N-Glycosyl-thiophene-2-carboxamides: Effects on endothelial cell growth in the presence and absence of bFGF - A significant increase in potency using per-O-acetylated sugar analogues

Article abstract of DOI:10.1016/j.bmcl.2005.11.058

Inhibitors of endothelial cell proliferation are of interest in development of therapies for angiogenesis related disease. N-Glucosyl-thiophene-2- carboxamides have been synthesized and evaluated for their effects on proliferation in bovine aortic endothelial cells. Per-O-acetylated-N-glucosyl- thiophene-2-carboxamides showed improved inhibition of both serum and bFGF stimulated uptake of [³H]thymidine, when compared to non-acetylated analogues.

Full text of DOI:10.1016/j.bmcl.2005.11.058

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1316–1319
N-Glycosyl-thiophene-2-carboxamides: Effects on endothelial
cell growth in the presence and absence of bFGF—
A significant increase in potency using per-O-acetylated
sugar analogues
a
b
b,
a,
*
*
Sarah L. Rawe, Violeta Zaric, Kathy M. OÕ Boyle and Paul V. Murphy
a
Centre for Synthesis and Chemical Biology, UCD School of Chemistry and Chemical Biology, UCD Conway Institute,
University College Dublin, Belfield, Dublin 4, Ireland
UCD School of Biomolecular and Biomedical Research, UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland
b
Received 13 October 2005; revised 16 November 2005; accepted 16 November 2005
Available online 15 December 2005
Abstract—Inhibitors of endothelial cell proliferation are of interest in development of therapies for angiogenesis related disease.
N-Glucosyl-thiophene-2-carboxamides have been synthesized and evaluated for their effects on proliferation in bovine aortic
endothelial cells. Per-O-acetylated-N-glucosyl-thiophene-2-carboxamides showed improved inhibition of both serum and bFGF
3
stimulated uptake of [ H]thymidine, when compared to non-acetylated analogues.
ꢀ
2005 Elsevier Ltd. All rights reserved.
The signal transduction processes that modulate cellular
behaviour are important biological events. For example,
angiogenesis provides new blood vessels to growing and
VEGF families, induce new blood vessel formation by
activating their cognate tyrosine kinase receptors
(FGFR) on the endothelial cell surface. Compounds
1
4
developing tissue including tumours, and it relies in part
on the up-regulation of endothelial cell proliferation.
Up-regulated angiogenesis is characteristic in rheuma-
toid arthritis, diabetic retinopathy, during tumour
that target the factors that promote proliferation of
endothelial cells or that target the cell signalling path-
way up-regulated by growth factors have potential as
therapeutics. Previously we set out to develop com-
pounds reduced in carbohydrate character (monosac-
charide conjugates) that might be mimetics of non
2
growth and metastasis. The tumour angiogenesis pro-
cess results from the production of the pro-angiogenic
factors basic fibroblast growth factor (bFGF) and vas-
cular endothelial growth factor (VEGF) in a signalling
cascade, and the down-regulation of negative modula-
tors, like angiostatin, in tissues with a quiescent vascula-
ture. Angiogenesis is not only a prerequisite for tumour
growth and expression but also a major factor affecting
the metastatic spread of malignant cells. Thus, the devel-
opment of angiogenic inhibitors may allow a new thera-
peutic strategy against malignant tumours. Inhibitors of
cell proliferation and consequently growth are of inter-
est. A number of strategies are being considered for
5
sulfated disaccharides and potential modulators of
6
bFGF-induced endothelial cell growth; this led to iden-
tification of N-(b-D-glucopyranosyl)-thiophene-2-car-
boxamide 1 and glucuronic acid derivative 2 as
inhibitors of bovine aortic endothelial cell (BAEC)
growth (see Chart 1). It was later determined that they
7
were not mimetics of the disaccharides.
The synthesis and investigation of the biological proper-
ties of structural analogues of 1 are thus of interest with
3
the development of anti-proliferative agents. Heparin-
binding endothelial growth factors, like bFGF and
HO
HO
HO
2
C
HO
HO
HO
O
H
N
O
H
N
OH
OH
S
S
O
O
Keywords: Endothelial cell proliferation; Angiogenesis; bFGF; Glyco-
conjugates; Glycosyl amides; Thiophene-2-carboxamides.
1
2
*
Corresponding authors. E-mail: paul.v.murphy@ucd.ie
Chart 1.
0
960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.11.058

S. L. Rawe et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1316–1319
1317
9
a view to development of more potent cell growth inhib-
itors. This could ultimately be helpful for the determina-
tion of the biological mechanism of action of 1, which is
as yet unknown, and could yield novel targets in cell sig-
nalling pathways. We have thus prepared a series of ana-
logues of 1 and have explored effects of these
compounds on endothelial cells grown in the presence
and absence of bFGF. N-Glucosyl-thiophene-2-carbox-
The effect of GTCs on proliferation of BAEs (BAECs)
induced by 10% foetal bovine serum (=complete culture
medium) was investigated. The inhibitory activities of
the thiophene–sugar conjugates were compared to that
of heparin–albumin (HA, positive control) on cell prolif-
3
eration by analysing [ H]thymidine incorporation into
DNA during DNA synthesis. Addition of HA (10 lg/
mL) to the complete culture medium resulted in reduced
8
3
[ H]thymidine incorporation by 37 ± 2.5% after 24 h
amides (GTCs) were prepared from glucosamine 3
(
Scheme 1). The reactions of 3, in the presence of sodi-
exposure, results similar to those previously reported
6
um carbonate, with acyl chlorides derived from thio-
phene-2-carboxylic acids afforded 4–9. Anomerisation
was observed during the coupling reaction leading to
minor amounts (a:b = 1:16) of the a-anomers being
by researchers from this group. One set of experiments
was conducted to determine whether the inhibition of
3
[ H]thymidine incorporation into BAECs by a thio-
phene conjugate was dose dependent. The 3-bromo
derivative 10 showed a dose-dependent decrease in
1
observed in the H NMR spectra of the products.
3
[ H]thymidine incorporation, 24 h after exposure
Deacetylation of per-O-acetylated derivatives 4–9 gave
the unprotected N-glucosyl-thiophene-2-carboxamides
(Fig. 1), reducing BAEC growth by 25 ± 3% at
500 lM and by 10 ± 3% at 100 lM (Fig. 1). A wider
range of GTCs (Table 1) were subsequently evaluated
for their effects on BAEC growth. Most GTCs
(500 lM) consistently produced a maximal suppressive
1
0–15.
The acetylene derivatives 16/17 were prepared from 4/6
by a Sonogashira coupling with ethynyltrimethylsilane
3
(
Scheme 2), which was carried out in a sealed reaction
response after 24 h exposure and inhibited [ H]thymi-
vessel at 80 ꢁC. The removal of the terminal TMS and
deacetylation from 16/17 was achieved by their reaction
with TBAF in THF at 0 ꢁC for 5 min and subsequent
treatment with potassium carbonate in methanol to give
dine uptake by 16–35%. The 5-bromothieno conjugate
13 had the greatest effect, showing 35% inhibition.
Thiophene conjugate 1 was found to be inactive under
these conditions.
1
8/19.
Further experiments explored the effect of the per-O-
acetylated GTCs (Fig. 2) on 10% serum-induced BAEC
proliferation. The protected derivative 9, applied at
the highest concentration of 80 lM, was found to
Na
2
CO
3
R1
R2
AcO
AcO
AcO
2
CH Cl , H O
AcO
O
2
2
O
H
N
AcO
NH
2
AcO
R1
R2
OAc
R3
OAc
S
3
O
Cl
4: R1
= Br, R2 = H, R3 = H, 88 %
5: R1 = Me, R2 = H, R3 = H, 41 %
R3
S
O
6
7
8
9
: R1 = H, R2 = Br, R3 = H, 88 %
: R1 = H, R2 = H, R3 = Br, 92 %
: R1 = H, R2 = H, R3 = Me, 73 %
: R1 = H, R2 = H, R3 = Et, 91 %
R1
R2
K
2
CO
3
(5 mol %) HO
H
N
O
HO
HO
MeOH,
R3
OH
S
rt, 30 min
O
1
1
1
1
1
1
0: R1 = Br, R2 = H, R3 = H, 88 %
1: R1 = Me, R2 = H, R3 = H, 89 %
2: R1 = H, R2 = Br, R3 = H, 79 %
3: R1 = H, R2 = H, R3 = Br, 73 %
4: R1 = H, R2 = H, R3 = Me, 83 %
5: R1 = H, R2 = H, R3 = Et, 71 %
Scheme 1.
Figure 1. Concentration-dependent inhibition by 10 on 10% serum-
induced BAEC proliferation. Proliferation of BAEC was assessed by
3
measuring [ H]thymidine incorporation. Cells were seeded (30,000
R1
R2
R1
R2
AcO
AcO
AcO
AcO
AcO
H
N
O
H
N
cells/well) in 24-well plates. After a 24 h adhesion period, cells were
treated with increasing concentrations of 10 for the following 20 h at
O
TMS
AcO
OAc
S
OAc
S
3
PdCl2(cat.),
37 ꢁC and 5% CO
added to each well, and incubation was continued for a further 4 h at
2
. Subsequently, [ H]thymidine (2 lCi/mL) was
O
O
1
2
1
C, R2 = H, 63%
C, 82%
4
: R = Br, R = H
CuI(cat.), PPh3
: R = H, R = Br NEt3, DMF, 80 o
16: R = TMS-C
1
2
17: R = H, R2 = TMS-C
1
6
C
37 ꢁC and 5% CO . Experiments were terminated with washing (2· in
PBS) and fixing the cells in trichloroacetic acid (10%). The cells were
2
R1
R2
3
then lysed with 0.1 N NaOH. The amount of incorporated [ H]thy-
. TBAF, THF, 0 oC, 5 min
HO
HO
HO
H
N
1
O
midine in cell lysates was measured by scintillation counting. Data are
presented as percentage of the values for the control-treated cells
(100%) and each bar is the mean ± SEM of three separate experiments
(each performed in triplicate). Statistical comparisons between groups
were performed using StudentÕs t test. **(p < 0.01); ***(p < 0.001) vs
control group.
OH
S
2
. K2CO3, MeOH, rt, 3h,
O
quant over 2 steps
1
C, R2 = H
1
8: R = H-C
1
2
1
9: R = H, R = H-C
C
Scheme 2.

1318
S. L. Rawe et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1316–1319
Table 1. Effect of GTCs (500 lM) and heparin–albumin (HA; 10 lg/
mL) on 10% serum-induced BAEC proliferation
80 lM (Fig. 2). The non-acetylated analogue 15 was
inactive at all concentrations up to 80 lM. Thus, there
is greater than sixfold improvement for 9 when com-
pared to the corresponding deacetylated analogue 15
and related compounds in Table 1. The GTCs 9 and
a
Compound
% of control
p versus control
HA
1
63 ± 2.5
na
0.001
—
1
bFGF (10 ng/mL) stimulated proliferation of BAECs
5 were also investigated (80 lM) for their effects on
1
1
1
1
1
1
1
0
1
3
4
5
8
9
75.4 ± 1.3
84 ± 3
0.001
0.001
0.001
0.001
0.001
0.001
0.001
3
over a period of 48 h incubation (Fig. 3). The [ H]thymi-
65 ± 5.3
83 ± 1.8
80.3 ± 2.5
79.75 ± 2.7
71 ± 3.3
dine incorporation increased by 30% ± 4 in the presence
of bFGF (Fig. 3) over 48 h. Whereas the per-O-acetylat-
10
ed derivative 9
[
(80 lM) inhibited bFGF-induced
H]thymidine uptake to control levels, the correspond-
3
3
11
Proliferation was measured by quantification of [ H]thymidine incor-
poration after 24 h.
ing deacetylated analogue 15 had no effect under iden-
tical conditions at the same concentration (Fig. 3). We
suggest that the reason for the improved activity of acet-
ylated conjugates is that the compounds may be exerting
their mechanism of action intracellularly and that their
membrane permeability is superior to polyhydroxylated
sugars. It is known that acetylated carbohydrates can
have increased absorption when compared to non-acet-
ylated compounds and that esterases can cleave the acyl-
protecting groups and presumably generate the active
glucoside once the compound is localized in the
a
The results are expressed as percentage of the values obtained for the
control-treated cells (100%) and means ± SEM of 3 independent
experiments are reported. na = not active.
1
2
cytosol.
In summary, a series of N-glucosyl-thiophene-2-carbox-
amides have been synthesized and evaluated for their ef-
fects on serum and bFGF stimulated bovine aortic
endothelial cell growth; per-O-acetylated-N-glucosyl-
thiophene-2-carboxamides showed significant inhibition
3
of [ H]thymidine incorporation when compared to the
non-acetylated analogues. Presumably the weaker inhib-
itory activities achieved with non-acetylated sugars are
due to the low transport of polar agents through the cell
membrane. Further investigations are underway to con-
firm this hypothesis. The results indicate that further
synthetic and biological studies with acetylated glyco-
conjugates related to those described herein offer prom-
ise of development of novel compounds that could
inhibit tumour angiogenesis or other pathological pro-
cesses induced by bFGF. Cell biological mechanistic
studies are currently underway for 9 and related conju-
gates and results will be presented in due course.
Figure 2. The effect of per-O-acetylation on biological activity. Per-O-
acetylated conjugate 9 and its deacetylated analogue 15 were evaluated
for effects on BAE cell proliferation, which was determined by
3
[
H]thymidine incorporation after 24 h exposure to compounds. Each
bar is the mean of triplicate values from three separate experiments.
**(p < 0.001) versus control.
*
3
significantly reduce incorporation of [ H]thymidine by
1 ± 2%. The per-O-acetylated 5-ethyl derivative 9
showed a dose-dependent inhibition of [ H]thymidine
4
3
uptake; it was inactive at 3.3 lM; inhibited uptake by
2
1% ± 2 at 16 lM (data not shown) and by 41 ± 2% at
Figure 3. Effect of compounds on bFGF-promoted proliferation of BAECs. The per-O-acetylated conjugate 9 showed significant inhibiton (80 lM)
of bFGF induced biosynthesis of DNA. The unprotected conjugate 15 was inactive at the same concentration. Cells were seeded at 30,000 cells/well
into 24-well plates. Cells were starved with 0.5% foetal bovine serum for 24 h. The cells were then incubated with or without indicated reagents and
3
3
bFGF for 48 h and harvested. Before the harvest, cells were incubated with [ H]thymidine (2 lCi/mL) for 4 h. The incorporated [ H]thymidine
radioactivity was measured as described previously (Fig. 1). Each bar is the mean of triplicate values from three separate experiments. Statistical
#
comparisons between groups were performed using StudentÔs t test. ***(p < 0.001) versus control, *** (p < 0.001) versus bFGF treatment.

S. L. Rawe et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1316–1319
1319
8
Acknowledgments
a biphasic mixture of 3 (0.5 g, 1.44 mmol) in CH
(
(
2
Cl
7 mL) and sodium carbonate (0.15 g, 1.44 mmol) in water
7 mL) and stirring was continued overnight at room
2
The authors thank Science Foundation Ireland for fund-
ing and Ms. Geraldine M. Fitzpatrick (NMR) and
Dr. Dilip Rai (MS) for analysis.
temperature. The mixture was then transferred to a
separating funnel, the organic layer removed and the
aqueous layer extracted with CH
2
Cl
2
(2·20 mL). The
combined organic layers were washed (satd NaHCO ,
3
20 mL), dried (MgSO ) and the solvent was removed in
4
vacuo. The residue was recrystallised from EtOAc and
References and notes
cyclohexane to afford 9 as a colourless crystalline solid
(0.42 g, 51%) and as an adduct with EtOAc (1:1);
1
. (a) Liekens, S.; de Clercq, E.; Neyts, J. Biochem. Pharm.
1
); H NMR
2
4
2
001, 61, 253; (b) Carmeliet, P.; Jain, R. K. Nature 2000,
07, 249; (c) Folkman, J.; Shing, Y. J. Biol. Chem. 1992,
67, 10931.
mp = 64–66 ꢁC; [a]
(300 MHz, CDCl
D
+12 (c 8.0, CDCl
) d = 7.33 (1H, d, J = 3.9 Hz, aromatic),
3
3
6.84 (1H, d, JNH,H1 = 9.3 Hz, NH), 6.77 (1H, dd,
J = 3.9 Hz, J = 0.9 Hz), 5.37 (2H, 2· overlapping t,
J = 9.3 Hz, H-1 and 3), 5.10 (1H, t, J = 9.3, H-4), 5.03
(1H, t, J = 9.3 Hz, H-2), 4.34 (1H, dd, J6a,6b = À12.6 Hz,
J6a,5 = 4.2 Hz, H-6a), 4.09 (1H, dd, J6b,6a = À12.3 Hz,
2
3
4
. Hanahan, D. Nat. Med. 1998, 4, 13.
. Brower, V. Nat. Biotechnol. 1999, 17, 963.
. (a) Iozzo, R. V.; San Antonio, J. D. J. Clin. Invest. 2001,
1
08, 349; (b) Sasisekharan, R.; Shriver, Z.; Venkataraman,
G.; Narayanasami, U. Nat. Rev. Cancer 2002, 2, 521.
. Some oligosaccharides have been shown to modulate bFGF
signalling pathways in a cell type expressing an FGF
receptor. Ornitz, D. M.; Herr, A. B.; Nilsson, M.; Westman,
J.; Svahn, C.-M.; Waksman, G. Science 1995, 268, 432.
. (a) Murphy, P. V.; Pitt, N.; OÕBrien, A.; Enright, P. M.;
Dunne, A.; Wilson, S. J.; Duane, R. M.; OÕBoyle, K. M.
Bioorg. Med. Chem. Lett. 2002, 12, 3287; (b)Pitt, N.;Duane,
R. M.; OÕBrien, A.; Bradley, H.; Wilson, S. J.; OÕBoyle, K.
M.; Murphy, P. V. Carbohydr. Res. 2004, 339, 1873.
. OÕBrien, A.; Lynch, C.; OÕBoyle, K. M.; Murphy, P. V.
Carbohydr. Res. 2004, 339, 2343.
J6b,5 = 2.1 Hz, H-6b), 3.88 (1H, ddd, J5,4 = 9.9 Hz,
5
6
J
5,6a = 4.2 Hz,
J5,6b = 2.1 Hz, H-5), 2.86 (2H, q,
CH ), 2.08, 2.044, 2.039, 2.03 (each 3H,
J = 7.5 Hz, CH
each s, each CH ), 1.32 (3H, t, J = 7.5 Hz, –CH CH );
2
3
1
3
C
3
2
3
3
NMR (75 MHz, CDCl ) d = 171.5, 170.7, 169.9, 169.6
(each s, each ester C@O), 161.8 (s, amide C@O), 154.9,
134.3 (each s), 129.5, 124.6 (each d), 78.9, 73.6, 72.6, 70.7,
68.3 (each d), 61.7 (t), 23.8 (t), 20.6, 20.7 (each q), 15.7 (q);
+
LR-ESMS m/z = 486 (M + H) , 324, 271, 169; HR-ESMS
(M + H)
+
calcd 486.1434, found: 486.1448; Anal.
Calcd for C H NO S (EtOAc adduct): C, 52.35, H,
25 35 12
7
8
6.15, N, 2.44, S, 5.59. Found: C, 52.22, H, 6.09, N, 2.49, S,
5.92.
. (a) Bertho, A.; Maier, J. Justus Liebigs Ann. Chem. 1932,
4
98, 55; (b) Murphy, P. V.; Bradley, H.; Tosin, M.; Pitt,
N.; Fitzpatrick, G. M.; Glass, W. K. J. Org. Chem. 2003,
8, 5693.
11. To a solution of per-O-acetylated compound in methanol
(0.2 M) was added K CO (10 mol %) and the mixture
2
3
6
was stirred at room temperature (30 min) and the solvent
was then removed in vacuo. The residue was immediately
purified by flash chromatography on (MeOH:CH CN,
9
. Procedure for measurement of DNA synthesis: BAE
cells (30,000 cells/well) were seeded on 24-well plates in
culture medium. After a 24 h adhesion period, the
compounds were added and cells were cultured for 24 h
in presence of serum or for 48 h in presence of bFGF.
During the last 4 h of the assay, the culture was pulsed
3
1:9) to afford a yellow syrup (0.14 g, 71%) which was a
mixture of anomers. Purification by reverse phase HPLC
(C-4 column) gave the pure b-anomer 15 for biological
evaluation. Analytical data for 15: [a]D À16.3 (c, 0.54,
3
1
MeOH); H NMR (300 MHz, D
with 2 lCi of [Me- H]thymidine/well. After being
2
O) d = 7.56 (1H, d,
washed two times with ice-cold PBS, the cells were
solubilized with 0.1 N NaOH and transferred to
counting vials containing 5 mL of liquid scintillant
J = 3.6 Hz), 6.84 (1H, d, J = 3.6 Hz), 5.07 (1H, d,
1,2 = 8.7 Hz, H-1), 3.83 (1H, dd, 6a,6b = 12.3 Hz,
J6a,5 = 2.1 Hz, H-6a), 3.68(1H, dd, J6b,6a = 12.3 Hz,
6b,5 = 4.8 Hz, H-6b), 3.56–3.37 (4H, overlapping signals,
H2-H5), 2.7913 (2H, q, J = 7.8 Hz), 1.20 (3H, t,
J = 7.8 Hz); NMR (75 MHz, D O) d = 165.3
J
J
(
ICN).
0. To a solution of 5-ethyl-thiophene-2-carboxylic acid
0.23 g, 1.44 mmol) in anhyd CH Cl (7 mL) was added
J
1
(
C
2
2
2
oxalyl chloride (0.185 g, 0.125 mL, 1.44 mmol) followed
by 1 drop of anhydrous DMF and the solution was stirred
at room temp for 2 h. The solvent was removed in vacuo
to afford the acid chloride as a yellow oil, which was used
immediately without further purification. This oil was
(C@O), 156.3, 131.2, 125.1, 79.9, 77.6, 76.5, 71.7, 69.3
(each d), 60.5 (t), 23.2 (t), 15.0 (q); ESMS m/z = 316
À
À
[M À H] 196, 119; HRMS [M À H] calcd: 316.0855;
found: 316.0840.
12. Luchansky, S. J.; Bertozzi, C. R. ChemBioChem 2004, 5,
1706.
dissolved in CH
2
Cl
2
(1 mL) and the solution was added to