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characterized, we synthesized molecules belonging to the flavone,
flavan, chalcone, stillbene, styrene, and isoquinoline scaffolds,
representing significant diversity at the three-dimensional level
(Fig. 1).19–21 Furthermore, the synthesized molecules contain 1–5
sulfate groups and are under 500 Da in size; therefore, they have
similar charge density, sulfate functionality, and size as HS/heparin
di-/tri-saccharides and should mimic HS/heparin functions.
These molecules were screened in an in vitro assay of
tumor-associated angiogenesis which utilizes reduced growth fac-
tor basement membrane extract (RGF-BME) derived from the
Englebreth-Holm-Swarm (EHS) mouse sarcoma. When bovine lung
microvascular endothelial cells (BLMVEC) are cultured on
RGF-BME, they spontaneously form tube-like structures. The
development of these ‘tubes’ in vitro mimics an important step
in the formation of blood vessels in vivo. An extensive tube-like
network with significant branching and tube length indicates nor-
mal angiogenesis. On the other hand, disjointed groups of cells
forming short tubes that are not interconnected exemplify the
inhibition of angiogenesis. Several previous studies have utilized
this matrigel tube formation assay.18,22
We initially screened the library at a number of different
concentrations and found that several molecules completely abol-
ished tube formation. Control wells contained either no compound
(positive control) or sulforaphane (negative control). Sulforaphane,
found in broccoli and other cruciferous vegetables, is a potent anti-
cancer agent provided by the assay manufacturer.23 Screening of
the 18 molecules (Fig. 1) led to the identification of 4, 5, 6, 7 and
9 as potent inhibitors of angiogenesis at 100 lM (Fig. 2). While un-
treated wells and inactive mimetics showed significant branching
and interconnectivity, endothelial cells treated with these sulfo-
nated molecules were dispersed and formed small cell clumps
without much network formation.
Based on our findings, it is possible to glimpse into structure–
activity relationships that play a role in vascular tube formation;
although, this process is considerably complex and involves a large
number of probable mechanisms. The active molecules, 4, 5, 6, 7
and 9, carry two, three, or four sulfate groups per scaffold
(Fig. 1). However, the inhibitory activity was not proportional to
number of sulfate groups as several tetra- and penta- sulfonated
molecules (e.g., 10–18) were found to be inactive. A comparison
of structures of the molecules that exhibit inhibitory activity
shows that the minimal ‘pharmacophore’ appears to be two sulfate
groups at an optimal distance of 5–10 Å as found in scaffolds 4, 5,
and 7. This suggests that structural selectivity is involved in the
process. Additionally, due to the highly charged nature of these
molecules, they probably inhibit tube formation via a chelation
or competition mechanism outcompeting cell-surface heparan sul-
fates for pro-angiogenic factors such as FGF. We have previously
shown that disruption of cell surface HS by heparitinases and by
GAG biosynthesis inhibitors halts tube-formation.9,18 The current
study presents an alternative approach which utilizes sulfonated
small molecules to inhibit tube formation by competing with the
functions of cell surface HS.
This work presents the first small, synthetic, non-saccharide,
highly sulfonated heparin/HS mimetics that possess anti-angio-
genic function. The compounds in the current study are likely to
be clinically superior to current carbohydrate-based high molecu-
lar weight drugs which have shown significant anti-cancer poten-
tial in clinical trails.13 Molecules 4, 5, 6, 7, and 9 have lower
molecular weights, can potentially modulate a variety of signalling
pathways, are easy to synthesize, and probably exert their activity
outside cells without the need for cell penetration. Due to these
properties, it is expected that the current molecules will have more
favorable pharmacokinetic properties and clinical application.
Additional scaffolds will be developed to identify more potent
angiogenesis inhibitors and to ascertain the mechanism of action
of these molecules. In vivo testing of these compounds will lead
to the identification of potential drug candidates for further
studies.
Experimental information
Tube formation assay: A premixed solution of 1 Â 105 BLMVEC,
heparin/HS mimetic inhibitors, and MCDB-131 media were added
to matrigel in a 96 well plate, in duplicate. After incubation for
16 h at 37 °C, cells were imaged with an Olympus IX81.
Synthesis: All tested compounds were synthesized in one step
from their phenolic and/or alcoholic precursors using microwave-
assisted synthesis, as described previously.19 Briefly, the precursor
and trimethylamine sulfur trioxide complex at a molar ratio of 1:6
per –OH group were mixed in acetonitrile and exposed to micro-
waves (50 W) at 90 °C for 30 min. The purity of the sulfonated
compounds was assayed using reverse polarity capillary electro-
phoresis, as previously described20 and found to be >95%. (see
Supplementary material for details).
Figure 1. Chemical structures of the library of sulfonated small molecules.
Raman, Karthik
