Hybrid angiogenesis inhibitors: Synthesis and biological evaluation of bifunctional compounds based on 1-deoxynojirimycin and aryl-1,2,3-triazoles

Article abstract of DOI:10.1016/j.bmcl.2007.12.034

Synthesis of hybrids of 1-deoxynojirimycin (DNJ) and 5-aryl-1,2,3-triazole as potential bifunctional inhibitors of angiogenesis is described. The DNJ component inhibits the biosynthesis of cell surface oligosaccharides necessary for angiogenesis, whereas the aryl-1,2,3-triazole inhibits methionine aminopeptidase II, a target in angiogenesis therapy. One bifunctional compound was a more potent inhibitor of angiogenesis in vitro than DNJ alone or the 5-aryl-1,2,3-triazole alone.

Full text of DOI:10.1016/j.bmcl.2007.12.034

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 954–958
Hybrid angiogenesis inhibitors: Synthesis and biological
evaluation of bifunctional compounds based on
1-deoxynojirimycin and aryl-1,2,3-triazoles
Ying Zhou,b Yunxue Zhao,a Kathy M. O’ Boylea and Paul V. Murphyb,*
aUCD School of Biomolecular and Biomedical Science and the UCD Conway Institute, University College Dublin,
Belfield, Dublin 4, Ireland
bUCD School of Chemistry and Chemical Biology and Centre for Synthesis and Chemical Biology, University College Dublin,
Belfield, Dublin 4, Ireland
Received 22 November 2007; revised 13 December 2007; accepted 16 December 2007
Available online 23 December 2007
Abstract—Synthesis of hybrids of 1-deoxynojirimycin (DNJ) and 5-aryl-1,2,3-triazole as potential bifunctional inhibitors of angio-
genesis is described. The DNJ component inhibits the biosynthesis of cell surface oligosaccharides necessary for angiogenesis,
whereas the aryl-1,2,3-triazole inhibits methionine aminopeptidase II, a target in angiogenesis therapy. One bifunctional compound
was a more potent inhibitor of angiogenesis in vitro than DNJ alone or the 5-aryl-1,2,3-triazole alone.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Angiogenesis1 provides new blood vessels to growing
and developing tissue including tumours. Pathological
angiogenesis occurs in tumour formation, and also in
a range of other ‘angiogenesis-dependent diseases’ that
include rheumatoid arthritis, diabetic retinopathy, ath-
erosclerosis, Crohn’s disease and diseases of the nervous
system.2 Angiogenesis is a major factor affecting the
metastatic spread of malignant tumour cells and so the
development of angiogenesis inhibitors is of interest in
cancer therapy. Drug discovery for angiogenesis depen-
dent disease has been recently reviewed by Folkman.3
Angiogenesis inhibitors that target a range of angiogenic
proteins and/or pathways will be less susceptible to drug
resistance than those agents that target a single protein
or pathway. Therefore, it will be important for synthetic
chemists to produce novel multifunctional pharmaceuti-
cal compounds that have potential to inhibit two or
more biological targets relevant to angiogenesis.
rently in clinical trials.5 Recently aryl-1,2,3-triazoles 1
have been identified as inhibitors of both MetAP2 and
angiogenesis.6 Glycosidases are another relevant target;
inhibitors of a-glucosidases such as N-methyl-1-deoxy-
nojirimycin 2b, castanospermine, or 1-deoxymannojiri-
mycin alter the biosynthesis of glycans on endothelial
cell surfaces that are required for angiogenesis.7 Herein
we describe the synthesis and evaluation of the hybrid
angiogenesis inhibitors 3–5 (Chart 1) which have been
designed to inhibit both a-glucosidase and MetAP2.
The design of 3–5 was based on combining the proper-
ties of glycosidase inhibition and MetAP2 inhibition
into a single molecule to generate a hybrid angiogenesis
OH
H
N
R
N
OH
R
N
Methionine aminopeptidase II (MetAP2) has been
indentified4 as the target for the angiostatic agents fum-
agillin and ovalicin and an inhibitor PPI-2458 is cur-
HO
OH
2a R = H
1
N
2b R = Me
2c R = Bu
H
OH
OH
N
(
)
O
N
n
N
HO
N
3 (n = 2)
4 (n = 4)
5 (n = 6)
Keywords: Angiogenesis; Inhibitor; Alpha-glucosidase; Methionine
aminopeptidase; Aryl-1,2,3-triazole; 1-Deoxynojirimycin, Bifunctional
compound; Hybrid.
OH
*
Chart 1. Structures of 1–5.
Corresponding author. E-mail: paul.v.murphy@ucd.ie
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.12.034

Y. Zhou et al. / Bioorg. Med. Chem. Lett. 18 (2008) 954–958
955
inhibitor. The ability of N-butyl-DNJ 2c to inhibit a-
glucosidases8 indicated that an alkyl chain linker be-
tween the DNJ nitrogen atom and the aromatic residue
would not have a deleterious effect on glycosidase activ-
ity. Secondly, the docking of a low energy conforma-
tional isomer of 5 to the active site of MetAP2, based
on the crystal structure of the complex between an
aryl-1,2,3-triazole derivative and MetAP2, indicated
that 5 would adopt a conformation that would preserve
the MetAP2 interactions and additionally bind the
DNJ moiety. The DNJ moiety can form VDW interac-
tions with the residues on the surface of the enzyme
(Fig. 1).
with benzyl azide or azidotrimethylsilane were unsuc-
cessful. Conversely, the reaction of benzyl azide and
acetylenes 13a–c, obtained by per-O-acetylation of the
DNJ hydroxyl groups of 12a–c, gave the triazoles 14a–
c as mixtures of regioisomers.14 Deacetylation of 14a–c
using sodium methoxide in methanol followed by cata-
lytic hydrogenolysis gave the hybrids 3–5.15 In addition
the aryl-1,2,3-triazole 15 was also prepared from the rel-
evant aromatic aldehyde by first converting the aldehyde
to the acetylene and the subsequent Huisgen reaction
with azidotrimethylsilane.
Compounds were evaluated for their ability to inhibit a-
glucosidase assay from bacillus stearothermophilus as
described by Hakamata et al.16 in order to test their en-
zyme inhibitory activity, and the results are provided in
Table 1. The IC50 for 2a was 1.67 lM. The hybrid com-
pound 5 (IC50 = 1.15 lM) was more potent than 2a,
The synthesis of 3–5 (Scheme 1) began with cleavage of
the aromatic methoxy group of 3-methoxybenzaldehyde
69 to give 7. This was best achieved using thiophenol,
potassium carbonate and N-methyl-pyrrolidinone.10
These conditions were superior to the use of boron tri-
chloride or to the use of boron trichloride–tetrabutylam-
monium iodide which have been applied previously for
the demethylation of aromatic ethers. Next the selective
alkylation of 7 using dialkyl bromides in acetone using
potassium carbonate gave the aldehydes 8a–c. The alde-
hyde groups of 8a–c were then converted to the acety-
lenes 10a–c, respectively, using the Ohira-Bestmann
reagent 911 under basic conditions.12
whereas compounds
3 and 4 were less potent
(IC50 = 6.07 lM and 2.41 lM, respectively) than 2a.
Thus, the structural modification of incorporating the
aryl triazole via an alkyl linker to 2a did not lead to a
significant reduction of the inhibition of a-glucosidase.
In the case of 5, the structural modification led to a
modest enhancement of inhibitory properties, indicating
that the N-substituent may have favourable interactions
with the glucosidase. The aryl-1,2,3-triazole 15 (Chart 2)
did not inhibit a-glucosidase.
The alkylation of 2a13 or per-O-acetylated 2a with the
bromides 10a–c under a variety of conditions (using
the bases K2CO3, KOH, DIPEA, NaH, K2CO3–CuI,
Et3N in solvents MeCN, DMF or MeOH) gave either
low yields or none of the desired products. To circum-
vent this problem the bromides 10a–c were converted
to the more reactive iodides 11a–c by reaction with so-
dium iodide in acetone. These iodides were then reacted
with DNJ 12 in DMF in the presence of K2CO3 to give
13a–c in moderate to good yield. Attempts to carry out
Huisgen 1,3-dipolar cycloaddition reactions of 12a–c
The inhibition of BAEC growth by compounds was next
determined using the MTT assay17 and the results are
also summarized in Table 1.18 The iminosugar 2a was
inactive with no significant inhibition of proliferation
being observed at concentrations up to 1 mM. However,
the aromatic triazole 15 (IC50 = 0.347 mM) was able to
inhibit growth of BAECs. The inhibition of human
(HUVEC) and mouse (MS-1) endothelial cell prolifera-
tion by aryl-1,2,3-triazoles is consistent with the ability
to inhibit MetAP2 and it can be inferred that inhibition
of MetAP2 in BAECs would account for the activity ob-
served for 15. It is worth noting that aryl-1,2,3-triazole
derivatives have IC50 values of 2–30 lM as inhibitors
of HUVEC and MS-1 growth.5 The hybrids 3–5 all
inhibited BAEC proliferation with 5 being the most po-
tent inhibitor (IC50 = 0.105 mM). Thus, introduction of
the alkylated DNJ substituent to the 3-position of the
benzene residue did not lead to a loss in ability of the
aryl-1,2,3-triazole to inhibit endothelial cell growth; in
the case of 5, the structural modification led to an in-
crease in potency.
As compounds 3–5 can inhibit a-glucosidase at the en-
zyme level, the effect on the cell surface oligosaccharide
structure was investigated. Accordingly, BAECs were
treated with compounds (50 lM) for 24 h and then the
binding of specific fluorescein (FITC)-labelled lectins
to the cells was monitored by fluorescence activated cell
sorting (FACS) analysis in a flow cytometer.19 In these
studies the two lectins, concanavalin A (ConA) and phy-
tohemagglutinin-L (L-PHA), were investigated. Con A
recognises mannose and glucose residues, whereas L-
PHA recognises complex branched chain oligosaccha-
rides containing tri- and tetra-antennary b-linked disac-
Figure 1. Hybrid 5 (CPK model) docked to the aryl-1,2,3-triazole
binding site (brown tubes) of MetAP2. The MetAP2 amino acid
˚
residues shown are those within 4 A of 5.

956
Y. Zhou et al. / Bioorg. Med. Chem. Lett. 18 (2008) 954–958
Br
K2CO3, MeOH
(
)
Br
PhSH, K2CO3, NMP
n
Br
n
Br
n
( )
( )
OHC
OH
OHC
OMe
OHC
O
O
O
P
O
heat
77%
K2CO3, acetone
heat
6
7
MeO
MeO
10a n=3 79%
10b n=5 78%
10c n=7 89%
8a n=3 73%
8b n=5 75%
8c n=7 71%
N2
9
OAc
OH
N
OAc
OAc
NaI, Acetone
heat
OH
OH
2a, K2CO3, DMF
Ac2O,Py
DMAP
I
N
( )
105 oC
( )
O
n
( )
n
O
O
n
11a n=3 94%
11b n=5 97%
11c n=7 98%
AcO
HO
12a n=3 50%
12b n=5 48%
12c n=7 48%
13a n=3 55%
13b n=5 74%
13c n=7 50%
OAc
OAc
OAc
OAc
OAc
PhCH2N3
toluene
3 100%
4 100%
5 80%
1. NaOMe, MeOH
+
N
N
)
(
OAc
)
O
(
O
BnN
N
N
n
n
Heat, 48 h
2. 10%Pd/C,
HCl/MeOH
N
NBn
N
AcO
AcO
14a n=3
14b n=5
14c n=7
Scheme 1.
Table 1. Inhibition of a-glucosidase and BAEC growth by compounds
Compound a-Glucosidase inhibition BAEC growth inhibition
three did this to a greater extent than 2a. The hybrid
5, the most potent a-glucosidase inhibitor, was the most
potent inhibitor of glycoprocessing as indicated by the
high level of ConA and low level of L-PHA binding to
BAECs following treatment. Although compounds 3
and 4 are less potent a-glucosidase inhibitors than 2a,
they alter surface oligosaccharide expression to a greater
extent than 2a. This apparent anomaly may be explained
by the possibility that they have increased cellular per-
meability compared to 2a, thereby facilitating diffusion
to the endoplasmic reticulum(ER) and enhancing access
to the glycoprocessing enzymes therein. Satisfactory cel-
lular permeability and its potency as an inhibitor of glu-
cosidase activity would account for the activity
displayed by 5.
IC50 (lM)
IC50 (mM)
2a
3
1.67
6.07
Not active
0.797
0.610
4
2.41
1.15
5
15
0.105
0.347
Not active
OMe
N
NH
N
15
Chart 2. Structure of 15.
Compounds 3–5 were next evaluated for their ability to
inhibit angiogenesis in vitro using the endothelial cell
tube formation assay.20 Endothelial cells which are in-
duced to undergo tube formation change their architec-
ture and form cell–cell contacts that lead to branched
networks that are similar to capillary-like blood vessels.
When BAECs are cultured on polymerized matrigel they
organise into such tube-like structures (Fig. 3(a)). How-
ever, when BAECs were cultured on polymerized matri-
gel in the presence of 5 (0.2 mM, Fig. 3(b)) the cells
failed to organise into these capillary-like structures.
At this concentration none of the other compounds
inhibited tube formation.
charides of N-acetyl-lactosamine on mannose residues
of asparagine linked oligosaccharides.7a
Following 24-h treatment with compounds, cells were
labelled with ConA-FITC or L-PHA-FITC and then
cells were sorted according to the intensity of the fluo-
rescence they emitted. Results of the analyses are shown
in Figure 2. As expected, BAECs that had been treated
with 2a for 24 h showed greater binding of ConA than
untreated control cells, indicating increased levels of
high mannose structures at the cell surface. The treat-
ment of BAECs with 3–5 also led to increased binding
of ConA to cell surfaces when compared to the control
and 5 and 4 increased ConA binding more than 2a, pro-
viding evidence that the hybrid compounds inhibit a-
glucosidase at the cellular level. Further evidence to sup-
port this conclusion was obtained when the binding of
L-PHA to BAECs was examined. It was found that,
as expected, treatments that increased ConA binding de-
creased L-PHA binding. Thus, L-PHA binding to BAE-
Cs was reduced in cells that had been treated with 2a
compared to control, untreated cells. Compounds 3–5
lowered levels of L-PHA binding to BAECs and all
In summary, the synthesis of novel hybrids of 2a and 5-
aryl-1,2,3-triazoles has been achieved by a sequence that
includes N-alkylation of 2a and subsequent Huisgen 1,
3-dipolar cycloadditions of azides and alkynes. These
novel hybrid compounds showed interesting biological
properties when compared with 2a and 15. One hybrid
showed increased glycosidase inhibitory activity and in-
creased ability to alter the biosynthesis of oligosaccha-
rides on endothelial cell surfaces when compared with
2a. The same compound showed increased ability to in-

Y. Zhou et al. / Bioorg. Med. Chem. Lett. 18 (2008) 954–958
957
Figure 2. Effect of glycosidase inhibitors on cell surface BAEC
oligosaccharide expression. BAECS were cultured for 24 h in the
absence (control) or presence of 5 · 10À5 M DNJ and novel com-
pounds. Cells were harvested with trypsin/EDTA, treated with Con A-
FITC (top) or L-PHA-FITC (bottom) and analyzed by flow cytom-
etry. Plots show the relationship between fluorescence intensity
(FL1Log, x-axis) and cell number (counts, y-axis) for populations of
cells treated with different compounds.
Figure 3. Tube formation. (a) BAECs cultured on polymerized
matrigel organise into tube-like structures. (b) Hybrid 5 inhibits tube
formation (2 · 10À4 M).
References and notes
1. (a) Liekens, S.; de Clercq, E.; Neyts, J. Biochem. Pharm.
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hibit the proliferation of BAECs when compared to 15.
Endothelial cell proliferation was not inhibited by 2a at
high concentrations and aryl-1,2,3-triazoles did not inhi-
bit either a-glucosidase at the enzyme level or glycopro-
tein processing at the cellular level. Hybrid 5 completely
inhibited capillary tube formation similar to capillary-
like blood vessels that are formed in vivo during angio-
genesis. Hybrid 5 displays properties desirable in the
development of bifunctional inhibitors of angiogenesis.
2. Hanahan, D. Nature Med. 1998, 4, 13.
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Acknowledgments
The authors thank staff at the UCD NMR Centre and
Dr. Dilip Rai. The research was funded by Science
Foundation Ireland (03/IBN/B352).
Supplementary data
8. For reviews on glycosidase inhibition, see (a) Winchester,
B.; Fleet, G. W. J. Glycobiology 1992, 2, 199; (b) Ganem,
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Vasella, A. T. Angew. Chem., Int. Ed. 1999, 38, 750; (d)
NMR spectra of 3–5, and additional figures. Supple-
mentary data associated with this article can be found,
in the online version, at doi:10.1016/j.bmcl.2007.12.034.

958
Y. Zhou et al. / Bioorg. Med. Chem. Lett. 18 (2008) 954–958
Lillelund, V. H.; Jensen, H. H.; Liang, X.; Bols, M. Chem.
10% heat-inactivated FBS, 25 mM glutamine, 100 U/mL
penicillin 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. BAECs (3 · 103 cells) were
seeded on 96-well microtiter plates in 1640 medium with
Rev. 2002, 102, 515; (e) Greimel, P.; Spreitz, J.; Stutz, A.
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Chem. 2002, 67, 6406.
11. Xu, Z. S.; Peng, Y. G.; Ye, T. Org. Lett. 2003, 5, 2821.
12. (a) Muller, S.; Liepold, B.; Roth, G. J.; Bestmann, H. J.
¨
10% FBS and incubated overnight. The compounds (10À6
–
10À3 M) were then added to the cells and cultured for
another 48 h. MTT was added directly to the cell superna-
tant 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 dis-
solved in 200 lL DMSO and the absorbance of the
formazan solution was measured at 570 nm. Each com-
pound was tested in quadruplicate and the experiments were
repeated at least twice. The IC50s are shown in Table 1.
18. For other selected carbohydrate derived compounds that
inhibit BAEC growth, see (a) Rawe, S. L.; Zaric, V.; O’
Boyle, K. M.; Murphy, P. V. Bioorg. Med. Chem. Lett.
2006, 16, 1316; (b) Murphy, P. V.; Pitt, N.; O’ Brien, A.;
Enright, P. M.; Dunne, A.; Wilson, S. J.; Duane, R. M.;
O’ Boyle, K. M. Bioorg. Med. Chem. Lett. 2002, 12, 3287.
19. Cultured BAECs (Ref. 17 ) were treated with compounds
(50 lM) for 24 h in 6 well plate. Cells were then harvested
and washed with DPBS. Approximately 5 · 105 cells were
resuspended in test tubes and incubated in DPBS/1% BSA
with 200 lL of 2 lg/ml Con A-FITC or L-PHA-FITC for
1 h at 4 ꢁC. Cells were washed twice with DPBS, and
fluorescence histogram profiles were determined using
flow cytometry. Each experiment was carried out at least
twice with each compound.
SynLett 1996, 6, 521; (b) Roth, G. J.; Liepold, B.; Muller,
S.; Bestmann, H. J. Synthesis 2004, 1, 59.
¨
13. (a) Danieli, E.; Lalot, J.; Murphy, P. V. Tetrahedron 2007,
63, 6827; (b) McDonnell, C.; Cronin, L.; O’ Brien, J. L.;
Murphy, P. V. J. Org. Chem. 2004, 69, 3565.
14. A regioselective reaction was not required as the benzyl
group was later removed.
15. Selected analytical data for 5: 1H NMR (CD3OD,
500 MHz) d 8.14 (s, 1H), 7.37 (m, 2H), 7.31 (t, 1H, J
7.8 Hz), 6.92 (d, 1H, J 7.7 Hz), 4.04 (t, 2H, J 6.40 Hz), 3.87
(d, 2H, J 2.6 Hz), 3.50 (dt, 1H, J 4.7 Hz, J 9.9 Hz), 3.38 (t,
1H, J 9.3 Hz), 3.16 (t, 1H, J 9.1 Hz), 3.04 (dd, 1H, J
4.9 Hz, J 11.3 Hz), 2.84 (m, 1H), 2.62 (m, 1H), 2.25 (t, 1H,
J 11.00 Hz), 2.21 (d, 1H, J 9.5 Hz), 1.82 (dt, 2H, J 6.5 Hz,
J 13.5 Hz), 1.55–1.29 (m, 10H); 13C NMR (CD3OD,
125 MHz) d 159.9 (C), 131.3 (C), 129.9 (CH), 118.1 (CH),
114.7 (CH), 111.7 (CH), 79.2 (CH), 70.7 (CH), 69.3 (CH),
67.9 (CH2), 66.2 (CH), 58.0 (CH2), 56.3 (CH2), 52.6
(CH2), 29.4 (CH2), 29.2 (CH2), 29.1 (CH2), 27.3 (CH2),
25.9 (CH2), 24.1 (CH2); IR (cmÀ1) ESI-HRMS: calcd for
C22H35N4O5 435.2607, found m/z 435.2602 [M + H]+.
16. Hakamata, W.; Nakanishi, I.; Masuda, Y.; Shimizu,
T.; Higuchi, H.; Nakamura, Y.; Saito, S.; Urano, S.;
Oku, T.; Ozawa, T.; Ikota, N.; Miyata, N.; Okuda,
H.; Fukuhara, K. J. Am. Chem. Soc. 2006, 128, 6524,
4-Nitrophenyl a-D-glucopyranoside (PNP-G, 40 lL of
3 mM) in DPBS (pH 7.0), selected compound (5 lL)
in DPBS and a-glucosidase (5 lL, Sigma, G-3651)
were mixed. After incubation of this mixture for
20 min at 37 ꢁC, the reaction mixture was added to
200 lL of 0.5 M Na2CO3 to stop the reaction, and the
absorbance of 4-nitrophenol released from PNP-G at
405 nm was then measured. Each compound was
tested in triplicate and the experiment was repeated
at least twice. The concentrations of compound
required to inhibit the a-glucosidase activity by 50%
(IC50) are shown in Table 1.
20. Tube-structure formation on Matrigel was conducted and
modified as described previously. See Akalu, A.; Roth, J.
M.; Caunt, M.; Policarpio, D.; Liebes, L.; Brooks, P. C.
Cancer Res. 2007, 67, 4353, 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
(Ref. 17) were treated with compounds (0.2 mM) for 24 h
in 6-well plate. BAE cells were then harvested and
suspended at a concentration of 3 · 104 cells/0.1 ml in
RPMI 1640 containing 10 ng/mL bFGF and 0.2 mM
compounds. Control cells were resuspended 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 photographed 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.
17. Mosmann, T. J. Immunol. Methods 1983, 65, 55, BAECs
were cultured in RPMI 1640 medium supplemented with