Development of a new class of aromatase inhibitors: Design, synthesis and inhibitory activity of 3-phenylchroman-4-one (isoflavanone) derivatives

Article abstract of DOI:10.1016/j.bmc.2012.02.042

Aromatase (CYP19) catalyzes the aromatization reaction of androgen substrates to estrogens, the last and rate-limiting step in estrogen biosynthesis. Inhibition of aromatase is a new and promising approach to treat hormone-dependent breast cancer. We present here the design and development of isoflavanone derivatives as potential aromatase inhibitors. Structural modifications were performed on the A and B rings of isoflavanones via microwave-assisted, gold-catalyzed annulation reactions of hydroxyaldehydes and alkynes. The in vitro aromatase inhibition of these compounds was determined by fluorescence-based assays utilizing recombinant human aromatase (baculovirus/insect cell-expressed). The compounds 3-(4-phenoxyphenyl)chroman-4- one (1h), 6-methoxy-3-phenylchroman-4-one (2a) and 3-(pyridin-3-yl)chroman-4-one (3b) exhibited potent inhibitory effects against aromatase with IC₅₀ values of 2.4 μM, 0.26 μM and 5.8 μM, respectively. Docking simulations were employed to investigate crucial enzyme/inhibitor interactions such as hydrophobic interactions, hydrogen bonding and heme iron coordination. This report provides useful information on aromatase inhibition and serves as a starting point for the development of new flavonoid aromatase inhibitors.

Full text of DOI:10.1016/j.bmc.2012.02.042

Bioorganic & Medicinal Chemistry 20 (2012) 2603–2613
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of a new class of aromatase inhibitors: Design, synthesis
and inhibitory activity of 3-phenylchroman-4-one (isoflavanone) derivatives
Kevin Bonfield, Erica Amato, Tony Bankemper, Hannah Agard, Jeffrey Steller, James M. Keeler, David Roy,
⇑
Adam McCallum, Stefan Paula, Lili Ma
Department of Chemistry, Northern Kentucky University, Nunn Drive, Highland Heights, KY 41099, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Aromatase (CYP19) catalyzes the aromatization reaction of androgen substrates to estrogens, the last and
rate-limiting step in estrogen biosynthesis. Inhibition of aromatase is a new and promising approach to
treat hormone-dependent breast cancer. We present here the design and development of isoflavanone
derivatives as potential aromatase inhibitors. Structural modifications were performed on the A and B
rings of isoflavanones via microwave-assisted, gold-catalyzed annulation reactions of hydroxyaldehydes
and alkynes. The in vitro aromatase inhibition of these compounds was determined by fluorescence-based
assays utilizing recombinant human aromatase (baculovirus/insect cell-expressed). The compounds 3-(4-
phenoxyphenyl)chroman-4-one (1h), 6-methoxy-3-phenylchroman-4-one (2a) and 3-(pyridin-3-yl)chro-
Received 13 November 2011
Revised 11 February 2012
Accepted 17 February 2012
Available online 27 February 2012
Keywords:
Breast cancer
Aromatase inhibitors
Isoflavanones
man-4-one (3b) exhibited potent inhibitory effects against aromatase with IC50 values of 2.4
lM, 0.26 lM
and 5.8 M, respectively. Docking simulations were employed to investigate crucial enzyme/inhibitor
l
interactions such as hydrophobic interactions, hydrogen bonding and heme iron coordination. This report
provides useful information on aromatase inhibition and serves as a starting point for the development of
new flavonoid aromatase inhibitors.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
therapy is largely dependent on effective medicines that are po-
tent, selective, and have favorable pharmacologic and pharmacoki-
Breast cancer is the leading cancer for women and is the second
leading cause of the deaths after lung cancer. Women in the US
have the highest incidence rate of developing breast cancer. Cur-
rently there are approximately 2.5 million women living with
breast cancer in the US. The estimated number of new breast can-
cer cases for the year 2010 was over 200,000.1 One out of eight wo-
men will develop breast cancer over the course of a lifetime, a
significant increase compared to 1 in 35 in the 1930s.2 The risk fac-
tors of breast cancer include age, personal health history, family
health history, diet, exercise and obesity.3 Various forms of treat-
ment are available for breast cancer, including surgery, radiation,
chemotherapy and hormonal therapy.4 Hormonal therapy, unlike
surgery, radiation and chemotherapy, is relatively facile and con-
trollable, and is therefore embraced by a majority of patients with
estrogen receptor positive breast cancer. The success of hormonal
netic profiles such as suitable absorption, distribution, metabolism
and excretion (ADME) properties.
Breast cancer tumor cell proliferation is stimulated by the signal
released when estrogen binds to estrogen receptors.5 In biosyn-
thetic pathways, estrogens (estrone, estradiol and estriol) are syn-
thesized from cholesterol through progestagens and androgens
(e.g., androstenedione Fig. 1a). Aromatase (CYP19) catalyzes the
aromatization reaction, the last and rate-limiting step in estrogen
biosynthesis. Therefore, aromatase is a new and promising phar-
maceutical target for hormonal-dependent breast cancer.6 Aroma-
tase inhibitors (AIs) have many advantages over the traditional
hormonal breast cancer therapy using selective estrogen receptor
modulators (SERMs). These advantages include reversibility, total
blockage and possibly reduced side effects.7 AI medicines are often
categorized as steroidal or non-steroidal aromatase inhibitors
(NSAIs) based on their structural similarity with steroids, or as
1st, 2nd and 3rd generations based on their evolution time.8 Inten-
sive ongoing studies are focused on the third generation non-ste-
roidal aromatase inhibitors (NSAIs), which can be further divided
into the following classes based on their scaffold. The first class
of NSAIs consists of analogs of third generation triazole AI drug
molecules such as letrozole,9 anastrozole10 (Fig. 1b), which were
approved by FDA and are used as first-line therapy in the treatment
Abbreviations: Phe 134, phenylalanine 134; Arg 192, arginine; Trp, tryptophan
224 phenylalanine 224; Asp 309, aspartic acid 309; Met 374, methionine 374; Ser
478, serine 478; CYP19, cytochrome P450 aromatase; AIs, aromatase inhibitors;
SERMs, selective estrogen receptor modulators; NSAIs, non-steroidal aromatase
inhibitors; NMR, nuclear magnetic resonance; MS, mass spectrometry; HPLC, high
performance liquid chromatography.
⇑
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 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.02.042

2604
K. Bonfield et al. / Bioorg. Med. Chem. 20 (2012) 2603–2613
Figure 1. Molecular structures of the aromatase natural substrate and selected inhibitors. (a) Androstenedione, the natural substrate; (b) anastrozole (ArimidexÒ),10
a
representative example of third-generation aromatase inhibitors in clinical use; (c), chrysin (from blue passion flower),16 a representative example of a naturally occurring
flavone aromatase inhibitor; (d), biochanin A (from red clover),17 the most potent naturally occurring isoflavone aromatase inhibitor.
of breast cancer in postmenopausal women. These AIs are charac-
terized by the presence of diarylmethyl and imidazole or triazole
functionalities. Representative examples include arylimidazole
and azolylbenzyl compounds.11 Since these aromatase inhibitors
are structural modifications of synthetic NSAI drug molecules, they
might have side effects similar to these of NSAI drugs when given
on a long-term basis. The second class of NSAIs consists of natural
products and their derivatives such as flavonoid, coumarin, resve-
ratrol and lignan.12 Despite the scarcity of knowledge as to their
mechanism of action, natural product drugs are believed to exhibit
reduced side effects, which might be the results of the combined
action of a mixture of constituents, or the multi-target function
of one constituent.13 As the people are becoming aware of side ef-
fects of synthetic AI drugs, natural product NSAIs are attracting
more and more attention recently for their future clinical uses.14
Some representative examples of naturally occurring flavonoid
aromatase inhibitors are shown in Figure 1c (5,7-dihydroxyflav-
one) and 1d (5,7-dihydroxy -40-methoxyisoflavone). In addition
to the NSAIs mentioned above, new NSAI entities such as morpho-
lino ethanone have recently been discovered using computational
high-throughput docking studies.15
lavanones revealed similar orientations in the enzyme pocket.
However, due to the non-planarity of its scaffold, isoflavanone
had an additional enzyme–ligand interaction with the highly
hydrophobic enzyme pocket through its C2 methylene group. This
CH2/
p
interaction26 might be responsible for the observed differ-
ence in the inhibitory activity between isoflavone and isoflava-
none. We envisioned that with proper functional groups (see
below) on this non-planar scaffold, isoflavanone could result
in enhanced anti-aromatase activities. To the best of our knowl-
edge, there is only one report that investigated aromatase inhibi-
tion by isoflavanone compounds such as 2-hydroxyisoflavanone
(IC50 = 170
l
M), 40-hydroxyisoflavanone (IC50 = 160
l
M) and
30,40-dihydroxyisoflavanone (IC50 ꢀ 200
l
M).20b Here we report
the design and synthesis of a series of isoflavanone derivatives
as aromatase inhibitors. A novel and convenient microwave-as-
sisted gold-catalyzed annulation reaction was utilized to synthe-
size isoflavanone derivatives. Various substituents such as
methyl, phenyl, methoxy and pyridyl were attached to the isoflav-
anone scaffold to explore hydrophobic interactions, hydrogen
bonding and heme iron coordination. Computational docking of
active isoflavanones into the aromatase active site was employed
to detect crucial enzyme/inhibitor interactions. The calculated
physicochemical properties of isoflavanone inhibitors are also dis-
cussed in order to obtain some preliminary insights on their long-
term pharmacokinetic effects.
Flavonoid compounds, which include flavones, isoflavones, flav-
anones, and isoflavanones, display diverse biological activities such
as antioxidant, anticancer, antiviral and anti-inflammatory activi-
ties.18 Natural flavones or their synthetic analogs have been re-
ported to show inhibitory effects against aromatase (Fig. 1c). A
common strategy to increase the potency of flavones is to intro-
duce appropriate functional groups. Flavones with functionalities
such as halides, nitro and amino have been synthesized and tested
for their aromatase inhibitory effects by different groups working
in this field.19 Specifically, methylated/hydroxylated flavanones,20
benzoflavanones,21 and imidazolyl flavones22 have been reported
to be potent aromatase inhibitors. On the other hand, isoflavones,
which differ from flavones in the phenyl substitution position on
ring C, are not well studied probably due to their significantly
poorer inhibitory effects against aromatase than flavones.23 Natu-
rally occurring isoflavones such as daidzein, glycitein and genistein
are either inactive or weak inhibitors against aromatase. Even
though conflicting results exist, the most potent known natural
isoflavone inhibitor is biochanin A (Fig. 1d) with reported IC50 val-
2. Results
2.1. Inhibitor design and synthesis
To explore the feasibility of isoflavanone compounds as aroma-
tase inhibitors, a design strategy was adopted in which the isoflav-
anone core could be modified to yield diverse compounds with
enhanced inhibitory potency. As revealed by the crystal structure
of aromatase, there are three unique structural features of the aro-
matase active site.6b First, the enzyme binding pocket is highly
hydrophobic. Therefore hydrophobic groups such as alkyl groups
and aromatic rings may be favorable for enhanced binding. Second,
hydrogen bonding plays an important role in enzyme–substrate
interaction, and the 17-keto oxygen in the androstenedione sub-
strate (Fig. 1a) forms a hydrogen bond with the Met 374 backbone
amide. The hydroxyl of Ser 478 was also reported to participate in
hydrogen bonding with carbonyl or cyano groups of the aromatase
inhibitors.27 Third, the Fe2+ in the heme group of aromatase is
capable of chelating to hetero atoms in inhibitors, especially N
atoms. Many recent studies on aromatase inhibitors utilized pyri-
dine,24,28 triazole29 or imidazole29,30 N-heterocyclic groups to en-
hance the coordination between the heme iron atom of the
enzyme and the heterocyclic nitrogen lone pair. Based on these
structural features, the design of isoflavanone aromatase inhibitors
ues around 113 l
M.17 Significant information on isoflavone AIs
was provided by Brueggemeier, including first examples of isoflav-
one-based pyridyl-modified aromatase inhibitors24 in 2004 and
subsequent improvements on isoflavone aromatase inhibitors with
azoles, alkoxys, etc.25
Isoflavanones (Scheme 1) are a rare class of flavonoids that dif-
fer from isoflavones by having the C2–C3 bond saturated. We no-
ticed that the isoflavone scaffold is inactive against aromatase,
whereas the isoflavanone scaffold showed weak inhibition.20b
A
comparison of docking-predicted poses of isoflavones with isof-

K. Bonfield et al. / Bioorg. Med. Chem. 20 (2012) 2603–2613
2605
Scheme 1. Structure and design strategy of isoflavanone aromatase inhibitors.
was centered on enhancement of hydrophobic interactions, hydro-
gen bonding and heme iron coordination (Scheme 1).
(Fig. 2). Since racemic compounds were synthesized, the activities
reported in this paper represent the lower limits of inhibitory po-
tency. Interestingly, the parent molecule 1a showed moderate
Isoflavanone compounds are normally synthesized in three
steps from commercially available materials. A typical procedure
involves a Lewis acid catalyzed Friedel–Crafts acylation of phenols,
followed by an annulation of 2-hydroxyphenylketone and aldehyde
to form isoflavone (3-phenylbenzopyrone), and finally hydrogena-
tion of C2–C3 double bond to produce isoflavanone compounds.31
However, these synthetic routes are laborious, and harmful or sen-
sitive reagents are utilized. Considering the commercial availability
of various hydroxyaldehydes and phenylacetylenes, we employed a
AuCN/nBu3P(1:25)-catalyzed annulation reaction of salicylalde-
hyde and phenylacetylene in toluene at 150 °C for 36 h to assemble
the isoflavanone scaffold in one step.32 However, the long reaction
time and the large amount of nBu3P ligand required motivated us to
search for new reaction conditions. Therefore we investigated this
reaction under microwave conditions and found that the reaction
time was greatly reduced from 36 h to only 10 min when subjected
to microwave irradiation at high temperatures. The reaction sol-
vent, toluene, poorly absorbs microwave irradiation, hence the
amount of AuCN catalyst was increased from 1% to 5% to facilitate
temperature increase. The final optimized reaction conditions to
synthesize isoflavanone derivatives under microwave irradiation
were 5% AuCN, 25% nBu3P, 200 °C for 10 min (Scheme 2). Utilizing
these new reaction conditions, twenty three isoflavanone deriva-
tives were synthesized and evaluated for their inhibitory effects
against aromatase (Table 1). These compounds were fully charac-
terized by 1H NMR, 13C NMR and mass spectroscopy. Three proton
signals in the range of 3.90–4.70 ppm were observed in the 1H NMR
spectra, indicative of the aliphatic protons attached to C2 and C3 in
the product isoflavanone scaffold.
inhibition against aromatase with an IC50 value of 29 lM (Table 1).
Under the same conditions, no significant inhibition was found
with compounds 1i and 1j, in which the B ring was attached to a
large aromatic group. All other compounds showed moderate to
good inhibitory effects, with 2a being the most potent compound
in this investigation (Table 1). Both enantiomers of synthesized
isoflavanones were docked into the aromatase active site, and
the enantiomer with the higher docking score was used to identify
crucial enzyme/inhibitor interactions (Fig. 3).
3. Discussion
3.1. Hydrophobic interactions
The aromatase (CYP19) active site is highly hydrophobic and is
dominated by aliphatic amino acid residues such as Met 374, Val
373, Val 370, Ile 305, Ala 306, Ile 133, Trp 224, Leu 372, Leu 477,
Phe 134 and Thr 310. Hence inhibitors with alkyl or aromatic
groups are expected to bind with high affinity.6b A methyl group
was introduced to the A or B rings of isoflavanones with 1c and
1e being the most potent with IC50 values of 18 lM and 20 lM.
There was no significant difference in activities between the meth-
ylated analogs and the model compound 1a. Overall, the presence
and the position of the methyl substituent on the isoflavanone
scaffold did not influence the inhibitory activity significantly. In
addition to the methyl group, benzo and phenoxy groups were also
installed for possible
p–p stacking interactions. The extension of
the isoflavanone A ring by a benzo group at positions 5 and 6 (com-
pound 1g) increased the inhibitory potency slightly in comparison
to 1a (IC50 = 20 lM). The docking pose of 5,6-benzoisoflavanone 1g
2.2. Aromatase inhibition
in the aromatase active site (Fig. 3a) indicated that the primary
interactions between 1g and the enzyme active site were the coor-
dination between the carbonyl O atom in 1g and the iron in the
heme group (distance = 2.5 Å). The benzo group attached to the
The evaluation of the synthesized isoflavanones for aromatase
inhibitory activity was performed using a fluorometric substrate
(7-methoxy-trifluoromethylcoumarin) and human CYP19 aroma-
tase with ketoconazole as a positive control.33 All experiments
were performed at least in triplicate to determine the IC50 value
isoflavanone A ring was predicted to have
tions with aromatase residue Trp 224.
p–p stacking interac-
Scheme 2. Microwave-assisted annulation reactions of hydroxyaldehydes and alkynes.

2606
K. Bonfield et al. / Bioorg. Med. Chem. 20 (2012) 2603–2613
Table 1
Chemical structures and aromatase inhibition of isoflavanone derivatives 1a–4d
R5'
O
O
R6'
O
O
R4'
R3'
S
O
O
R5
A
O
B
O
O
N
R6
R7
C
O
R2'
R8
1a-1f, 1h, 1i,
2a-2g, 4a-4d
1g
1j
3a
3b
a
0
0
0
0
0
Compd
R5
R6
R7
R8
R2
R3
R4
R5
R6
IC50
29
(lM)
1a
1b
1c
1d
1e
1f
1g
1h
1i
H
H
H
H
H
H
Benzo
H
H
H
CH3
H
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
4
44 11
18
N.A.b
7
CH3
H
20
41
20
7
7
9
H
CH3
H
PhO
Ph
H
H
H
H
H
H
2.4 2.2
Inactive
Inactive
1j
H
H
H
90-Phenanthryl
2a
2b
2c
2d
2e
2f
2g
3a
3b
4a
4b
4c
4d
H
H
H
H
H
H
H
H
H
H
H
H
CH3O
H
H
H
H
H
H
H
H
H
CH3O
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3O
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3O
H
H
H
H
CH3O
H
CH3O
H
H
H
H
H
CH3O
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0.26 0.06
41 16
N.A.b
97 70
99 27
24 14
H
H
H
CH3O
11
30
7
9
30-Thiophene
30-Pyridyl
5.8 1.8
32
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
7
Cl
Br
tBu
5.1 3.4
29 10
61 15
H
a
IC50 values are average of at least three runs and standard deviations.
N.A. = not available. Compounds exhibit fluorescence which interferes with the bioassay.
b
compared to the model compound 1a with an IC50 of 2.4 lM, while
1i was inactive. The differences in their activities may be attributed
to the flexibilities of these two molecules. The presence of the oxy-
gen bridge in compound 1h gives the molecule more flexibility so
that it can bend and undergo
p–p stacking interactions. On the
other hand, compound 1i with a biphenyl group is rigid and cannot
fit into the pocket. The same reason might also apply for compound
3-(phenanthren-9-yl)chroman-4-one 1j. Therefore, there were no
anti-aromatase activities observed for 1h and 1j.
As mentioned above, the key interactions identified from com-
puter modeling of 1h into the enzyme active site were
interactions (Fig. 3b). The distance between the PhO benzene ring of
1h and aromatase residue Phe 221 was 2.7 Å, indicating strong p–p
p–p stacking
stacking interactions. The Asp 309 side chain was in close proximity
with the oxygen atom of the 40-phenoxide group. However, no H-
bonds between 1h and Asp 309 were predicted by the docking re-
sults. The p–p stacking interactions between the A ring of inhibitor
1h and the heme group were also predicted (distance = 3.0 Å), as
can be seen from the docking pose. Additionally, the carbonyl O
atom in 1h was positioned to coordinate to the heme iron, similar
to the heme coordination predicted for compound 1g (Fig. 3a).
Figure 2. Aromatase inhibition by selected isoflavanone compounds and ketocon-
azole. Relative fluorescence is proportional to enzyme activity. Logistic fit reveals
the IC50, a measure of potency (inhibitor concentration required to inhibit half of
the enzyme’s activity).
Compounds 40-phenoxyisoflavanone 1h and 40-phenylisoflava-
none 1i have some structural similarity by bearing an additional
aromatic group on ring B, while their inhibitory potencies are sur-
prisingly different. The inhibition potency of 1h increased 12-fold
3.2. Hydrogen bonds
In order to increase hydrogen bonding between inhibitor and
enzyme, isoflavanone derivatives with methoxy groups capable

K. Bonfield et al. / Bioorg. Med. Chem. 20 (2012) 2603–2613
2607
Figure 3. Representations of molecular docking performed in this study. The inhibitor is depicted as green 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) 5,6-Benzoisoflavanone 1g; (b) 40-phenoxyisoflavanone 1h; (c) 6-methoxyisoflavanone 2a; (d) 30,50-dimethoxyisoflavanone 2g; and (e) inhibitor 3-(pyridin-
3-yl)chroman-4-one 3b.
of acting as H-bond acceptors at various positions were synthe-
sized (compounds 2a–2f). The most potent inhibitor was 6-meth-
oxyisoflavanone 2a which showed an about 100-fold increase in
inhibition potency (IC50 = 0.26 lM). The Met 374 backbone NH
and the 6-methoxy group in 2a is in close proximity (Fig. 3c),
tive site for both compounds, there was no benefit from increasing
the electron density on ring A.
The B ring methoxylated isoflavanone did not show much prom-
ise at first (compounds 2d–2f). When a dimethoxylated compound
30,50-dimethoxyisoflavanone 2g was synthesized, however, the cor-
which might have favorable hydrogen bonding interactions. In
responding IC50 value was 11 lM, about a threefold increase com-
addition, the
p
–
p
stacking interactions (distance = 3.2–4.1 Å) be-
pared to the parent molecule 1a. Docking indicated that the
aromatase heme iron might coordinate to the carbonyl oxygen atom
in 2g (Fig. 3d). A careful examination of the enzyme pocket around
the heme group revealed that this was mostly a hydrophobic area
which was composed of non-polar amino acids such as Ile 133, Leu
301, Ala 306, Ile 305, Phe 221, Trp 224 and Val 370. Therefore, the
tween the B ring of 2a and the heme porphyrin may also contribute
to the observed high affinity. One should point out that both com-
pounds 2a and 2b have increased electron density on ring A due to
the presence of the electron-donating methoxy group. Since ring A
was not involved in p–p stacking interactions with the enzyme ac-

2608
K. Bonfield et al. / Bioorg. Med. Chem. 20 (2012) 2603–2613
hydrophobic interactions between 30,50-dimethoxyisoflavanone 2g
and the enzyme active site also contributed to the enhanced inhibi-
tory potency. It should be pointed out that hydrogen bonding inter-
actions were predicted between the other enantiomer of 2g and
aromatase. Specifically, the NH in aromatase residue Arg 192 formed
a hydrogen bond (2.0 Å) with the oxygen in 30-methoxy group, and
the -OH of the Ser 478 side chain formed a second hydrogen bond
(2.1 Å) with the 50-methoxy group. However, this enantiomer did
not fit into the enzyme active site very well due to reduced hydro-
phobic interactions and received a lower docking score. Even though
enhanced inhibitory potency was observed for the B-ring methoxy-
lated compound 2g, one should keep in mind that the introductionof
polar groups (e.g., –OH, –OCH3) on ring B of isoflavanone might also
decrease activity, as it seems to be the case for 20-methoxyisoflava-
Table 2
Calculated physicochemical properties of isoflavanone derivatives and anastrozoleÒ
Compd Id.
cLogPa
MWb
Hac
Hdd
TPSAe (Å2)
IC50 (lM)
1g
1h
2a
2g
4.55
5.15
3.43
3.44
1.69
2.85
274.3
316.4
254.3
284.3
225.3
293.4
2
3
3
4
3
5
0
0
0
0
0
0
26.3
35.5
35.5
44.8
39.2
78.3
20
2.4
0.26
11
3b
5.8
AnastrozoleÒ
0.01010
a
b
c
LogP, calculated logarithm of partition coefficient between n-octanol and water.
MW, molecular weight.
Ha, hydrogen bond acceptor.
Hd, hydrogen bond donor.
TPSA, topological polar surface area.
d
e
none 2d (IC50 = 97
40-hydroxyisoflavanone (IC50 = 160
lavanone (IC50 = 200
M).20b
l
M), 30-methoxyisoflavanone 2e (IC50 = 99
M)20b and 30,40-dihydroxyisof-
lM),
spacious and polar due to the presence of Ser 478 and Arg 115 lin-
ing the wall of the pocket. A polar functional group with reasonable
size might fit into this region and interact favorably by steric and
electronic effects. Therefore, the modulation at C6 of isoflavanone
with chlorine improved the binding affinity while that with the
l
l
3.3. Heme iron coordination
t
non-polar butyl decreased the binding interactions.
The coordination of the inhibitor with the iron atom of the
heme moiety is an important feature of potent and selective inhib-
itors of aromatase. Functional groups such as imidazole, triazole
and pyridine are able to form nitrogen heme iron coordination,
and others such as phenol or thiophene may coordinate to the
heme iron.34 3-(thiophen-3-yl)chroman-4-one 3a and 3-(pyridin-
3-yl)chroman-4-one 3b were synthesized in order to probe the
heme iron coordination. Compound 3a with a thiophene group
which might coordinate to heme iron did not exhibit a significant
increase in potency in the inhibition assay (Table 1). Compound 3b
with a pyridine group showed fivefold increase in aromatase
3.5. Calculated physicochemical properties
In order to explore the bioavailability of isoflavanone deriva-
tives, theoretical calculations were carried out to predict some
physicochemical properties of synthesized compounds. Lipinski’s
rule of five36 and the later addition of other parameters such as po-
lar surface area (PSA),37 well known in pharmaceutical industry,
outline the basic principles for orally active small molecules:
molecular weight (MW) should be under 500, lipophilicity which
is expressed as logarithm of partition coefficient between n-octa-
nol and water (cLogP) should be less than 5, the number of hydro-
gen bond donors (Hd) should be no more than 5, the number of
hydrogen bond acceptors (Ha) should be no more than 10, and
the topological polar surface area (TPSA) should be smaller than
90 Å.2 Among these parameters, LogP, the measure of the com-
pound’s solubility and permeability, is believed to be very impor-
tant. Very high lipophilicity and the resulted large LogP values
cause poor absorption or permeation and should be avoided.
The calculated physicochemical properties38 of some of the
most active isoflavanone inhibitors together with those for anas-
trozole are listed in Table 2. Based on these calculations, com-
pounds 5,6-benzoisoflavanone 1g and 40-phenoxyisoflavanone 1h
had very large cLogP values, 4.55 and 5.15, respectively, which
might be disadvantageous with regard to the pharmacokinetic
properties of these molecules in biological systems. Compounds
2a, 2g and 3b, exhibited favorable cLogP values. The polar surface
areas of these molecules were relatively small ranging from 26.3 to
44.8 Å2 in comparison with the average value (90 Å2) for approved
drug molecules. This provides an excellent opportunity to modify
these isoflavanone derivatives with additional functional groups.
Figure 4 summarizes our current understanding as to how isof-
lavanones bind to the active site of aromatase. These compounds
bind to the active site in an orientation such that their A, C and B
rings mimic D, C and A rings of the steroids, respectively. The ami-
no acids responsible for important interactions with isoflavanone
derivatives are Phe 221, Met 374, Arg 192, Ser 478 and Asp 309.
The modification on the A and B rings of isoflavanone compounds
has resulted in aromatase inhibitors exhibiting stronger binding
affinity and enhanced activities.
inhibitory potency (IC50 = 5.8 lM), likely due to the coordination
to the heme iron. This observation was supported by our docking
study, which suggested that the pyridine ring was about 2.4 Å
away from heme group, and the pyridyl nitrogen coordinated to
the iron in the heme group (Fig. 3e). Our attempts to synthesize
3-(pyridin-2-yl)chroman-4-one and 3-(1-methyl-1H-imidazol-5-
yl)chroman-4-one were not successful, probably due to the re-
duced the activities of gold(I) catalysts upon their coordination
to the N-heteroaromatic alkynes.35 Information on heme iron
coordination with other N-heterocyclic rings such as pyridine
and imidazole could not be provided for the current study. Since
the enzyme pocket surrounding the heterocyclic ring of chro-
man-4-one inhibitors are hydrophobic and sterically restricted,
further optimization on this series of molecules has to take these
factors into consideration by avoiding large substituents (e.g., –
OPh, biphenyl) or polar substituents (e.g., –OH, –OCH3).
3.4. Further exploration of 6-substituted isoflavanones
Prompted by the inhibitory effect of 6-methoxyisoflavanone, we
examined the effects of different substituents at the C6 position of
the parent molecule. Comparison of compounds 1b, 1g and 2a re-
vealed that a relatively big polar group at this position might en-
hance the interactions between isoflavanone and the aromatase
pocket. Specifically, introduction of an electron-donating substitu-
ent such as a methoxy group or electron-withdrawing substituent
such as a benzo at the C5–C6 position on the A ring increased the
aromatase inhibitory effect. Following these results, isoflavanones
with F, Cl, Br and tbutyl at C6 position were prepared and tested for
their aromatase inhibition (Table 1 4a–4d). Enhanced inhibitory ef-
fects were observed for compounds 4b which had a chlorine group.
t
4. Conclusion
The presence of a butyl group at C6 (compound 4d) reduced the
binding affinity. These observations could be explained from the
electronic and steric properties of the amino acid residues around
C6 position of isoflavanone. As mentioned previously, this region is
In conclusion, we have demonstrated in this work that isoflav-
anones can be used as aromatase inhibitors through appropriate

K. Bonfield et al. / Bioorg. Med. Chem. 20 (2012) 2603–2613
2609
Figure 4. (a) Structural features of isoflavanones required for effective inhibition of aromatase; (b) structural similarity between isoflavanone scaffold and aromatase natural
substrate—androstenedione.
structural modulation. For the first time, a small set of isoflavanon-
es with various functionalities was synthesized via a one-step
annulation reaction and tested for their aromatase inhibitory ef-
fects. Compared to other flavanoid aromatase inhibitors, our syn-
thesis of isoflavanone derivatives was straightforward utilizing
microwave irradiation and readily available hydroxyaldehydes
and aromatic alkynes as starting materials. In addition, the most
active compounds identified from this study showed favorable
physicochemical properties which are promising for further devel-
opment of this series of compounds.
In comparison to isoflavones, isoflavanone derivatives with
functional groups such as methoxy, phenoxy, chloride, pyridyl,
etc. demonstrated good inhibitory potencies against aromatase,
revealing that the non-planarity configuration of the isoflavanone
scaffold might play an important role in enzyme–ligand binding.
Specifically, compound 2a bearing a methoxy group at C6 position
stood out as the most potent inhibitor with a 100-fold enhance-
ment in potency compared to model compound 1a. Our data also
showed that the key structural features that enable isoflavanones
bind to the aromatase active site with high affinity are as follows:
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).
(4
A Nova-Pak C18 column
m, 3.9 ꢁ 150 mm), also from Waters, was used with a mobile
l
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-
tion and 10 lL solution was injected for HPLC test. The purity of all
the compounds was assessed by HPLC under 254 nm. All final com-
pounds were confirmed to be ꢀ95% purity by analysis of their peak
area. Mass spectra were obtained on a Water TQD Tandem Quadra-
pole Mass Spectrometer, and data was collected in electrospray po-
sitive mode (ESI+). High resolution mass spectra were recorded on
a Micromass Q-TOF 2 or a Thermo Scientific LTQ-FT™ mass spec-
trometer operating in electrospray (ES) mode. Calculation 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.
(1) Isoflavanone rings A or B may participate in p–p stacking inter-
actions with aromatase’s porphyrin ring or amino acid residues,
such as Phe 221, Trp224 and Phe 134. (2) Methoxy groups could in-
crease binding affinity by forming hydrogen bonds with aromatase
residues, such as Met 374, Arg 192 and Ser 478. (3) Heterocyclic
nitrogen or carbonyl oxygen atom of the inhibitors can coordinate
to the heme iron atom and thereby enhance the binding interac-
tions. The calculated physicochemical properties of these com-
pounds indicated that it is possible to obtain more potent and
selective aromatase inhibitors by appropriate modification on the
isoflavanone scaffold. Compounds 2a, 2g and 3b showed not only
high potency against aromatase but also favorable physicochemi-
cal properties, demonstrating that the isoflavanone scaffold can
be further optimized for the development of new therapeutic
agents for hormone-dependent breast cancer.
5.1.1. General procedure for isoflavanone formation
5.1.1.1. Method A (Thermal Process). To a 5-mL micro reaction
vial equipped with a stirring bar was added AuCN (0.03 mmol,
0.03 equiv, 6.6 mg), Bu3P(0.75 mmol, 0.75 equiv, 185.1 lL), alde-
hyde (1 mmol, 1 equiv), alkyne (3 mmol, 3 equiv) and 4 mL of
freshly distilled toluene. The reaction mixture was stirred at
150 °C for 36 h. After cooling down to room temperature, the crude
mixture was loaded directly on silica gel and was purified by Med-
ium Performance Liquid Chromatography, eluding with an ethyl
acetate/hexanes gradient to afford the desired products.
5. Experimental
5.1. Chemistry
5.1.1.2. Method B (Microwave Irradiation Process). The micro-
wave-assisted reactions were conducted on a single-mode Discover
System from CEM Corporation. To an oven-dried standard micro-
wave 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,
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
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.
0.25 equiv, 61.7 lL), aldehyde (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 mixture was loaded directly on silica gel and was purified by
Medium Performance Liquid Chromatography eluding with an ethyl
acetate/hexanes gradient to afford the desired products.

2610
K. Bonfield et al. / Bioorg. Med. Chem. 20 (2012) 2603–2613
5.1.1.3. 6-Methyl-3-phenylchroman-4-one (1b). Synthesized
from 2-hydroxy-5-methyl-benzaldehyde (1 mmol, 1 equiv,
5.1.1.8.
(1g)39
2-Phenyl-2,3-dihydro-1H-benzo[f]chromen-1-one
Synthesized from 2-hydroxy-1-naphthaldehyde (1 mmol,
.
136.15 mg) and phenylacetylene (3 mmol, 3 equiv, 330
l
L) accord-
1 equiv, 175.4 mg) and phenylacetylene (3 mmol, 3 equiv,
330 L) according to the general procedure for the synthesis of
ing to the general procedure for the synthesis of isoflavanone
derivatives Method A (Thermal Process) described above. Light yel-
low solid. Rf = 0.39 (10% EtOAc/Hex). Yield, 26.4%. Purity, 96.5%. 1H
NMR (CDCl3, 500 MHz, ppm): d 7.76 (s, 1H, 5-H), 7.28 (m, 6H, Ar-
H), 6.91 (d, J = 4.4 Hz, 1H, 8-H), 4.64 (m, 2H, 2-H), 3.96 (t,
J = 7.1 Hz, 1H, 3-H), 2.32 (s, 3H, CH3). 13C NMR (CDCl3, 125 MHz,
ppm): d 192.5, 159.8, 137.3, 135.3, 131.2, 129.0, 128.71, 127.8,
127.4, 120.7, 117.8, 71.6, 52.5, 20.5. HRMS Calcd for C16H14O2Na
[M+Na] 261.0891. Found: 261.0890.
l
isoflavanone derivatives Method B (Microwave Irradiation Process)
described above. Light yellow solid. Rf = 0.42 (10% EtOAc/Hex).
Yield, 17.1%. Purity, 96.1%. 1H NMR (CDCl3, 500 MHz, ppm): d
9.48 (d, J = 8.7 Hz, 1H), 7.95 (d, J = 9.2, 1H), 7.76 (d, J = 7.8, 1H),
7.62 (t, J = 7.8, 1H), 7.43 (t, J = 7.6 Hz, 1H), 7.37–7.25 (m, 5H, Ar-
H), 7.13 (d, J = 9.2 Hz, 1H), 4.80 (d, J = 7.4 Hz, 2H, 2-H), 4.07 (t,
J = 6.9 Hz, 1H, 3-H). 13C NMR (CDCl3, 125 MHz, ppm): d 193.3,
163.6, 137.7, 135.7, 131.9, 129.8, 129.4, 128.9, 128.6, 128.5,
127.8, 126.1, 125.0, 118.7, 112.6, 71.3, 52.9. HRMS Calcd for
5.1.1.4. 7-Methyl-3-phenylchroman-4-one (1c). Synthesized
C
19H14O2Na+ [M+Na] 297.0891. Found: 297.0888.
from
2-hydroxy-4-methylbenzaldehyde
(1 mmol,
1 equiv,
138.9 mg) and phenylacetylene (3 mmol, 3 equiv, 330
l
L) accord-
5.1.1.9. 3-(4-Phenoxyphenyl)chroman-4-one (1h). Synthesized
from salicylaldehyde (0.5 mmol, 1 equiv, 52.4 L) and 1-ethynyl-
4-phenoxybenzene (1.5 mmol, 3 equiv, 271 L) according to the
general procedure for the synthesis of isoflavanone derivatives
Method (Thermal Process) described above. Yellow solid.
ing to the general procedure for the synthesis of isoflavanone
derivatives Method B (Microwave Irradiation Process) described
above. Light yellow solid. Rf = 0.41 (10% EtOAc/Hex). Yield, 31.6%.
l
l
Purity, 98.3%. 1H NMR (CDCl3, 500 MHz, ppm):
d
7.85 (d,
A
J = 8.3 Hz, 1H, 5-H), 7.25–7.31 (m, 5H, Ar-H), 6.86 (d, J = 8.3 Hz,
1H, 6-H), 6.82 (s, 1H, 8-H), 4.64 (d, J = 9.2, 2H, 2-H), 3.96 (t,
J = 7.8, 1H, 3-H), 2.37 (s, 3H, CH3). 13C NMR (CDCl3, 125 MHz,
ppm): d 191.9, 161.7, 147.7, 135.4, 128.9, 128.7, 127.8, 127.7,
123.1, 118.9, 117.9, 71.6, 52.3, 22.0. HRMS Calcd for C16H14O2Na+
[M+Na] 261.0891. Found: 261.0892.
Rf = 0.38 (10% EtOAc/Hex). Yield, 22.0%. Purity, 98.2%. 1H NMR
(CDCl3, 500 MHz, ppm): d 7.97 (d, J = 6.8 Hz, 1H, 5-H), 7.51 (t,
J = 7.8 Hz, 1H, 7-H), 7.34 (t, J = 8.0, 2H, Ar-H), 7.24 (d, J = 10.5, 2H,
Ar-H), 6.98–7.06 (m, 7H, Ar-H), 4.65 (m, 2H, 2-H), 3.99 (dd,
J = 8.7, 5.5 Hz, 1H, 3-H). 13C NMR (CDCl3, 125 MHz, ppm): d
192.3, 161.7, 157.1, 157.0, 136.2, 130.0, 129.9, 129.7, 127.9,
123.6, 121.7, 121.1, 119.3, 119.1, 118.0, 71.6, 51.7. HRMS Calcd
for C21H16O3Na [M+Na] 339.0997, found 339.1002.
5.1.1.5. 8-Methyl-3-phenylchroman-4-one (1d). Synthesized
from 2-hydroxy-3-methyl-benzaldehyde (1 mmol, 1 equiv,
120.0
lL) and phenylacetylene (3 mmol, 3 equiv, 330
lL) according
5.1.1.10. 3-(Biphenyl-4-yl)chroman-4-one (1i)39
from salicylaldehyde (1 mmol, 0.5 equiv, 52.4 L) and 4-ethynylbi-
. Synthesized
to the general procedure for the synthesis of isoflavanone deriva-
tives Method A (Thermal Process) described above. Yellow solid.
Rf = 0.51 (10% EtOAc/Hex). Yield, 31.4%. Purity 98.8%. 1H NMR
(CDCl3, 500 MHz, ppm): d 7.84 (d, J = 7.3 Hz, 1H, 5-H), 7.28–7.40
(m, 6H, Ar-H), 6.96, (t, J = 7.5, 1H, 6-H), 4.68 (m, 2H, 2-H), 4.00
(dd, J = 9.1, 5.5 Hz, 1H, 3-H), 2.29 (s, 3H, CH3). 13C NMR (CDCl3,
125 MHz, ppm): d 192.9, 159.8, 137.0, 135.3, 129.0, 128.8, 127.8,
127.2, 125.4, 121.1, 71.5, 52.3, 15.8. HRMS Calcd for C16H14O2Na
[M+Na] 261.0891. Found: 261.0882.
l
phenyl (3 mmol, 1.5 equiv, 267.3 mg) according to the general pro-
cedure for the synthesis of isoflavanone derivatives Method A
(Thermal Process) described above. Light yellow solid. Rf = 0.40
(10% EtOAc/Hex). Yield, 26.2%. Purity 97.0%. 1H NMR (CDCl3,
500 MHz, ppm): d 8.00 (d, J = 7.8 Hz, 1H, 5-H), 7.25–7.60 (m, 10H,
Ar-H), 7.03–7.09 (m, 2H, Ar-H), 4.71 (d, J = 6.4, 2H, 2-H), 4.06 (t,
J = 7.3 Hz, 1H, 3-H). 13C NMR (CDCl3, 125 MHz, ppm): d 192.4,
180.0, 161.7, 140.9, 140.8, 136.2, 134.1, 129.1, 128.9, 127.9,
127.7, 127.5, 127.2, 121.8, 118.0, 71.6, 52.1. HRMS Calcd for
5.1.1.6. 3-m-Tolylchroman-4-one (1e). Synthesized from salicyl-
C21H16O2Na [M+Na] 323.1048. Found: 323.1038.
aldehyde (1 mmol, 1 equiv, 105.0
lL) and 1-ethylyn-3-methylben-
zene (3 mmol, 3 equiv, 387 L) according to the general procedure
l
5.1.1.11. 3-(Phenanthren-9-yl)chroman-4-one (1j). Synthe-
sized from salicylaldehyde (0.5 mmol, 1 equiv, 52.4 L) and 9-eth-
for the synthesis of isoflavanone derivatives Method A (Thermal
Process) described above. Light yellow solid. Rf = 0.42 (10% EtOAc/
Hex). Yield, 38.0%. Purity, 99.2%. 1H NMR (CDCl3, 500 MHz, ppm):
d 7.97 (d, J = 9.6 Hz, 1H, 5-H), 7.50 (t, J = 7.8 Hz, 1H, 7-H), 7.24 (t,
J = 7.8 Hz, 1H, 6-H), 7.01–7.13 (m, 5H, Ar-H), 4.66 (d, J = 6.8 Hz,
2H, 2-H), 3.97 (t, J = 7.3 Hz, 1H, 3-H), 2.34 (s, 3H, CH3). 13C NMR
(CDCl3, 125 MHz, ppm): d 192.7, 161.7, 138.6, 136.1, 135.0, 129.5,
128.9, 128.7, 127.9, 125.7, 121.7, 121.2, 117.7, 71.6, 52.4, 21.5.
HRMS Calcd for C16H14O2Na [M+Na] 261.0891. Found: 261.0879.
l
ynylphenanthrene (1.5 mmol, 3 equiv, 303.4 mg) according to the
general procedure for the synthesis of isoflavanone derivatives
Method B (Microwave Irradiation Process) described above. Light
yellow solid. Rf = 0.36 (10% EtOAc/Hex). Yield 16.2%. Purity,
98.2%. 1H NMR (CDCl3, 500 MHz, ppm): d 8.77 (d, J = 8.3 Hz, 1H,
Ar-H), 8.67 (d, J = 8.3 Hz, 1H, Ar-H), 8.05–8.10 (m, 2H, Ar-H), 7.80
(d, J = 8.3, 1H, Ar-H), 7.63–7.71 (m, 4H, Ar-H), 7.55–7.59 (m, 2H,
Ar-H), 7.08–7.13 (m, 2H, Ar-H), 4.82–4.93 (m, 3H, 2-H and 3-H).
13C NMR (CDCl3, 125 MHz, ppm): d 192.3, 161.7, 136.2, 131.3,
131.1, 130.6, 130.3, 129.9, 128.7, 128.0, 127.6, 127.1, 127.0,
126.9, 127.7, 124.1, 126.6, 122.6, 121.9, 124.5, 118.1, 71.5, 49.0.
HRMS Calcd for C23H16O2Na [M+Na] 347.1048, found 347.1047.
5.1.1.7. 3-p-Tolylchroman-4-one (1f)39
cylaldehyde (1 mmol, 1 equiv, 104.7 L), and 4-ethynyltoluene
(3 mmol, 3 equiv, 380.5 L) according to the general procedure
. Synthesized from sali-
l
l
for the synthesis of isoflavanone derivatives Method A (Thermal
Process) described above. Light yellow. Light yellow solid.
Rf = 0.26 (10% EtOAc/Hex). Yield, 28.7%. Purity, 99.4%. 1H NMR
(CDCl3, 500 MHz, ppm): d 7.98 (d, J = 7.8 Hz, 1H, 5-H), 7.51 (t,
J = 7.8 Hz, 1H, 7-H), 7.18 (m, 4H, Ar-H), 7.02–7.08 (m, 2H, 6-H, 8-
H), 4.65 (d, J = 8.5 Hz, 2H, 2-H), 3.97 (t, J = 6.0 Hz, 1H, 3-H), 2.35
(s, 3H, CH3). 13C NMR (CDCl3, 125 MHz, ppm): d 192.5, 179.7,
161.7, 137.6, 136.1, 132.0, 129.7, 128.5, 127.8, 121.6, 117.9, 71.6,
52.0, 21.2. HRMS Calcd for C16H14O2Na [M+Na] 261.0891. Found:
261.0889.
5.1.1.12. 6-Methoxy-3-phenylchroman-4-one (2a). Synthesized
from 2-hydroxy-5-methoxy-benzaldehyde (1 mmol, 1 equiv,
123.7 lL) and phenylacetylene (3 mmol, 3 equiv, 330 lL) accord-
ing to the general procedure for the synthesis of isoflavanone
derivatives Method A (Thermal Process) described above. Light yel-
low solid. Rf = 0.28 (10% EtOAc/Hex). Yield, 30.4%. Purity, 98.2%. 1H
NMR (CDCl3, 500 MHz, ppm): d 7.26–7.38 (m, 6H, Ar-H), 7.11 (d,
J = 4.6 Hz, 1H, 7-H), 6.95 (d, J = 4.6 Hz, 1H, 8-H), 4.64 (m, 2H, 2-
H), 3.97 (d, J = 4.1 Hz, 1H, 3-H), 3.81 (s, 3H, OCH3). 13C NMR (CDCl3,

K. Bonfield et al. / Bioorg. Med. Chem. 20 (2012) 2603–2613
2611
125 MHz, ppm): d 192.2, 156.4, 154.3, 135.4, 129.0, 128.7, 127.9,
125.4, 120.9, 119.3, 108.1, 71.8, 55.9, 52.4. HRMS Calcd for
NMR (CDCl3, 125 MHz, ppm): d 192.3, 161.6, 159.2, 136.0, 129.7,
127.8, 127.0, 121.6, 121.1, 117.9, 114.4, 71.6, 55.3, 51.6. HRMS
Calcd for C16H14O3Na [M+Na] 277.0841. Found: 277.0826.
C16H14O3Na [M+Na] 277.0841. Found: 277.0828.
5.1.1.13. 7-Methoxy-3-phenylchroman-4-one (2b)39
.
Synthe-
5.1.1.18. 3-(3,5-Dimethoxyphenyl)chroman-4-one (2g). Syn-
sized from 2-hydroxy-4-methoxybenzaldehyde (1 mmol, 1 equiv,
159.2 mg) and phenylacetylene (3 mmol, 3 equiv, 330 L) accord-
thesized from salicylaldehyde (0.5 mmol, 1 equiv, 52.4 lL), and
l
1-ethynyl-3,5-dimethoxybenzene (1.5 mmol, 3 equiv, 243.5 mg)
according to the general procedure for the synthesis of isoflava-
none derivatives Method A (Thermal Process) described above. Yel-
low solid. Rf = 0.21 (10% EtOAc/Hex). Yield 24.6%. Purity, 99.3%. 1H
NMR (CDCl3, 500 MHz, ppm): d 7.95 (d, J = 7.8 Hz, 1H, 5-H), 7.49 (t,
J = 7.8 Hz, 1H, 7-H), 6.99–7.05 (m, 2H, 6-H and 8-H), 6.44 (d,
J = 1.85 Hz, 2H, Ar-H), 6.40 (s, 1H, Ar-H), 4.66 (d, J = 7.3 Hz, 2H, 2-
H), 3.91 (t, J = 7.1 Hz, 1H, 3-H), 3.76 (s, 6H, OCH3). 13C NMR (CDCl3,
125 MHz, ppm): d 197.2, 161.7, 161.1, 137.3, 136.0, 127.8, 121.6,
121.0, 117.9, 106.8, 99.76, 71.3, 55.3, 52.5. HRMS Calcd for
ing to the general procedure for the synthesis of isoflavanone
derivatives Method A (Thermal Process) described above. Light yel-
low solid. Rf = 0.22 (10% EtOAc/Hex). Yield, 37.1%. Purity, 97.7%. 1H
NMR (CDCl3, 500 MHz, ppm): d 7.90 (d, J = 8.8 Hz, 1H, 5-H), 7.25–
7.34 (m, 5H, Ar-H), 6.61 (d, J = 8.7 Hz, 1H, 6-H), 6.45 (s, 1H, 8-H),
4.65 (m, 2H, 2-H), 3.92 (dd, J = 7.8, 5.5 Hz, 1H, 3-H), 3.85 (s, 3H,
OCH3). 13C NMR (CDCl3, 125 MHz, ppm): d 190.9, 166.2, 163.6,
135.5, 129.6, 128.9, 128.7, 127.8, 115.0, 110.3, 100.8, 72.0, 55.7,
52.0. HRMS Calcd for
277.0830.
C16H14O3Na [M+Na] 277.0841. Found:
C17H16O4Na [M+Na] 307.0946. Found: 307.0939.
5.1.1.14. 8-Methoxy-3-phenylchroman-4-one (2c). Synthesized
from o-vanillin (1 mmol, 1 equiv, 166 mg), and phenylacetylene
5.1.1.19. 3-(Thiophen-3-yl)chroman-4-one (3a). Synthesized
from salicylaldehyde (1 mmol, 1 equiv, 104.7 L) and 3-ethynylthi-
ophene (3 mmol, 3 equiv, 295.5 L) according to the general proce-
l
(3 mmol, 3 equiv, 330
l
L) according to the general procedure for
l
the synthesis of isoflavanone derivatives Method A (Thermal Pro-
cess) described above. Yellow solid. Rf = 0.51 (10% EtOAc/Hex).
Yield, 43.7%. Purity, 95.5%. 1H NMR (CDCl3, 500 MHz, ppm): d
7.82 (d, J = 2.3 Hz, 1H, 5-H), 7.55 (d, J = 4.7 Hz, 1H, 7-H), 7.23–
7.36 (m, 6H, Ar-H), 4.74–4.79 (m, 2H, 2-H), 4.00–4.03 (m, 1H, 3-
H), 3.92 (s, 3H, OCH3). 13C NMR (CDCl3, 125 MHz, ppm): d 190.3,
155.8, 135.6, 133.9, 129.1, 128.6, 128.2, 126.9, 125.8, 12.0, 60.5,
51.7, 14.3. MS Calcd for C16H15O3 [M+H] 255.29. Found: 255.04.
dure for the synthesis of isoflavanone derivatives Method A
(Thermal Process) described above. Light yellow oil. Rf = 0.26
(10% EtOAc/Hex). Yield, 28.7%. Purity, 99.8%. 1H NMR (CDCl3,
500 MHz, ppm): d 7.98 (d, J = 7.8 Hz, 1H, 5-H), 7.50 (t, J = 6.1 Hz,
1H, 7-H), 7.01–7.18 (m, 5H, Ar-H), 4.65 (d, J = 6.9 Hz, 2H, 2-H),
3.96 (t, J = 7.3 Hz, 1H, 3-H). 13C NMR (CDCl3, 125 MHz, ppm): d
192.5, 161.7, 137.5, 136.1, 132.0, 129.7, 128.5, 127.8, 121.6,
121.1, 117.9, 71.6, 60.5, 52.0, 21.2. HRMS Calcd for C13H10O2SNa
[M+Na] 253.0299. Found: 253.0291.
5.1.1.15. 3-(2-Methoxyphenyl)chroman-4-one (2d)40
sized from salicylaldehyde (1.5 mmol, 3 equiv, 59.3 L) and 2-eth-
ynylanisole (0.5 mmol, 1 equiv, 64.66 L) according to the general
. Synthe-
l
5.1.1.20. 3-(Pyridin-3-yl)chroman-4-one (3b). Synthesized from
salicylaldehyde (0.5 mmol, 1 equiv, 52.4 lL) and 3-ethylnylpyri-
l
procedure for the synthesis of isoflavanone derivatives Method B
(Microwave Irradiation Process) described above. Yellow solid.
Yield, 35.5%. Purity, 95.5%. Rf = 0.38 (10% EtOAc/Hex). 1H NMR
(CDCl3, 500 MHz, ppm): d 7.80 (d, J = 7.8 Hz, 1H, 5-H), 7.48–7.54
(m, 1H, 7-H), 7.29 (t, J = 7.9 Hz, 1H, 6-H), 6.92–7.12 (m, 5H, Ar-
H), 4.66 (t, J = 11.5 1H, 2-H), 4.53 (dd, J = 11.0, 5.5 Hz, 1H, 3-H),
4.35–4.40 (m, 1H, 3-H), 3.78 (s, 3H, OCH3). 13C NMR (CDCl3,
125 MHz, ppm): d 192.6, 161.9, 157.5, 135.7, 130.4, 129.2, 127.8,
123.6, 121.7, 121.5, 121.0, 117.9, 111.3, 70.8, 55.6, 48.6. HRMS
Calcd for C16H14O3Na [M+Na] 277.0835. Found: 277.0835.
dine (3 mmol, 1.5 equiv, 267.3 mg) according to the general proce-
dure for the synthesis of isoflavanone derivatives Method A
(Thermal Process) described above. Light yellow solid. Rf = 0.22
(40% EtOAc/Hex). Yield, 28.0%. Purity 99.7%. 1H NMR (CDCl3,
500 MHz, ppm): d 8.55 (s, 2H, Pyr-H), 7.93 (d, J = 2.4, 1H, 5-H),
7.58 (d, J = 7.8, 1H, Pyr-H), 7.51 (t, J = 7.3, 1H, 7-H), 7.25–7.28 (m,
1H, 6-H), 6.99–7.06 (m, 2H, Ar-H and Pyr-H), 4.62–4.68 (m, 2H,
2-H), 4.02 (dd, J = 9.1, 5.0 Hz, 1H, 3-H). 13C NMR (CDCl3,
125 MHz, ppm): d 191.1, 161.6, 150.0, 149.2, 136.5, 136.2, 130.9,
127.8, 123.8, 122.0, 120.8, 118.0, 71.0, 50.0. HRMS Calcd for
C14H11NO2Na [M+Na] 248.0687. Found: 248.0682.
5.1.1.16. 3-(3-Methoxyphenyl)chroman-4-one (2e). Synthe-
sized from salicylaldehyde (1 mmol, 1 equiv, 104.7
lL) and 3-eth-
5.1.1.21. 6-Fluoro-3-phenylchroman-4-one (4a). Synthesized
ynylanisole (3 mmol, 3 equiv, 387.95 L) according to the general
l
from 5-fluorosalicylaldehyde (0.5 mmol, 1 equiv, 70.1 mg) and
procedure for the synthesis of isoflavanone derivatives Method B
(Microwave Irradiation Process) described above. Yellow solid.
Yield, 51.4%. Purity, 97.7%. Rf = 0.34 (10% EtOAc/Hex). 1H NMR
(CDCl3, 500 MHz, ppm): d 7.96 (d, J = 8.3 Hz, 1H, 5-H), 7.49 (t,
J = 7.8 Hz, 1H, 7-H), 7.27 (t, J = 7.3 Hz, 1H, 6-H), 7.00–7.06 (m, 2H,
Ar-H), 6.85 (t, J = 11.7 Hz, 3H, Ar-H), 4.67 (d, J = 6.87 Hz, 2H, 2-H),
3.96 (t, J = 7.1 Hz, 1H, 3-H), 3.78 (s, 3H, OCH3). 13C NMR (CDCl3,
125 MHz, ppm): d 192.0, 161.7, 160.0, 136.5, 136.1, 130.0, 127.9,
121.7, 120.9, 118.0, 114.6, 113.2, 71.5, 55.3, 52.4. HRMS Calcd for
phenylacetylene (1.5 mmol, 3 equiv, 164.7 lL) according to the
general procedure for the synthesis of isoflavanone derivatives
Method B described above. Light yellow solid. Rf = 0.44 (10%
EtOAc/Hex). Yield, 21.7%. Purity, 99.5%. 1H NMR (CDCl3, 500 MHz,
ppm): d 7.60 (d, J = 8.3 Hz, 1H, 5-H), 7.38–7.20 (m, 6H, Ar-H),
7.00 (dd, J = 9.2, 4.1 Hz, 1H, 8-H), 4.65 (d, J = 6.4 Hz, 2H, 2-H),
3.99 (t, J = 7.3 Hz, 1H, 3-H). 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 Calcd for
265.0641. Found: 265.0631.
C15H11FO2 [M+Na]
C16H14O3Na [M+Na] 277.0835. Found: 277.0835.
5.1.1.17. 3-(4-Methoxyphenyl)chroman-4-one (2f)39
.
Synthe-
5.1.1.22. 6-Chloro-3-phenylchroman-4-one (4b)39
from 5-chlorosalicylaldehyde (1 mmol, 1 equiv, 156.6 mg and
phenylacetylene (3 mmol, 3 equiv, 330 L) according to the gen-
. Synthesized
sized from salicylaldehyde (1 mmol, 1 equiv, 104.7 L), and 4-eth-
l
ynylanisole (3 mmol, 3 equiv, 389 L) according to the general
l
l
procedure described above. Yellow solid. Rf = 0.14 (10% EtOAc/
Hex). Yield, 36.5%. Purity, 99.5%. 1H NMR (CDCl3, 500 MHz, ppm):
d 7.95 (d, J = 7.8 Hz, 1H, 5-H), 7.49 (t, J = 7.8 Hz, 1H, 7-H), 7.20 (d,
J = 8.7 Hz, 2H, Ar-H), 6.87–7.04 (m, 4H, Ar-H), 4.62–4.65 (m, 2H,
2-H), 3.95 (dd, J = 8.7, 6.0 Hz, 1H, 3-H), 3.79 (s, 3H, OCH3). 13C
eral procedure for the synthesis of isoflavanone derivatives Method
A (Thermal Process) described above. Light yellow solid. Rf = 0.44
(10% EtOAc/Hex). Yield 32.8%. Purity, 98.8%. 1H NMR (CDCl3,
500 MHz, ppm): d 7.91 (d, J = 2.8 Hz, 1H, 5-H), d 7.43 (dd, J = 9.0,
6.0 Hz, 1H, 7-H), 7.30–7.38 (m, 3H, Ar-H), 7.26 (d, J = 6.0 Hz, 2H,

2612
K. Bonfield et al. / Bioorg. Med. Chem. 20 (2012) 2603–2613
Ar-H), 6.98 (d, J = 9.2 Hz, 1H, 8-H), 4.67 (d, J = 3.7 Hz, 2H, 2-H), 3.99
(t, J = 7.1 Hz, 1H, 3-H). 13C NMR (CDCl3, 125 MHz, ppm): d 190.9,
160.0, 135.9, 134.5, 129.0, 128.6, 128.0, 127.3, 127.1, 121.9,
substrate MFC was substituted by dibenzylfluorescein (DBF) which
was converted by aromatase to fluorescein (kex = 485 nm;
kem = 530 nm).41 The protocol was the same as the one above, ex-
cept for a longer incubation time (2 h) and the use of a different
stop reagent (2 M NaOH).
119.7, 71.6, 52.0. HRMS Calcd for
281.0345. Found: 281.0349.
C15H11O2ClNa [M+Na]
5.1.1.23. 6-Bromo-3-phenylchroman-4-one (4c). Synthesized
5.3. Molecular modeling
from 5-bromosalicylaldehyde (1 mmol, 1 equiv, 201 mg) and
phenylacetylene (3 mmol, 3 equiv, 330
l
L) according to the gen-
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 protein structure. Partial charges were as-
signed according 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.42
Energy minimization was performed with the Powell method in
combination with the Amber7 FF99 force field, a distance-depen-
dent dielectric constant 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).
Inhibitor structures were computationally docked into the en-
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.43
All four scoring functions (GoldScore, ChemScore, ASP, and Chem-
PLP) were tested and various sizes and centers of the docking
sphere were evaluated. 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 is-
lands: 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.
eral procedure for the synthesis of isoflavanone derivatives Method
B (Microwave Irradiation Process) described above. Light yellow
solid. Yield, 31.8%. Purity, 97.4%. Rf = 0.46 (10% EtOAc/Hex). 1H
NMR (CDCl3, 500 MHz, ppm): d 8.06 (d, J = 2.8 Hz, 1H, 5-H), 7.56
(dd, J = 8.7, 2.3 Hz, 1H, 7-H), 7.25–7.44 (m, 5H, Ar-H), 6.92 (d,
J = 8.7 Hz, 1H, 8-H), 4.66 (d, J = 7.8 Hz, 2H, 2-H), 3.99 (t, J = 3.9 Hz,
1H, 3-H). 13C NMR (CDCl3, 125 MHz, ppm): d 191.1, 160.6, 138.8,
134.5, 130.2, 129.1, 128.6, 128.1, 122.3, 120.1, 114.4, 71.6, 52.0.
HRMS Calcd for
324.9833.
C
15H11O2BrNa+ [M+Na] 324.9840. Found:
5.1.1.24. 6-tert-Butyl-3-phenylchroman-4-one (4d). Synthe-
sized from 5-tert-butyl-2-hydroxy-benzaldehyde (0.5 mmol,
1 equiv, 85.8
lL), and phenylacetylene (1.5 mmol, 3 equiv,
164.7 L) according to the general procedure for the synthesis of
l
isoflavanone derivatives Method B (Microwave Irradiation Process)
described above. A yellow oil product was formed. Light yellow so-
lid. Yield, 14.4%. Purity, 94.4%. Rf = 0.49 (10% EtOAc/Hex). 1H NMR
(CDCl3, 500 MHz, ppm): d 7.95 (t, J = 2.1 Hz, 1H, 5-H), 7.57 (d,
J = 8.7 Hz, 1H, 7-H), 7.35–7.28 (m, 5H, Ar-H), 6.96 (d, J = 8.7 Hz,
1H, 8-H), 4.65 (d, J = 5.95 Hz, 2H, 2-H), 3.97 (t, J = 6.9 Hz, 1H, 3-
H), 1.31 (s, 9H, C(CH3)3). 13C NMR (CDCl3, 125 MHz, ppm): d
192.1, 159.7, 144.6, 135.4, 133.9, 128.9, 128.7, 127.8, 123.7,
117.6, 71.6, 52.5, 34.5, 31.4, 22.7, 14.2. HRMS Calcd for
C
19H20O2Na+ [M+Na] 303.13555. Found: 303.13560.
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.33 This fluorescence-based assay mea-
sures the rate at which recombinant human aromatase
(baculovirus/insect cell-expressed) converts the substrate 7-meth-
oxy-trifluoromethylcoumarin (MFC) into a fluorescent product
(kex = 409 nm, kem = 530 nm) in a NADPH regenerating system.
Briefly, concentrated stock solutions of test compounds were pre-
Acknowledgments
This project was supported by Grants from the National Center
for Research Resources (5P20RR016481-12) and the National Insti-
tute of General Medical Sciences (8 P20 GM103436-12) from the
National Institutes of Health, the Center for Integrated Science
and Mathematics (CINSAM) and Northern Kentucky University Of-
fice of Research, Grants and Contracts Seed Grant. We thank Dr.
Stuart Oehrle for the assistance with LC/MS, Dr. Grant Edwards
and Dr. Heather Bullen for their help with HPLC and Dr. Isabelle
Lagadic for the assistance with Microwave Reactor. We also thank
Dr. Peter Crooks for his advice regarding the preparation of the
manuscript.
pared in acetonitrile. 100 lL samples containing serial dilutions
of test compounds (dilution factor of 3 between samples) and
cofactor mixture (0.4 U/mL glucose-6-phosphate dehydrogenase;
16.2
50
incubating the plate for 10 min at 37 °C, 100
P450 reductase/substrate solution (105 g protein/mL enzyme;
50 M MFC; 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 0.5 M Tris base were then added to stop the reaction
l
M NADP+; 825
l
M MgCl2; 825
M citrate buffer, pH 7.5) were prepared in a 96 well plate. After
L of an aromatase/
lM glucose-6-phosphate;
l
l
l
l
l
References and notes
and the fluorescence of the formed de-methylated MFC was mea-
sured 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.
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