Novel 3-hydroxy vinylboronates influence sphingolipid metabolism, cause apoptosis in Jurkat cells and prevent tumor development in nude mice

Article abstract of DOI:10.1016/j.bmcl.2012.11.028

A series of novel 3-hydroxy vinylboronates which share structural similarities with sphingolipids were synthesized and tested in vitro and in vivo as anticancer agents. The molecules reduced cancer cell survival in vitro by influencing their sphingolipid metabolism. In a cancer model in nude mice the lead compound E7 prevented the development of tumor as long as the treatment period continued. Moreover, it delayed tumor growth after the treatment was finished.

Full text of DOI:10.1016/j.bmcl.2012.11.028

Bioorganic & Medicinal Chemistry Letters 23 (2013) 507–512
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel 3-hydroxy vinylboronates influence sphingolipid metabolism, cause
apoptosis in Jurkat cells and prevent tumor development in nude mice
Alina Botvinik Livshits a, , , Abed Al Aziz Al Quntar a,b, Zhanna Yekhtin c, Morris Srebnik a,à, Arie Dagan d,
⇑
⇑
a Institute for Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, 91120, Israel
b Department of Material Engineering, Faculty of Engineering, Al Quds University, Abu Dies, East Jerusalem, Israel
c Department of Bone Marrow Transplantation and Cancer Immunotherapy, Hadassah University Hospital Ein Kerem, The Hebrew University of Jerusalem, 91120, Israel
d Department of Developmental Biology and Cancer Research, Faculty of Medicine, The Hebrew University of Jerusalem, 91120, Israel
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 3-hydroxy vinylboronates which share structural similarities with sphingolipids were
synthesized and tested in vitro and in vivo as anticancer agents. The molecules reduced cancer cell sur-
vival in vitro by influencing their sphingolipid metabolism. In a cancer model in nude mice the lead com-
pound E7 prevented the development of tumor as long as the treatment period continued. Moreover, it
delayed tumor growth after the treatment was finished.
Received 30 August 2012
Revised 1 November 2012
Accepted 8 November 2012
Available online 22 November 2012
Ó 2012 Elsevier Ltd. All rights reserved.
The Letter is dedicated to the memory of
Professor Morris Srebnik
Keywords:
Cancer
Sphingolipid metabolism
Boronates
Sphingolipids
Ceramide
3-Hydroxy vinylboronates
Sphingolipids are complex lipids composed of a hydrophilic
head group and a lipophilic backbone. Cells use them for the
regulation of the fluidity and the sub-domain structure of the lipid
bilayers.1 Besides providing structural integrity in cell membranes,
sphingolipids play crucial roles in signal transduction and gene
regulation, and are thusly involved in many processes: apoptosis,
transport, endocytosis, migration, senescence, proliferation and
inflammation.1–3 In particular, ceramide, its metabolites and the
enzymes involved in sphingolipid metabolism participate in
tumorigenesis and cancer progression and influence the efficacies
of cancer therapies.1–4 In cells the major sources of ceramide are:
The de novo synthesis from serine and palmitoyl CoA; from its
metabolite sphingosine (the salvage pathway); or by enzymatic
hydrolysis of sphingomyelin and glycosphingolipids. All these
pathways and some others minor ones result in ceramide accumu-
lation. Ceramide is metabolized by ceramidase to sphingosine.
Sphingomyelin and glycosphingolipids are synthesized from cera-
mide. Cells control the homeostasis of sphingolipids by regulating
the activity of metabolic enzymes. Cancer cells change the sphin-
golipid metabolism regulation in order to bypass apoptosis, partic-
ularly by up-regulating the synthesis of sphingolipids that are
generated from ceramide and by down-regulating the metabolism
of these sphingolipids.3–7 This is done in order to decrease the con-
centration of the pro-apoptotic sphingolipid ceramide in cancer
cells.
All these data suggest that many interventions in the metabolic
pathways of ceramide should influence cancer development and
therapy. Different approaches are being taken in order to influence
the metabolism of sphingolipids.1,8,9
Vinylboronates are valuable but relatively inert compounds10
and can be synthesized by various methods.11 Their utility lies in
the ability to transfer the vinyl group from the boron to other
elements10 and metals.12–17 We synthesized 3-hydroxy-1-alkenyl-
boronates via tributylphosphine stabilized borylzirconacycloprop-
enes.18 Boron is appealing as a pharmacore since it can inhibit
many of the activities of the carboxyl group, by mimicking it to a
point when the biological system recognizes the exchange. Boranes
have been at the focus of our laboratory as pharmacologically active
chemical agents.19,20 The use of boron-containing compounds to
⇑
Corresponding authors. Tel.: +972 0526 422237/972 2 6758683; fax: +972 2
6757175 (A.B.L.); tel.: +972 2 6758355; fax: +972 2 6411663 (A.D.).
E-mail addresses: alinabo@ekmd.huji.ac.il (A. Botvinik Livshits), dagan@cc.hu-
ji.ac.il (A. Dagan).
The results reported here are included in the dissertation project of Alina Botvinik
Livshits in partial fulfillment of her Ph.D. degree requirements at The Hebrew
University of Jerusalem. A.B.L. was the recipient of a student fellowship from The
Hebrew University of Jerusalem and expresses her gratitude.
à
Deceased.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.028

508
A. Botvinik Livshits et al. / Bioorg. Med. Chem. Lett. 23 (2013) 507–512
O
O
O
a
b
O
B
c
O
O
R
B
R
R
B
d
R1
H
ZrCp2
OH
R2
(n-Bu)3P
Scheme 1. Method of 3-hydroxy vinylboronates synthesis.
treat various cancers has until recently been restricted to Boron
Neutron Capture Therapy (BNCT).21 While appealing, BNCT is none-
theless impractical at this stage since it requires a nuclear facility.
This may however change in the next decade as a new generation
of neutron sources is made available based on various tabletop par-
ticle accelerators. In 2003, however, Millenium Pharmaceuticals
introduced VelcadeÒ (bortezomib) into the clinic for use against
refractory and recurring myelomas.22 VelcadeÒ is the first entirely
synthetic boron-containing compound to be introduced in clinical
practice. Its success has prompted intense research into developing
other synthetic boranes. To the best of our knowledge, highly substi-
tuted vinylboronates have not been tested for anticancer activity of
any kind.
chain from an alkyne boronate together with the chain from a ke-
tone. Compound E8, with its greatest length (16 carbon atoms)
gave higher IC50 values. This suggests that a longer hydrocarbon
chain, given that the 3-substituted carbon has only hydroxyl and
a linear hydrocarbon chain as ligands, results in decreased activity.
We have tried to synthesize alkyne boronates using tetra- and
octadecynes, in order to combine cyclopropyl phenyl hydroxyl 3-
substitution with a longer hydrocarbon chain, but with no success.
We assume that the long chains of the alkynes interfere in the
course of the reaction.
Compound E4, the only molecule that was synthesized using an
aldehyde (and not a ketone like others) was not active on myeloma
cells and gave IC50 value of greater than 100 lM on colon cancer
We synthesized 3-hydroxy vinylboronates for in vitro studies
by the method, presented in Scheme 1, which was developed in
cells. This result led us to conclude that it was not enough for
the 3-substituted carbon to have both a hydroxyl and a phenyl
groups as ligands (the hydroxyl and the phenyl are present in
E4), but it should also have another ligand that is not hydrogen.
Comparing all the molecules in Table 1 we conclude that in or-
der to lower the IC50 of the molecule it should contain a long
hydrocarbon chain and 3-substitution with a cyclopropyl phenyl
hydroxyl. The molecule that follows these requirements and re-
duces cancer cells survival the most is E7. So E7 was selected for
further investigations. Compound E7 was also tested by a MTT as-
say on Melanoma-624 and leukemia (Jurkat) cells. After 72 h incu-
bation the IC50 on Melanoma-624 and on Jurkat cells were
our laboratory and published earlier18
:
3-Hydroxy vinylboronates that were synthesized for in vitro
studies are presented in Figure 1.
The anticancer activity of the synthesized 3-hydroxy vinylboro-
nates was evaluated by the MTT assay23 on several cancer cell
lines. The IC50 values are presented in Table 1.
The novel 3-hydroxy vinylboronates reduced cancer cells sur-
vival in the MTT assays (Table 1). Compounds E1, E2 and E7 have
a similar 3-substitution, but differ in the length of the hydrocarbon
chain, with E7 exhibiting greatest length (8 carbon atoms). Com-
pound E7 gave lower IC50 values, suggesting that a longer hydro-
carbon chain, in molecules with cyclopropyl phenyl hydroxyl 3-
substitution, results in increased activity. Compounds E5, E6 and
E8 have hydrocarbon chains of different lengths, composed of the
32.31 4.78 and 16.33 0.82 lM, respectively.
Our synthesized 3-hydroxy vinylboronates share several struc-
tural similarities with the sphingolipids, particularly sphingosine:
an allylic hydroxyl and linear hydrophobic alkyl chains. Figure 2
Figure 1. 3-Hydroxy vinylboronates that have been synthesized and examined in this study.

A. Botvinik Livshits et al. / Bioorg. Med. Chem. Lett. 23 (2013) 507–512
509
Table 1
IC50 values of vinylboronates after 72 h incubation with colon cancer (HT-29) and
myeloma (ARH-77) cells
Compound
Cell type
HT-29 (
l
M)
ARH-77 (lM)
E1
E2
E3
E4
E5
E6
E7
E8
Not active
83.93 1.33
86.83 2.42
>100
57.19 3.33
69.62 3.25
18.32 0.26
>100
10.38 0.97
55.22 2.83
64.91 2.92
Not active
33.85 0.17
54.61 1.64
6.36 1.10
79.07 2.27
Figure 3. Percentage of Jurkat cells at each stage of cell cycle after incubation with
different E7 concentrations or vehicle (0.2% DMSO) for 24 h. Values are mean, error
bars indicate s.d. Asterisks indicate that data are statistically significant compared
to a naïve control using a t test (⁄P <0.05, ⁄⁄P <0.01, ⁄⁄⁄P <0.001).
O
B
O
NH2
HO
HO
CH2OH
E7
Sphingosine
Figure 2. Structures of E7 and sphingosine.
presents the structure of our lead compound, E7, together with the
structure of sphingosine.
Compound E7 gave the best results in MTT assays and thus was
chosen for further investigations. Norman Radin published charac-
teristic structural features of many anticancer molecules: an allylic
O, N or S; the allylic oxygen is in a carbonyl or an alcohol group;
double bonds conjugated to the allylic bond; two or more allylic
moieties in each molecule and linear chains.24 Those structural
characteristics are common to many anticancer drugs and sphingo-
lipids and Radin suggests that those structural similarities affect
the mechanism of action of the drugs. We tend to agree with
him and assume that even if some of the reasons for E7 activity
superiority in MTT assays are maybe not target related (improved
cell permeability for example), some are related to better sphingo-
lipid mimicking.
Jurkat cells treated with E7 for 24 h were examined by FACS.
Their distribution in the cell cycle stages is presented in Figure 3.
DNA degradation that takes place during apoptosis leads to lower
cellular DNA content. This sub G1 DNA content is detected by flow
cytometry25 and apoptotic cells are represented by a sub G1 pop-
ulation.26,27 FACS examination of Jurkat cells indicate that E7 at
Figure 4. Percentage of ceramide and sphingomyelin in Jurkat cells after 72 h
incubation with different concentrations of E7 or vehicle (0.1% DMSO, signposted as
0 lM E7) and a constant concentration of Bodipy-12-Ceramide. Values are mean,
error bars indicate s.d. Asterisks indicate that data are statistically significant
compared to vehicle control using a t test (⁄P <0.01).
and 0.2% of DMSO. The percentages of the sphingolipids were alike
in the control groups, however at the group of 100 lM treatment
100
l
M causes cells to stop at the sub G1 phase more than twice
only ceramide without sphingomyelin was detected by the HPLC.
Ceramide is an important suppressor of cancer development. It is
a proapoptotic and antiproliferative sphingolipid.29–31 Ceramide
is a metabolite of sphingomyelin and also its building block
(Scheme 2).5
Compound E7 increases the percentage of ceramide and de-
creases the percentage of sphingomyelin in Jurkat cells in a con-
centration dependent manner (Fig. 4). The effect is seen already
as much as the controls or lower concentrations of E7 (Fig. 3). Thus
more leukemic cells degrade their DNA and less are involved in cell
proliferation in stages G1, S and G2.
In order to track the influence of 3-hydroxy vinylboronates on
sphingolipid levels in cancer cells Jurkat cells were incubated with
E7 and a fluorescent ceramide (Bodipy-12-Ceramide)28 for 72 h.
Low concentration (0.25 lM) of fluorescent ceramide (Bodipy-12-
Ceramide) is added to the cells at the beginning of the experiment,
30 min after the tested compound. After 72 h of incubation we
investigate the fate of this fluorescent ceramide – how much was
consumed for sphingomyelin synthesis and how much remained
as ceramide. We examine how the tested compound influenced
the fate of the fluorescent ceramide. Figure 4 shows the relative
percentages of ceramide and sphingomyelin as was detected by
at 30 lM and increases at 50 lM. One hundred micromolars of
E7 reduced the concentration of sphingomyelin to a level that
was not detected by the HPLC.
The MTT assays that their results are presented in Table 1 were
conducted in medium free of FCS. The sphingolipid metabolism
study was conducted in medium containing 10% FCS. The IC50 of
E7 after 72 h incubation with Jurkat cells in medium containing
HPLC. In another experiment a concentration of 100
lM of E7
10% FCS is 84.01 0.94
lM. The effect of E7 on percentage of
was tested together with naïve cells and vehicle controls of 0.1%
ceramide and sphingomyelin is seen already at 30
lM of E7 and

510
A. Botvinik Livshits et al. / Bioorg. Med. Chem. Lett. 23 (2013) 507–512
The possible synergistic effect that 3-hydroxy vinylboronates
Sphingomyelin synthase
Sphingomyelinase
may have together with a commercially available anticancer drug
was examined by a MTT assay. Jurkat cells were incubated with
E7 together with Doxorubicin for 24 h. Figure 6 shows the results
of the assay. Drug combinations are commonly used in cancer ther-
apy. Combinations of E7 with Doxorubicin led to lower Jurkat cells
survival than treatment with each of the compounds by itself
(Fig. 6). In anticancer therapy that combines several drugs each
of them is given at lower concentration (than if it were given so-
lely) in order to minimize their adverse effects.
The anticancer activity of E7 was also evaluated in a prelimin-
ary study in vivo in female nude mice. During the experiment
(50 days) the mice gained weight and except for the tumors were
in good health condition, assessed by weighing and clinical exam-
ination. The therapeutic effect that E7 caused in treated mice is
presented in Figure 7. Compound E7 (50 mg/kg) prevented the
development of tumors for 3 weeks of the injections. Moreover, it
delayed tumor growth after the treatment was finished (last injec-
tion was at day 21). Only at day 33 small tumors were measured in
two of the E7-treated mice. At day 37 an additional mouse from
vehicle treated group was measured with a tumor. All mice from
50 mg/kg treatment group survived in good general health condi-
tion (except for small tumors in two mice) until the end of the
experiment at day 50. From the vehicle treatment group two mice
were sacrificed until day 50 because of the animals’ well-being
requirements of the ethics approval for the experiment.
Ceramide
Sphingomyelin
Scheme 2. The connection between ceramide and sphingomyelin.
increases at higher concentrations. Compound E7 increases the
percentage of ceramide and decreases the percentage of sphingo-
myelin while its concentrations are below its IC50 in 10% FCS con-
taining medium.
We assume that E7 is able to inhibit the enzyme sphingomyelin
synthase and thus inhibit sphingomyelin synthesis from ceramide.
We think this is the reason the concentration of ceramide rises and
the concentration of sphingomyelin becomes lower and lower as
the concentration of E7 increases. (From data not shown, E1 also
interfered with sphingolipid metabolism in a similar manner.)
The activity of caspase-3 in several cell lines treated with E7
was tested with the Z-DEVD–R110 substrate. Caspases are cysteine
proteases that attain apoptotic cell death by the cleavage of several
substrates.32 Different incubation times did not change caspase-3
activity in the cells (data not shown). Treatment periods were cho-
sen with the consideration of the fact that there is a time window
of caspase activity that ceases with time. Published studies suggest
that programmed cell death can be achieved dependently and also
independently of caspase activity.33,34
In conclusion, we synthesized highly substituted vinyl boro-
nates in a sterio and regioselective manner from stable borylzirc-
onacyclopropenes and evaluated their anticancer activity. The
molecules reduce the survival of tested cancer cell lines. The se-
lected molecule – E7 – triggers apoptotic events in Jurkat cells.
Compound E7 interferes with sphingolipid metabolism in Jurkat
cells. It increases the concentration of the pro-apoptotic sphingo-
lipid – ceramide, and decreases the concentration of sphingomye-
lin, that is synthesized from ceramide. Concomitant treatments of
Jurkat cells with E7 and Doxorubicin led to a higher anticancer
Jurkat cells treated with E7 for 24 h were stained with propidi-
um iodide and Annexin V and checked by FACS to measure the per-
centage of living non-apoptotic cells (PI and Annexin negative),
cells at the early stage of apoptosis (PI negative, Annexin positive),
cells at the late apoptotic stage (PI and Annexin positive) and
necrotic cells (PI positive, Annexin negative). Representative FACS
results are shown in Figure 5. Compound E7 at 100 lM caused
more Jurkat cells to become apoptotic compared to naïve or vehicle
treated cells.
Figure 5. FACS results of Jurkat cells stained with PI and Annexin V after 24 h treatment with the vehicle (0.2% DMSO) or with 100 lM of E7 or kept at 4 °C overnight or naïve
cells.

A. Botvinik Livshits et al. / Bioorg. Med. Chem. Lett. 23 (2013) 507–512
511
Cell cycle analysis. Jurkat cells (1 Â 106 cells/ml) were plated in
96-wells round bottom plates (100 ll/well). Compound E7 solu-
tions or vehicle were added (5 ll/well). After 24 h the cells were
harvested and fixed in 70% cold ethanol. The cell cycle analysis
was performed in LSR-II flow cytometer after the addition of
50 lg/ml of RNAse and PI to cell suspension in PBS.
Sphingolipid metabolism. Jurkat cells (0.75 Â 106 cells/ml) were
plated in 24-wells flat bottom plates (750 ll/well). Compound E7
solutions or vehicle were added (250
l
l/well). After 30 min the
fluorescent Bodipy-12-Ceramide28 was added (0.25
lM). After
72 h the cells were harvested. Cells pellets were extracted once
with dichloromethane/methanol 1:2 and once with dichlorometh-
ane/methanol 1:1. The extractions were dried from solvents,
dissolved in ethanol and the concentrations of ceramide and sphin-
gomyelin were determined by the HPLC instrument (WatersÒ)
equipped with Luna 5
l C18(2) 100A column and fluorescence
detector (w474) (Ex 505 nm, Em 530 nm).
Figure 6. Percentage of survival of Jurkat cells that underwent concomitant
treatments with E7 and Doxorubicin for 24 h. Values are mean, error bars indicate
s.d. Asterisks indicate that data are statistically significant compared to same
Doxorubicin concentration without E7 using a t test (⁄P <0.05, ⁄⁄P <0.01, ⁄⁄⁄P
<0.001).
Caspase-3 activity. Cells (2 Â 106 cells/ml) were plated in 24-
wells flat bottom plates (500 ll/well). Media used were RPMI
1640 and supplemented RPMI 1640. Compound E7 solutions or
vehicle were added (25 l/well). After different incubation times
l
cells were harvested. Caspase-3 activity was measured with the
EnzChekÒ Caspase-3 Assay Kit #2 (Molecular ProbesÒ).
Annexin V and PI staining. Jurkat cells (1 Â 106 cells/ml) were
effect than treatment with each of the compounds alone. In cancer
model in nude mice the lead compound E7 (50 mg/kg) prevented
the development of tumors as long as the treatment period contin-
ued. Moreover, it delayed tumor growth after the treatment was
finished. These findings are supported by the claim, published in
several articles, that hydrophobic allyl alcohol derivatives (which
are sphingolipid analogues) are efficient as anticancer drugs.24,35
Cell culture. HT-29, ARH-77, Melanoma-624 and Jurkat cells
were maintained in an incubator at 37 °C in 5% CO2/95% air atmo-
sphere in RPMI 1640 supplemented medium: supplemented with
10% fetal calf serum and also glucose, glutamine, pyruvate, Pen-
Strep and HEPES. Same full medium was used for experiments un-
less otherwise stated. Media and supplements were purchased
from Biological Industries, Kibbutz Beth HaEmek, Israel.
plated in 96-wells round bottom plates (100 ll/well). Compound
E7 solutions or vehicle were added (5 ll/well). After 24 h the cells
were harvested, worked-up with MebcytoÒ Apoptosis Kit (Annexin
V-FITC Kit) (MBL) and checked in LSR-II flow cytometer.
Synergism studies. Jurkat cells (1 Â 106 cells/ml) were plated in
96-wells flat bottom plates (100 ll/well). Medium used was RPMI
1640. Compound E7 and Doxorubicin were added simultaneously
(5
l/well). After 24 h the MTT assay was performed.23
In vivo study in nude mice. The experiment was approved by the
l
Committee for Ethics in Animal Experimentation of The Hebrew
University of Jerusalem. Eleven female nude mice 5–6 weeks old
were kept under specific pathogen-free conditions in an environ-
ment with a controlled temperature (ꢀ22 °C) and a 12-h light–dark
cycle. The animals had free access to chow and water. Mice were
injected sc with 1 Â 106 HT-29 cells. At the same day they were
randomly divided into two groups according to the intended treat-
ment: 50 mg E7/kg (5 mice) or the vehicle: cremophor/ethanol/
water for inj. (10:10:80) (6 mice). Mice were injected ip once a
MTT assay. Cells (1 Â 106 cells/ml) were plated in 96-wells flat
bottom plates (100 ll/well). Medium used was RPMI 1640.
HT-29, ARH-77 and Melanoma-624 were left in the incubator over-
night for attachment. Then 3-hydroxy vinylboronates were added
(5 l
l/well). After 24, 48 or 72 h the MTT assay was performed.23
Figure 7. Percentage of tumor-free mice in vehicle (n = 6) and E7 50 mg/kg (n = 5) treated mice.

512
A. Botvinik Livshits et al. / Bioorg. Med. Chem. Lett. 23 (2013) 507–512
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mouse). After the treatment period the mice were followed up
for another 4 weeks. During the experiment general health condi-
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weighed and tumor volume was measured.
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