Synthesis and evaluation of a novel nonsteroidal-specific endothelial cell proliferation inhibitor

Article abstract of DOI:10.1021/jm034007d

The identification of agents with specific antiproliferative or cytostatic activity against endothelial cells has significant value for the treatment of pathologies associated with angiogenesis, including solid tumors. Here, we describe a novel substitute

Full text of DOI:10.1021/jm034007d

J . Med. Chem. 2003, 46, 1289-1292
1289
liminary in vitro biological activity that demonstrates
that they are specific inhibitors of proliferating endot-
helial cells.
Syn th esis a n d Eva lu a tion of a Novel
Non ster oid a l-Sp ecific En d oth elia l Cell
P r olifer a tion In h ibitor
Syn th esis. There are a number of methods that have
been utilized to prepare dibenzo[b,d]pyran-6-ones.15-17
Our initial efforts with these methods were unsuccess-
ful, although we did not explore them exhaustively. We
subsequently turned our attention to the report where
a NO2 containing dibenzo[b,d]pyran-6-one was synthe-
sized.18 In this case, Suzuki coupling of an arylboronic
acid with the electron-withdrawing NO2 containing
bromobenzene generated the requisite biphenyl that
cyclized efficiently to the lactone.
J onathan M. Schmidt,† Gilles B. Tremblay,‡
Martine Page´,‡ J ulie Mercure,†,§ Miklos Feher,†
Robert Dunn-Dufault,† Markus G. Peter,† and
Peter R. Redden*,†
SignalGene Inc., Unit 2, 335 Laird Road,
Guelph, Ontario N1G 4P7, Canada, and SignalGene Inc.,
Bureau 1000, 8475 Avenue Christophe-Colomb,
Montreal, Quebec H2M 2N9, Canada
Received J anuary 16, 2003
Since methyl 5-acetylsalicylate was commercially
available, we modified the procedure slightly and the
route to the dibenzo[b,d]pyran-6-ones 9a and 10 is given
in Scheme 1. Both compounds still relied on Suzuki
coupling but now with a common aryl triflate and an
arylboronic acid. The requisite arylboronic acids were
either commercially available (5a ) or prepared as shown
in Scheme 1 (5b). 2-Bromo-5-hydroxy-4-methoxyben-
zaldehyde was treated with K2CO3 and benzyl bromide
in acetonitrile to give the benzyl-protected aryl aldehyde
2 that was then oxidized with mCPBA in CH2Cl2 to the
phenol 3. To set up for the future lactone formation, 3
was treated with NaH and CH3I in anhydrous THF to
give the aryl methyl ether 4. Then, by use of metal
halogen exchange conditions, 4 was treated with nBuLi
in dry THF at -78 °C followed by triisopropyl borate,
which gave the boronic acid 5b. The other partner for
the Suzuki coupling reactions was prepared by making
the triflate 6 from methyl 5-acetylsalicylate using triflic
anhydride in CH2Cl2 containing pyridine at 0 °C. Suzuki
coupling of the arylboronic acids 5a ,b with 6 using Na2-
CO3 and a catalytic amount of Pd(PPh3)4 in absolute
ethanol and DME yielded the biphenyl esters 7a ,b.
Saponification with KOH gave the free acids 8a ,b that
upon reaction with thionyl chloride and AlCl3 gave the
dibenzo[b,d]pyran-6-ones 9a ,b. We have since deter-
mined that AlCl3 is not required for lactone formation.
The 3-hydroxy dibenzo[b,d]pyran-6-one 10 was obtained
by in situ catalytic hydrogenation of 9b with NH4HCO2
and 10% Pd/C in absolute ethanol and ethyl acetate. The
yield of this debenzylation reaction was very low (27%),
and we subsequently improved the yield to greater than
80% and will be reporting the methodology elsewhere.
The structures of the intermediates and final com-
pounds were confirmed by 1H NMR with elemental
analysis determined on the final compounds.
Abstr a ct: The identification of agents with specific antipro-
liferative or cytostatic activity against endothelial cells has
significant value for the treatment of pathologies associated
with angiogenesis, including solid tumors. Here, we describe
a novel substituted dibenzo[b,d]pyran-6-one scaffold, exempli-
fied by structures 9a and 10, and report preliminary in vitro
activity data indicating that this scaffold is a promising lead
for the development of specific inhibitors of endothelial cell
proliferation.
In tr od u ction . Angiogenesis, the growth of new blood
vessels, involves the proliferation of endothelial cells in
response to specific growth stimuli such as vascular
endothelial growth factor (VEGF) of basic fibroblast
growth factor (bFGF). The growth and maintenance of
solid tumors is highly dependent on neovascularization
and can be regulated by compounds that interfere with
either the stimulation or proliferation of endothelial
cells.1 Consequently, the control of angiogenesis con-
tinues to be an attractive area for the development of
novel therapeutic agents.2
One such agent is 2-methoxyestradiol (2-ME2, Pan-
zem), an endogenous metabolite of estradiol (E2) that
has been demonstrated to inhibit both tumor growth
and angiogenesis in vivo.3,4,7-13 However, in addition to
its antiangiogenic activity, 2-ME2 induces apoptosis in
actively proliferating cells and exhibits general nonspe-
cific antiproliferative effects against a wide range of
human cancer cell cultures. It also has limited oral
bioavailability in mice14 and is rapidly cleared following
administration.
In light of these potential pharmacological and thera-
peutic limitations, there remains an ongoing interest
in the identification of more potent and bioavailable
nonsteroidal analogues of 2-ME2 with specific inhibitory
activities against proliferating endothelial cells. Hence,
in the course of our drug discovery programs, we were
intrigued by the structural similarities between a series
of substituted dibenzo[b,d]pyran-6-ones and 2-ME2 and
were curious to determine whether they had selective
antiangiogenic activity. Here, we report the synthesis
of substituted dibenzo[b,d]pyran-6-ones along with pre-
Although the dibenzo[b,d]pyran-6-one scaffold has not
been used frequently in pharmaceuticals, it is found in
naturally occurring compounds exemplified by alterna-
riol,15 ellagic acid,15 and the benzo[d]naphthopyran-6-
one gilvocarcin V19 as shown in Scheme 2. Ellagic acid
inhibits the mutagenicity of polycyclic aromatic hydro-
carbons in vivo, and gilvocarcin V has significant in vitro
antitumor activity when activated by low-energy light.
In Vitr o Biology. The antiproliferative effects of 9a
and 10 compared to 2-ME2 were evaluated using
primary human umbilical vein endothelial cells (HU-
VECs) in the presence of bFGF20 as shown in Figure 1.
* To whom correspondence should be addressed. Phone: (519)-823-
9088. Fax: (519)-823-9401. E-mail: peter.redden@signalgene.com.
† Unit 2.
‡ Bureau 1000.
§ Present address: SYNX Pharma Inc., 6354 Viscount Road, Miss.,
Ontario, L4V 1H3, Canada.
10.1021/jm034007d CCC: $25.00 © 2003 American Chemical Society
Published on Web 03/15/2003

1290 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8
Letters
Sch em e 1
Sch em e 2
Both 9a and 10 caused dose-dependent inhibition of
bFGF-stimulated endothelial cell proliferation. The
activity of these compounds was comparable to that of
2-ME2. At a concentration of 10 µM, 2-ME2 reduced cell
counts below the level of the untreated controls, indicat-
ing a possible cytotoxic effect. In contrast, 9a and 10
did not reduce cell counts below the control level,
suggesting that they may be cytostatic rather than
cytotoxic agents over this concentration range.
Under the same treatment concentrations, neither 9a
nor 10 significantly inhibited or stimulated the prolif-
eration of MDA-MB-231 human breast cancer cells,
estradiol-responsive MCF-7 human breast cancer cells,
or HCT-116 human colorectal carcinoma cells.21 In the
same assays, 2-ME2 was significantly antiproliferative.
These observations suggest that the inhibitory effects
of 9a and 10 are more specific than those of 2-ME2. The
ability to selectively inhibit endothelial cell proliferation
has important therapeutic implications, especially for
the treatment of nononcological diseases and the use of
these compounds in combination therapy.
F igu r e 1. Inhibition of the proliferation of human endothelial
cells by 9a , 10, and 2-Me2. Top panel: 10 vs 2-ME2 in the
HUVEC proliferation assay. The cells were incubated with
bFGF alone or in the presence of increasing amounts of 10 or
2-ME2 as indicated. The maximum growth factor response was
set to 100%, and the results are expressed as the percentage
of cells remaining compared to the maximal response. The
amount of cells remaining in the wells that were not treated
with bFGF (starved) was set to 0 (not shown). The asterisk
(/) indicates that the amount of cells remaining was below that
of the unstimulated cells. The data represent the mean ( SEM
(n ) 11). Bottom panel: as in the top except 9a is compared
to 2-ME2 (n ) 5).
In light of the structural similarities between 9a and
10 with estradiol (E2), it was important to examine the
ability of these compounds to bind with the estrogen
receptor (ER). In a competitive binding assay, neither
9a nor 10 significantly inhibited the binding of 0.5 nM
radiolabeled E2 with either ERR (Figure 2, left panel)
or ERâ (Figure 2, right panel) at 10 µM. At the same
concentration, the positive control DES (diethylstil-
besterol) inhibited E2 binding by >75%. At 10 µM,
2-ME2 inhibited E2 binding to ERR by 60% and to ERâ
by 25%. Measurable binding of 2-ME2 at this high dose
is consistent with previously reported values.22 The
observations that 9a and 10 do not compete with E2 for
the estrogen receptor and do not have a stimulatory
effect on E2-responsive MCF-7 breast cancer cells pro-
vide strong evidence that these compounds are unlikely
to have estrogenic liabilities such as uterotrophic activ-
ity.
Com p u t a t ion a l Mech a n ism of Act ion St u d ies.
The mechanism of action of 2-ME2 is not well under-
stood, and the molecular target remains unknown.
Several hypotheses have been proposed including the

Letters
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8 1291
Further studies are underway in an attempt to identify
the putative molecular target for these molecules.
We also thought it might be the hydrolyzed lactones
that may be responsible for the biological activity;
however, the precursor esters 7a ,b and acids 8a ,b were
inactive in the HUVEC assay.
Con clu sion s. In view of its oral in vivo efficacy
against solid tumors, low toxicity, and lack of estroge-
nicity,35 it has been argued that the natural metabolite
2-ME2 is close to being an ideal antiproliferative agent.
However, low bioavailability in mice,14 rapid clearance,
and lack of specificity for endothelial cells35 may limit
the potential therapeutic applications of 2-ME2. The
acetylated dibenzo[b,d]pyran-6-ones 9a and 10 appear
to provide an opportunity to improve upon the thera-
peutic profile of 2-ME2. These compounds are nearly as
effective inhibitors of stimulated HUVEC proliferation
as 2-ME2. They are also unlikely to be estrogenic. At
the same time, the nonsteroidal scaffolds of 9a and 10
open a route for the identification of analogues that are
not subject to the same well-established clearance
mechanisms as 2-ME2. More importantly, our in vitro
studies indicate that the antiproliferative activity of 9a
and 10 is more selective for endothelial cells than that
of 2-ME2.
This finding is of practical significance for the man-
agement of diseases in which angiogenesis and neovas-
cularization are the primary or exclusive targets of
treatment, e.g., retinopathies and inflammatory condi-
tions. In oncological therapy, compounds that act specif-
ically against endothelial cells are likely to have the
greatest clinical value when administered in combina-
tion with other cytotoxic agents. By exploitation of
independent mechanisms of action in distinct cell types,
it may be possible to mitigate against the rapid onset
of resistance to more general antimitotic treatments.
The discovery that the dibenzo[b,d]pyran-6-ones are
specific inhibitors of stimulated endothelial cells and not
generally active against proliferating cells is a signifi-
cant step in the development of such a therapeutic
approach.
F igu r e 2. Effect of 9a , 10, and 2-ME2 with the human
estrogen receptors. In vitro transcribed-translated ERR and
ERâ were incubated with 0.5 nM [3H]E2 in the presence of 10
µM diethylstilbestrol (DES), 9a , 10, or 2-ME2. The maximum
amount of specifically bound radioligand was determined in
the presence of vehicle and expressed as 100% (control). The
data represent the mean ( SEM (n ) 3).
inhibition of tubulin polymerization,23 the up-regulation
of p53,24,25 the induction of caspases,26 and the inhibition
of superoxide dismutase activity.27 In each of these
proposed mechanisms, treatment of cells with 2-ME2
ultimately leads to either cell arrest or apoptosis by
interfering with the regulation of the cell cycle.28 Despite
its low affinity to E2, a recent report has documented
that the action of 2-ME2 is independent of the ERs.22
Similarly, we have ruled out ERs as potential targets
for the dibenzo[b,d]pyran-6-ones because we have ob-
served no significant interaction between ERR and ERâ
with 9a and 10.
Although we have only just begun to address the issue
of a mechanism of action, preliminary studies indicate
that they do not induce the expression of caspases,29
suggesting that they may inhibit endothelial cell pro-
liferation by a mechanism other than apoptosis. It was
also determined that the effect of 9a was not dependent
on p53.21
On the basis of flexible overlays of 2-ME2 and several
colchicine analogues, it appears unlikely that 2-ME2
would inhibit tubulin polymerization by binding to the
colchicine site,30 a mechanism that has been invoked
for 2-ME2.23 For several other potential targets, we
performed pairwise flexible molecular alignments31 of
2-ME2/9a /10 with low nanomolar inhibitors of these
targets to evaluate the likelihood of any of the three
molecules competing for the corresponding binding site.
In all cases 1000 overlays were carried out using a
Cartesian perturbation of 0.5 Å, with all other condi-
tions being equal to those previously described.31 On the
basis of alignment with indanocene, PD153035, and
SU5416, it appears unlikely that 9a and 10 share the
binding site of these molecules on microtubules, the
EGFR-TK receptor, and the VEGF2 (Flk-1/KDR) recep-
tor, respectively. In a similar manner, flexible align-
ments of 9a and 10 with colchicine analogues confirm
that the colchicine site is an unlikely target for these
molecules.
We will be reporting the results of in vivo efficacy
studies separately.21 In addition, since with steroids,
clearance and metabolism via phenol conjugation and
demethylation are well-established mechanisms, we will
be evaluating these pathways for 9a and 10 as well.
Also, further research is in progress to identify the mode
of action and molecular target of these compounds.
Ack n ow led gm en t. The authors acknowledge Dal-
ton Chemical Laboratories, Inc. and MCR Research,
Inc., which conducted the initial syntheses.
Su p p or tin g In for m a tion Ava ila ble: Detailed chemistry
and biological experimental procedures for all compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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