Hybrids of 1-deoxynojirimycin and aryl-1,2,3-triazoles and biological studies related to angiogenesis

Article abstract of DOI:10.1016/j.bmc.2008.05.012

Hybrids of 1-deoxynojirimycin (DNJ) and aryl-1,2,3-triazole have been synthesized with a view to identifying an inhibitor of both α-glucosidase and methionine aminopeptidase 2 (MetAP2). One compound was a potent inhibitor of α-glucosidase at both the enzyme and cellular level, and this agent also inhibited bovine aortic endothelial cell (BAEC) growth and tube formation. The anti-proliferative activity of this hybrid is due to its ability to induce cell-cycle arrest in the G₁phase. The novel agent caused a reduction in the expression of cyclin D1 but did not promote apoptosis or inhibit the phosphorylation of ERK1/2. These observations indicate that its mechanism of action is distinct from fumagillin and its analogues, which inhibit MetAP2. Stress-fibre assembly in BAECs was abolished by the novel agent indicating that the inhibition of BAEC tube formation observed is partially a result of a reduction in cell motility.

Full text of DOI:10.1016/j.bmc.2008.05.012

Bioorganic & Medicinal Chemistry 16 (2008) 6333–6337
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Hybrids of 1-deoxynojirimycin and aryl-1,2,3-triazoles and biological
studies related to angiogenesis
Yunxue Zhao a, Ying Zhou b, Kathy M. O’Boyle a, Paul V. Murphy b,
*
a UCD School of Biomolecular and Biomedical Science and the UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland
b UCD School of Chemistry and Chemical Biology and Centre for Synthesis and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
Hybrids of 1-deoxynojirimycin (DNJ) and aryl-1,2,3-triazole have been synthesized with a view to iden-
tifying an inhibitor of both a-glucosidase and methionine aminopeptidase 2 (MetAP2). One compound
was a potent inhibitor of a-glucosidase at both the enzyme and cellular level, and this agent also inhibited
bovine aortic endothelial cell (BAEC) growth and tube formation. The anti-proliferative activity of this
hybrid is due to its ability to induce cell-cycle arrest in the G1phase. The novel agent caused a reduction
in the expression of cyclin D1 but did not promote apoptosis or inhibit the phosphorylation of ERK1/2.
These observations indicate that its mechanism of action is distinct from fumagillin and its analogues,
which inhibit MetAP2. Stress-fibre assembly in BAECs was abolished by the novel agent indicating that
the inhibition of BAEC tube formation observed is partially a result of a reduction in cell motility.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 29 February 2008
Revised 27 April 2008
Accepted 5 May 2008
Available online 7 May 2008
Keywords:
1-Deoxynojirimycin
Aryl-triazoles
Angiogenesis
Cell cycle
Cyclin D1
Inhibitor
Cell migration
Hybrid compounds
1. Introduction
and these glycosidase inhibitors inhibit angiogenesis.6 With a goal
to obtain novel anti-angiogenic agents that can target more than
Angiogenesis1 provides new blood vessels to growing and
developing tissue including tumours. Pathological angiogenesis oc-
curs in tumour formation and metastasis and in a range of other
‘angiogenesis-dependent diseases’ that includes rheumatoid
arthritis and diabetic retinopathy.2 Accordingly, the design and
synthesis of angiogenesis inhibitors is of interest and progress in
this area has been recently reviewed.3 It is emerging that angio-
genesis inhibitors that target a range of angiogenic proteins and/
or pathways may be less susceptible to drug resistance than those
agents that target a single protein or pathway. One strategy to
counteract this mode of resistance to angiogenesis inhibitors is to
produce new molecules that inhibit two or more biological targets
relevant to angiogenesis.
one biological process, we have designed and synthesized the hy-
brids 3–5 (Fig. 1) that can block both a-glucosidase and MetAP2.
The design and synthesis of 3–5 and some biological properties
have been outlined in a previous communication,7 and one of these
agents, 5, has been shown to inhibit angiogenesis in vitro.7 Cell bio-
logical studies show that 5 reduced the expression of cyclin D1, a
key modulator of cell proliferation. The hybrid 5 also abolished
stress-fibre assembly in BAECs that may lead to reduced cell
motility.
OH
There are a number of cellular targets of interest in angiogenesis
therapy, and herein we focused on two of these: methionine ami-
nopeptidase II (MetAP2) and a-glucosidase. MetAP2 has been iden-
tified as a cellular target for angiostatic agents4 such as fumagillin
and TNP-470 as well as aryl-1,2,3-triazoles 1.5 Inhibitors of a-glu-
cosidases such as N-methyl-1-deoxynojirimycin 2b, castanosper-
mine or 1-deoxymannojirimycin alter the biosynthesis of glycans
on endothelial cell surfaces that are required for angiogenesis,
H
H
N
R
N
OH
OMe
N
2a R = H
2b R = Me
2c R = Bu
R
N
N
N
1a
HO
OH
1
N
H
N
OH
OH
(
)
O
N
n
N
3 (n = 2)
4 (n = 4)
5 (n = 6)
HO
N
OH
* Corresponding author. Tel.: +353 1 7162504; fax: +353 1 7162501.
E-mail address: paul.v.murphy@ucd.ie (P.V. Murphy).
Figure 1. Structures of 1–5.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.05.012

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Y. Zhao et al. / Bioorg. Med. Chem. 16 (2008) 6333–6337
the endoplasmic reticulum (ER).12 The treatment of BAECs with
2. Results and discussion
3–5 led to increased binding of Con A to cell surfaces when com-
pared to the control. As expected treatments that increased Con
A binding decreased L-PHA binding; thus L-PHA binding to BAECs
was reduced in cells treated with 2a and 3–5. Thus, all novel DNJ
derivatives inhibited a-glucosidase at the cellular level.
The design of 3–5 was based on combining the properties of
both a-glucosidase8 and MetAP2 in a single molecule. The ability
of N-butyl-DNJ 2c and other N-alkyl-DNJ derivatives to inhibit a-
glucosidases indicated that an alkyl chain linker between the DNJ
nitrogen atom and the aromatic residue would not lead to a loss
of glycosidase inhibitory activity. Molecular modelling7 indicated
that the compunds could also inhibit MetAP2.
2.4. Effects of compounds on angiogenesis in vitro
Hybrids 3–5 were evaluated for their ability to inhibit angio-
genesis in vitro using the endothelial cell tube formation assay13
given that they could inhibit BAEC growth and alter the biosyn-
thesis of cell surface oligosaccharides important for angiogenesis.
Endothelial cells which are induced to undergo tube formation
change their architecture and form cell–cell contacts that lead
to branched networks similar to capillary-like blood vessels.
When BAECs were cultured on polymerized matrigel they orga-
nized into such tube-like structures (Figure S2 in supporting
information). However, when BAECs were cultured on polymer-
ized matrigel in the presence of 5 (0.2 mM, the cells failed to
organize into the capillary-like structures. At these concentra-
tions no other compounds tested inhibited tube formation and
5 was inactive at 0.1 mM.
2.1. Inhibition of glycosidases
Compounds 1a, 2a, and 3–5 were evaluated for their ability to
inhibit a-glucosidase from Bacillus stearothermophilus as de-
scribed by Hakamata et al.9 in order to test their enzyme inhibitory
activity, and the results are shown in Table 1. The IC50 value for 2a
was 1.67 lM. The hybrid compound 5 (IC50 = 1.15 lM) was a more
potent glycosidase inhibitor than 2a, whereas the other com-
pounds tested were less potent than 2a and 5. The modest
enhancement of inhibitory properties observed for 5 indicated that
the N-substituent may have favourable interactions with the glu-
cosidase. The aryl-1,2,3-triazole 1a did not inhibit the a-glucosi-
dase, as was expected.
2.5. Cell-cycle analysis
2.2. Inhibition of BAEC growth
Inhibitors of MetAP2, fumagillin and TNP-470, have previously
been shown to inhibit the proliferation of endothelial cells by
arresting these cells at the G1 phase of the cell cycle.14 It can
be hypothesized that the aryl-1,2,3-triazole residue of 5 inhibits
MetAP2 at the cellular level, which leads to the inhibition of
BAEC proliferation observed for 5 (Table 1). To gain further in-
sights into the mechanism of the anti-proliferative activity of 5
and to investigate if 5 induces G1 arrest similar to other inhibi-
tors of MetAP2 its effect on cell-cycle distribution was analyzed
and the result of a typical experiment is shown in Figure 2. As
The inhibition of BAEC growth by compounds was next deter-
mined using the MTT assay10 and the results are shown in Table
1.11 The iminosugar 2a was inactive with no significant inhibition
of proliferation being observed at concentrations up to 1 mM.
However, the aromatic triazole 1a (IC50 = 0.347 mM) inhibited
the growth of BAECs. The inhibition of human (HUVEC) and mouse
(MS-1) endothelial cell proliferation by aryl-1,2,3-triazoles is con-
sistent5 with an ability to inhibit MetAP2, and it can be inferred
that inhibition of MetAP2 in BAECs would account for the activity
observed for 1a. It is worth noting that aryl-1,2,3-triazole deriva-
tives had IC50 values of 2–30 lM as inhibitors of HUVEC and MS-
1 cell growth.5 Compounds 3–5 all inhibited BAEC growth with 5
being the more potent inhibitor (IC50 = 105 lM).
determined by flow cytometry, exposure of BAECs to
5
(200 lM) after 24 h, resulted in an increase of the percentage
of cells in the G0/G1 phase and a decrease in the percentage of
cells in the S phase compared with control cells, consistent with
an induction of G1 arrest by 5.
2.3. Effects on cell surface oligosaccharide structure
2.6. Effect of 5 on apoptosis
As hybrids 3–5 can inhibit a-glucosidase at the enzyme level,
their effect on the bioprocessing of cell surface oligosaccharides
was also investigated. Thus, BAECs were treated with compounds
(50 lM) for 24 h and the binding of specific fluorescein (FITC)-la-
The inhibitor of MetAP2, TNP-470, has been reported to induce
apoptosis in both tumour15 and vascular endothelial cells.16 The
ability of 5 to promote apoptosis in BAECs was thus evaluated by
treatment of cells with 5 (100 and 200 lM doses) for 24, 48, and
72 h, subsequent staining with annexin V–propidium iodide and
flow cytometry analysis, in which only annexin V-stained cells
were considered as apoptotic cells (data not shown). These exper-
iments did not show any considerable promotion of apoptosis
when compared to the control whereas cell growth inhibition
was observed at the concentrations evaluated.
belled lectins, concanavalin A (Con A) and phytohemagglutinin-
-PHA), to the cells was monitored by fluorescence activated cell
sorting (FACS) analysis in a flow cytometer. Con A recognizes man-
nose and glucose residues, whereas -PHA recognizes complex
L
(L
L
branched chain oligosaccharides containing N-acetyl-lactos-
amine.6a BAECs that had been treated with 2a for 24 h showed
greater binding of Con A (Figure S1 in supporting information) than
untreated control cells, indicating increased levels of glucose or
mannose structures at the cell surface; this is due to inhibition
by 2a of a-glucosidases involved in glycoprotein processing in
2.7. Effect of 5 on expression of cyclinD1 and the
phosphorylation level of extracellular signal-regulated kinases
(ERK) in BAECs
Table 1
Inhibition of a-glucosidase and BAEC growth by compounds
The effect of 5 on the expression of protein molecules important
for cell-cycle regulation was evaluated in order to elucidate the
molecular mechanisms involved in the observed cell-cycle altera-
tions. Underlying mechanisms for other MetAP2 inhibitors have
been studied previously. The inhibitor of MetAP2 and angiogenesis,
TNP-470, is reported to decrease levels of cyclin D1 in tumours.17
Increased levels of cyclin D1 are linked with the promotion of
Compound
a-Glucosidase inhibition
IC50 (lM)
BAEC growth inhibition
IC50 (mM)
1a
2a
3
4
5
Not active
1.67
6.07
2.41
1.15
0.347
Not active
0.797
0.610
0.105

Y. Zhao et al. / Bioorg. Med. Chem. 16 (2008) 6333–6337
6335
Figure 3. Western blot analyses for levels of cyclin D1 and activated Erk(phos-
phorylation of Erk) in BAECs. BAECs were incubated in the absence or presence of 5
at 100 and 200 lM or control for 24 h. Cell lysates were prepared from the treated
cells, and Western blot analyses were performed.
2.8. Effect of 5 on stress-fibre assembly in BAECs
It is well established that actin polymerization that leads to
stress-fibre assembly and depolymerization plays a crucial function
in cell motility. In the presence of some angiogenesis inhibitors such
as fumagillin and its analogues actin stress-fibre densities are in-
creased, indicating that these angiogenesis inhibitors induce endo-
thelial cells to assume an adhesive state that is not conducive to
motility.21 Other inhibitors of cell motility such as fausidil lead to
a loss of stress-fibre formation.22 It is possible that 5 also inhibited
BAEC motility, given thatit inhibitedtubeformation in vitro. The for-
mation of the actin cytoskeleton in BAECs was assessed by staining
with FITC-phalloidin, in order to specifically detect F-actin, in an at-
tempt to identify morphological traits in response to treatment with
5. The results (Fig. 4) show that stress-fibre assembly in BAECs was
abolished by 5, indicating that 5 inhibited BAEC tube formation, at
least partially through its ability to alter the behaviour of actin.
Figure 2. Effect of 5 on cell-cycle progression of BAECs. BAEC cells were treated
with DMSO (control) or 5 for 24 h, respectively. Cells were then stained with pro-
pidium iodide, and nuclei were analyzed for DNA content by flow cytometry. A total
of 10,000 nuclei were analyzed from each sample, and the percentages of cells
within G0/G1, S, and G2/M were determined. Representative profiles are shown and
the numbers given in the upper right corner of the profiles.
3. Conclusions
The synthesis of novel hybrids of DNJ and aryl-1,2,3-triazoles has
led 5 being identified as an inhibitor of angiogenesis in vitro. The hy-
brid 5 had a number of interesting biological effects. It inhibited
a-glucosidase at both the enzyme and cellular level, altering the bio-
synthesis of oligosaccharides on endothelial cell surfaces important
to angiogenesis. It inhibited the proliferation of BAECs by inducing
cell-cycle arrest. The novel hybrid 5 caused a reduction in the
expression of cyclin D1 but did not promote apoptosis or inhibit
the phosphorylation of ERK1/2. The hybrid 5 thus displayed mecha-
nistic differences to other classes of inhibitors of MetAP2, such as
fumagillin and its analogues, which have been reported to inhibit
the phosphorylation of ERK1/2 and induce apoptosis. Stress fibre
assembly in BAECs was abolished by the hybrid compound indicat-
ing the inhibition of BAEC tube formation by 5 is a result of a reduc-
tion in cell motility. Hybrid 5 displays biological properties of
interest in the development of multifunctional inhibitors of angio-
genesis and the research provides a basis for efforts to development
hybrid angiogenesis inhibitors with increased potency.
cell-cycle progression.18 Compounds that reduce the expression of
cyclin D1 would be expected to reduce endothelial cell growth and
angiogenesis in vitro. The levels of expression of cyclin D1 in BAECs
treated with 5 were established using Western blot analysis with
antibodies specific for cyclin D1. The expression of cyclin D1 was
found to be reduced in the presence of 5 at 100 and 200 lM doses
(Fig. 3).
The phosphorylation of ERK1/2 also occurs during cell prolifer-
ation.19 Reduction in the phosphorylation of ERK1/2 is associated
with reduced proliferation, and it has been reported that fumagillin
increases affinity of MetAP2 to ERK1/2 and causes the inhibition of
the phosphorylation of ERK1/2.20 There was the possibility that 5
could display similar behaviour to fumagillin. BAECs were there-
fore treated with 5 and the levels of both phosphorylated and
non-phosphorylated ERK1/2 were established using antibodies
and Western blot analysis under conditions where b-actin was
used as a loading control. The hybrid compound 5 (at 100 and
200 lM doses) was found not to alter either ERK1/2 phosphoryla-
tion or the total ERK1/2 level. Thus, 5 inhibited the expression of
cyclin D1 in endothelial cells but not a signalling pathway that in-
volves ERK1/2 phosphorylation.
4. Experimental
4.1. Cell culture
BAE cells were cultured in RPMI 1640 medium supplemented
with 10% heat inactivated FBS, 2 mM glutamine, 100 U/mL penicil-

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Y. Zhao et al. / Bioorg. Med. Chem. 16 (2008) 6333–6337
4.4. Tube-formation assay
Tube-structure formation on Matrigel was conducted and mod-
ified as described previously.13 Briefly, 70 ll growth factor-reduced
Matrigel was added to 96-well plates at 4 °C and then allowed to
polymerize at 37 °C for 1 h. Cultured cells were treated with com-
pounds (0.2 mM) for 24 h in 6-well plate. BAE cells were then har-
vested and suspended at a concentration of 3 Â 104cells/0.1 ml in
RPMI 1640 containing 10 ng/ml bFGF and 0.2 mM compounds.
Control cells were re-suspended with 10 ng/mL bFGF alone. Cells
were carefully layered on top of the polymerized gel and incubated
for 8 h at 37 °C in 5% CO2. Tube formation was observed and pho-
tographed under a microscope. At least five visual fields were
counted and the average number of tubes per field was calculated
using light microscope. The experiments were repeated at least
twice for each compound.
4.5. Cell-cycle analysis
Cells were seeded on 6-well plates in 1640 medium with 10%
FBS overnight. Then the compounds were added to the cells and
cultured for another 24 h. Cells were collected with trypsin–EDTA
and then washed three times with DPBS. The cells were resus-
pended and fixed for at least 2 h at À20 °C with 70% Ethanol. After
washing twice with DPBS, cells were incubated at 25 °C with
200 lg/ml RNase A for 30 min. The resulting cells were incubated
with 50 lg/ml propidium iodide for 30 min at 4 °C. The treated
cells were subjected to flow cytometry and the percentage of cells
at each phase of the cell cycle was analyzed. Each experiment was
repeated at least twice.
Figure 4. The effect of 5 on actin stress-fibre polymerization in BAEC was assessed
by fluorescence staining with FITC phalloidin, a substance which specifically detects
F-actin. In the control, stress fibres are present (red arrow). Incubation of BAECs
with 5 (200 lM) leads to marked disruption of stress-fibre assembly.
4.6. Quantitative apoptosis assay
The ability of 5 to induce apoptotic death of BAEC was quantified
by annexin V and PI staining and flow cytometry, as described re-
cently.23 Briefly, after treatment with 5 (0, 100, and 200 lM for 24,
48, and 72 h), cells were collected and washed with PBS twice, and
subjected to annexin V and propinium iodide staining using annexin
V FITC apoptosis kit following the step-by-step protocol provided by
the manufacturer. After staining, flow cytometry was performed for
the quantification of apoptotic cells. Each group was tested in tripli-
cate and the experiments were repeated at least twice in each case.
lin, and 100 lg/mL streptomycin and maintained at 37 °C in a
humidified atmosphere of 5% CO2. Subcultures were created by
passaging using a trypsin/EDTA mixture in phosphate buffered
saline.
4.2. Flow cytometry analysis of cell surface oligosaccharides
Cells were stained with fluorescin-labelled plant lectins and
analyzed by flow cytometry. Cultured cells were treated with com-
pounds (50 lM) for 24 h in 6-well plate. Cells were then harvested
and washed with DPBS. Approximately 5 Â 105 cells were resus-
pended in test tubes and incubated in DPBS/1% BSA with 200 ll
4.7. Immunoblot analysis
BAEC cells were treated with 5 (0, 100, and 200 lM) for 24 h in
6-well plate for the western blot studies. Cells were washed twice
with ice-cold PBS and lysed with RIPA buffer with protease inhib-
itors. After centrifuged at 13,000 rpm for 15 min, protein concen-
trations of the lysates were determined by micro-BCA protein
assay kit. The total cellular protein extracts were boiled with 2Â
Laemmli sample buffer and separated by SDS–PAGE and trans-
ferred to PVDF membrane. Membranes were blocked with 5%
non-fat dry milk in TBS containing 0.1% Tween 20 for 1 h at room
temperature and incubated with antibodies against cyclin D1, Erk,
phosphor-Erk, and b-actin overnight at 4 °C. Blots were washed
three times in TBS-T buffer, followed by incubation with the appro-
priate HRP-linked secondary antibodies for 1 h at room tempera-
ture. The specific proteins in the blots were visualized using the
enhanced chemiluminescence reagent. The experiments were re-
peated at least twice in each case.
2 lg/mL of Con A-FITC or -PHA-FITC for 1 h at 4 °C. Cells were
L
washed twice with DPBS, and fluorescence histogram profiles were
determined using flow cytometric FACS analysis. Each experiment
was carried out at least twice with each compound.
4.3. Inhibition of proliferation of BAECs
BAECs (3 Â 103 cells) were seeded on 96-well microtiter plates
in 1640 medium with 10% FBS and incubated overnight. The com-
pounds (10À6–10À3 M) were then added to the cells and cultured
for another 48 h. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyl-tetrazolium bromide] reagent was added directly to the
cell supernatant so as to give a final concentration of 0.5 mg/mL
of MTT. After 3 h at 37 °C the cell culture medium was removed.
Formazan crystals in adherent cells were dissolved in 200 ll DMSO,
and the absorbance of the formazan solution was measured at
570 nm.10 Each compound was tested in quadruplicate and the
experiments were repeated at least twice.
4.8. Immunofluorescence
Stress fibre of BAEC cells were detected using fluorescent phal-
loidin and analyzed by confocal microscopy. Cells were seeded to

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Acknowledgment
Funding was provided by Science Foundation Ireland (03/IBN/
B352).
Supplementary data
Synthesis of 3–5 and Figures S1 and S2. Supplementary data
associated with this article can be found, in the online version, at
doi:10.1016/j.bmc.2008.05.012.
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