Investigation of fluorinated and bifunctionalized 3-phenylchroman-4-one (isoflavanone) aromatase inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.11.045

Fluorinated isoflavanones and bifunctionalized isoflavanones were synthesized through a one-step gold(I)-catalyzed annulation reaction. These compounds were evaluated for their in vitro inhibitory activities against aromatase in a fluorescence-based enzym

Full text of DOI:10.1016/j.bmc.2013.11.045

Bioorganic & Medicinal Chemistry 22 (2014) 126–134
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Investigation of fluorinated and bifunctionalized
3-phenylchroman-4-one (isoflavanone) aromatase inhibitors q
Erica Amato a, Tony Bankemper a, Rebecca Kidney a, Thuy Do a, Alma Onate a, Fathima Shazna Thowfeik b,
Edward J. Merino b, Stefan Paula a, Lili Ma a,
⇑
a Department of Chemistry, Northern Kentucky University, Nunn Drive, Highland Heights, KY 41099, United States
b Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Fluorinated isoflavanones and bifunctionalized isoflavanones were synthesized through a one-step
gold(I)-catalyzed annulation reaction. These compounds were evaluated for their in vitro inhibitory activ-
ities against aromatase in a fluorescence-based enzymatic assay. Selected compounds were tested for
their anti-proliferative effects on human breast cancer cell line MCF-7. Compounds 6-methoxy-3-(pyri-
din-3-yl)chroman-4-one (3c) and 6-fluoro-3-(pyridin-3-yl)chroman-4-one (3e) were identified as the
Received 10 September 2013
Revised 15 November 2013
Accepted 23 November 2013
Available online 5 December 2013
most potent aromatase inhibitors with IC50 values of 2.5
have great potential for the development of pharmaceutical agents against breast cancer.
Ó 2013 Elsevier Ltd. All rights reserved.
lM and 0.8 lM. Therefore, these compounds
Keywords:
Breast cancer
Estrogen
Aromatase inhibitors
Fluorine
Isoflavanones
Functional groups
1. Introduction
tissues.4 Since 75% of breast cancer tumors are hormone-depen-
dent, interfering with the production of estrogen using aromatase
inhibitors (AIs) is a validated target. In this work, we expand on
the types of AIs currently available by reporting for the first time more
potent isoflavanone AIs that have not been explored previously.
There are two types of AIs: steroidal (type I inhibitors) and non-
steroidal (type II inhibitors), based on their chemical structure.5
Steroidal AIs (e.g., exemestane in Fig. 1) are mechanism-based
inhibitors which bind to the enzyme active site irreversibly. On
the other hand, many nonsteroidal AIs (e.g., letrozole and anan-
strozole in Fig. 1) bind to the enzyme’s active site reversibly by
non-covalent interactions such as heme iron coordination, hydro-
gen bonding, etc. Both types of AIs serve to block the aromatase
function in order to prevent its estrogen production. AIs have been
used in the treatment of hormone-dependent breast cancer and
have shown clear superiority compared with the traditional treat-
ment selective estrogen receptor modulators (SERMs) such as
tamoxifen.6
Breast cancer is the most common type of cancer found in wo-
men worldwide, and it is reported that one in every eight women
develops metastatic breast cancer in their lifetime.1 There is a high
concentration of estrogen in breast tissue, therefore the risk of
developing breast cancer increases greatly.2 In addition, immature
breast tissue cells allow stronger binding of carcinogens to breast
tissue cells and they possess lower DNA repair capacity. Aromatase
is a cytochrome P450 enzyme that has been found to catalyze the
last and rate-limiting step of endogenous estrogen synthesis.
Chemically, aromatase catalyzes the oxidation and aromatization
of androgens (testosterone and androstenedione) to estrogens
(estrone and estradiol).3 Aromatase is present in breast tissue
and is the source of local estrogen production in breast cancer
Abbreviations: Arg 115, arginine 115; Ile 133, isoleucine 133; Phe 221,
phenylalanine 221; Trp 224, tryptophan 224; Asp 309, aspartic acid 309; Thr 310,
threonine 310; Val 370, valine 370; Leu 477, leucine 477; CYP19, cytochrome P450
aromatase; AIs, aromatase inhibitors; SERMs, selective estrogen receptor modula-
tors; NSAIs, non-steroidal aromatase inhibitors; NMR, nuclear magnetic resonance;
MS, mass spectrometry; HPLC, high performance liquid chromatography.
Flavonoids have been shown to have antiviral, anti-inflamma-
tory, antimutagenic and anticarcinogenic activities. Some naturally
occurring flavones, flavanones and isoflavones have been reported
to inhibit aromatase and affect breast cancer cell proliferation.
Being a rare class of natural products, the aromatase inhibition
effects of isoflavanones were scarcely reported. The aromatase
inhibition potencies of flavonoid core structures are summarized
q
Abbreviations used for amino acids and designation of peptides follow the rules
of the IUPACIUB Commission of Biochemical Nomenclature in J. Biol. Chem. 1972,
247, 977–983.
⇑
Corresponding author. Tel.: +1 859 572 6961; fax: +1 859 572 5162.
E-mail address: mal1@nku.edu (L. Ma).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.11.045

E. Amato et al. / Bioorg. Med. Chem. 22 (2014) 126–134
127
N
useful information to develop more potent yet less toxic aromatase
inhibitors.
O
N
N
N
N
N
2. Results
N
O
NC
CN
N
2.1. Design and synthesis of fluorinated and bifunctionalized
isoflavanone compounds
exemestane
anastrozole
letrozole
Figure 1. Structures for steroidal (exemestane) and nonsteroidal (letrozole, anas-
trozole) aromatase inhibitors.
For the beneficial effects of fluorine mentioned above, eight
fluorinated isoflavanones 2a–2h were synthesized according to
Scheme 1 and evaluated for their biological activities. A micro-
wave-assisted, gold(I)-catalyzed annulation reaction of fluorinated
hydroxyaldehydes and phenylacetylenes was utilized to synthesize
these isoflavanones.
in Figure 2. It is interesting to note the different activities exhibited
by flavone, flavanone and isoflavone. Unlike flavone or flavanone
which showed moderate inhibition against aromatase, isoflavone
is inactive toward aromatase.7,8 In our previous study, we showed
for the first time that an isoflavonone-based AI 1a was active
Compared to AI clinical drugs, the molecular weights and polar
surface areas of the isoflavanone AIs previously synthesized in our
lab were relatively small. For example, the polar surface areas
(PSA) for 6-bromo-3-phenylchroman-4-one 1b (PSA = 26.3 Å2),
3-(biphenyl-4-yl)chroman-4-one 1c (PSA = 26.3 Å2), 3-(4-Phen-
oxyphenyl)chroman-4-one 1d (PSA = 35.3 Å2) and 3-(pyridin-3-
yl)chroman-4-one 1e (PSA = 39.2 Å2) were only about half of that
for letrozole (PSA = 78.3 Å2). This provided additional opportuni-
ties to modify these AIs with additional functional groups. The
development of an aromatase inhibitor with multiple functional
groups is an attractive concept as this single agent should inhibit
estrogen biosynthesis by binding to the aromatase through two
or more of the following interactions: hydrophobic interactions,
hydrogen bonding and heme iron coordination. In addition, the ex-
tra functional groups can be used to fine tune the physicochemical
properties of AIs so that desirable bioavailability and pharmacoki-
netic properties can be achieved. Therefore, five bifunctionalized
isoflavanones 3a–3e were synthesized according to Scheme 1
and tested for their inhibitory effects against aromatase. The struc-
tures of fluorinated isoflavanones 2a–2h and bifunctionalized isof-
lavanones 3a–3e are listed in Table 1.
against aromatase with an IC50 value of 29 lM, probably due to in-
creased hydrophobic interactions between isoflavanone and the
aromatase active site.9 This indicated the potential medicinal value
of this subclass of flavonoid compounds.
Fluorinated drugs have been a very popular topic in the field of
pharmaceutical science.10 In the past 50 years, approximately 20%
of pharmaceuticals contain one or more fluorine atoms.11 The rate
of introduction of fluorinated drugs has increased compared to
past years. Fluorine has effects on several properties of drug mole-
cules including: drug–target interactions and specificity, metabolic
stability, acidity or basicity, membrane permeability and toxicity.
It has been used as a bioisostere for H, OH and NH2 groups to mod-
ulate bioactivity and the pharmacological properties of medi-
cines.12 The fluorine atom is highly electronegative and small.
The addition of fluorine to an aryl ring or other p-system increases
the lipophilicity as a result of electron donation of fluorine lone
pair electrons by resonance.13 The short contacts involving fluorine
atoms between proteins and fluorinated ligands are very frequent,
and they may increase the fluorinated molecule’s binding affinity
for the enzyme.11,14 Thus, fluorine has played and will continue
its important role in drug design.15
2.2. Aromatase inhibition activities of fluorinated and
biofunctionalized AIs
To expand on this important new isoflavanone scaffold, we
chose to explore fluorinated and bifunctionalized AIs. Herein we
report our effort to optimize isoflavanone AIs through structure-
activity relationship analysis and enhanced binding interaction
design identified via computation. The enzymatic and cell-based
assays were used to evaluate the biological activities. Computa-
tional docking of the AIs into the aromatase active site was used
to determine the crucial interactions that occur within the enzyme
pocket. The physiochemical properties such as LogP, number of
hydrogen bond donors, number of hydrogen bond acceptors and
polar surface area were also calculated to gain some insight into
their pharmacokinetic effects. Selected compounds from isoflava-
none AIs were tested further for their anti-proliferative effects on
human breast cancer cells. The bioactivity and physicochemical
properties exhibited by these isoflavanone compounds provide
Aromatase inhibition was determined by using a fluorogenic
substrate (7-methoxy-trifluoromethylcoumarin) and human CYP
19 aromatase with ketoconazole as positive control.16 Each com-
pound was tested in triplicate measurements and the average
IC50 value can be seen in Table 1. Fluorinated isoflavanone 2b
showed stronger inhibitory effects against aromatase compared
to the parent molecule 1a while compounds 2c and 2h were inac-
tive. No significant inhibition occurred with bifunctionalized
isoflavanones 3a and 3b. Bifunctionalized isoflavanones 3c and
3e showed improved inhibition against aromatase.
Also shown in Table 1 are the calculated important physico-
chemical properties (partition coefficient between n-octanol and
water logP, number of hydrogen bond donors Hd, number of
5'
2'
5'
O
O
5
8
5
8
6'
6'
4'
3'
4'
3'
4
4
O
O
3
2
3
2
5
8
5
A
8
6
7
6
7
B
4
4
3
2
3
2
6
7
6
7
1'
1'
C
2'
O
1
O
1
O
1
O
1
Flavone
IC50 = 10 µM7
Flavanone
IC50 = 8 µM8
Isoflavone
Isoflavanone 1a
inactive7
IC50 = 29 µM9
Figure 2. Structures and aromatase inhibitory effects of flavonoid compounds.

128
E. Amato et al. / Bioorg. Med. Chem. 22 (2014) 126–134
Scheme 1. Synthesis of fluorinated isoflavanone AIs (2a–2h) and bifunctionalized isoflavanone AIs (3a–3e).
hydrogen bond acceptors Ha and polar surface area PSA) and
predicted toxicity profiles for these AIs. The calculation of
physicochemical properties was performed using Molinspiration
Cheminformatics software at URL http://www.molinspiration.com.
Prediction of mutagenic, tumorigenic, irritating and reproductive
toxicities was achieved utilizing OSIRIS Property Explorer software
at URL http://www.organic-chemistry.org/prog/peo/.17
assays. Unfortunately, these two compounds turned out to be inac-
tive in the cell assay. Since the presence of the pyridyl group in-
creased the bioactivity as seen in AI 1e, the observed inactivity
for compounds 3c and 3e is probably related to the substituents
at the C6 position. On the other hand, the C8 substituted com-
pound 3d showed good anti-proliferative activity in the MCF-7 cell
assay with an IC50 value of 43
6 lM.
2.3. Estrogen-dependent MCF-7 growth inhibition
2.4. Docking poses for isoflavanone AIs
Examination in human breast cancer cells of the synthesized
compounds was accomplished. As a starting point, compounds
1b–e were examined for activity in MCF-7 breast cancer cells using
letrozole as a positive control.18 To examine activity, MCF-7 cells
were grown under two different conditions. First, as is standard
MCF-7 cells were raised in estrogen, low picomole, which has been
shown to increase both estrogen and estrogen receptor content.19
With added estrogen the doubling time was twice as fast and sta-
bilized over 10 days. The doubling rate increased more than 2-fold
during this period. In contrast, MCF-7 cells raised without estrogen
and phenol red displayed an altered phenotype, grew more slowly,
and importantly had reduced sensitivity towards anti-estrogen
molecules including flavenoids.20 Examination of the positive con-
trol, letrozole, a well-known AI with estrogen-dependent activity
in the low nanomolar range was performed. These cells were incu-
bated with letrozole at low nanomolar concentrations. The IC50
value for letrozole is 7 3 nM and is similar to literature values
of 20 nM.21 The novel AIs were then examined for differential
activity against estrogen-dependent MCF-7 cells. Viability plots
with and without estrogen for compound 1e are shown in
Figure 3a. When non-estrogen dependent MCF-7 cells were incu-
bated with 3c, no growth inhibition was observed. In fact, cells
tended to grow faster than controls likely due to non-specific
estrogen mimicking since MCF-7 is known to overexpress estrogen
receptors. Similar experiments using estrogen-dependent MCF-7
Both enantiomers of synthesized isoflavanones were docked
into the aromatase active site (PDB code 3EQM),22 and the enantio-
mer with the higher docking score was used to identify crucial en-
zyme/inhibitor interactions. The factors which were taken into
account in the docking score included external hydrogen bonds,
external van der Waals interactions, internal hydrogen bonds,
internal torsion and internal van der Waals interactions. The top
docking scores for active isoflavanone AIs 2a, 2b, 2g and 3e were
55.2, 53.9, 56.3 and 56.9 kJ/mol. The dominant interactions for 6-
fluoroisoflavanone 2a (Fig. 4a) and 30, 50-difluoroisoflavanone 2g
(Fig. 4c) predicted by docking poses were p–p stacking interactions
between the isoflavanone B ring and the heme group in the en-
zyme active site.
The binding interactions between 8-fluoroisoflavanone 2b
(Fig. 4b) and the aromatase active site were more complicated than
those of 2a and 2g. At least three different types of interactions
were noticed from our study: heme iron coordination,
p–p stack-
ing interactions and hydrogen binding. Specifically, the C4 car-
bonyl of 2b coordinates to the heme iron with a predicted
distance of 2.9 Å. Carbonyl-heme iron coordination is common
for isoflavanone AIs, and it has been observed for 2-phenyl-2,3-
dihydro-1H-benzo[f]chromen-1-one,3-(4-phenoxyphenyl)chro-
man-4-one and 3-(3,5-dimethoxyphenyl)chroman-4-one in our
previous study.9 The
p–p stacking interactions are frequently in-
volved in binding modes between the ligand and aromatase amino
cells showed high activity. The IC50 value for 1e was 31
2 lM.
acid residues such as Phe 221, Trp 224 or the heme group.
Thus, the new AIs in this report require estrogen-dependent
Compound 2b showed p–p stacking interactions with Trp 224
growth to induce anti-cancer activity.
(distance = 3.3 Å). The hydrogen binding interactions between
the C7-H of 2b and the enzyme amino acid residue Asp309
(distance = 2.4 Å) were also predicted in the docking study.
The bifunctionalized isoflavanone 3e was docked into the en-
zyme active site and its docking pose is shown in Figure 4d. The
crucial interactions between compound 3e and the enzyme active
site included the following: first, the coordination between the 3e
pyridyl nitrogen and the heme iron atom (distance = 2.5 Å); sec-
ond, the interaction between the A ring of 3e and the enzyme ami-
no acid residue Arginine 115 (distance = 3.8 Å). The pyridyl
nitrogen and heme iron coordination is the most important inter-
action for heterocyclic isoflavanones as it was also observed in
our previous study for compounds such as 3-(pyridin-3-yl)chro-
man-4-one.9 Even though the amino acid residue Thr 310 showed
ligand exposure in the docking study, there were no obvious
hydrogen bonding interactions between the Thr 310 side chain
OH and the 3e pyridyl nitrogen.
The estrogen-dependent IC50 values for 1b, 1c, 1d and 1e were
obtained. The relationship between aromatase inhibition and anti-
cancer cell activity was examined (Fig. 3b). For example, the inac-
tive AI 1c did not show anti-cancer cell activity. Thus, 1c was given
a value of 150 as this is the upper detection limit. The moderate AI
1b showed moderate anti-proliferative activity on MCF-7 cell assay
with an IC50 value of 100 11
lM. The cellular IC50 values for good
AIs 1d and 1e are 17 M and 44
l
lM, respectively (Fig. 3b). The cor-
relation coefficient for this set of five molecules is 0.96 and the p
value is less than 0.05. In addition it should be noted that the
letrozole shows the same trend with correspondingly lower
in vitro and cellular activities. This data demonstrate that the cel-
lular anti-cancer activity of these new AIs correlates with in vitro
aromatase inhibition.
The most active AIs from the enzymatic assay 3c and 3e were
also tested for their anti-proliferative activities on MCF-7 cell

E. Amato et al. / Bioorg. Med. Chem. 22 (2014) 126–134
129
Table 1
Structure, calculated physicochemical properties, predicted toxicity and experimental aromatase inhibitory potencies of isoflavanone AIs 2a–2h and 3a–3e
Structure
ID
2a
2b
LogPa
3.54
Hab
Hdc
PSA (Å2)d
Toxicitye
IC50 (lM)
mut/tum/irr/rep
O
O
F
2
0
26.3
L/L/L/L
L/L/L/L
32 7f
O
3.51
2
0
26.3
15 9f
O
F
O
O
F
2c
3.63
2
0
26.3
L/L/L/L
N.A.g
F
O
2d
2e
3.04
3.54
2
2
0
0
26.3
26.3
L/L/L/M
L/L/L/M
24 8f
F
O
O
35 5f
F
F
O
O
2f
3.56
3.65
2
2
0
0
26.3
26.3
L/L/L/M
L/L/L/M
33 12f
O
O
F
2g
20 11f
F
O
O
CF3
2h
3a
4.29
6.31
2
3
0
0
26.3
35.5
L/L/L/L
H/L/L/L
N.A.g
O
O
O
O
190 25f
O
O
O
3b
3c
5.19
1.71
4
4
0
0
44.8
48.4
H/L/L/L
L/L/L/L
N.A.g
O
O
O
N
2.5 1.1f
O
O
N
3d
3e
1.83
3
0
39.2
L/L/L/L
67.1 4f
O
F
O
O
F
N
1.83
2.15
3
5
0
0
39.2
78.3
L/L/L/L
0.8 0.5f
Letrozole
H/L/M/M
0.0028 0.0006f
a
b
c
d
e
f
LogP, calculated logarithm of partition coefficient between n-octanol and water.
Ha, hydrogen bond acceptor.
Hd, hydrogen bond donor.
PSA, polar surface area.
Toxicity includes mutagenic, tumorigenic, irritating and reproductive effects. L, low; M, medium; H, high.
IC50 values are average of three runs.
Inactive in enzymatic assay.
g

130
E. Amato et al. / Bioorg. Med. Chem. 22 (2014) 126–134
3. Discussion
3.1. The effect of fluorine on isoflavanone AIs
The addition of fluorine atoms on the isoflavanone scaffold did
not improve the calculated physicochemical properties and pre-
dicted toxicity profiles significantly when compared with their
parent molecule 1a. Among the physicochemical properties listed
in Table 1, the most important factor is Log P which measures
the solubility and permeability of orally-active drug molecules. A
drug-like molecule usually has a LogP value between 0 and 3.23
Most of the fluorinated isoflavanones had logP values around
3.50 (Table 1), similar to the parent molecule 1a which had a logP
value of 3.40, indicating potential poor bioavailability. Thus, the
physicochemical properties of these compounds could be further
modulated by installing additional polar functional groups. The
fluorinated isoflavanone AIs usually exhibited low to medium pre-
dicted toxicity profiles. The active fluorinated isoflavanones 2b and
2g were predicted to have low mutagenic, tumorigenic and irritat-
ing toxicity profiles (Table 1).
The unique hydrogen binding pattern between the C7-H of
8-fluoroisoflavanone 2b and the enzyme amino acid residue
Asp309 indicates the increased acidity of isoflavanone compounds
in the presence of fluorine. Namazian reported that fluorine
substituents could significantly increase the gas-phase acidity of
benzene,24 which might make the aromatic ring more capable of
Figure 3. Potency of AIs in estrogen-dependent MCF-7. (a) Estrogen-dependent
(grey) and estrogen independent (black) anti-cancer activity of Compound 1e.
Compound 1e requires estrogen-dependent cells to inhibit cell growth (compare
grey and black). (b) The relationship of anti-cancer anti-aromatase activity. The
correlation coefficient is 0.96 and p less than 0.05 showing strong correlation
between in vitro and cellular experiments.
Figure 4. Representations of molecular docking performed in this study. The inhibitor is depicted in yellow sticks. The heme group is shown in orange. Selected relevant
amino acid residues in aromatase are shown as sticks (white, H; black, C; blue, N; red, O). Lines are drawn between the atoms likely involved in hydrogen bonding or heme
coordination. (a) 6-fluoroisoflavanone 2a; (b) 8-fluoroisoflavanone 2b; (c) 30,50-difluoroisoflavanone 2g. (d) 6-fluoro-3-(pyridin-3-yl)chroman-4-one 3e.

E. Amato et al. / Bioorg. Med. Chem. 22 (2014) 126–134
131
forming hydrogen bonds. Also, it has been noticed that b-fluorina-
tion invariably increases C–H acidity due to the stabilization of the
carbanion by the inductive effect and hyperconjugative reso-
nance.13a Given these facts and the strong electronegativity of fluo-
rine, the observed hydrogen bonding might result from the
increased acidity of C7-H and the close proximity with Asp309.
The active site of aromatase (CPY19) is highly hydrophobic. By
the addition of fluorine the binding affinity might increase as seen
with the most potent compound 8-fluoroisoflavanone 2b.
than 2-fold when compared with its precursor 3-(pyridin-3-
yl)chroman-4-one 1e. Encouraged by this discovery and our
findings on fluorinated compounds, 8-fluoro-3-(pyridin-3-yl)chro-
man-4-one 3d and 6-fluoro-3-(pyridin-3-yl)chroman-4-one 3e
were prepared and tested for their enzymatic activity. Being the
most active compound in this study, isoflavanone 3e (IC50 = 0.8 -
l
M) showed a 7-fold increase in potency when compared with
its precursor 3-(pyridin-3-yl)chroman-4-one 1e (IC50 = 5.8 M),
and a 36-fold increase in potency when compared with its parent
molecule 1a (IC50 = 29 M). Coupled with its desirable physico-
l
The hydrophobic interactions between the B ring of 30,50-diflu-
oroisoflavanone 2g and the heme porphyrin (distance = 3.4–3.9 Å,
Fig. 4c) increased when compared with compound 2a which has
no fluorine on its B ring (distance = 3.5–4.1 Å, Fig. 4a). This can
be explained from the effect of fluorine substituents on lipophilic-
ity. Although there are might be exceptions, aromatic fluorination
l
chemical properties, predicted low toxicity profile and the benefi-
cial effects of fluorine, compounds 3d and 3e represented a highly
interesting class of potential anti-breast cancer agents.
3.3. Estrogen-dependent MCF-7 growth inhibition
or fluorination adjacent to atoms with
p bonds usually increases
lipophilicity due to the resonance donor nature of fluorine.13a
The effects of fluorine substituents are additive and with each
additional fluorine, the compound becomes increasingly lipo-
philic15 and its LogP value becomes larger. The logP values for
non-, mono- and difluorinated isoflavanones 1a, 2a and 2g are
3.40, 3.51 and 3.65, respectively. Therefore, the hydrophobic inter-
actions increased from compound 2a to 2g.
Under physiological conditions, typical estrogen levels are 20–
400 pM.26 Therefore, the use of a baseline level of estrogen may
be more meaningful for the assessment of anti-proliferative activ-
ities of isoflavanone AIs. The estrogen used in our cell assays was b-
estradiol and the concentration required to stimulate MCF-7 cell
proliferation was determined to be 12 pM. Using this concentra-
tion, the cytotoxicity of selected isoflavanone AIs was tested
against the human breast cancer MCF-7 cell line to estimate their
IC50 values. The investigation of the antiproliferative effect on hu-
man breast cancer cells showed interesting cytotoxic activity of
some isoflavanone compounds such as 1d, 1e and 3d. For mono-
functionalized isoflavanones, these results are mostly in agreement
with their aromatase inhibitory potencies, even though the IC50
values for their cytotoxic effects are about 2 to 7 times higher than
their aromatase inhibitory potencies. For bifunctionalized isoflava-
nones, the results were more complicated, likely due to the inclu-
sion of more factors in the cell assays and in the substrate
structure. The cellular anti-cancer activity of these new AIs may
be at least in part due to the aromatase inhibition, though other
mechanisms might also be possible.
3.2. The effect of bifunctional groups on isoflavanone AIs
The addition of a second functional group serves the following
purposes: first, to enhance the enzyme/substrate binding interac-
tions through multiple binding sites; second, to fine-tune the phar-
macokinetic and pharmacodynamics properties of potential drug
molecules. Methoxyl, phenoxyl, benzo, halogens and pyridyl
groups on the isoflavanone scaffold have resulted in favorable
interactions with aromatase.9 Since the C6 position is very impor-
tant for enzymatic bioactivity of isoflavanone AIs, the second func-
tional group was mostly installed at the C6 position.
Compounds 3a and 3b were designed based on the structure-
activity relationships of their active precursors. Specifically, we
found in our previous study that 40-PhO, 6-CH3O and 5,6-benzo
groups significantly improved the aromatase inhibitory potencies
of isoflavanone compounds. It was expected that the enzyme/
inhibitor binding interactions could be enhanced by bringing the
favorable functional groups together. Even though the calculated
physicochemical properties of these compounds were not perfect,
as seen by the poor Log P values and predicted high mutagenic tox-
icity in Table 1, the high activity of their precursors in enzyme as-
says justify the synthesis and evaluation these compounds.
Unfortunately, these two compounds lost their activity in the en-
zyme inhibition assay. This might arise from the increased volumes
of compounds 3a and 3b which no longer fit into the enzyme pock-
et. Being the end product of cholesterol degradation, estrogens are
small in size. Correspondingly, the aromatase active pocket is only
400 Å3, considerably smaller compared to other CYP 450 enzymes
which normally have an active site of about 530 Å3.25 The volumes
for AI clinical drugs and aromatase natural substrates are in the
range of 254.5–285.7 Å3. Compounds 3a and 3b have significantly
larger volumes (380.1 and 356.4 Å3, respectively), which might
render them too sterically bulky to fit into the aromatase active
site. The best docking score for compound 3a was only 40.0 kJ/
mol, indicating its poor binding interactions with the enzyme.
A great improvement was achieved when a methoxy group was
added to compound 1e to result in compound 3c (Table 1): the
LogP value increased from 1.69 to 1.71, the PSA increased from
39.2 to 48.4 Å2, the volume of this molecule increased from 201.1
to 227.6 Å3 and the risk of reproductive toxicity was reduced. More
importantly, the IC50 value of bifunctionalized compound 6-meth-
oxy-3-(pyridin-3-yl)chroman-4-one 3c (Table 1) decreased more
4. Conclusion
In conclusion, we have described the synthesis and biological
evaluation of two subsets of isoflavanone AIs: fluorinated isoflava-
nones and bifunctionalized isoflavanones. Fluorine substituents on
the isoflavanone scaffold might enhance the hydrogen bonding,
heme iron coordination and/or hydrophobic interactions between
aromatase and its inhibitor. The proper choice and installation of
a second functional group on the isoflavanone could improve the
physicochemical properties and bioactivities of isoflavanone AIs.
These new AIs exhibited aromatase inhibition, cellular anti-cancer
activity and desirable drug-like properties. Therefore, isoflavanone
AIs appear as interesting candidates for future investigation of
their effects to reduce cancer cell proliferation in hormone depen-
dent breast cancer.
5. Experimental
5.1. Chemistry
Reagents and solvents were obtained from Aldrich and used
without further purification unless otherwise noted. Toluene was
freshly distilled from CaH2 prior to use. The microwave-assisted
reactions were conducted on a single-mode Discover System from
CEM Corporation. Power cooling was turned off manually during
the reaction to ensure that the reaction temperature reached
200 °C. Thin-layer chromatography was performed using pre-
coated silica gel F254 plates (Whatman). Column chromatography

132
E. Amato et al. / Bioorg. Med. Chem. 22 (2014) 126–134
was performed using pre-packed RediSep Rf Silica columns on a
CombiFlash Rf Flash Chromatography system (Teledyne Isco).
NMR spectra were obtained on a Joel 500 MHz spectrometer.
Chemical shifts were reported in parts per million (ppm) relative
to the tetramethylsilane (TMS) signal at 0.00 ppm. Coupling con-
stants, J, were reported in Hertz (Hz). The peak patterns were indi-
cated as follows: s, singlet; d, doublet; t, triplet; dt, doublet of
triplet; dd, doublet of doublet; m, multiplet; q, quartet. Analytical
reverse-phase HPLC was carried out using a system consisting of a
1525 binary HPLC pump and 2996 photodiode array detector
(Waters Corporation, Milford, MA). A Nova-Pak C18 column
d 7.72 (d, J = 8.3 Hz, 1H), 7.37–7.25 (m, 6H), 6.99–6.95 (m, 1H),
4.76 (d, J = 6.4 Hz, 2H), 4.04 (t, J = 7.1 Hz, 1H), 13C NMR (CDCl3,
125 MHz, ppm): d 191.2, 150.7, 134.4, 129.1, 128.6, 128.1, 123.1,
122.8, 121.9, 121.8, 121.1, 72.1, 52.3. HRMS Calculated for
C15H11O2FNa [M+Na] 265.0641, found 265.0630.
5.1.1.3. 6,8-Difluoro-3-phenylchroman-4-one (2c).
sized from 3,5-difluorosalicylaldehyde (0.5 mmol, 1 equiv,
80.8 mg) and phenylacetylene (1.5 mmol, 3 equiv, 164.7 L)
Synthe-
l
according to the general procedure for the synthesis of isoflava-
none derivatives described above. Orange solid. Yield: 30.9%. Pur-
ity: 98.9%. Rf = 0.31 (10% EtOAc/Hex). 1H NMR (CDCl3, 500 MHz,
ppm): d 7.46–7.21 (m, 7H), 4.76 (d, J = 7.3 Hz, 2H), 4.05 (t,
J = 7.1 Hz, 1H). 13C NMR (CDCl3, 125 MHz, ppm): d 190.3, 134.0,
129.1, 128.6, 128.2, 111.2, 111.0, 110.8, 108.1, 107.9, 72.2, 52.3.
HRMS Calculated for C15H10O2F [MꢁH] 259.0571, found 259.0580.
(4
lm, 3.9 ꢀ 150 mm), also from Waters, was used with a mobile
phase of methanol and water (60:40, vol./vol.) plus 0.25% acetic
acid, flow rate 1.2 mL/min and UV detection wavelength at
250 nm. Control and data acquisition was done using the Empower
2 software (Waters Corporation, Milford, MA). Synthesized com-
pounds were prepared in methanol to make 1.0 mg/mL stock solu-
tions and 10
l
L of solution was injected for the HPLC test. The
5.1.1.4. 3-(2-Fluorophenyl)chroman-4-one (2d).
sized from 1-ethynyl-2-fluorobenzene (1 mmol, 1 equiv,
113.3 L) and salicylaldehyde (3 mmol, 3 equiv, 314.2 L) accord-
ing to the general procedure for the synthesis of isoflavanone
derivatives described above. White solid. Yield: 25.1%. Purity:
98.4%. Rf = 0.45 (10% EtOAc/Hex). 1H NMR (CDCl3, 500 MHz,
ppm): d 7.99 (d, J = 7.8 Hz, 1H), 7.52 (t, J = 7.8 Hz, 1H), 7.32–7.02
(m, 6H), 4.65–4.58 (m, 2H), 4.33 (dd, J = 10.5, 6.9 Hz, 1H). 13C
Synthe-
purity of all the compounds was assessed by HPLC at 254 nm. All
final compounds were confirmed to be P95% purity by analysis
of their peak area. Mass spectra were obtained on a Waters TQD
Tandem Quadrapole Mass Spectrometer, and data was collected
in electrospray positive mode (ESI+). High resolution mass spectra
were recorded on a Micromass Q-TOF 2 or a Thermo Scientific LTQ-
FT™ mass spectrometer operating in electrospray (ES) mode. Cal-
culation of important physicochemical properties (logP, number
of hydrogen bond donors and acceptors and polar surface area)
was performed using Molinspiration Cheminformatics software
at URL http://www.molinspiration.com. Prediction of mutagenic,
tumorigenic, irritating and reproductive toxicities and drug score
was achieved utilizing OSIRIS Property Explorer software at URL
http://www.organic-chemistry.org/prog/peo/.
l
l
NMR (CDCl3, 125 MHz, ppm):
d
191.2, 161.8, 161.2 (d,
J = 245 Hz), 136.2, 130.7, 129.7, 127.8, 124.6, 122.1, 121.8, 117.9,
116.0, 115.8, 70.7, 47.6. HRMS Calculated for
[M+Na] 265.0641, found 265.0630.
C
15H11O2FNa
5.1.1.5. 3-(3-Fluorophenyl)chroman-4-one (2e).
Synthe-
sized from salicylaldehyde (1 mmol, 1 equiv, 105
lL) and 1-ethy-
nyl-2-fluorobenzene (3 mmol, 3 equiv, 340 L) according to the
l
5.1.1. General procedure for isoflavanone synthesis
general procedure for the synthesis of isoflavanone derivatives de-
scribed above. Light yellow, flakey solid. Yield: 30.5%. Purity:
96.9%. Rf = 0.41 (10% EtOAc/Hex). 1H NMR (CDCl3, 500 MHz,
ppm): d 7.95 (d, J = 7.8 Hz, 1H), 7.52 (t, J = 6.9 Hz, 1H), 7.32 (m,
1H), 7.08–7.00 (m, 5H), 4.67 (d, J = 3.65 Hz, 2H), 3.99 (d,
J = 3.2 Hz, 1H). 13C NMR (CDCl3, 125 MHz, ppm): d191.4, 163.9,
161.5, 137.4, 136.3, 130.4, 127.9, 124.4, 121.9, 120.9, 118.0,
The microwave-assisted isoflavanone syntheses were con-
ducted on a single-mode Discover System from CEM Corporation.
To an oven-dried standard microwave reaction vial (capacity
10 mL) equipped with a stirring bar was added AuCN (0.05 mmol,
0.05 equiv, 11.0 mg), Bu3P (0.25 mmol, 0.25 equiv, 61.7 lL), alde-
hyde (1 mmol, 1 equiv), alkyne (3 mmol, 3 equiv) and 1 mL of
freshly distilled toluene. The reaction vial was then sealed with a
Teflon septum cap, and the sample was subjected to microwave
irradiation at a power of 200 W for 10 min (hold time) at 200 °C.
After being cooled down, the vial was opened, and the crude mix-
ture was loaded directly on silica gel and was purified by Medium
Performance Liquid Chromatography eluding with an ethyl ace-
tate/hexanes gradient to afford the desired products.
115.8, 115.0, 71.2, 52.0. HRMS Calculated for
[M+Na] 265.06353, found 265.06357.
C
15H11O2FNa
5.1.1.6. 3-(4-Fluorophenyl)chroman-4-one (2f).
Synthe-
sized from 1-ethynyl-4-fluorobenzene (1 mmol, 1 equiv,
114.4 L) and salicylaldehyde (3 mmol, 3 equiv, 314.2 L) accord-
l
l
ing to the general procedure for the synthesis of isoflavanone
derivatives described above. Yellow solid. Yield: 28.3%. Purity:
99.5%. Rf = 0.38 (10% EtOAc/Hex). 1H NMR (CDCl3, 500 MHz,
ppm): d 7.95 (d, J = 7.8 Hz, 1H), 7.57 (t, J = 8.7 Hz, 1H), 7.26–7.23
(m, 2H), 7.06-7.00 (m, 4H), 4.64 (m, 2H), 3.97 (dd, J = 9.2, 5.0 Hz,
1H), 13C NMR (CDCl3, 125 MHz, ppm): d 192.0, 161.6, 136.3,
130.3, 130.2, 127.8, 121.8, 120.9, 117.9, 116.0, 115.8, 71.5, 51.6.
5.1.1.1. 6-Fluoro-3-phenylchroman-4-one (2a)9.
sized from 5-fluorosalicylaldehyde (0.5 mmol, 1 equiv, 70.1 mg),
and phenylacetylene (1.5 mmol, 3 equiv, 164.7 L) according to
Synthe-
l
the general procedure for the synthesis of isoflavanone derivatives
described above. Light yellow solid. Yield: 21.7%. Purity: 99.5%.
Rf = 0.43 (10% EtOAc/Hex). 1H NMR (CDCl3, 500 MHz, ppm): d
7.60 (d, J = 8.3 Hz, 1H), 7.38- 7.20 (m, 6H), 7.00 (dd, J = 9.2,
4.1 Hz, 1H), 4.65 (d, J = 6.4 Hz, 2H), 3.99 (t, J = 7.3 Hz, 1H). 13C
NMR (CDCl3, 125 MHz, ppm): d 191.5, 158.2, 156.4, 134.7, 129.0,
128.6, 128.0, 123.7, 121.5, 119.7, 112.7, 71.7, 52.2. HRMS Calcu-
lated for C15H11O2FNa [M+Na] 265.0641, found 265.0631.
HRMS Calculated for
265.0630.
C15H11O2FNa [M+Na] 265.0641, found
5.1.1.7. 3-(3,5-Difluorophenyl)chroman-4-one (2g).
sized from 1-ethynyl-3,5-difluorobenzene (1 mmol, 1 equiv,
118.8 L) and salicylaldehyde (3 mmol, 3 equiv, 314.2 L) accord-
Synthe-
l
l
ing to the general procedure for the synthesis of isoflavanone
derivatives described above. A light yellow solid was formed from
this reaction. Yield: 35.8%. Purity: 96.8%. Rf = 0.43 (10% EtOAc/Hex).
1H NMR (CDCl3, 500 MHz, ppm): d 7.94 (d, J = 7.8 Hz, 1H), 7.53 (t,
J = 7.8 Hz, 1H), 7.08–6.74 (m, 5H), 4.70–4.62 (m, 2H), 3.82 (dd,
J = 8.3, 5.1 Hz, 1H). 13C NMR (CDCl3, 125 MHz, ppm): d 190.7,
161.5, 136.5, 127.9, 122.0, 120.7, 118.0, 111.8, 111.6, 103.7,
5.1.1.2. 8-Fluoro-3-phenylchroman-4-one (2b).
from 3-fluorosalicylaldehyde (0.5 mmol, 1 equiv, 70.1 mg) and
phenylacetylene (1.5 mmol, 3 equiv, 164.7 L) according to the
general procedure for the synthesis of isoflavanone derivatives de-
scribed above. Orange yellow solid. Yield: 21.5%. Purity: 99.2%.
Rf = 0.32 (10% EtOAc/Hex). 1H NMR (CDCl3, 500 MHz, ppm):
Synthesized
l

E. Amato et al. / Bioorg. Med. Chem. 22 (2014) 126–134
133
103.5, 70.8, 51.7. HRMS Calculated for C15H10O2F [MꢁH] 259.0571,
122.8, 122.4, 122.2, 121.4, 71.6, 49.9. HRMS Calculated for C14H11-
FNO2 [M+H] 244.0781, found 244.0774.
found 259.0568.
5.1.1.8. 3-(4-(Trifluoromethyl)phenyl)chroman-4-one (2h)27-
5.1.1.13.
6-Fluoro-3-(pyridin-3-yl)chroman-4-one
(3e).
.
Synthesized from salicylaldehyde (1 mmol, 1 equiv,
L), and 1-ethynyl-4-trifluoromethylbenzene (3 mmol,
L) according to the general procedure for the syn-
Synthesized from 5-fluororsalicylaldehyde (0.5 mmol, 1 equiv,
70.1 mg) and 3-ethynylpyridine (1.5 mmol, 3 equiv, 154.7 mg)
according to the general procedure for the synthesis of isoflava-
none derivatives described above. Light yellow solid. Yield:
27.4%. Purity: 99.5%. Rf = 0.21 (30% EtOAc/Hex). 1H NMR (CDCl3,
500 MHz, ppm): d 8.56–8.54 (m, 2H), 7.58 (dd, J = 8.2, 2.8 Hz,
2H), 7.30–7.24 (m, 2H), 7.01 (dd, J = 9.2, 4.1 Hz, 1H), 4.66–4.62
(m, 2H), 4.01 (dd, J = 9.6, 5.0 Hz, 1H). 13C NMR (CDCl3, 125 MHz,
ppm): d 190.5, 157.9, 157.6, 149.9, 149.4, 136.2, 130.6, 124.1,
123.8, 121.2, 119.8, 112.7, 71.3, 49.8. HRMS Calculated for C14H11-
FNO2 [M+H] 244.0781, found 244.0774.
104.7
3 equiv, 489.4
l
l
thesis of isoflavanone derivatives described above. Light yellow so-
lid. Yield: 19.6%. Purity: 97.5%. Rf = 0.37 (10% EtOAc/Hex). 1H NMR
(CDCl3, 500 MHz, ppm): d 7.95 (d, J = 7.8 Hz, 1H), 7.61 (d, J = 7.8 Hz,
2H), 7.52 (t, J = 8.3, 1H), 7.41 (d, J = 8.3, 2H), 7.00–7.08 (m, 2H), 4.68
(d, J = 7.3 Hz, 2H), 4.06 (t, J = 8.0 Hz, 1H). 13C NMR (CDCl3, 125 MHz,
ppm): d 191.4, 161.6, 139.1, 136.5, 129.1, 127.9, 125.9, 121.9,
120.9, 118.0, 71.1, 52.1, 13.6. HRMS Calculated for C16H11O2F3Na
[M+Na] 315.0609, found 315.0604
5.1.1.9.
men-1-one (3a).
hyde (0.4 mmol,
phenoxybenzene (1.2 mmol, 3 equiv, 217
eral procedure for the synthesis of isoflavanone derivatives de-
scribed above. White solid. Yield: 35.1%. Purity: 98.5%. Rf = 0.39
(10% EtOAc/Hex). 1H NMR (CDCl3, 500 MHz, ppm): d 9.49 (dd,
J = 8.7 Hz, 1H), 7.95 (dd, J = 8.7, 1H), 7.25–7.65 (m, 4H), 6.98–7.15
(m, 2H), 4.79 (m, 2H), 4.04 (dd, J = 7.8, 4.6 Hz, 1H). 13C NMR (CDCl3,
125 MHz, ppm): d 193.3, 163.6, 157.0, 137.8, 131.9, 130.3, 129.9,
129.8, 129.4, 128.6, 126.1, 125.0, 123.5, 119.2, 119.0, 118.7, 71.3,
52.3. HRMS Calculated for C25H18O3Na [M+Na] 389.1154, found
389.1140.
2-(4-Phenoxyphenyl)-2,3-dihydro-1H-benzo[f]chro-
Synthesized from 2-hydroxy-1-naphthalde-
1 equiv, 68.9 mg) and 1-ethynyl-4-
L) according to the gen-
5.2. Aromatase activity assay
Inhibitory potencies of compounds were determined according
to an established procedure using a commercially available aroma-
tase test kit from BD Gentest.16 This fluorescence-based assay
measures the rate at which recombinant human aromatase
(baculovirus/insect cell-expressed) converts the substrate 7-meth-
oxy-trifluoromethylcoumarin (MFC) into a fluorescent product 7-
ethynyl-trifluoromethylcoumarin (HFC) (kex = 409 nm, kem = 530 nm)
in a NADPH regenerating system. Briefly, concentrated stock
solutions of test compounds were prepared in acetonitrile.
l
100
(dilution factor of 3 between samples) and cofactor mixture
(0.4 U/mL glucose-6-phosphate dehydrogenase; 16.2
M NADP+;
825 M MgCl2; 825 M glucose-6-phosphate; 50 M citrate buffer,
pH 7.5) were prepared in a 96 well plate. After incubating the plate
lL samples containing serial dilutions of test compounds
l
5.1.1.10.
(3b).
6-Methoxy-3-(4-phenoxyphenyl)chroman-4-one
Synthesized from 2-hydroxy-5-methoxybenzaldehyde
l
l
l
(0.4 mmol, 1 equiv, 49.8
zene(1.2 mmol, 3 equiv, 217.04
l
L), and 1-ethynyl-4-phenoxyben-
for 10 min at 37 °C, 100
substrate solution (105
l
g
L of an aromatase/P450 reductase/
protein/mL enzyme; 50 MFC;
lL) according to the general proce-
l
lM
dure described above. Light yellow solid. Yield 36.3%. Rf = 0.30 (10%
EtOAc/Hex). 1H NMR (CDCl3, 500 MHz, ppm): d 7.37 (d, J = 3.2 Hz,
1H), 7.32 (t, J = 8.0 Hz, 3H), 7.23 (t, J = 8.7 Hz, 2H), 7.10–7.12 (m,
2H), 6.94–7.02 (m, 5H), 4.57–4.64 (m, 2H), 3.96 (dd, J = 8.25,
5.05 Hz, 1H). 13C NMR (CDCl3, 125 MHz, ppm): d 192.3, 157.0,
156.3, 154.4, 130.0, 129.9, 125.5, 123.7, 119.3, 119.2, 119.1,
108.1, 71.8, 55.9, 51.7.
20 mM phosphate buffer, pH 7.4) were added to each well. The
plate was covered and incubated for 30 min at 37 °C. 75 L of
l
0.5 M Tris base were then added to stop the reaction and the
fluorescence of the formed de-methylated MFC was measured with
a plate reader (SpectraMax Gemini, Molecular Devices).
Fluorescence intensities, which were proportional to the
amount of reaction product generated by aromatase, were graphed
as a function of inhibitor concentration and then fit to a 3-param-
eter logistic function. Inhibitory potencies were expressed in terms
of the IC50 value, the inhibitor concentration necessary to reduce
the enzyme activity by half. Each experiment was performed at
least in triplicate.
5.1.1.11. 6-methoxy-3-(pyridin-3-yl)chroman-4-one (3c).
Synthesized from 2-hydroxy-5-methoxybenzaldehyde (1 mmol,
1 equiv, 136.04 lL), and 3-ethynylpyridine (3 mmol, 3 equiv,
309.3 mg) according to the general procedure for the synthesis of
isoflavanone derivatives described above. Light yellow solid. Yield:
26.9%. Purity: 98.2%. Rf = 0.28 (40% EtOAc/Hex). 1H NMR (CDCl3,
500 MHz, ppm): d 8.55 (d, J = 1.9 Hz, 2H), 7.57–7.59 (m, 1H), 7.34
(d, J = 3.2 Hz, 1H), 7.25–7.29 (m, 1H), 7.13 (dd, J = 8.7, 3.2 Hz, 1H),
6.95 (d, J = 9.2 Hz, 1H), 4.59–4.64(m, 2H), 3.98 (dd, J = 9.2, 5 Hz,
1H), 3.79 (s, 3H). 13C NMR (CDCl3, 125 MHz, ppm): d 191.2,
156.3, 154.5, 149.9, 149.2, 136.1, 131.1, 125.8, 123.7, 120.6,
119.3, 107.9, 71.3, 55.9, 49.9. HRMS Calculated for C15H13O3NNa
[M+Na] 256.0974, found 256.0967.
5.3. Molecular modeling
The coordinates of the X-ray crystal structure of the aromatase/
androstenedione complex (PDB code 3EQM) were downloaded from
the Protein Databank (http://www.rcsb.org) and imported into the
modeling program SYBYL (version 8.0; Tripos, St. Louis, MO). All
non-protein components were deleted and hydrogen atoms were
added to the proteinstructure. Partialcharges were assignedaccord-
ing to the Amber library and the positions of the added hydrogen
atoms were optimized by molecular mechanics minimization that
kept the positions of the heavy atoms static.28 The energy minimiza-
tion was performed with the Powell method in combination with
the Amber7 FF99 force field, a distance-dependent dielectric con-
stant of 4, and a convergence criterion of 0.05 kcal/(mol Å). The
molecular structures of inhibitors were also prepared in SYBYL and
the conformational energy of each structure was minimized by
molecular mechanics (MMFF94s force field, MMFF94 charges, and
distance-dependent dielectric constant of 4) using the conjugate
gradient method and a termination criterion of 0.01 kcal/(mol Å).
5.1.1.12.
8-Fluoro-3-(pyridin-3-yl)chroman-4-one
(3d).
Synthesized from 3-fluorosalicylaldehyde (1.0 mmol, 1 equiv,
140.1 mg) and 3-ethynylpyridine (3 mmol, 3 equiv, 309.4 mg)
according to the general procedure for the synthesis of isoflava-
none derivatives described above. Light yellow solid. Yield:
23.3%. Purity: 99.5%. Rf = 0.40 (60% EtOAc/Hex). 1H NMR (CDCl3,
500 MHz, ppm): d 8.55–8.53 (m, J = 2.3, 2H), d 7.70 (d, J = 6.8 Hz,
1H), 7.58 (d, J = 6.2, 1H), 7.33–7.26 (m, 2H), 6.99–6.95 (m, 1H),
4.76–4.70 (m, 2H), 4.08–4.06 (m, 1H). 13C NMR (CDCl3, 125 MHz,
ppm): d 190.2, 152.7, 150.6, 149.9, 149.4, 136.3, 130.3, 123.9,

134
E. Amato et al. / Bioorg. Med. Chem. 22 (2014) 126–134
2. Gray, J.; Evans, N.; Taylor, B.; Rizzo, J.; Walker, M. Int. J. Occ. Environ. Health
Inhibitor structures were computationally docked into the en-
2009, 15, 43.
zyme’s binding site using the program GOLD (version 5.0.1; CCDC,
Cambridge, UK). GOLD operates with a genetic search algorithm
and allows for complete ligand and partial binding site flexibility.29
The scoring function GoldScore was used and the size and center of
the docking sphere were as reported before.9 The settings for the
genetic algorithm runs were kept at their default values (population
size: 100, selection pressure: 1.1; number of operations: 100,000,
the number of islands: 5, niche size: 2, probability for migration,
mutation, and crossover: 10%, 95%, and 95%, respectively). For each
ligand, 30 runs were performed under identical conditions.
3. (a) Muftuoglu, Y.; Mustata, G. Bioorg. Med. Chem. Lett. 2010, 20, 3050; (b)
Recanatini, M.; Cavalli, A.; Valenti, P. Med. Res. Rev. 2002, 22, 282.
4. Brueggemeier, R. W.; Hackett, J. C.; Diaz-Cruz, E. S. Endocr. Rev. 2005, 26, 331.
5. Furr, B. J. A. Milestones in Drug Therapy. Aromatase Inhibitors, 2nd ed.;
Birkhauser, 2006. p 182.
6. Christodoulou, M. S.; Fokialakis, N.; Passarella, D.; Garcia-Argaez, A. N.; Gia, O.
M.; Pongratz, I.; Dalla Via, L.; Haroutounian, S. A. Bioorg. Med. Chem. 2013, 21,
4120.
7. Ibrahim, A. R.; Abul-Hajj, Y. J. J. Steroid Biochem. Mol. Biol. 1990, 37, 257.
8. Brueggemeier, R. W.; Richards, J. A.; Joomprabutra, S.; Bhat, A. S.; Whetstone, J.
L. J. Steroid Biochem. Mol. Biol. 2001, 79, 75.
9. Bonfield, K.; Amato, E.; Bankemper, T.; Agard, H.; Steller, J.; Keeler, J. M.; Roy,
D.; McCallum, A.; Paula, S.; Ma, L. Bioorg. Med. Chem. 2012, 20, 2603.
10. Purser, S.; Moore, P. R.; Swallow, S.; Gouverneur, V. Chem. Soc. Rev. 2008, 37,
320.
5.4. Cell assay
11. Zhou, P.; Zou, J.; Tian, F.; Shang, Z. J. Chem. Inf. Model. 2009, 49, 2344.
12. Meanwell, N. A. J. Med. Chem. 2011, 54, 2529.
MCF-7 cells were obtained from the NCI-Federick national lab.
The cells were cultured at 37 °C in a 5% CO2 humidified atmosphere
in RPMI-1640 medium containing 10% FBS, 0.01 mg/ml insulin and
10 nM b-estradiol (Sigma). Assays were accomplished by seeding
cells at a density of 15,000 cells/well in a 24 well plate and incu-
bated at 37 °C overnight. Then cells were treated for 72 h with indi-
cated concentrations of freshly dissolved compounds. The medium
containing compounds was discarded; fresh medium containing
13. (a) Smart, B. E. J. Fluorine Chem. 2001, 109, 3; (b) Banks, Ronald Eric; Smart, B.
E.; Tatlow, J. C. Organofluorine Chemistry: Principles and Commercial Applications,
1st ed.; Springer, 1994. p 669.
14. Ahuja, R.; Samuelson, A. G. Cryst. Eng. Commun. 2003, 5, 395.
15. Hagmann, W. K. J. Med. Chem. 2008, 51, 4359.
16. (a) Maiti, A.; Reddy, P. V. N.; Sturdy, M.; Marler, L.; Pegan, S. D.; Mesecar, A. D.;
Pezzuto, J. M.; Cushman, M. J. Med. Chem. 2009, 52, 1873; (b) Stresser, D. M.;
Turner, S. D.; McNamara, J.; Stocker, P.; Miller, V. P.; Crespi, C. L.; Patten, C. J.
Anal. Biochem. 2000, 284, 427.
17. (a)
Molinspiration,
http://www.molinspiration.com/cgi-bin/properties
(accessed 6-6-2013).; (b) OSIRIS Property Explorer, http://www.organic-
chemistry.org/prog/peo/ (accessed 6-6-2013).
20
for an additional 4 h. The medium was removed after adding
500 L of DMSO to each well the optical densities at 570 nm were
determined. The IC50 values were calculated using Kaleida-
Graph(Synergy Software, USA) software.
lL of MTT (5 mg/ml) was added to each well and incubated
18. (a) Bell-Horwath, T. R.; Vadukoot, A. K.; Thowfeik, F. S.; Li, G.; Wunderlich, M.;
Mulloy, J. C.; Merino, E. J. Bioorg. Med. Chem. Lett. 2013, 23, 2951; (b) Jones, A.
R.; Bell-Horwath, T. R.; Li, G.; Rollmann, S. M.; Merino, E. J. Chem. Res. Toxicol.
2012, 25, 2542; (c) Li, G.; Bell, T.; Merino, E. J. ChemMedChem 2011, 6, 869.
19. (a) Welshons, W. V.; Craig Jordan, V. Eur. J Cancer Clin. Oncol. 1987, 23, 1935; (b)
Le Bail, J. C.; Varnat, F.; Nicolas, J. C.; Habrioux, G. Cancer Lett. 1998, 130, 209.
20. Kijima, I.; Itoh, T.; Chen, S. J. Steroid Biochem. Mol. Biol. 2005, 97, 360.
21. Mitropoulou, T. N.; Tzanakakis, G. N.; Kletsas, D.; Kalofonos, H. P.; Karamanos,
N. K. Int. J. Cancer 2003, 104, 155.
l
Acknowledgments
This project was supported by an Institutional Development
Award (IDeA) from the National Institute of General Medical Sci-
ences of the National Institutes of Health under grant number
P20GM103436. The content is solely the responsibility of the
authors and does not necessarily represent the official views of
the National Institutes of Health. This study was also supported
by the Center for Integrative Science and Mathematics (CINSAM)
and Northern Kentucky University Office of Research, Grants and
Contracts Seed Grant. We thank Dr. Grant Edwards for his help
with the HPLC and Dr. Isabelle Lagadic for the assistance with
the microwave reactor.
22. Ghosh, D.; Griswold, J.; Erman, M.; Pangborn, W. Nature 2009, 457, 219.
23. Kerns, E. H.; Di, L. Drug-like Properties: Concepts, Structure Design and Methods.
from ADME to Toxicity Optimization, 2nd ed.; Elsevier Inc., 2008. p 552.
24. Namazian, M.; Coote, M. L. J. Fluorine Chem. 2009, 130, 621.
25. (a) Rowland, P.; Blaney, F. E.; Smyth, M. G.; Jones, J. J.; Leydon, V. R.; Oxbrow, A.
K.; Lewis, C. J.; Tennant, M. G.; Modi, S.; Eggleston, D. S.; Chenery, R. J.; Bridges,
A. M. J. Biol. Chem. 2006, 281, 7614; (b) Williams, P. A.; Cosme, J.; Vinkovic´, D.
M.; Ward, A.; Angove, H. C.; Day, P. J.; Vonrhein, C.; Tickle, I. J.; Jhoti, H. Science
2004, 305, 683.
26. Imhof, M.; Molzer, S. Toxicol. In Vitro 2008, 22, 1452.
27. (a) Bellina, F.; Masini, T.; Rossi, R. Eur. J. Org. Chem. 2010, 2010, 1339; (b) Lessi,
M.; Masini, T.; Nucara, L.; Bellina, F.; Rossi, R. Adv. Synth. Catal. 2011, 353, 501.
28. Paula, S.; Ball, W. J., Jr. Proteins 2004, 56, 595.
29. (a) Jones, G.; Willett, P.; Glen, R. C. J. Mol. Biol. 1995, 245, 43; (b) Jones, G.;
Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R. J. Mol. Biol. 1997, 267, 727.
References and notes
1. American Cancer Society Cancer Facts & Figures 2013; American Cancer Society:
Atlanta, 2013.