Synthesis and biological evaluation of aromatic enones related to curcumin

Article abstract of DOI:10.1016/j.bmc.2005.03.054

Curcumin, a natural product isolated from the spice turmeric, has been shown to exhibit a wide range of pharmacological activities including certain anti-cancer properties. It has been specifically shown to be an effective inhibitor of angiogenesis both in vitro and in vivo. Using curcumin as a lead compound for anti-angiogenic analog design, a series of structurally related compounds utilizing a substituted chalcone backbone have been synthesized and tested via an established SVR cell proliferation assay. The results have yielded a wide range of compounds that equal or exceed curcumin's ability to inhibit endothelial cell growth in vitro. Due to both their commercial availability and their fairly straightforward synthetic preparation, these low molecular weight compounds are attractive leads for developing future angiogenic inhibitors.

Full text of DOI:10.1016/j.bmc.2005.03.054

Bioorganic & Medicinal Chemistry 13 (2005) 4007–4013
Synthesis and biological evaluation of aromatic enones related
to curcumin
Thomas Philip Robinson,b Richard B. Hubbard, IV,b Tedman J. Ehlers,b
Jack L. Arbiser,c David J. Goldsmithd and J. Phillip Bowena,*
aCenter for Drug Design, Department of Chemistry and Biochemistry, University of North Carolina at Greensboro,
400 New Science Building PO Box 26170, Greensboro, NC 27402, USA
bCenter for Biomolecular Structure and Dynamics, Department of Chemistry, University of Georgia, Athens, GA 30602, USA
cDepartment of Dermatology, Emory University School of Medicine, 5007 Woodruff Memorial Building, Atlanta, GA 30322, USA
dDepartment of Chemistry, Emory University, Atlanta, GA 30322, USA
Received 9 March 2005; revised 30 March 2005; accepted 30 March 2005
Available online 28 April 2005
Abstract—Curcumin, a natural product isolated from the spice turmeric, has been shown to exhibit a wide range of pharmacological
activities including certain anti-cancer properties. It has been specifically shown to be an effective inhibitor of angiogenesis both in
vitro and in vivo. Using curcumin as a lead compound for anti-angiogenic analog design, a series of structurally related compounds
utilizing a substituted chalcone backbone have been synthesized and tested via an established SVR cell proliferation assay. The
results have yielded a wide range of compounds that equal or exceed curcuminÕs ability to inhibit endothelial cell growth in vitro.
Due to both their commercial availability and their fairly straightforward synthetic preparation, these low molecular weight
compounds are attractive leads for developing future angiogenic inhibitors.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
ulceration, psoriasis, and tumor growth. Although it is
not essential during the initial stages of tumor forma-
tion, neovascularization is vital for the continued sur-
vival and growth of a developing tumor mass. Without
an adequate blood supply to provide it with oxygen
and an efficient means of disposing of waste products,
the tumor will be unable to grow larger than 1–2 mm
in diameter. Along with a stable supply of blood, fresh
capillary invasion of the tumor mass also introduces
the possibility of metastasis.
Cancer is a general term used to describe many disease
states, each of which is characterized by abnormal cell
proliferation. The causes, which bring about this abnor-
mal cellular behavior are specific to each type of cancer.
The success of tumor-targeted therapy is limited by this
inherent dissimilarity. One common denominator for all
types of cancer is the requirement of a stable blood sup-
ply. Therefore, tumor vasculature has been identified as
a potential target for therapeutic intervention.
The process of angiogenesis is multifaceted. Prospective
therapeutic strategies can focus on several key aspects of
the process: blocking the degradation of the basement
membrane (metalloproteinase inhibitors), maintaining
the equilibrium between biochemical angiogenic pro-
moters and inhibitors, and the direct inhibition of
endothelial cell proliferation (TNP-470, thalidomide,
squal- amine, and endostatin).
Angiogenesis is the process by which new capillaries are
formed from the bodyÕs existing vascular system. It is
typically a quiescent process that is only made active
in conjunction with menstruation and wound healing
in adults. When it does take place outside of these lim-
ited boundaries, it is typically due to a disease process.
Angiogenesis is present, and in fact instrumental, in a
wide range of disease states including arthritis, corneal
Curcumin, also known as diferuloylmethane, is a carot-
enoid pigment isolated from the spice turmeric. Its struc-
ture is provided in Figure 1. Turmeric itself is originally
obtained from the powdered rhizome of the plant
Curcuma longa L.1,2 Curcumin, the major bioactive
Keywords: Curcumin; Angiogenesis; Chalcone; Enone.
*
Corresponding author. Tel.: +1 336 334 4257; fax: +1 336 334
5402; e-mail: jpbowen@uncg.edu
0968-0896/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.03.054

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T. P. Robinson et al. / Bioorg. Med. Chem. 13 (2005) 4007–4013
Figure 1. Structure of curcumin.
component of turmeric, has been shown to exhibit a
wide range of interesting biological activities including
anti-oxidant,3,4 anti-inflammatory,5,6 and anti-HIV
properties.7,8 Perhaps most importantly, it has also
exhibited significant anti-tumor activity. Curcumin has
been shown to inhibit the progression of artificially
induced skin and colon cancers in animal models.9–13
Both of these forms of cancer differ significantly in their
genetic basis, which lead researchers to postulate that
curcuminÕs ability to hinder the progression of these dis-
tinct cancer types is based on its ability to inhibit angio-
genesis. Arbiser et al. discovered soon after that
curcumin inhibits basic fibroblast growth factor
(bFGF)-induced endothelial cell proliferation in vitro
and does, in fact, inhibit angiogenesis in vivo.14
Figure 2. Key structural regions of curcumin.
minÕs original b-diketone to a more compact singular
enone moiety (B0) [see Fig. 3]. Figure 4 is a generalized
representation of the pharmacophore substitution pat-
terns. Working within this new molecular framework
(A0, B0, C0), a variety of mixed aromatic systems and
substitution patterns were either obtained from com-
mercial sources or synthesized in the laboratory. The
biological efficacy as an in vitro inhibitor of endothelial
cell proliferation was evaluated for each compound via
the previously described Arbiser SVR cell assay.
Curcumin has previously been shown to inhibit in vitro
SVR endothelial cell growth by 41.5% at 1 lg/ml, 37.8%
at 3 lg/ml, and 56.2% at 6 lg/ml. These values were used
as a benchmark by which to judge the relative effective-
ness of the synthesized derivatives. The prototypical
aromatic enone, chalcone (1), was the first enone analog
tested, as it possessed all of the fundamental character-
istics required for the proposed pharmacophore.
Although the specific mechanism by which curcumin en-
acts its regulation of the angiogenic process is not cur-
rently known, its efficacy in doing so, coupled with its
relatively straightforward chemical structure, has made
it an attractive lead compound for structure modifica-
tion and drug development studies. The efforts to date
have centered mostly on the truncation of the central re-
gion of the molecule from a b-diketone to a more com-
pact enone linker. This, coupled with modifications to
the size and substitution patterns of the two aromatic
ring moieties present in the molecule, has yielded a num-
ber of highly effective curcumin analogs, which show
impressive anti-proliferative activity against SVR endo-
thelial cells in vitro.15–17
Chalcone inhibits SVR cell growth by 71.6% at 1 lg/ml,
92.8% at 3 lg/ml, and 94.4% at 6 lg/ml. This was a very
encouraging piece of datum as it proved that even with
2. Results and discussion
It has been postulated that the biological efficacy of cur-
cumin arises from the electrophilic nature of its central
b-diketone component.15,16 The enone derivatives se-
lected for investigation retain this essential characteris-
tic, while at the same time simplifying the overall
structure of the molecule. In an effort to develop a ro-
bust pharmacophoric model and understand the basis
for biological activity, the molecular structure of curcu-
min can be subdivided into three key regions: two
substituted aromatic moieties (A and C) joined together
by a conjugated b-diketone linker (B) [see Fig. 2].
Figure 3. Proposed modification of curcuminÕs b-diketone linker
region.
By dividing the molecule into specific and modifiable re-
gions of interest, a simple, yet effective, pharmacophore
model for the curcumin analogs was developed. Each
individual region can be modified independently of the
others in order to aid in the systematic refinement of
the search for increasingly effective curcumin deriva-
tives. In the current study, compounds with an abbrevi-
ated linker between the two aromatic regions comprised
the primary focus. The linker was shortened from curcu-
Figure 4. Generalized substitution patterns of the aromatic enone
analogs investigated. R = Cl, F, CH3, NO2, isopropyl, OBn, phenyl,
OCH3, and OH; X = C, N; A0 and C0 = benzene, pyridine, furan,
pyrrole, naphthalene, anthracene, biphenyl, and benzo[1,3]dioxole.

T. P. Robinson et al. / Bioorg. Med. Chem. 13 (2005) 4007–4013
4009
Table 1 (continued)
the simplified framework, the aromatic enone analogs
still retain the fundamental characteristics required for
biological potency. In fact, a large number of the enone
analogs investigated either matched or exceeded curcu-
minÕs levels of in vitro cell inhibition. Overall, 63 differ-
ent analogs were obtained and tested. Their structures
and the corresponding inhibition data are presented in
Tables 1–5. Each table represents a specific subset of
the enone analogs. Tables 1–4 contain derivatives differ-
ing in either the substitution pattern of the benzene rings
or the type of aromatic moiety utilized (benzene, pyr-
role, pyridine, furan, naphthyl, anthryl, biphenyl, and
benzo[1,3]dioxole). Table 5 presents the data obtained
from the tetralone analogs included in the study. The
tetralone derivatives introduced a measure of rigidity
to the central enone moiety of the compounds. The
2-naphthyl analog (61) showed very promising results.
Compd R1
R2
SVR growth inhibition
1 lg/ml 3 lg/ml 6 lg/ml
13
14
48.3
43.5
75.3
38.1
93.7
69.1
15
29.1
63.4
85.2
16
17
18
4.6
2.2
61.0
51.1
43.7
94.0
88.7
52.3
Table 1. Percent inhibition of in vitro endothelial cell proliferation for
enone analogs
23.2
Compd R1
R2
SVR growth inhibition
1 lg/ml 3 lg/ml 6 lg/ml
19
20
31.8
25.8
56.4
39.8
60.8
63.5
1
2
71.6
92.8
94.4
29.9
40.2
58.5
21
36.2
49.2
39.2
3
73.7
98.2
98.1
22
23
40.7
30.3
89.1
31.7
96.9
83.2
4
5
19.9
19.5
10.4
14.2
84.7
59.2
24
25
26
0.0
12.6
13.7
53.4
0.0
85.2
5.3
6
7
47.7
36.3
57.9
67.3
89.6
89.5
3.8
29.7
8
9
41.7
29.6
80.4
25.3
87.3
73.4
27
28
29
31.1
47.6
0.0
88.6
70.3
0.0
88.6
84.5
0.0
10
11
13.8
42.3
11.7
87.4
31.1
96.9
30
6.6
55.6
88.7
12
22.3
24.5
46.5

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T. P. Robinson et al. / Bioorg. Med. Chem. 13 (2005) 4007–4013
Table 2. Percent inhibition of in vitro endothelial cell proliferation for
naphthyl/anthryl/p-phenyl analogs
Table 3. Percent inhibition of in vitro endothelial cell proliferation for
5-benzo[1,3]dioxole analogs
Compd R1
R2
SVR growth inhibition
Compd R1
R2
SVR growth inhibition
1 lg/ml 3 lg/ml 6 lg/ml
1 lg/ml 3 lg/ml 6 lg/ml
47
1.9
24.2
48.1
31
32
0.0
0.0
78.7
75.4
88.6
66.2
O
48
5.7
21.3
1.0
1.4
0.0
11.9
0.0
O
33
34
35
10.4
19.2
24.9
34.7
0.0
0.0
0.0
49
50
51
52
53
54
14.4
12.7
34.3
32.5
44.1
45.5
24.5
80.8
68.9
78.4
36.5
31.0
33.0
28.3
12.9
41.4
36
12.2
35.3
96.8
37
38
25.6
18.4
4.4
5.1
62.9
17.0
Table 4. Percent inhibition of in vitro endothelial cell proliferation for
aromatic five membered ring analogs
39
25.5
46.4
69.0
40
41
42
43
54.6
52.5
10.9
0.0
53.5
57.3
33.2
0.0
71.3
48.1
68.2
4.7
Compd
R1
R2
SVR growth inhibition
1 lg/ml
3 lg/ml
6 lg/ml
55
56
7.7
9.8
8.1
12.0
0.0
1.0
57.7
11.4
21.5
57
58
59
28.2
8.3
90.2
19.4
0.4
19.0
44
45
0.0
0.0
24.9
22.4
50.3
52.0
A number of the most active chalcone analogs, 2,6-di-
chloro-40-methylchalcone (3) for one, bear electron
withdrawing groups on the ortho position(s) of their aro-
matic rings. Several of the other top rated analogs pos-
sess either an ortho positioned heterocycle or, in the case
of the tetralone derivatives, an extra cyclohexanone ring
that could result in significant steric strain for the mole-
cule if it were to adopt a planar orientation. This sug-
gests that such substitution may force the aromatic
rings out of conjugation with the enone moiety, thereby
isolating it and increasing its overall electrophilic char-
46
12.0
55.5
60.3
Even at the lowest concentration, it exhibited over 80%
inhibition.

T. P. Robinson et al. / Bioorg. Med. Chem. 13 (2005) 4007–4013
4011
Table 5. Percent inhibition of in vitro endothelial cell proliferation for
tetralone analogs
traditional Claisen–Schmidt reaction conditions. The re-
ported a,b-unsaturated ketone products are a result of a
base induced condensation of the appropriate aromatic
aldehydes and ketones.
General synthetic procedure: Equimolar portions of the
appropriately substituted aromatic aldehydes (10 mmol,
1 equiv) and ketones (10 mmol, 1 equiv) were dissolved
in approximately 15 ml of ethanol. The mixture was al-
lowed to stir for several minutes at 5–10 °C (ice bath). A
10 ml aliquot of a 40% aqueous potassium hydroxide
solution was then slowly added dropwise to the reaction
flask via a self-equalizing addition funnel. The reaction
solution was allowed to stir at room temperature for
approximately 4 h. Most commonly, a precipitate
formed and was then collected by suction filtration.
Recrystallization from the appropriate solvent afforded
the pure product. In certain instances, a solid product
was not obtained by the end of the reaction period
(monitored by TLC). Two alternate strategies were em-
ployed in order to isolate the target compounds. First,
the reaction was neutralized with a dilute acid solution
in an effort to aid the productÕs precipitation from solu-
tion. If this method was not successful in producing
a precipitate, the solution was then extracted with
either anhydrous ether or chloroform (three portions,
ꢀ10 ml). The organic layers were collected and concen-
trated using a rotoevaporator to afford the crude prod-
uct. Recrystallization and/or column chromatography
was then utilized to purify the desired product (Scheme
1).
Compd
R1
SVR growth inhibition
1 lg/ml
3 lg/ml
6 lg/ml
60
61
5.7
11.4
40.8
78.8
84.7
84.9
37.0
62
63
32.8
1.0
53.7
52.1
76.4
acter, subsequently making it more susceptible to nucleo-
philic attack. As a further test of the necessity of the
electrophilic enone moiety in the test compounds, 1,3-di-
phenyl-1-propanone was synthesized and evaluated. Its
comparatively low level of activity (5.1% at 1 lg/ml,
0% at 3 lg/ml, and 11.5% at 6 lg/ml) lends credence to
the purported importance of the enone moietyÕs pres-
ence in the curcumin analogs.
2.1. Conclusions and future research
With the recent discovery that curcumin may achieve its
antiangiogenic activity via the inhibition of an amino-
peptidase,18 the potential for future fine tuning of the
pharmacophore utilizing molecular modeling packages
is very promising. With a possible active site identified,
modeling efforts could greatly aid in the search for the
ideal angiogenic inhibitor. Potential drug choices could
be assessed via docking studies prior to their synthesis
in order to pre-evaluate their binding potential. Compu-
tational studies are currently underway in an effort to
build a structure–activity relationship profile utilizing
the current crop of inhibitors described herein.
Several variations of this general procedure were utilized
in order to obtain the test compounds. Each variation
has been illustrated with a specific example.
3.1. 20,60-Dimethoxychalcone (20)
To a solution of benzaldehyde (295 mg, 2.77 mmol) and
2,6-dimethoxyacetophenone (500 mg, 2.77 mmol) in
4.1 ml of methanol was added 0.1 ml of 40% sodium
hydroxide in water at 10 °C. After stirring for 1 h at
room temperature, a solid appeared and was filtered.
The precipitate was recrystallized from methanol to
afford 371 mg (49.9%) of white flakes: mp 125.5–
1
3. Experimental
126.6 °C (lit. mp 123–124 °C). H NMR (250 MHz) d
7.55–7.51 (m, 2H), 7.39–7.30 (m, 5H), 7.00–6.94 (d,
1H), 6.64–6.61 (d, 2H), 3.79 (s, 6H); 13C NMR
(62.7 MHz) 213.8, 176.1, 163.9, 153.4, 149.4, 149.0,
147.4, 147.3, 147.0, 122.6, 74.5 ppm.
The chalcone derivatives presented in Tables 1–5 were
each prepared according to a similar synthetic proce-
dure. The synthesis of the target compounds employed
Scheme 1. Generalized synthetic scheme for the preparation of enone derivatives. Reagents and conditions: (i) 10–40% KOH, EtOH, 5–10 °C.
Let stir ꢀ4 h at room temperature. X = H, Cl, F, OCH3, CF3, OH, NH2, CH3, CH(CH3)2, NO2, phenyl, OBn, and CH2COOH; A and C
ring systems = benzene, 1-napthalene, 2-napthalene, 2-pyridine, 3-pyridine, 5-benzo[1,3]dioxole, 2-furan, 2-pyrrole, and 9-anthracene.

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T. P. Robinson et al. / Bioorg. Med. Chem. 13 (2005) 4007–4013
3.2. 1-Phenyl-3-(3-pyridyl)propenone (23)
Benzoylmethylene triphenylphosphorane
concentrated under reduced pressure, the oil remained
and purified with silica gel using a 9:1 hexanes:ether
solution to afford 850 mg (32.9%) of yellow flakes: mp
105.5–106.7 °C. H NMR (250 MHz) d 8.37–8.33 (m,
1H), 8.03–8.00 (d, 1H), 7.95–7.90 (m, 1H), 7.81–7.78
(d, 1H), 7.68–7.49 (m, 6H), 7.45–7.41 (m, 2H), 7.36–
7.29 (d, 1H); 13C NMR (62.7 MHz) 195.9, 146.1,
137.3, 134.8, 134.0, 132.5, 131.8, 130.9, 129.2, 128.9,
127.5, 126.7, 125.6, 124.7 ppm.
(1.95 g,
1
5.14 mmol) was added to a stirring solution of pyri-
dine-3-carboxaldehyde (500 mg, 4.67 mmol) in 7.0 ml
of dry methylene chloride. The solution was allowed
to stir at room temperature overnight. It was then con-
centrated on a rotoevaporator, which afforded the crude
product. Recrystallization from diethyl ether resulted in
295 mg (31.9%) of the expected product (light-yellow
powder): mp 102.7–103.7 °C. 1H NMR (250 MHz) d
8.86 (s, 1H), 8.64–8.63 (d, 1H), 8.06–7.93 (m, 3H),
7.83–7.76 (d, 1H), 7.63–7.49 (m, 4H), 7.39–7.34 (m,
1H); 13C NMR (62.7 MHz) 190.0, 151.3, 150.1, 141.1,
137.9, 134.8, 133.3, 130.6, 126.9, 126.7, 124.0 ppm.
3.6. 1-(1-Naphthyl)-3-(2-naphthyl)propenone (44)
An aqueous 40% potassium hydroxide solution (10 ml)
was added dropwise to an ice cold (5–10 °C), stirring
flask containing a mixture of 1-naphthalen-1-yl-etha-
none (1.70 g, 10 mmol) and naphthalene-2-carbaldehyde
(1.56 g, 10 mmol) in 15 ml of ethanol. After stirring for
approximately 3 h, a viscous oil formed. The oil was al-
lowed to sit at room temperature for 2 days, at which
time a solid formed. The crude product was recrystallized
(two times) from ethyl acetate to afford 1.39 g (45.1%) of
3.3. 1,3-Di(2-pyridyl)propenone (24)
Concentrated hydrochloric acid (2.1 ml) was added
dropwise to an ice-cold (5-10 °C) solution of pyridine-
2-carboxaldehyde (442 mg, 4.13 mmol) and 2-acetylpyri-
dine (500 mg, 4.13 mmol) in 10.3 ml ethanol. The
reaction mixture was stirred for 1.5 h at 5–10 °C. The
flask was then removed from the ice and allowed to stir
at room temperature for an additional 6 h. At this time,
the solution was placed back into the ice bath and neu-
tralized using 40% sodium hydroxide. A standard ether
extraction/workup yielded the crude product, which was
chromatographed on a silica gel column using 2:1 hex-
ane/ethyl acetate to afford 253 mg (29.1%) of a light-
yellow solid that turned light green upon standing
(purity was unaffected): mp 66.2–67.2 °C. 1H NMR
(250 MHz) d 8.68–8.60 (m, 3H), 8.12–8.09 (d, 1H),
7.88–7.40 (m, 5H), 7.23–7.19 (m, 1H); 13C NMR
(62.7 MHz) 189.9, 154.0, 153.9, 148.1, 143.1, 137.1,
136.9, 127.2, 125.1, 124.8, 124.4, 122.2 ppm.
1
yellow flakes: mp 83.5–84.3 °C. H NMR (250 MHz) d
8.41–8.37 (m, 1H), 8.05–7.74 (m, 9H), 7.63–7.51 (m,
5H), 7.47–7.40 (d, 1H); 13C NMR (62.7 MHz) 195.7,
146.2, 137.3, 134.8, 134.1, 133.5, 132.3, 131.8, 131.0,
130.7, 129.0, 128.8, 128.7, 128.0, 127.7, 127.4, 127.3,
127.0, 126.7, 125.6, 124.7, 123.8 ppm.
3.7. 1-Phenyl-3-(2-pyrrolyl)propenone (58)
To a solution of acetophenone (630 mg, 5.26 mmol) and
pyrrole-2-carboxaldehyde (500 mg, 5.26 mmol) in 2.1 ml
of ethanol was added 0.5 ml of an aqueous 10% sodium
hydroxide solution at room temperature. After stirring
overnight, the solution was filtered. The resulting precip-
itate was recrystallized from 90% ethanol to afford
500 mg (48.5%) of the pure product: mp 136.6–
1
3.4. 2,20,3,30,4,40,5,50,6,60-Decafluorochalcone (27)
137.4 °C. H NMR (250 MHz) d 9.19 (b s, 1H), 8.01–
7.97 (m, 2H), 7.81–7.75 (d, 1H), 7.60–7.45 (m, 3H),
7.22–7.16 (d, 1H), 7.01 (s, 1H), 6.73 (s, 1H), 6.36–6.33
(m, 1H); 13C NMR (62.7 MHz) 190.9, 135.1, 132.6,
128.8, 128.5, 123.5, 116.0, 115.6, 111.7 ppm.
Sodium hydroxide (100 mg) was added to 4.2 ml of
water and 3.2 ml of 95% ethanol. This solution
(1.9 ml) was placed in an ice bath and 2,3,4,5,6-pentaflu-
oroacetophenone (500 mg, 2.38 mmol) along with
2,3,4,5,6-pentafluoroaldehyde (467 mg, 2.38 mmol) was
added to the reaction flask. After stirring for approxi-
mately 2 h at room temperature, a yellow solid precipi-
tated and was filtered. The solid was recrystallized
from ethanol (two times) to afford 550 mg (59.5%) of a
3.8. 2-(2,6-Dichlorobenzylidene)tetralone (62)
An aqueous 40% potassium hydroxide solution (10 ml)
was added dropwise to an ice cold (5–10 °C), stirring
flask containing a mixture of 1-tetralone (1.46 g,
10 mmol) and 2,6-dichloro-benzaldehyde (1.75 g,
10 mmol) in 30 ml of ethanol. The solution turned pur-
ple following the addition of the basic solution. After
stirring for approximately 4 h, a viscous purple oil
formed. Column chromatography with silica gel using
9:1 hexane/ethyl acetate as the eluent afforded a yellow
oil. Upon standing at 4 °C, a solid formed and was
recrystallized from ethanol yielding 2.00 g (66.0%) of
faint-yellow platelets: mp 73.8–75.6 °C. 1H NMR
(250 MHz) d 8.20–8.17 (d, 1H), 7.60 (s, 1H), 7.50 (t,
1H), 7.40–7.37 (m, 3H), 7.26–7.19 (m, 2H), 2.97 (t,
2H), 2.86 (t, 2H); 13C NMR (62.7 MHz) 187.0, 144.1,
139.7, 134.8, 134.2, 133.7, 133.4, 130.8, 129.6, 128.5,
128.2, 127.2, 28.8, 28.0 ppm.
1
white powder: mp 58.4–59.0 °C. H NMR (250 MHz)
d 7.66–7.59 (d, 1H), 7.37–7.30 (d, 1H); 13C NMR
(62.7 MHz) 183.1, 148.3, 146.9, 144.2, 142.6, 140.1,
136.1, 132.1, 130.3, 114.2, 109.7 ppm.
3.5. 1-(1-Naphthyl)-3-phenylpropenone (42)
An aqueous 40% potassium hydroxide solution (10 ml)
was added drop wise to a chilled (5–10 °C), stirring flask
containing a mixture of 1-napthylethanone (1.2 g,
10 mmol) and benzaldehyde (1.56 g, 10 mmol) in 15 ml
of ethanol. A viscous oil formed after stirring for
3 hours and the reaction mixture was extracted with
ether (3 portions, ꢀ10 ml). After the organic layers were

T. P. Robinson et al. / Bioorg. Med. Chem. 13 (2005) 4007–4013
4013
3.9. SVR cell studies
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 CoulterÕs
Isoton II balanced electrolyte solution and the total
number of cells/well was counted using a Coulter Z1 cell
counter. The total cell count of each test well was then
compared to the DMSO control well for each concentra-
tion. The percent inhibition of cell growth was calcu-
lated from these values.
The endothelial cell proliferation assays used in this
study were developed by Arbiser et al.19 at Emory Uni-
versity. Primary murine endothelial cells, garnered from
adult C57BL/6 mice, were infected with an ecotropic ret-
rovirus encoding the simian virus 40 (SV40) large T 58-3
allele coupled with a resistance to neomycin. The result-
ing cells were selected in a solution of DulbeccoÕs mod-
ified EagleÕs medium (DMEM), 5% fetal calf serum, and
0.6 mg/ml geneticin. The clone cells were stained with
1,10-dioctadecyl-3,3,30,30-tetramethylindocyanine per-
chlorate coupled to acetylated low density lipoprotein
(diI-Ac-LDL). Fluorescence microscopy was used to
identify the positive clones. The clone MS1 was ex-
panded and infected with an additional retrovirus,
which encoded for activated H-ras (mutant allele at gly-
cine-12 and threonine-59) as well as hygromycin resis-
tance. The resulting, immortalized endothelial cell line
was designated SVR.
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For use in the in vitro assays, the SVR cells were plated
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