzed by flow cytometry (Table 4) in order to
quantify the DNA in the sub-G0/G1 cell cycle phase (Fig. 6).
The 2,2-diphenyl-1,3,2-oxazaborolidin-5-one obtained from
Fig. 6. Effect of 3a on DNA distribution. DNA histograms of L5178Y cells: A)
untreated and B) treated with 3a 100 μM. Results are expressed as a percentage
of the sub G0/G1.
glycine (3a) induced the greatest increase in L5178Y cells in
the sub-G0/G1 cell cycle phase. 3a induced 21.2 4.2%
apoptotic cells while for the control cells only 4.35 2.1%
were observed to be apoptotic.
Discussion
The induction of apoptosis, or programmed cell death, is
thought to be one of the most interesting therapeutic strategies
to specifically target cancer cells. Until now chemo- and
radiotherapy treatments have not been selective for tumor cells,
making the development of new therapeutic agents necessary.
Apoptosis is a programmed cell death that involves genetically
controlled morphological and biochemical events, including
phosphatidylserine externalization, cytochrome c leakage from
the mitochondria, caspase activation, reduction of cellular and
nuclear volume, chromatin condensation and internucleosomal
DNA fragmentation (Schultz and Harrington, 2003). Apoptotic
cells exhibit some morphological modifications that are readily
detected by flow cytometry according to their light scattering
properties (FSC/SCC) (Lecouer et al., 1997). Thus by using this
Fig. 5. Apoptotic induction by 3a. FL1-H vs FL2-H dot-plot of L5178Y cells
incubated 24 h and stained with Annexin-FITC and PI after treatment with:
A) untreated and B) treated with 3a 100 μM.

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B. Velasco et al. / Life Sciences 80 (2007) 1007–1013
technique, a series of ten 2,2-diphenyl-1,3,2-oxazaborolidin-5-
ones was evaluated in murine L5178Y lymphoma cells. Our
results are consistent with those reported previously by Trejo-
Solís et al. (2005), who modified both the amino and carboxylic
hydroxyl groups of glycine, which was then used to treat glioma
C6 cells and resulted in cell death by apoptosis.
The cytotoxic effect induced in the murine L5178Y lymphoma
cells by compound 3a (2,2-diphenyl-1,3,2-oxazaborolidin-5-one)
is highly potent, when compared with the other amino acid
derivatives, as measured by the MTT assay (Table 2). According
to our hypothesis, the heterocyclic ring is the active moiety in
inducing apoptosis when both the amino and carboxylic hydroxyl
groups are modified. The results of this study show that the
modification of the fourth position of this ring, by inserting a
heterocyclic indole ring or any of the R-groups (Table 1), clearly
decreased the observed effect in comparison with the lead
compound (3a) (Table 2).
sured by the MTT assay), externalizing phosphatidylserine (as
measured by Annexin V), and inducing the sub-G0/G1 peak (as
measured by PI staining). The mechanism of action of this
compound is now being evaluated in our lab and we will report
them in detail in an coming paper. At the moment we can say that
this kind of compounds induced transmembrane potential
reduction, caspase-3 activation and DNA fragmentation evalu-
ated by TUNEL and DNA electrophoresis. The results described
above indicate that this 2,2-diphenyl-1,3,2-oxazaborolidin-5-
ones activate the intrinsic apoptotic pathway.
Acknowledgements
This research was financially supported by SIP grants
20040289 and 20050322. Benjamín Velasco thanks CONACyT
for a scholarship; and Cátedra IN1-71 FESC-UNAM, for financial
support. Luvia Enid Sánchez-Torres, José G. Trujillo-Ferrara and
Luis Fabila Castillo are fellows of COFAA-IPN, EDI-IPN.
In spite of the fact that 3a was more active than the other nine
amino acid derivatives (Fig. 3), the latter showed a significant
concentration dependent growth inhibition of L5178Y cells. In
order to find out if the cells died due to apoptosis or necrosis, the
pro-apoptotic effect of DPOXB was evaluated in L5178Y cells
exposed to 3a–j at 100 μM. Apoptosis was quantified by flow
cytometry using Annexin V to evaluate the flip-flop of
phosphatidylserine (an indicator that the cell is entering the
apoptotic process), which showed that cell death caused by 3a
was by apoptosis and not by necrosis (Fig. 5B). Phosphatidyl-
serine externalization was exhibited by 6.1 1.3% of the control
cells, whereas 17.7 1.7% (p≤0.05) of cells treated with 3a
showed the same event in the apoptotic process (Table 3). Cell
shrinkage characteristic of apoptotic cells was also evident in the
FSC vs SSC dot plots generated by 3a. This cell shrinkage was
observed as a reduction in the FSC (Fig. 4B). To confirm that the
induction of cell death was by apoptosis, the DNA distribution
was evaluated in cells treated with each of the tested compounds
at a concentration of 100 μM for 24 h and stained with PI as
described in the Materials and methods section. This technique
detects the fragmentation of DNA, which occurs in the later part
of the apoptotic process. The presence of the sub-G0/G1 peak in
the histogram of the DNA distribution indicates that a population
of cells is dying by apoptosis (Fig. 5B). Particularly important is
that 21.2 4.2% (p≤0.018) of the cells died by apoptosis when
treated with 3a as opposed to 4.35 2.1% when the cells were
untreated. When the cells were incubated with compounds 3b–j,
the difference in the percentage of apoptosis with respect to the
control was not significant (p≥0.05, Table 4), whereas with 3a
the difference was clearly significant (Table 4). A reduction in
cell volume was also observed with this technique, and it was
possible to establish that the cells with a reduced FSC were the
ones that appear in the sub-G0/G1 peak (Fig. 6B). In conclusion,
as indicated in the literature, glycine and its N-derivatives show
cyto-protective effects. Our results confirm that the modification
of both the amino and carboxylic hydroxyl groups gives rise to
apoptotic inducing compounds, unlike the effect of glycine,
which acts as an apoptotic protector. Our study points out the
importance of glycine derivatives as apoptotic inducers, inhi-
biting the growth of murine L5178Y lymphoma cells (as mea-
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