Design, synthesis, and biological evaluation of angiogenesis inhibitors: Aromatic enone and dienone analogues of curcumin

Article abstract of DOI:10.1016/S0960-894X(02)00832-6

The quest to find new antitumor compounds is an ongoing research endeavor in many laboratories around the world. The use of small-molecule angiogenesis inhibitors promises to be a potentially effective method for cancer treatment and possible prevention. Many antiangiogenic compounds are in various stages of laboratory evaluations and clinical trials. Curcumin is a natural product that has exhibited potent antiangiogenic properties. Based on a simple pharmacophore model, using standard drug design concepts, aromatic enone and aromatic dienone analogues of curcumin were prepared and/or obtained commercially. These compounds were screened for antiangiogenic properties via an in vitro SVR assay and were found to inhibit cell proliferation.

Full text of DOI:10.1016/S0960-894X(02)00832-6

Bioorganic & Medicinal Chemistry Letters 13 (2003) 115–117
Design, Synthesis, and Biological Evaluation of Angiogenesis
Inhibitors: Aromatic Enone and Dienone Analogues of Curcumin
Thomas Philip Robinson,a Tedman Ehlers,a Richard B. Hubbard, IV,a Xianhe Bai,b
Jack L. Arbiser,b David J. Goldsmithc and J. Phillip Bowena,*
aComputational Center for Molecular Structure and Design, Department of Chemistry, University of Georgia, Athens,
GA 30602-2556, USA
bDepartment of Dermatology, Emory University School of Medicine, 5007 Woodruff Memorial Building, Atlanta, GA 30322, USA
cDepartment of Chemistry, Emory University, Atlanta, GA 30322, USA
Received 3 July 2002; accepted 19 September 2002
Abstract—The quest to find new antitumor compounds is an ongoing research endeavor in many laboratories around the world.
The use of small-molecule angiogenesis inhibitors promises to be a potentially effective method for cancer treatment and possible
prevention. Many antiangiogenic compounds are in various stages of laboratory evaluations and clinical trials. Curcumin is a nat-
ural product that has exhibited potent antiangiogenic properties. Based on a simple pharmacophore model, using standard drug
design concepts, aromatic enone and aromatic dienone analogues of curcumin were prepared and/or obtained commercially. These
compounds were screened for antiangiogenic properties via an in vitro SVR assay and were found to inhibit cell proliferation.
# 2002 Elsevier Science Ltd. All rights reserved.
Cancer is a general term used to describe many disease
states, each of which are characterized by abnormal cell
proliferation. The causes that bring about this abnormal
cellular behavior are specific to each type of cancer.
Success of tumor-targeted therapy is limited by this
diversity. One common denominator for all types of
cancer is the requirement of a suitable blood supply.1
Therefore, tumor vasculature has emerged as a potential
target for therapeutic intervention.
mobilize angiogenic promoters from the extracellular
matrix, and/or stimulate normal host cells to inappro-
priately produce angiogenic substances. The goal of
antiangiogenic therapy is to interfere with these
mechanisms and prevent tumor cells from developing a
viable blood supply. Success here has the potential to
induce a persistent dormant state.
Curcumin is a natural product isolated from the spice
turmeric. The compound has been shown to have anti-
angiogenic properties in vitro and in vivo.3 Although
the specific mechanism of action for its activity is not
fully understood, this small molecule carotenoid pig-
ment is a promising lead compound for structure modi-
fication. Our efforts toward the design of novel
curcumin analogues have focused on the truncation of
the general structure to either a single enone or dienone
system (Fig. 1).
New blood vessel growth from preexisting vasculature
stimulated by biochemical signals is termed angiogenesis
or neovascularization. Tumor masses require a constant
supply of oxygen and nutrients, as well as efficient waste
removal for development. Diffusion from nearby capil-
laries supplies adequate nutrition for tumors less than
2 mm,2 but for continued growth the tumors must
develop their own blood supply. Alteration of the deli-
cate balance of angiogenic stimulating factors and
angiogenic inhibitors results in the phenotypic change
from quiescence to active endothelial proliferation. This
angiogenic switch is not completely understood. Angio-
genic tumors produce positive regulators of angiogenesis,
*Corresponding author. Fax: +1-706-542-2673; e-mail: bowen@
ccmsd.chem.uga.edu
Figure 1. Structure of curcumin.
0960-894X/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00832-6

116
T. P. Robinson et al. / Bioorg. Med. Chem. Lett. 13 (2003) 115–117
endothelial cells, garnered from adult C57BL/6 mice,
were infected with an ecotropic retrovirus encoding the
simian virus 40 (SV40) large T 58-3 allele coupled with a
resistance to neomycin. The resulting cells were then
selected in a solution of Dulbecco’s modified Eagle’s
medium (DMEM), 5% fetal calf serum and 0.6 mg/mL
geneticin. The clone cells were then stained with 1,10-
dioctadecyl-3,3,30,30-tetramethylindocyanine perchlorate
coupled to acetylated low density lipoprotein (diI-Ac-
LDL). Fluorescence microscopy was used to identify the
positive clones. The clone MS1 was expanded and
infected with an additional retrovirus which encoded for
activated H-ras (mutant allele at glycine-12 and threo-
nine-59) as well as hygromycin resistance. The resulting,
immortalized endothelial cell line was designated SVR.
Scheme 1. Generalized synthetic scheme for the preparation of enone
and dienone analogues of curcumin: (i) 40% KOH, EtOH, 5 ꢀC. Let
stir 10 h (rt). X=C, N. R=OH, OMe, Cl, F.
All of the compounds presented in Tables 1 and 2 were
prepared according to the same general synthetic pro-
cedure.4 The utilization of classic Claisen–Schmidt
reaction conditions allows for the condensation of an
aromatic aldehyde with a ketone to afford an a,b unsa-
turated ketone product (Scheme 1).
For use in the in vitro assays, SVR cells were plated
10,000 cells/well on a multi-well gelatinized dish. The
cells were then cultured for 24 h at 37 ꢀC in 1 mL/well of
5% DMEM under an atmosphere of 10% CO2. After
24 h of incubation, the medium was aspirated and
replaced with 1 mL of a fresh 5% DMEM solution
containing either 3 or 6 mg of our individual test com-
pounds. In order to prepare these test solutions each
compound was dissolved in a minimal amount of dime-
thylsulfoxide (DMSO). A measured amount of these
stock (1 mg/mL) solutions was added to the fresh incu-
bation medium in order to achieve our desired con-
centrations for testing. For this reason, each plate
utilized one column as a DMSO control group for each
concentration. After the addition of the test solutions,
the dishes were allowed to culture under the same con-
ditions for an additional 72 h. At the end of the three
day incubation period, the wells were aspirated. Each
well was then rinsed with 1 mL of Dulbecco’s Phosphate
Buffered Saline (DPBS) and aspirated again in order to
remove any dead cells or lingering DMEM. The
remaining, live cells were then removed from the bottom
of each well using 0.5 mL of a 1ꢁ trypsin-EDTA solu-
In the case of our enone analogues the following proce-
dure was used: A mixture of the aldehyde (10 mmol,
1 equiv) and the appropriately substituted aromatic
ketone (10 mmol, 1 equiv) is dissolved in 15 mL of etha-
nol and allowed to stir for several min at 5 ꢀC (ice bath).
10 mL of a 40% KOH solution in water is added drop
wise to the flask over several min. The mixture is then
allowed to stir at room temperature for approximately
10 h. The reaction is next neutralized with a dilute solu-
tion of hydrochloric acid. A precipitate forms and is
collected by suction filtration. Recrystallization from
ethanol affords the pure product. In the event that a
solid was not formed, the solution was extracted with
anhydrous ether (3ꢁ, ꢂ10 mL). The organic layers are
collected, consolidated, and concentrated under reduced
pressure to afford the crude product. Recrystallization
and or column chromatography was then utilized in order
to isolate the pure product. The experimental procedure
for isolating the dienone analogues is identical save for the
use of a 2:1 ratio of aldehyde/ketone in the reaction flask.
The endothelial cell proliferation assays used in this
study were developed by Arbiser et al.5 Primary murine
Table 2. Percent inhibition of in vitro endothelial cell proliferation
for the dienone analogues of curcumin
Compd structure
3 mg/mL
6 mg/mL
Table 1. Percent inhibition of in vitro endothelial cell proliferation
for the enone analogues of curcumin
7
8
9
96.6
97.7
Structure
3 mg/mL
6 mg/mL
2
3
4
5
6
98.2
98.1
94.4
92.9
97.7
96.7
92.9
92.8
92.2
89.1
97.5
94.4
94.7
96.9
10
11
90.1
87.1
96.0
90.4

T. P. Robinson et al. / Bioorg. Med. Chem. Lett. 13 (2003) 115–117
117
effort to explore stereoelectronic effects, aromatic
substituents were explored, represented by compounds
2 and 3. Initially, assuming potential nucleophilic
addition with the enone, it was hypothesized that
substituents in positions 2 and 6 of the aromatic ring
in Region A might affect inhibition via stereoelec-
tronic effects resulting from a conformational change.
Adjusting the aromatic rings in Regions A and C led
to 5. Substitution of the phenyl ring in Region A
with a pyridyl ring, 6, yielded a compound with
excellent inhibition.
Figure 2.
Figure 3.
Table 2 displays another five compounds, 7–11, with
excellent inhibition. The parent aromatic dienone, 8, is a
cyclohexanone derivative, and 7 is an acetone deriva-
tive. As before with the enone series, the importance of
stereoelectronic effects and the affect of conformational
changes were explored by altering the aromatic sub-
stituents in Regions A and C. This approach led to 11.
Substitution of both phenyl rings, Regions A and C,
with pyridyl rings, 8, yielded a compound with excellent
inhibition. Since curcumin has a 3-methoxy 4-hydroxy
aromatic substitution pattern, it was decided to examine
compounds with similar functional group patterns,
represented by 10.
tion (0.5% trypsin, 0.53 mM EDTA-4Na in HBSS
without Ca, Mg, or NaCO3). The enzymatic activity of
the trypsin was neutralized by the addition of 0.5 mL of
fresh 5% DMEM after approximately 8 min. After each
well was thoroughly mixed to insure homogeny, 0.5 mL
of the media from each well was mixed with 9.5 mL of
Isotonin Coulter Electrolyte Solution and the number
of cells/well were counted using a Coulter Cell Counter.
The total cell count of each compound test well was
then compared to the DMSO control well for each
concentration. The percent inhibition of cell growth was
calculated from these values.
Aromatic enone and aromatic dienone analogues of
curcumin are excellent antiangiogenic compounds, hav-
ing inhibition patterns equivalent or better than the
parent natural product. The compounds can easily be
prepared using classic Claisen–Schmidt reaction condi-
tions, which is important for future animal model stud-
ies. Molecular modeling coupled with structure–activity
studies are currently underway to help refine the pre-
liminary pharmacophore model.
Small-molecule antiangiogenic analogues of curcumin
are attractive targets. Examination of curcumin suggests
that the two aromatic regions might be critical for
potential ligand–receptor binding.
A
reasonable
approach following standard medicinal chemistry
design concepts was to explore compounds with sys-
tematic differences in the carbon chain connecting the
two aromatic regions (Fig. 2). A preliminary pharmaco-
phore model has curcumin divided into three regions.
Region A requires an aromatic ring, Region B is com-
posed of a symmetrical dienedione linker, and Region C
also requires an aromatic ring. Based on this simple
model, various linkers in Region B were prepared or
obtained commercially. By modifying the linker in
Region B to an enone or a dieneone, two new series
possessing antiangiogenic properties were discovered. In
Figure 3, the aromatic enone and dienone lead com-
pounds may also be divided into three regions.
References and Notes
1. Folkman, J. N. Engl. J. Med. 1971, 285, 1182.
2. Denekamp, J. Cancer Metastasis Rev. 1990, 9, 267.
3. Arbiser, J. L.; Klauber, N.; Rohan, R.; van Leeuwen, R.;
Huang, M. T.; Fisher, C.; Flynn, E.; Byers, H. R. Mol. Med.
1998, 4, 376.
4. Maitra, S.; Singh, R.; Sinha, A.; Lahiri, S. Synth. Commun.
1989, 19, 2363.
5. Arbiser, J. L.; Moses, M. A.; Fernandez, C. A.; Ghiso, N.;
Cao, Y.; Klauber, N.; Frank, D.; Brownlee, M.; Flynn, E.;
Parangi, S.; Byers, H. R.; Folkman, J. Proc. Natl. Acad. Sci.
U.S.A. 1997, 94, 861.
Table 1 displays five compounds, 2–6 with excellent
inhibition. The parent aromatic enone, 4, is chalcone,
a well-known natural product that has previously
been reported as having antitumor properties.6 In an
6. Shibata, S. Stem Cells 1994, 12, 44.