Synthesis and biological evaluation of (6- and 7-Phenyl) coumarin derivatives as selective nonsteroidal inhibitors of 17β-hydroxysteroid dehydrogenase type 1

Article abstract of DOI:10.1021/jm101104z

17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) is an enzyme that catalyzes NADPH-dependent reduction of the weak estrogen, estrone, into the most potent estrogen, estradiol, which exerts proliferative effects via the estrogen receptors. Overexpression of 17β-HSD1 in estrogen-responsive tissues is related to the development of hormone-dependent diseases, such as breast cancer and endometriosis; thus, 17β-HSD1 represents an attractive target for the development of new therapies. We have discovered that simple coumarines 1 and 2 significantly inhibit 17β-HSD1 in a recombinant enzyme assay, with high selectivity against 17β-HSD2. We postulated that the introduction of various p-substituted phenyl moieties to position 6 or 7 of the coumarin core using the Suzuki-Miyaura cross-coupling reaction would provide mimetics of steroidal structures with improved inhibition of 17β-HSD1. The best inhibitor in the series proved to be 6a, with an IC₅₀ of 270 nM, and with exceptional selectivity for 17β-HSD1 over 17β-HSD2 and against the α and β estrogen receptors.

Full text of DOI:10.1021/jm101104z

248 J. Med. Chem. 2011, 54, 248–261
DOI: 10.1021/jm101104z
Synthesis and Biological Evaluation of (6- and 7-Phenyl) Coumarin Derivatives as Selective
Nonsteroidal Inhibitors of 17β-Hydroxysteroid Dehydrogenase Type 1
†
†
Stefan Starcevic, Petra Brozic, Samo Turk, Jozko Cesar, Tea Lanisnik Rizner, and Stanislav Gobec*
‡
,†
ꢁ †
ꢀ ꢀ ‡
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
†
‡
Faculty of Pharmacy, University of Ljubljana, Askerceva 7, 1000 Ljubljana, Slovenia, and Institute of Biochemistry, Faculty of Medicine,
ꢀ
ꢀ
University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia
Received August 25, 2010
17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) is an enzyme that catalyzes NADPH-depen-
dent reduction of the weak estrogen, estrone, into the most potent estrogen, estradiol, which exerts
proliferative effects via the estrogen receptors. Overexpression of 17β-HSD1 in estrogen-responsive
tissues is related to the development of hormone-dependent diseases, such as breast cancer and endo-
metriosis; thus, 17β-HSD1 represents an attractive target for the development of new therapies. We have
discovered that simple coumarines 1 and 2 significantly inhibit 17β-HSD1 in a recombinant enzyme
assay, with high selectivity against 17β-HSD2. Wepostulated that the introduction of various p-substituted
phenyl moieties to position 6 or 7 of the coumarin core using the Suzuki-Miyaura cross-coupling reaction
would provide mimetics of steroidal structures with improved inhibition of 17β-HSD1. The best inhibitor in
the series proved to be 6a, with an IC50 of 270 nM, and with exceptional selectivity for 17β-HSD1 over
17β-HSD2 and against the R and β estrogen receptors.
Introduction
Chart 1. Reactions Catalyzed by 17β-HSD1 and 17β-HSD2
Abnormal activities of enzymes that are involved in the
local biosynthesis of steroid hormones can significantly con-
tribute to the development of many hormone-related diseases.
Hormone-dependent breast cancer and prostate cancer are
the most common malignancies in western women and men,
respectively.1,2 The incidence of hormone-dependent cancers
is continuously rising, and they still represent one of the major
causes of death.
Among the enzymes thatact atthe final stages of biosynthe-
sis of the steroid sex hormones, the 17β-hydroxysteroid dehy-
drogenases (17β-HSDs) have important roles through their
catalysis of redox reactions at position 17 of the steroidal core,
thus regulating the biological potencies of these hormones.3,4
The 17β-hydroxy forms of androgens and estrogens have
higher affinities toward their corresponding receptors.3 At
present, 14mammalian17β-HSDshave beenidentified.4 With
the exception of 17β-HSD type 5 (17β-HSD51), which is a
member of the aldo-keto reductase superfamily, all of the 17β-
HSDs belong to the short-chain alcohol dehydrogenase re-
ductase superfamily, although their overall homologies are
low.5 The activities of the 17β-HSDs are related to the patho-
genesis of a number of diseases,4 and therefore, they represent
important targets for the design of new therapies. Their roles
in the control of cell proliferation are particularly important
for the development and progression of hormone-dependent
breast and prostate cancers.6-8 However, only 17β-HSD types
1, 2, and 3 appear to have roles solely in steroid metabolism,4,9
while the other 17β-HSDs share substrates with enzymes in-
volved in other metabolic pathways, e.g., lipid metabolism.10
The most extensively characterized of the 17β-HSDs is 17β-
HSD1,11-15 which catalyzes NADPH-dependent reduction of
the weakly active estrone (E1) into the potent estrogen estradiol
(E2) (Chart 1).9 In this manner, 17β-HSD1 can regulate the
occupancy of the estrogen receptors (ERs) and thus the pro-
liferative effects of the estrogens.16,17
17β-HSD1 is expressed mainly18 in ovaries,19 placenta,20
and breast tissue,21 and also in ectopic endometrium.22 Thus,
it is related to the development and progression of hormone-
dependent breast cancer,8 as well as endometriosis.22 E2 stim-
ulates the growth of breast cancers,23 and the intratumoral
E2/E1 ratio in ER-positive breast cancer tumors that depends
on 17β-HSD1 activity is significantly higher in postmenopau-
sal patients than in premenopausal patients.24 17β-HSD1
expression has also been reported for more aggressive hor-
mone-dependent breast cancers that show poor prognosis and
shorter remission times of the disease.8,25 The incidence of
breast cancer is higher in postmenopausal patients, where
the production of estrogens is limited to only peripheral
tissues. There, the estrogens are formed and act on the same
*To whom correspondence should be addressed. Mailing address:
ꢀ
ꢀ
Faculty of Pharmacy, University of Ljubljana, Askerceva 7, 1000 Ljubljana,
Slovenia. Tel: þ386-1-4769501. Fax: þ386-1-4258031. E-mail: stanislav.
gobec@ffa.uni-lj.si.
1 Abbreviations: 17β-HSD1, 17β-hydroxysteroid dehydrogenase Type 1;
17β-HSD2, 17β-hydroxysteroid dehydrogenase Type 2; 17β-HSD5, 17β-
hydroxysteroid dehydrogenase Type 5; E1, estrone; E2, estradiol; ER,
estrogen receptor; NAD(H), nicotinamide dinucleotide; NADP(H), nicotin-
amide dinucleotide phosphate; RBA, relative binding affinity; SM, Suzuki-
Miyaura.
r
pubs.acs.org/jmc
Published on Web 12/07/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 249
Chart 2. Reported Structures of the Most Potent Nonsteroidal Inhibitors of 17β-HSD1
(intracrine action) and neighboring (paracrine action) cells.26,27
The intracrine concept for lowering the concentrations of
active hormones in target cells has already been successfully
applied for the treatment of prostatic diseases bythe use of 5R-
reductase inhibitors. Steroidal 5R-reductase inhibitors, e.g.,
finasteride and dutasteride, have been drugs of choice for the
treatment of benign prostatic hyperplasia. A great effort was
also put in the development of nonsteroidal 5R-reductase
inhibitors, which would exert fewer side effects than steroidal
inhibitors.28-30
Inhibitors of 17β-HSD1 that are based on a benzothieno-
pyrimidinone and dihydrobenzothiophene core (Chart 2 A,B,
respectively) were discovered in lead-finding processes based
on the structures of known natural inhibitors, like coumestrol
and kaempferide. Compounds A40 and B41 of Chart 2 showed
remarkable inhibitory potencies for 17β-HSD1, at very low
nanomolar concentrations. They also lacked estrogenic effects,
and in addition, compound A reduced tumor growth in vivo.
However, their selectivities for 17β-HSD1 must be improved.40,41
Bis(hydroxyphenyl) substituted azoles and thiophenes showed
potent 17β-HSD1 inhibitory activities with 50 nM and 8 nM
IC50 values for C42 and D43 of Chart 2, respectively. These were
selective against 17β-HSD2 (selectivity factor of nearly 100)
and had negligible affinities for the ERs, with no stimulation
of cell proliferation in the T-47D cell line. Furthermore, they
reduced estrogen-responsive cell growth of T-47D cells44 and
showed excellent pharmacokinetic parameters.43 The princi-
ple feature of these compounds is the fixed distance of approx-
The selective inhibition of the local 17β-HSD1 activity
would thus provide specific therapies that would be beneficial
and supplemental to current treatments with selective ER
modulators and aromatase inhibitors.
Although clear structure-activity relationships have been
established for 17β-HSD1, no successful clinical drug candi-
dates have been designed yet.31 To be suitable as a potential
drug candidate, 17β-HSD1 inhibitors must meet two require-
ments:32 they should lack affinity toward ERs, to avoid estro-
genic effects; and they should show significant selectivity for
17β-HSD1 over 17β-HSD2, the latter of which has a protec-
tive role by catalyzing the opposite reaction in estrogen respon-
sive tissues (NADþ-dependent oxidation of E2 to E1).33-35
Many potent steroidal inhibitors of 17β-HSD1 that show
nanomolar IC50 values have been reported and extensively
reviewed.5,36-38 However, the majority of these steroidal
inhibitors are not selective enough for 17β-HSD1, and they
also have estrogenic properties.39 There is thusgreatinterestin
the identificationof new nonsteroidal and selective 17β-HSD1
inhibitors.
Recently, several groups have reported selective nonste-
roidal inhibitors of 17β-HSD1 with nanomolar or low micro-
molar IC50 values that have been determined in enzyme-based
or cell-based assays. Most of these are lipophilic, rigid, aro-
matic molecules that occupy the steroid substrate-binding
region in the active site of 17β-HSD1 (Chart 2). Of note here,
the inhibitory potencies of different inhibitors reported by
various research groups are difficult to compare, since differ-
ent assay conditions are often used, especially regarding the
various enzyme sources (purified enzymes, transfected or intact
cells containing the enzyme), the various substrate concentra-
tions, and the various cofactor species. Therefore, for more
comparable results, it is recommended to use an inhibitor
that has been previously characterized by at least one other
research group as a positive control in any inhibition assays.
˚
imately 12 A between the two hydroxyl groups on both of the
phenyl rings, which resembles the distance between the two
oxygens at positions 3 and 17 of E2.
(Hydroxyphenyl)naphthalenes (e.g., Chart 2 E)45 strongly
inhibited 17β-HSD1 with nanomolar IC50 values and good
selectivities against 17β-HSD2 and the ERs. These com-
pounds act as steroidal mimetics, with a flat lipophilic bicyclic
core that is substituted with two polar groups in the appro-
priate positions, to resemble the steroid O(3) to O(17) dis-
tance. These structures allow polar interactions with the
His221/Glu282 and Ser142/Tyr155 that are located at the
opposite ends of the mostly hydrophobic substrate-binding
cleft of 17β-HSD1.45,46 Recently, a combination of ligand-
and structure-based design resulted in heterocyclic substi-
tuted biphenylols and their aza-analogues as new 17β-HSD1
inhibitors.47
Here, we describe the design, chemical synthesis, and 17β-
HSD1 inhibitory activities of new coumarin derivatives. We
discuss their selectivities against 17β-HSD2, ERR, and ERβ,
and explain the biological data with reference to their inhibi-
tion of 17β-HSD1 through computer docking studies.
Results and Discussion
Design of new 17β-HSD1 Inhibitors with the Coumarin
Core. We postulated that a coumarin can be used as a planar
bicyclic core structure allowing for the introduction of

250 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Starcꢀevicꢁ et al.
different substituents to obtain steroidal mimetics with
potential for selective 17β-HSD1 inhibition. A lactone group
with two proton acceptor oxygens lead to additional favor-
able interactions in the active site. Although the coumarin
core is a part of many natural and synthethetic compounds
with wide pharmacological activity,48 coumarin structures
that inhibit 17β-HSD1 are rare. Cinnamic acids, which are
natural precursors of the coumarins, were recently reported
to be modest inhibitors of 17β-HSD1.49 Phytoestrogen cou-
mestrol contains the coumarin core, and along with some
structurally related flavanoids, these have also been reported
as inhibitors of 17β-HSD1.37,49 Therefore, we tested some
simple 3-acetylcoumarin derivatives from our in-house li-
brary of compounds for inhibition of 17β-HSD1. This pro-
vided us with two 3-acetylcoumarins that had promising
17β-HSD1 inhibitory activity (Chart 3).
as those seen for the C17 keto group of E1. To better mimic the
steroidal structure and to achieve the correct distance between
the steroidal O(3) to O(17) mimicking groups, we introduced
various para-substituted phenyl substituents to positions 6 and
7 of this 3-acetylcoumarin scaffold (Chart 4). The calculated
distances between the O(3) and O(17) mimicking groups
confirmed that these 6-phenyl and 7-phenyl coumarin deri-
vates are mimetics of E1, and they have the potential for
inhibition of 17β-HSD1. With this design approach, we
simultaneously wanted to avoid the affinity toward ERs, since
it is known that phytoestrogens substituted at positions 2 or
3 (e.g., coumestrol, Chart 4) possess estrogenic activity.50
Chemistry
The synthetic preparation of coumarin derivatives 1-38 is
presented in Schemes 1-4. The key reaction in all of these
cases was the Suzuki-Miyaura (SM)51,52 cross-coupling reac-
tion of various commercially available, p-substituted, phenyl-
boronic acids with either 7-triflylcoumarins 3, 10, and 17
(Schemes 1, 2, and 3, respectively) or 6-bromocoumarins 22,
28, and 34 (Scheme 4).
Compounds 1 and 2 showed modest inhibition of 17β-
HSD1 in the low micromolar range, and they did not act as
inhibitors of 17β-HSD2. We hypothesized that either the oxy-
gen of the keto lactone group or the oxygen of the 3-acetyl
substituent form similar binding interactions in the active site
The synthesis of the 3-acetylcoumarin derivatives 1-8
(Scheme 1) started by condensation53 of commercially avail-
able 2-hydroxy-4-methoxybenzaldehyde and ethylacetoace-
tate, which leads to 3-acetyl-7-methoxy coumarin 1, which
was further demethylated with AlCl354 to obtain the 3-acetyl-
7-hydroxy coumarin 2 in a satisfactory yield (68%). AlCl3
proved better than BBr3, since demethylation with BBr3 gave
only a 30% yield. The direct, one-step preparation of 2 from
2,4-dihydroxybenzaldehyde and ethylacetoacetate was ham-
pered by the poor yield of the reaction. Triflation55 of 2
provided compound 3, which was used in SM reactions to
obtain 7-phenyl cumarins 4-8. The 40-hydroxyphenyl deri-
vative 6a was prepared by demethylation of 6 with AlCl3,
again in an acceptable yield, which was close to 60%.
Chart 3. Simple 3-Acetylcoumarins 1 and 2 that can Inhibit
17β-HSD1 (the Numbering of the Coumarin Ring System is
Shown)
Chart 4. 6-Phenyl and 7-Phenyl Coumarins as Mimetics of E1
and Structurally Related Phytoestrogen Coumestrol
Scheme 2 shows the preparation of coumarins 9-14, which
started with condensation of commercially available 2,4-dihy-
droxybenzaldehyde and diethyl malonate,56 to obtain 9 in an
almost 50% yield. After the triflation, compound 10 was subjected
to SM reactions with various p-substituted phenylboronic acids,
to obtain the target 7-phenyl-3-ethoxycarbonyl cumarins 11-14.
Preparation of coumarins 15-21 (Scheme 3) that lack the
substitution on position 3 started with the Wittig reaction
Scheme 1. Synthesis of Compounds 1-8a
a Reagents and conditions: (a) ethyl acetoacetate, piperidine, EtOH, reflux, 4 h; (b) AlCl3, toluene, reflux, 3 h; (c) trifluormethanesulfonic anhydride,
pyridine, CH2Cl2, 0 °C, 1 h; (d) Pd(PPh3)4, K2CO3, toluene, reflux, overnight; (e) AlCl3, toluene, reflux, 3 h.

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 251
Scheme 2. Synthesis of Compounds 9-14a
a Reagents and conditions: (a) diethyl malonate, piperidine, AcOH, EtOH, reflux, 4 h; (b) trifluormethanesulfonic anhydride, pyridine, CH2Cl2, 0 °C,
1 h; (c) Pd(PPh3)4, K2CO3, toluene, reflux, overnight.
Scheme 3. Synthesis of Compounds 15-21a
a Reagents and conditions: (a) PhN(Et)2, 210 °C, 2 h; (b) AlCl3, toluene, reflux, 3 h; (c) trifluormethanesulfonic anhydride, pyridine, CH2Cl2, 0 °C, 1 h;
(d) Pd(PPh3)4, K2CO3, toluene, reflux, overnight; (e) AlCl3, toluene, reflux, 3 h.
Scheme 4. Synthesis of Compounds 22-38a
a Reagents and conditions: (a) ethylacetoacetate, piperidine, EtOH, reflux, 4 h; (b) Pd(PPh3)4, K2CO3, toluene, reflux, overnight; (c) diethylmalonate,
piperidine, AcOH, EtOH, reflux, 4 h; (d) Ac2O, K2CO3, N,N-dimethylformamide, 120 °C, overnight; (e) AlCl3, toluene, reflux, 3 h.
between 2-hydroxy-4-methoxybenzaldehyde and commer-
cially available [(methoxycarbonyl)methylene]triphenylphos-
phorane.57 The 7-methoxycoumarin 15 obtained was then
demethylated with AlCl3, to obtain 7-hydroxy coumarin 16;
this was further triflated to compound 17, which was used in
SM reactions to obtain the desired 7-phenyl coumarins
18-21. Compound 20a was obtained by demethylation of 20
with AlCl3. Triflate 3 in general gave poorer yields of SM
reactions than 10 and 17.
Reactions of commercially available 5-bromo-2-hydroxy-
benzaldehyde with ethylacetoacetate, diethylmalonate, and
acetic anhydride provided 6-bromocoumarins 22, 28, and 34,

252 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Starcꢀevicꢁ et al.
Table 1. Effects of Nonaromatic Substituents at Positions 3, 6, and 7 of the Coumarin Scaffold on Inhibition of 17β-HSD1 and 17β-HSD2
inhibition of 17β-HSD1 (%)a
inhibition of 17β-HSD2 (%)a
compound
R1
R2
R3
0.6 μMb
6 μM
0.6 μM
6 μM
1
-COCH3
-COCH3
-COCH3
-COOEt
-COOEt
-H
-H
-H
-H
-H
-H
-H
-H
-H
-Br
-Br
-Br
-OMe
-OH
-OTf
-OH
-OTf
-OMe
-OH
-OTf
-H
23
26
25
57
n.i.c
n.i.
n.i.
n.i.
2
3
60
100
17
n.i.
n.i.
9
10
n.i.
n.i.
n.i.
n.i.
n.i.
8
n.d.d
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.i.
n.i.
n.i.
n.i.
9
15
16
-H
-H
17
22
-COCH3
-COOEt
-H
19
28
34
-H
-H
n.i.
n.i.
81
n.i.
n.i.
100
Equilin
a Recombinant human enzyme overexpressed in E. coli, [3H]E1 þ E1 [67 nM] and NADPH[100 μM] for 17β-HSD1, [3H]E2 þ E2 [300 nM] and NADþ
[100 μM] for 17β-HSD2; data are mean values of two determinations, with RSD less than 10%. b Concentration of inhibitor. c No inhibition. d Not
determined.
respectively. These were then used in SM reactions with var-
ious p-substituted phenylboronic acids, as shown in Scheme 4.
SM reactions with 22 to obtain 23-27 proceeded with gen-
erally the lowest yields, while SM reactions of 34 to provide
compounds 35-38 gave the highest yields. Perkin conden-
sation58 of 5-bromo-2-hydroxybenzaldehyde with acetic an-
hydride to obtain 34 proceeded with poor yield, and the
above-mentioned Wittig reaction with [(methoxycarbonyl)
methylene]triphenylphosphorane appeared to be the more
convenient choice for improving the yield. Demethylation of
37 with AlCl3 gave 37a.
estrogen, is known for its potent inhibition of 17β-HSD1, at
submicromolar concentrations.59
The triflicgroupislabilein anaqueous environment, and its
reactivity can also lead to unfavorable covalent interactions
with other enzymes and receptors, which means that triflates
are not desirable as drug candidates. However, the triflic
group is one of the most reactive groups in SM cross-coupling
reactions with arylboronic and alkylboronic acids, which
allowed us to introduce different p-substituted phenyl groups
to position 7 of the coumarin scaffold. 7-Phenyl coumarins
with the 3-acetyl group 4-8 (Table 2) showed the most
promising inhibition of 17β-HSD1, which was strongly influ-
enced by the nature of the substituent in the para position of
the 7-phenyl group. Compounds 5, 6, and 6a, which all have
40-electron-donor substituents, showed excellent 17β-HSD1
inhibition. Conversely, halogen groups in 7 (Cl) and 8 (F) led
to significant reductions in the inhibitory activity. Interest-
ingly, the unsubstituted 7-phenyl group in compound 4
retained strong inhibitory potency, with 57% inhibition of
17β-HSD1 at 0.6 μM. Coumarin derivative 6a showed re-
markable 76% and 100% 17β-HSD1 inhibition at 0.6 μM and
6 μM, respectively, and was the most potent 17β-HSD1
inhibitor arising from this study. The phenolic OH group of
6a mimics the 3-OH group of E1, and this suggests that 6a
might work as a steroidal substrate mimetic, interacting in the
same way with the polar amino acid residues in the substrate-
binding region as for E1. The replacement of the 3-acetyl
group with 3-ethoxycarbonyl group (compounds 11-14) or
leaving position 3 of the coumarin unsubstituted (compounds
19-21) led to significant reductions in 17β-HSD1 inhibitory
activity, suggesting that the 3-acetyl group is favorable for
optimal interactions with the enzyme. None of the most
potent inhibitors 4, 5, 6, and 6a showed inhibition of 17β-
HSD2 at 60 μM, thereby confirming the excellent selectivity
for 17β-HSD1 over 17β-HSD2 of inhibitors from this struc-
tural class.
Biological Activity
All of these coumarin derivatives were evaluated for inhibi-
tion of recombinant human 17β-HSD1 and recombinant hu-
man 17β-HSD2. 3-Acetyl-7-methoxycoumarin 1 was a modest
inhibitor of the recombinant 17β-HSD1 enzyme, while its
demethylated derivative 2 showed a significant improvement
of the inhibitory potency, with 57% inhibition of 17β-HSD1
at 6 μM (Table 1). Further introduction of a large triflic group
to position 7 of the coumarin scaffold led to the potent 17β-
HSD1 inhibitor 3, with 60% and 100% inhibition at 0.6 μM
and 6 μM, respectively. None of compounds 1-3 showed any
inhibition of 17β-HSD2 (Table 1), and compound 3 did not
inhibit 17β-HSD2 even at 60 μM (data not shown), confirm-
ing the high selectivity of this class of inhibitors for 17β-HSD1
over 17β-HSD2. Replacement of the 3-acetyl group with a
3-ethoxycarbonyl substituent (compounds 9 and 10) led to
almost complete loss of inhibitory activity, and the same result
was seen with coumarins without substituents at position 3
(compounds 15-17). Introduction of bromine to position 6 of
coumarin while keeping position 7 unsubstituted (compounds
22, 28, 34) resulted in very low 17β-HSD1 inhibition (22)
(Table 1). These results suggested that 3-acetyl coumarin
derivatives with large substituents on position 7 can contri-
bute to optimal interactions in the active site of 17β-HSD1,
which leads to significant improvements of the inhibitory
activity;thisalmost reachedthe inhibitorypotency of equiline,
which was used as a positive control. Equilin, or equine
Introduction of various p-substituted phenyl groups to
position 6 of the coumarin scaffold did not result in potent
inhibitors of 17β-HSD1, and the concomitant variation of
substituents in position 3 did not result in significant differ-
ences in the inhibitory activities of this series (Table 3). These

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 253
Table 2. Effects of 7-Phenyl Coumarin Derivatives on Inhibition of 17β-HSD1 and 17β-HSD2
inhibition of 17β-HSD1 (%)a
inhibition of 17β-HSD2 (%)a
6 μM
compound
R1
R2
-H
0.6 μMb
6 μM
0.6 μM
60 μM
4
-COCH3
-COCH3
-COCH3
-COCH3
-COCH3
-COCH3
-COOEt
-COOEt
-COOEt
-COOEt
-H
57
40
63
72
86
100
17
13
7
n.d.c
n.d.
n.i.
n.i.d
n.i.
n.i.
n.i.
5
-Me
-OMe
-OH
-Cl
6
48
76
n.i.
n.i.
n.i.
n.i.
6a
7
n.i.
12
6
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
8
-F
-H
11
12
13
14
18
19
20
20a
21
n.i.
11
-Me
-OMe
-Cl
9
n.i.
n.i.
n.i.
10
1
n.i.
10
29
10
43
9
-H
-H
-H
-Me
-OMe
-OH
-Cl
n.i.
n.i.
n.i.
-H
-H
a Recombinant human enzyme overexpressed in E. coli, [3H]E1 þ E1 [67 nM] and NADPH[100 μM] for 17β-HSD1, [3H]E2 þ E2 [300 nM] and NADþ
[100 μM] for 17β-HSD2; data are mean values of two determinations, with RSD less than 10%. b Concentration of inhibitor. c Not determined. d No
inhibition.
Table 3. Effects of 6-Phenyl Coumarin Derivatives on Inhibition of
17β-HSD1
Table 4. IC50 Values for Compounds 3-6 and 6a Determined on
Bacterial Homogenates of Recombinant Human 17β-HSD1, and Cal-
culated Ki Values
17β-HSD1a (bacterial homogenate)
compound
IC50b (nM)
Kic (nM)
3
360
1928
1237
1135
270
173
911
585
585
143
4
5
inhibition of 17β-HSD1 (%)a
6
6a
compound
R1
R2
-H
0.6 μMb
6 μM
a Recombinant human enzyme overexpressed in E. coli. b Data are
mean values of three determinations, with RSD less than 10%. c Ki
estimated from IC50 by Cheng-Prusoff equation for competitive inhi-
bitor.60
23
24
25
26
27
29
30
31
32
33
35
36
37
37a
38
-COCH3
-COCH3
-COCH3
-COCH3
-COCH3
-COOEt
-COOEt
-COOEt
-COOEt
-COOEt
-H
n.i.c
n.i.
n.i.
n.i.
15
n.i.
n.i.
n.i.
n.i.
59
-Me
-OMe
-Cl
-F
-H
3-6 and 6a are presented in Table 4. The Ki values were
calculated by the Cheng-Prusoff equation,60 presuming that
these compounds are competitive reversible inhibitors. These
results again show the importance of proton donor OH-group
in 6a for 17β-HSD1 inhibitory activity of the 3-acetyl-
7-phenyl coumarin series. Compounds 3 and 6a are potent
inhibitors of 17β-HSD1 in the nanomolar range, with IC50
values of 360 and 270 nM, respectively. For the most potent
inhibitor 6a, the estimated Ki value is just about 2-fold higher
than the Km value for E1 (60 nM) that we experimentally
determined with the same assay, which led us to conclusion
that binding affinities of substrate and inhibitor 6a toward
17β-HSD1 are comparable.
n.i.
6
21
16
-Me
-OMe
-Cl
n.i
10
7
14
-F
-H
21
21
18
n.i.
n.i.
7
-H
-H
-Me
-OMe
-OH
-Cl
n.i.
26
-H
-H
4
n.i.
30
10
a Recombinant human enzyme overexpressed in E. coli, [3H]E1 þ E1
[67 nM] and NADPH [100 μM]; data are mean values of two determina-
tions, with RSD less than 15%. b Concentration of inhibitor. c No
inhibition.
In addition to selectivity against 17β-HSD2, 17β-HSD1
inhibitors to be used as potential drug therapies should not
show high affinities toward ERR and ERβ, as the binding to
these receptors can counteract their therapeutic efficiency.
The relative binding affinities (RBAs) of the most potent 17β-
HSD1 inhibitors 3, 6, and 6a were determined using recombi-
nant human ERs of both subtypes in a competitive binding
assay, using recombinant human receptors and [3H]-E2.61
None of the coumarins tested showed any detectable affinity
results lead to the conclusion that 6-phenyl coumarins do not
bind to the active site of 17β-HSD1 with the high affinities of
the 7-phenyl coumarins. The only exception here was com-
pound 27, which showed moderate inhibition of 17β-HSD1 at
6 μM and almost no inhibition at 0.6 μM; the rest of the
compounds from this series were practically inactive.
The experimentally determined IC50 values and estimated Ki
values for 17β-HSD1 inhibition of the most potent compounds

254 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Starcꢀevicꢁ et al.
Table 5. Binding affinities of selected compounds for recombinant
human ERR and ERβ
RBAa (%)
b
b
compound
ERR
ERβ
3
<10-8
<10-8
<10-8
<10-8
<10-8
<10-8
6
6a
Equilin
0.15
1.40
a RBA (relative binding affinity): E2, 100%, mean values of three
determinations, with RSD less than 10%. b Recombinant human protein
[0.9 nM], incubation with 9 nM [3H]-E2 and inhibitor for 16 h.
Figure 2. Docking of 6a (blue) and 25 (orange) to the active site of
the ternary 17β-HSD1 complex (PDB entry 1A27). For clarity, the
structure of E2 has been removed from the complex. Hydrogen
bonds between 6a and the polar residues are shown as dashed lines.
The nicotinamide moiety of NADPþ (yellow thick sticks) is also
shown. Hydrophobic residues are shown as yellow spheres.
region, the phenolic OH group of 6a forms a H-bond with
Glu282. The docking program did not predict H-bonding of
the 6a phenolic OH to His221, which has also been postulated
to be important for substrate binding. The reason for this
˚
could be the 13 A distance between the acetyl O and phenolic
˚
O in 6a, which exceeds the 12 A distance between O(C3) and
O(C17) in E2, and thus prevents the correct orientation of the
phenolic OH in 6a for H-bonding to His221. Although some
17β-HSD1 crystal structures of ternary complexes15,65 and
kinetic experiments with 17β-HSD1 mutants66,67 have not con-
firmed the role of Glu282 through the formation of a H-bond
to the substrate 3-OH group, the published crystal structures
of ternary complexes of 17β-HSD1 with the steroidal 17β-
HSD1 inhibitors equilin59 and E2B31 clearly show that the
phenolic 3-OH group of these steroidal inhibitors forms a
H-bond with Glu282. A similar pose was also predicted for
our inhibitor 6a. The coumarin core of 6a, which is accom-
modated in the hydrophobic pocket, overlaps closely with the
B and C rings of E2, which suggests the formation of similar
van der Waals contacts to the active-site lipophilic residues.13
It appears that the intracyclic oxygen is not included in the
formation of H-bonding with polar residues in the active site.
The absence of inhibitory activities for the 6-phenyl cou-
marin derivatives can also be explained by molecular docking
(Figure 2). Due to their different geometry, 6-phenyl couma-
rins cannot be accommodated in the active site in a similar man-
ner as the 7-phenyl coumarins, and consequently, H-bonds
with the catalytic Ser142 and Tyr155 are not feasible. Com-
pound 25 shows a distorted orientation when compared to 6a,
with both carbonyl groups pointing away from the catalytic
residues, which prevents the formation of the H-bonds.
To further explain the SAR and devise rules for 17β-HSD1
inhibition by 3-acetyl-7-phenyl coumarins, a pharmacophore
model was built (Figure 3). First, a superposition of active
compounds was performed and pharmacopohoric points
were detected and visualized. For potent inhibitory activity
of this series of coumarins, the H-bond donor (A) that forms
interaction with Glu282 and two H-bond acceptors interact-
ing with Ser142 (B) and Tyr155 (C) are important. The H-bond
Figure 1. Docking of 6a (blue) to the active site of the ternary 17β-
HSD1 complex (PDB entry 1A27). Comparison of the binding
modes between 6a and E2 (magenta) is shown. Hydrogen bonds
between 6a and the polar residues are shown as dashed lines. The
nicotinamide moiety of NADPþ (yellow thick sticks) is also shown.
Hydrophobic residues are shown as yellow spheres.
for the ERs (Table 5). Their binding affinities to the ERs were
compared to the ER affinity of equilin, which is the equine E1
that is used in hormone replacement therapy.59 As a side
effect, this estrogen stimulates proliferation of human breast
cancer cells via the ERs, although this stimulation is lower
than that with E2.23,62 It has also been reported that equilin
shows a significantly higher affinity toward ERβ than ERR,62
which was also confirmed in our assays. The RBAs toward the
ERs of the coumarins and equilin tested are not comparable,
and we can conclude that our compounds have no affinity for
the ERs. These results suggest that our most promising cou-
marins do not have ER agonistic or antagonistic activities,
which could counteract their potential therapeutic benefits.
Molecular Docking
The plausible binding modes of the 6-phenyl and 7-phenyl
coumarin derivatives in the active site of 17β-HSD1 were
assessed by molecular docking of selected compounds, using
FlexX 3.1.63 In the crystal structure of 17β-HSD1 (PDB entry
1A27),64 the active site was defined as the area within 6.5 A of
˚
the co-crystallized E2. In Figure 1, the predicted binding con-
formation of the 7-phenyl coumarin 6a in the active site of
17β-HSD1 is shown. The inhibitor 6a occupies a similar region
to the co-crystallized E2, where the carbonyl oxygen of its
3-acetyl group overlaps closely with the C-17 oxygen of E2, thus
showing a similar role for the forming of a bifurcated H-bond
to the catalytic residues Ser142 and Tyr155,13,15 while the oxy-
gen of its lactone carbonyl group forms an additional H-bond
to Ser142. On the opposite side of the substrate-binding
˚
acceptors B and C should be approximately 3 A apart and
˚
˚
11 A and 12 A away from H-bond donor A, respectively.
H-bond donor (A) and H-bond acceptors (B and C) should be

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 255
Dichloromethane was kept over calcium hydride and distilled
immediately prior to use. Analytical TLC was performed on
Merck 60 F254 silica gel plates (0.25 mm), using visualization
with ultraviolet light, 2,4-dinitrophenyl hydrazine, and FeCl3.
Flash chromatography was carried out on silica gel 60 (particle
size, 240-400 mesh). The melting points were determined on a
Reichert hot stage microscope and are uncorrected. The IR
spectra were recorded on a Perkin-Elmer 1600 FT-IR spectro-
meter. The 1H NMR and 13C NMR spectra were recorded at 300
and 75 MHz, respectively, on a Bruker AVANCE DPX300
spectrometer in CDCl3 or DMSO-d6 solutions, with TMS as
the internal standard. The microanalyses were performed on a
240 C Perkin-Elmer C, H, N analyzer. The analyses indicated by
the symbols of the elements were within (0.4% of the theore-
tical values. The mass spectra were obtained using a VG-
Analytical Autospec Q mass spectrometer. Reversed-phase
high-performance liquid chromatography (HPLC) analyses
were run on Agilent 1100 system (Agilent Technologies, Santa
Clara, CA, USA) equipped with quaternary pump and multiple-
wavelength detector using Agilent Eclipse Plus C18, 5 μm (150 ꢀ
4.6 mm) column. Compounds were dissolved in 50% acetoni-
trile/water at 0.1 mg/mL final concentration, and 10 μL was
injected onto the column. Acetonitrile was used as an organic
modifier and 0.1% TFA in water as an aqueous buffer. The
elution was performed using linear gradient from 20% to 80%
of acetonitrile over 15 min with 1.0 mL/min flow rate and
monitored at 210 nm. The relative purity of all tested com-
pounds was above 95.0%.
Method A. A general procedure for preparation of com-
pounds 1 and 34:53 The corresponding salicylaldehyde (1 equiv)
and ethyl acetoacetate (1 equiv) were dissolved in ethanol.
Piperidine (0.02 equiv) was added, and the mixture was refluxed
for 4 h. Filtration of the cooled mixture and the subsequent
washing of product with cold ethanol provided analytically pure
crystals.
Method B. A general procedure for preparation of com-
pounds 2, 6a, 16, and 20a:54 The methoxyaryl compound and
AlCl3 (3.3 equiv) were suspended in toluene and stirred under
reflux for 3 h. Toluene was removed in vacuo, and the residue
was purified by flash chromatography (0-5% MeOH in
dichloromethane).
Method C. A general procedure for preparation of com-
pounds 3, 10, and 17:55 a suspension of the corresponding phenol
in dry dichloromethane and anhydrous pyridine (1.4 equiv) was
cooled to -10 °C. Trifluormethanesulfonic anhydride (1.2 equiv)
was added dropwise over a period of 15 min, under Ar. After 1 h,
the reaction was quenched with a saturated solution of aqueous
NaHCO3 in an ice bath (caution: exothermic) and washed three
times with water. The organic layer was filtered over silica beads,
the solvent was evaporated in vacuo, and the dry residue was
recrystallized from ethyl acetate.
Method D. SM cross-coupling general procedure for prepara-
tion of compounds 4-8, 11-14, and 18-21:52 a mixture of the
7-triflylcoumarin derivative, the appropriate phenylboronic
acid (1.6 equiv), potassium carbonate (2.5 equiv), and tetrakis-
(triphenylphosphine) palladium (4.5% by weight of triflate) in
deoxygenated toluene was stirred at 100 °C overnight, under Ar.
After cooling to room temperature, the mixture was concen-
trated in vacuo to dryness, and the residue was purified by flash
chromatography (0-40% ethyl acetate in hexane).
Method E. SM cross-coupling general procedure for prepa-
ration of compounds 23-27, 29-33, and 35-38:52 a mixture
of the 6-bromocoumarin derivative, the appropriate phenyl-
boronic acid (1.6 equiv), potassium carbonate (2.5 equiv), and
tetrakis(triphenylphosphine) palladium (4.5% by weight of
bromide) in deoxygenated toluene was stirred at 100 °C over-
night, under Ar. After cooling to room temperature, the mix-
ture was concentrated in vacuo to dryness, and the residue
was purified by flash chromatography (0-20% ethyl acetate
in hexane).
Figure 3. Pharmacophore model derived from SAR of coumarin
derivatives in the active site of the ternary 17β-HSD1 complex (PDB
entry 1A27). Proton donor group (A) is shown as a blue sphere,
while the proton acceptor groups (B and C) are shown as red
spheres. The lipophilic core (D) is shown as a green ellipsoid.
Distances between proton donor and proton acceptor groups are
shown as yellow dashed lines.
connected by the hydrophobic region (D) of appropriate size
to interact with the central hydrophobic part of the active site.
The proper distances between pharmacophoric points are
presented in Figure 3.
Conclusions
The SM cross-coupling methodology was used for the gener-
ation of a library of 6-phenyl and 7-phenyl coumarin deriva-
tives, as potential inhibitors of 17β-HSD1. We established
clear structure-activity relationships, where 7-(4-substituted)-
phenyl coumarin derivatives with an acetyl group in position 3
of the coumarin scaffold provided highly active inhibitors of
17β-HSD1, with the most potent compound 6a showing an
IC50 of 270 nM. Moving the phenyl substitutionfrom position
7 to position 6 or altering the acetyl substituent on position 3
of the coumarins resulted in a drastic reduction in the 17β-
HSD1 inhibitory activity. Docking studies have revealed that
the 7-phenyl coumarin derivatives can occupy a similar bind-
ing region as E2, where the phenolic 4-OH group of 6a can
form polar interactions with Glu282, while on the other side,
both the keto groups of the coumarin ring and the 3-acetyl
substituent can form H-bonds with the residues of the cata-
lytic triad. From SAR results, a simple pharmacophore model
was established. None of the active 7-phenyl coumarins showed
inhibition of 17β-HSD2 at 60 μM, which makes them excep-
tionally selective inhibitors for 17β-HSD1 over 17β-HSD2.
Furthermore, none of the 7-phenyl coumarins tested showed
detectable affinity toward either subtype of the ERs, which
proved ourrationaldesign conceptsfor devoiding ER affinity.
The absence of any ER affinity suggests that these inhibitors
lack estrogenic and anti-estrogenic effects. The 7-phenyl cou-
marins described here demonstrate that the coumarin deriva-
tives are an important structural class that can be used for the
further development of potent and very selective nonsteroidal
17β-HSD1 inhibitors.
Experimental Section
Chemical Methods. The chemical names follow the IUPAC
nomenclature. The starting chemicals were purchased from Acros,
Aldrich, or Fluka, and were used without further purification.

256 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Starcꢀevicꢁ et al.
Method F. A general procedure for the preparation of com-
pounds 9 and 28:56 the mixture of the appropriate derivative of
2-hydroxybenzaldehyde and diethyl malonate (1.1 equiv) in
ethanol, piperidine (0.1 equiv), and glacial acetic acid (0.0005
equiv) was heated under reflux for 3 h. After the addition of hot
water (60 °C) and cooling of the solution, the crystals of the
product were collected by filtration.
HRMS (ESI) calculated for C17H13O4 [MH]þ 281.0814, found
281.0809; anal. (C17H12O4) C, H, N.
3-Acetyl-7-(4-chlorophenyl)-2H-chromen-2-one (7). The title
compound was prepared from 3 (250 mg, 0.74 mmol),
p-chlorophenylboronic acid (186 mg, 1.19 mmol), potassium car-
bonate (257 mg, 1.86 mmol), and tetrakis(triphenylphosphine)
palladium (11 mg, 12 μmol) in 10 mL toluene, according to
method D. The product was purified by flash chromatography
(ethyl acetate in hexane: 0-15%). Yield: 74 mg (33%), yellow
crystals. 1H NMR (300 MHz, CDCl3) δ: 8.55 (s, 1H, H-4), 7.73 (d,
J = 8.1 Hz, 1H), 7.61-7.48 (m, 6H), 2.76 (s, 3H) ppm. HRMS
(ESI) calculated for C17H12ClO3 [MH]þ 299.0475, found
299.0470; anal. (C17H12ClO3) C, H, N.
3-Acetyl-7-(4-fluorophenyl)-2H-chromen-2-one (8). The title
compound was prepared from 3 (250 mg, 0.74 mmol), p-
fluorophenylboronic acid (176 mg, 1.19 mmol), potassium car-
bonate (257 mg, 1.86 mmol), and tetrakis(triphenylphosphine)
palladium (11 mg, 12 μmol) in 10 mL toluene, according to
method D. The product was purified by flash chromatography
(ethyl acetate in hexane: 0-15%). Yield: 20 mg (10%) gray
powder. 1H NMR (300 MHz, CDCl3) δ: 8.70 (s, 1H), 7.85-7.79
(m, 2H), 7.58-7.54 (m, 2H), 7.46 (d, J = 8.4 Hz, 1H), 7.23-7.17
(m, 2H), 2.77 (s, 3H) ppm. HRMS (ESI) calculated for
C17H12FO3 [MH]þ 283.0770, found 299.0773.
3-Acetyl-7-methoxy-2H-chromen-2-one (1). The title com-
pound was prepared from 2-hydroxy-4-methoxy benzaldehyde
(3.0 g, 19.7 mmol) in ethanol (100 mL), according to method A.
Yield: 4.30 g (93%), yellow crystals. 1H NMR (300 MHz,
CDCl3) δ: 8.05 (s, 1H), 7.55 (d, J = 8.70 Hz, 1H), 6.91 (dd,
J = 8.70 Hz, 2.3 Hz, 1H), 6.84 (d, J = 2.30 Hz, 1H), 3.93 (s,
3H), 2.71 (s, 3H) ppm. MS (ESI): m/z 219.0 [MþH]þ.
3-Acetyl-7-hydroxy-2H-chromen-2-one (2). The title com-
pound was prepared from 1 (5.0 g, 22.9 mmol) in toluene (250 mL),
according to method B. The product was purified by flash chro-
matography (0-5% MeOH in dichloromethane). Yield: 3.21 g
(68%), yellow powder. 1H NMR (300 MHz, DMSO) δ: 11.10 (bs,
1H), 8.58 (s, 1H), 7.78 (d, J = 8.4 Hz, 1H), 6.85 (dd, J = 8.6 Hz, 2.4
Hz, 1H), 6.75 (d, J = 2.4 Hz, 1H), 2.54 (s, 3H) ppm. MS (ESI): m/z
205.0 [MþH]þ.
3-Acetyl-2-oxo-2H-chromen-7-yl trifluoromethanesulfonate
(3). The title compound was prepared from 2 (8.20 g, 40.2 mmol)
in dry dichloromethane (200 mL), according to method C.
Yield: 10.01 g (86%), yellow crystals. 1H NMR (300 MHz,
CDCl3) δ: 8.50 (s, 1H), 7.78 (d, J = 8.7 Hz, 1H), 7.35 (d, J =
2.1 Hz, 1H), 7.30 (partly overlapped by the solvent signal, 1H),
2.74 (s, 3H) ppm. HRMS (ESI) calculated for C12H8F3O6S
[MH]þ 336.9994, found 336.9994.
Ethyl 7-Hydroxy-2-oxo-2H-chromene-3-carboxylate (9). The
title compound was prepared from 2,4-dihydroxybenzaldehyde
(4.0 g, 29.0 mmol) in ethanol (20 mL), according to method F.
Yield: 3.14 g (46%), white crystals. 1H NMR (300 MHz, CDCl3)
δ: 11.05 (s, 1H), 8.67 (s, 1H), 7.75 (d, J = 8.6 Hz, 1H), 6.84 (dd,
J = 8.6 Hz, 2.3 Hz, 1H), 6.73 (d, J = 2.1 Hz, 1H), 4.26 (q, J =
7.1 Hz, 2H), 1.30 (t, J = 7.1 Hz, 3H) ppm. MS (ESI): m/z 235.1
[MþH]þ.
3-Acetyl-7-phenyl-2H-chromen-2-one (4). The title compound
was prepared from 3 (500 mg, 1.49 mmol), phenylboronic acid
(290 mg, 2.38 mmol), potassium carbonate (514 mg, 3.72 mmol),
and tetrakis(triphenylphosphine) palladium (22.5 mg, 25 μmol)
in 20 mL toluene, according to method D. The product was
purified by flash column chromatography (ethyl acetate in
hexane: 0-15%). Yield: 134 mg (34%), yellow crystals. 1H
NMR (300 MHz, CDCl3) δ: 8.57 (s, 1H), 7.76-7.745 (m, 8H),
2.77 (s, 3H) ppm. HRMS (ESI) calculated for C17H12O3Na
[MNaþ] 287.0684, found 287.0686.
Ethyl 2-Oxo-7-(trifluoromethylsulfonyloxy)-2H-chromene-
3-carboxylate (10). The title compound was prepared from 9
(2.0 g, 8.54 mmol) in dry dichloromethane (40 mL), according
to method C. Yield: 1.61 g (52%), white crystals. 1H NMR
(300 MHz, CDCl3) δ: 8.52 (s, 1H), 7.74 (d, J =8.5 Hz, 1H),
7.30-7.33 (m, 2H), 4.45 (q, J = 7.1 Hz, 2H); 1.44 (t, J = 7.1 Hz,
3H) ppm. HRMS (ESI) calculated for C13H10F3O7S [MH]þ
367.0099, found 367.0105.
3-Acetyl-7-p-tolyl-2H-chromen-2-one (5). The title compound
was prepared from 3 (1.0 g, 2.97 mmol), p-tolylboronic acid (647
mg, 4.76 mmol), potassium carbonate (1.03 g, 7.44 mmol), and
tetrakis(triphenylphosphine) palladium (45 mg, 50 μmol) in
50 mL toluene, according to method D. The product was purified
by flash chromatography (ethyl acetate in hexane: 0-15%).
Yield: 140 mg (17%), yellow crystals. 1H NMR (300 MHz,
CDCl3) δ: 8.56 (s, 1H), 7.70 (d, J = 8.4 Hz, 1H), 7.61-7.56
(m, 4H), 7.33 (d, 2H, J = 7.9 Hz, 2H), 2.76 (s, 3H), 2.45 (s, 3H)
ppm. HRMS (ESI) calculated for C18H15O3 [MH]þ 279.1021,
found 279.1009.
3-Acetyl-7-(4-methoxyphenyl)-2H-chromen-2-one (6). The title
compound was prepared from 3 (500 mg, 1.49 mmol), p-methoxy-
phenylboronic acid (362 mg, 2.38 mmol), potassium carbonate
(514 mg, 3.72 mmol), and tetrakis(triphenylphosphine) palladium
(22.5 mg, 25 μmol) in 20 mL toluene, according to method D. The
product was purified by flash chromatography (ethyl acetate/
hexane: 0-15%). Yield: 360 mg (72%), yellow crystals. 1H NMR
(300 MHz, CDCl3) δ: 8.55 (s, 1H), 7.70-7.56 (m, 5H), 7.05 (d, J =
8.7 Hz, 2H), 3.90 (s, 3H), 2.76 (s, 3H) ppm. HRMS (ESI) cal-
culated for C18H15O4 [MH]þ 295.0970, found 295.0980.
3-Acetyl-7-(4-hydroxyphenyl)-2H-chromen-2-one (6a). The ti-
tle compound was prepared from 6 (200 mg, 0.68 mmol) in
toluene (10 mL), according to method B. The product was puri-
fied by flash chromatography (0-3% MeOH in dichloromethane).
Yield: 110 mg (59%), yellow crystals. 1H NMR (300 MHz,
DMSO) δ: 9.86 (s, 1H), 8.68 (s, 1H), 7.96 (d, J = 8.7 Hz, 1H),
7.73-7.70 (m, 4H), 6.90 (d, J = 8.6 Hz, 2H), 2.60 (s, 3H) ppm.
13C NMR (DMSO) δ: 195.8, 159.6, 159.6, 156.3, 147.8, 147.1,
132.0, 129.5, 129.3, 123.9, 123.4, 117.3, 116.9, 113.2, 30.9 ppm.
Ethyl 2-Oxo-7-phenyl-2H-chromene-3-carboxylate (11). The
title compound was prepared from 10 (200 mg, 0.55 mmol),
phenylboronic acid (107 mg, 0.87 mmol), potassium carbonate
(189 mg, 1.37 mmol), and tetrakis(triphenylphosphine) palla-
dium (9 mg, 10 μmol) in 10 mL toluene, according to method D.
The product was purified by flash chromatography (ethyl
acetate in hexane: 0-25%). Yield: 148 mg (92%), pale yellow
crystals. 1H NMR (300 MHz, CDCl3) δ: 8.58 (s, 1H), 7.65-7.70
(m, 3H), 7.58-7.61 (m, 2H), 7.45-7.55 (m, 3H), 4.45 (q, J =
7.1 Hz, 2H), 1.45 (t, J = 7.1 Hz, 3H) ppm. HRMS (ESI)
calculated for C18H15O4 [MH]þ 295.0970, found 295.0974.
Ethyl 2-Oxo-7-p-tolyl-2H-chromene-3-carboxylate (12). The
title compound was prepared from 10 (200 mg, 0.55 mmol),
p-tolylboronic acid (119 mg, 0.88 mmol), potassium carbonate
(187 mg, 1.37 mmol), and tetrakis(triphenylphosphine) palla-
dium (9 mg, 10 μmol) in 10 mL toluene, according to method D.
The product was purified by flash chromatography (ethyl
acetate in hexane: 0-20%). Yield: 116 mg (69%), pale yellow
1
crystals. H NMR (300 MHz, CDCl3) δ: 8.57 (s, 1H), 7.66 (d,
J = 8.6 Hz, 1H), 7.55-7.60 (m, 4H), 7.33 (d, J = 7.9 Hz, 2H),
4.45 (q, J = 7.1 Hz, 2H), 2.36 (s, 3H), 1.44 (t, J = 7.1 Hz, 3H)
ppm. HRMS (ESI) calculated for C19H17O4 [MH]þ 309.1127,
found 309.1128.
Ethyl 7-(4-Methoxyphenyl)-2-oxo-2H-chromene-3-carboxy-
late (13). The title compound was prepared from 10 (600 mg,
1.64 mmol), p-methoxyphenylboronic acid (399 mg, 2.62
mmol), potassium carbonate (566 mg, 4.10 mmol), and tetrakis-
(triphenylphosphine) palladium (27 mg, 30 μmol) in 30 mL
toluene, according to method D. The product was purified
by flash chromatography (ethyl acetate in hexane: 0-25%).

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 257
Yield: 204 mg (46%), yellow crystals. 1H NMR (300 MHz,
CDCl3) δ: 8.56 (s, 1H), 7.54-7.66 (m, 5H), 7.04 (d, J = 8.8 Hz,
2H), 4.45 (q, J = 7.1 Hz, 2H), 3.82 (s, 3H), 1.44 (t, J = 7.1 Hz,
3H) ppm. HRMS (ESI) calculated for C19H17O5 [MH]þ
325.1076, found 325.1077.
Ethyl 7-(4-Chlorophenyl)-2-oxo-2H-chromene-3-carboxylate
(14). The title compound was prepared from 10 (200 mg,
0.55 mmol), p-chlorophenylboronic acid (136.7 mg, 0.87 mmol),
potassium carbonate (189 mg, 1.37 mmol), and tetrakis(tri-
phenylphosphine) palladium (9 mg, 10 μmol) in 10 mL toluene,
according to method D. The product was purified by flash
chromatography (ethyl acetate in hexane: 0-30%). Yield:
142.7 mg (80%), pale yellow crystals. 1H NMR (300 MHz,
CDCl3) δ: 8.57 (s, 1H), 7.69 (d, J = 8.5 Hz, 1H), 7.48-7.60
(m, 6H), 4.45 (q, J = 7.1 Hz, 2H), 1.44 (t, J = 7.1 Hz, 3H) ppm.
HRMS (ESI) calculated for C18H14O4Cl [MH]þ 329.0581,
found 329.0582.
7-(4-Methoxyphenyl)-2H-chromen-2-one (20). The title com-
pound was prepared from 17 (600 mg, 2.04 mmol), p-methoxy-
phenylboronic acid (496 mg, 3.26 mmol), potassium carbonate
(705 mg, 5.10 mmol), and tetrakis(triphenylphosphine) palla-
dium (27 mg, 29 μmol) in 30 mL toluene, according to method
D. The product was purified by flash chromatography (ethyl
acetate in hexane: 0-40%). Yield: 492 mg (96%), gray crystals.
1H NMR (300 MHz, CDCl3) δ: 7.74 (d, J = 9.0 Hz, 1H),
7.59-7.61 (m, 2H), 7.51-7.53 (m, 3H), 7.02-7.05 (m, 2H), 6.42
(d, J = 9.6 Hz, 1H), 3.89 (s, 3H) ppm. HRMS (ESI) calculated
for C16H13O2 [MH]þ 253.0865, found 253.0862.
7-(4-Hydroxyphenyl)-2H-chromen-2-one (20a). The title com-
pound was prepared from 20 (458 mg, 1.82 mmol) in toluene
(23 mL), according to method B. The product was purified by
flash chromatography (0-1% MeOH in dichloromethane).
Yield: 283 mg (66%), pale brown crystals. 1H NMR (300 MHz,
DMSO) δ: 9.75 (s, 1H), 8.07 (d, J = 9.5 Hz, 1H), 7.73 (d, J =
8.6 Hz, 1H), 7.61-7.66 (m, 4H), 6.88 (d, J = 8.6 Hz, 2H), 6.44
(d, J = 9.5 Hz, 1H) ppm. HRMS (ESI) calculated for C15H11O3
[MH]þ 239.0708, found 239.0711.
7-(4-Chlorophenyl)-2H-chromen-2-one (21). The title com-
pound was prepared from 17 (200 mg, 0.68 mmol), p-chloro-
phenylboronic acid (170 mg, 1.09 mmol), potassium carbonate
(235 mg, 1.70 mmol), and tetrakis(triphenylphosphine) palla-
dium (9 mg, 10 μmol) in 10 mL toluene, according to method D.
The product was purified by flash chromatography (ethyl acetate
in hexane: 0-25%). Yield: 85 mg (62%), gray crystals. 1H NMR
(300 MHz, CDCl3) δ: 7.75 (d, J=9.6 Hz, 1H), 7.46-7.59 (m,
7H), 6.46 (d, J = 9.6 Hz, 1H) ppm. HRMS (ESI) calculated for
C15H10O2Cl [MH]þ 257.0369, found 257.0362.
7-Methoxy-2H-chromen-2-one (15). The title compound was
prepared by a modified procedure for preparation of coumarins
using the Wittig reaction.57 A mixture of 2-hydroxy-4-methoxy-
benzaldehyde (1.0 g, 6.58 mmol) and [(methoxycarbonyl)-
methylene]triphenylphosphorane (2.75 g, 7.89 mmol) was heated
in N,N-diethylaniline (65 mL) at 210 °C for 2 h. After cooling to
room temperature, the reaction mixture was diluted with 5%
HCl solution (70 mL) and extracted with ether (3 ꢀ 50 mL). The
combined organic phases were dried over Na2SO4, filtered, and
evaporated in vacuo. The oily residue was purified by flash
column chromatography (0-20% ethylacetate in hexane).
Yield: 697 mg (60%), brown powder. 1H NMR (300 MHz,
CDCl3) δ: 7.65 (d, J = 9.5 Hz, 1H), 7.39 (d, J = 8.3 Hz, 1H),
6.84-6.88 (m, 2H), 6.27 (d, J = 9.5 Hz, 1H), 3.90 (s, 3H) ppm.
MS (ESI): m/z 177.1 [MþH]þ.
3-Acetyl-6-bromo-2H-chromen-2-one (22). The title com-
pound was prepared from 5-bromosalicylaldehyde (6.0 g, 29.9
mmol) in 200 mL ethanol, according to method A. Yield: 7.50 g
7-Hydroxy-2H-chromen-2-one (16). The title compound was
prepared from 15 (492 mg, 2.79 mmol) in toluene (25 mL),
according to method B. The product was purified by flash
chromatography (0-1% MeOH in dichloromethane). Yield:
390 mg (86%), brown powder. 1H NMR (300 MHz, DMSO) δ:
7.93 (d, J = 9.4 Hz, 1H); 7.53 (d, J = 8.5 Hz, 1H), 6.79 (dd, J =
8.5 Hz, 2.3 Hz, 1H), 6.71 (d, J = 2.3 Hz, 1H), 6.20 (d, J = 9.4 Hz,
1H) ppm. MS (ESI) m/z 161.0 [MþH]þ.
1
(81%), pale yellow crystals. H NMR (300 MHz, DMSO) δ:
8.60 (s, 1H), 8.22 (d, J = 2.4 Hz, 1H), 7.89 (dd, J = 9.0 Hz,
2.4 Hz, 1H), 7.45 (d, J = 9 Hz, 1H), 2.58 (s, 3H) ppm. MS (ESI):
m/z 267.0 [MþH]þ.
3-Acetyl-6-phenyl-2H-chromen-2-one (23). The title com-
pound was prepared from 22 (400 mg, 1.50 mmol), phenylboro-
nic acid (292 mg, 2.40 mmol), potassium carbonate (518 mg,
3.74 mmol), and tetrakis(triphenylphosphine) palladium (18 mg,
20 μmol) in 10 mL toluene, according to method E. The
product was purified by flash chromatography (ethyl acetate
in hexane: 0-15%). Yield: 160 mg (40%), yellow crystals. 1H
NMR (300 MHz, CDCl3) δ: 8.59 (s, 1H), 7.89 (dd, J = 8.6
Hz, 2.1 Hz, 1H), 7.84 (d, J = 2.1 Hz, 1H), 7.62-7.59 (m, 2H),
7.54 (m, 4H), 2.77 (s, 3H) ppm. HRMS (ESI) calculated for
2-Oxo-2H-chromen-7-yl trifluoromethanesulfonate (17). The
title compound was prepared from 16 (5.0 g, 30.7 mmol) in dry
dichloromethane (100 mL), according to method C. Yield: 8.0 g
(88%), white crystals. 1H NMR (300 MHz, CDCl3) δ: 7.74 (d,
J = 9.6 Hz, 1H); 7.60 (d, J = 8.6 Hz, 1H), 7.30 (d, J = 2.3 Hz,
1H), 7.24 (dd, J = 8.6, 2.4 Hz, 1H), 6.51 (d, J = 9.6 Hz, 1H)
ppm. HRMS (ESI) calculated for C10H6O5F3S [MH]þ
294.9888, found 294.9886.
C
17H13O3 265.0865 [MH]þ, found 265.0871.
7-Phenyl-2H-chromen-2-one (18). The title compound was
prepared from 17 (200 mg, 0.68 mmol), phenylboronic acid
(133 mg, 1.09 mmol), potassium carbonate (235 mg, 1.70 mmol),
and tetrakis(triphenylphosphine) palladium (9 mg, 10 μmol)
in 10 mL toluene, according to method D. The product was
purified by flash chromatography (ethyl acetate in hexane:
0-40%). Yield: 143 mg (95%), pale brown crystals. 1H
NMR (300 MHz, CDCl3) δ: 7.75 (d, J = 9.5 Hz, 1H),
7.64-7.67 (m, 2H), 7.42-7.58 (m, 6H), 6.45 (d, J = 9.5 Hz,
1H) ppm. HRMS (ESI) calculated for C15H11O2 [MH]þ
223.0759, found 223.0760.
7-(p-Tolyl)-2H-chromen-2-one (19). The title compound was
prepared from 17 (250 mg, 0.85 mmol), p-tolylboronic acid (185
mg, 1.36 mmol), potassium carbonate (294 mg, 2.13 mmol), and
tetrakis(triphenylphosphine) palladium (11 mg, 12 μmol) in
12 mL toluene, according to method D. The product was puri-
fied by flash chromatography (ethyl acetate in hexane: 0-25%).
Yield: 104 mg (52%), white crystals. 1H NMR (300 MHz,
CDCl3) δ: 7.74 (d, J = 9.5 Hz, 1H), 7.54-7.56 (m, 5H), 7.31
(d, J = 8.0 Hz, 2H), 6.43 (d, J = 9.5 Hz, 1H), 2.44 (s, 3H) ppm.
HRMS (ESI) calculated for C16H13O2 [MH]þ 237.0916, found
237.0905.
3-Acetyl-6-p-tolyl-2H-chromen-2-one (24). The title com-
pound was prepared from 22 (400 mg, 1.50 mmol), p-tolylboro-
nic acid (326 mg, 2.40 mmol), potassium carbonate (518 mg,
3.74 mmol), and tetrakis(triphenylphosphine) palladium (18 mg,
20 μmol) in 10 mL toluene, according to method E. The product
was purified by flash chromatography (ethyl acetate in hexane:
0-15%). Yield: 143 mg (34%), yellow crystals. 1H NMR
(300 MHz, CDCl3) δ: 8.58 (s, 1H), 7.88 (dd, J = 8.7 Hz, 2.1 Hz,
1H), 7.82(d, J = 2.1 Hz, 1H), 7.50 (d, J= 8.1 Hz, 2H), 7.45 (d, J=
8.7 Hz, 1H), 7.31 (d, J= 7.8 Hz, 2H), 2.77 (s, 3H), 2.44 (s, 3H) ppm.
HRMS (ESI) calculated for C18H15O3 279.1021 [MH]þ, found
279.1023.
3-Acetyl-6-(4-methoxyphenyl)-2H-chromen-2-one (25). The
title compound was prepared from 22 (400 mg, 1.50 mmol),
4-methoxyphenylboronic acid (364 mg, 2.40 mmol), potassium
carbonate (518 mg, 3.74 mmol), and tetrakis(triphenylphosphine)
palladium (18 mg, 20 μmol) in 10 mL toluene, according to
method E. The product was purified by flash chromatography
(ethyl acetate in hexane: 0-15%). Yield: 110 mg (25%), yellow
crystals. 1H NMR (300 MHz, CDCl3) δ: 8.57 (s, 1H), 7.85 (dd,
J = 8.7 Hz, 2.1 Hz, 1H), 7.79 (d, J = 2.1 Hz, 1H), 7.53 (d, J =
8.7 Hz, 2H), 7.44 (d, J = 8.7 Hz, 1H), 7.03 (d, J = 8.7 Hz, 2H),

258 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Starcꢀevicꢁ et al.
3.89 (s, 3H), 2.76 (s, 3H) ppm. HRMS (ESI) calculated for
C18H15O4 295.0970 [MH]þ, found 295.0965.
Ethyl 6-(4-Chlorophenyl)-2-oxo-2H-chromene-3-carboxylate
(32). The title compound was prepared from 28 (250 mg, 0.84
mmol), 4-chlorophenylboronic acid (205 mg, 1.35 mmol), po-
tassium carbonate (291 mg, 2.10 mmol), and tetrakis(triphenyl-
phosphine) palladium (11.3 mg, 13 μmol) in 10 mL toluene,
according to method E. The product was purified by flash
chromatography (ethyl acetate in hexane: 0-15%). Yield: 170 mg
(62%), white crystals. 1H NMR (300 MHz, DMSO) δ: 8.80 (s,
1H), 8.28 (d, J = 2.3 Hz, 1H), 8.06 (dd, J = 8.7 Hz, 2.3 Hz, 1H),
7.76 (d, J = 8.6 Hz, 2H), 7.58 (d, J = 8.6 Hz, 2H), 7.54 (d,
J = 8.7 Hz, 1H), 4.32 (q, J = 7.1 Hz, 2H), 1.33 (t, J = 7.1 Hz,
3H) ppm. HRMS (ESI) calculated for C18H13ClO4Na 351.0400
[MNa]þ, found 351.0407; anal. (C18H13ClO4) C, H, N.
3-Acetyl-6-(4-chlorophenyl)-2H-chromen-2-one (26). The title
compound was prepared from 22 (250 mg, 0.94 mmol), 4-chlor-
ophenylboronic acid (228 mg, 1.50 mmol), potassium carbonate
(323 mg, 2.34 mmol), and tetrakis(triphenylphosphine) pallad-
ium (11 mg, 12 μmol) in 10 mL toluene, according to method E.
The product was purified by flash chromatography (ethyl acetate
in hexane: 0-15%). Yield: 87 mg (31%), yellow solid. 1H NMR
(300 MHz, CDCl3) δ: 8.57 (s, 1H), 7.85 (dd, J = 8.7 Hz, 2.1 Hz,
1H), 7.81 (d, J = 2.1 Hz, 1H), 7.55-7.46 (m, 5H), 2.77 (s, 3H)
ppm. HRMS (ESI) calculated for C17H11ClO3Na 321.0294
[MNa]þ, found 321.0287.
Ethyl 6-(4-Fluorophenyl)-2-oxo-2H-chromene-3-carboxylate
(33). The title compound was prepared from 28 (250 mg, 0.84
mmol), 4-fluorophenylboronic acid (188 mg, 1.35 mmol), po-
tassium carbonate (291 mg, 2.10 mmol), and tetrakis(triphenyl-
phosphine) palladium (11 mg, 13 μmol) in 10 mL toluene,
according to method E. The product was purified by flash
chromatography (ethyl acetate in hexane: 0-15%). Yield: 142 mg
(54%), white crystals. 1H NMR (300 MHz, DMSO) δ: 8.80 (s,
1H), 8.24 (d, J = 2.3 Hz, 1H), 8.03 (dd, J = 8.7 Hz, 2.3 Hz, 1H),
7.77 (m, 2H), 7.52 (d, J = 8.7 Hz, 1H), 7.35 (m, 2H), 4.32
(q, J = 7.1 Hz, 2H), 1.33 (t, J = 7.1 Hz, 3H) ppm. HRMS (ESI)
calculated for C18H14FO4 313.0876 [MH]þ, found 313.0873;
anal. (C18H13FO4) C, H, N.
6-Bromo-2H-chromen-2-one (34). The title compound was
prepared by a modified procedure for Perkin condensation.58
5-Bromo-2-hydroxybenzaldehyde (3.00 g, 14.92 mmol), potas-
sium carbonate (2.27 g, 16.42 mmol), and acetic anhydride (9.10 g,
89.14 mmol) were mixed in N,N-dimethylformamide. The slurry
was heated to 70 °C, and water (0.3 mL) was added. The mixture
was then heated to 120 °C and mixed overnight. After cooling
the solution to 30 °C, water (30 mL) was added and the resulting
precipitate was filtered, dried, and purified with flash chroma-
tography (ethylacetate in hexane: 0-18%). Yield: 493 mg
(15%), white crystals. 1H NMR (300 MHz, CDCl3) δ:
7.62-7.70 (m, 3H), 7.25 (d, J = 9.4 Hz, 1H), 6.49 (d, J =
9.6 Hz, 1H). HRMS (ESI) calculated for C9H6O2Br 224.9551
[MH]þ, found 224.9558.
3-Acetyl-6-(4-fluorophenyl)-2H-chromen-2-one (27). The title
compound was prepared from 22 (250 mg, 0.94 mmol), 4-fluor-
phenylboronic acid (210 mg, 1.50 mmol), potassium carbonate
(323 mg, 2.34 mmol), and tetrakis(triphenylphosphine) palla-
dium (11 mg, 12 μmol) in 10 mL toluene, according to method E.
The product was purified by flash chromatography (ethyl acetate
in hexane: 0-15%). Yield: 110 mg (44%), brown powder.
1H NMR (300 MHz, DMSO) δ: 8.70 (s, 1H), 8.02 (d, J =
8.1 Hz, 1H), 7.93-7.88 (m, 2H), 7.79-7.74 (m, 2H), 7.39-7.33
(m, 2H), 2.60 (s, 3H) ppm. HRMS (ESI) calculated for
C17H12FO3 283.0770 [MH]þ, found 283.0775.
Ethyl 6-Bromo-2-oxo-2H-chromene-3-carboxylate (28). The
title compound was prepared from 5-bromo-2-hydroxybenzal-
dehyde (10.0 g, 49.75 mmol) in 200 mL ethanol, according
to method F. Yield: 12.0 g (81%), white crystals. 1H NMR
(300 MHz, DMSO) δ: 8.69 (s, 1H), 8.16 (bs, 1H), 7.86 (dd, J =
8.8 Hz, 0.6 Hz, 1H), 7.40 (d, J = 8.8 Hz, 1H), 4.31 (q, J = 7.1 Hz,
2H), 1.31 (t, J = 7.1 Hz, 3H). HRMS (ESI) calculated for
C12H9BrO4Na 318.9582 [MNa]þ, found 318.9587.
Ethyl 2-Oxo-6-phenyl-2H-chromene-3-carboxylate (29). The
title compound was prepared from 28 (2.00 g, 6.73 mmol),
phenylboronic acid (1.31 g, 10.77 mmol), potassium carbonate
(2.33 g, 16.83 mmol), and tetrakis(triphenylphosphine) palla-
dium (90 mg, 101 μmol) in 50 mL toluene, according to method
E. The product was purified by flash chromatography (ethyl
acetate in hexane: 0-15%) and recrystallized from ethylacetate/
1
hexane. Yield: 650 mg (33%), yellow crystals. H NMR (300
MHz, DMSO) δ: 8.82 (s, 1H), 8.26 (d, J = 2.1 Hz, 1H), 8.05 (dd,
J = 8.7 Hz, 2.1 Hz, 1H), 7.75-7.72 (m, 2H), 7.54-7.49 (m, 3H),
7.44-7.39 (m, 1H), 4.32 (q, J = 6.9 Hz, 2H), 1.33 (t, J = 6.9 Hz,
3H) ppm. HRMS(ESI) calculatedfor C18H15O4 295.0970 [MH]þ,
found 295.0973; anal. (C18H14O4) C, H, N.
6-Phenyl-2H-chromen-2-one (35). The title compound was
prepared from 34 (100 mg, 0.44 mmol), phenylboronic acid
(87 mg, 0.71 mmol), potassium carbonate (153 mg, 1.11 mmol),
and tetrakis(triphenylphosphine) palladium (4.5 mg, 5 μmol) in
5 mL toluene, according to method E. The product was purified
by flash chromatography (ethyl acetate in hexane: 0-10%).
Yield: 82 mg (83%), brown crystals. 1H NMR (300 MHz,
CDCl3) δ: 7.76-7.80 (m, 2H), 7.69 (d, J = 2.2 Hz, 1H), 7.59-
7.62 (m, 2H), 7.39-7.52 (m, 4H), 6.49 (d, J = 9.5 Hz, 1H) ppm.
HRMS (ESI) calculated for C15H12O2 223.0759 [MH]þ, found
223.0763.
Ethyl 2-Oxo-6-p-tolyl-2H-chromene-3-carboxylate (30). The
title compound was prepared from 28 (2.00 g, 6.73 mmol),
p-tolylboronic acid (1.46 g, 10.77 mol), potassium carbonate
(2.33 g, 16.83 mmol), and tetrakis(triphenylphosphine) palladium
(90 mg, 101 μmol) in 50 mL toluene, according to method E. The
product was purified by flash chromatography (ethyl acetate in
1
hexane: 0-15%). Yield: 493 mg (24%), gray crystals. H NMR
6-(p-Tolyl)-2H-chromen-2-one (36). The title compound was
prepared from 34 (100 mg, 0.44 mmol), phenylboronic acid (97 mg,
0.71 mmol), potassium carbonate (153 mg, 1.11 mmol), and
tetrakis(triphenylphosphine) palladium (4.5 mg, 5 μmol) in 5 mL
toluene, according to method E. The product was purified by
flash chromatography (ethyl acetate in hexane: 0-15%). Yield:
(300 MHz, DMSO) δ: 8.81 (s, 1H), 8.23 (d, J = 1.8 Hz, 1H), 8.02
(dd, J = 8.7 Hz, 1.8 Hz, 1H), 7.62 (d, J = 8.1 Hz, 2H), 7.51 (d,
J = 8.7 Hz, 1H), 7.32 (d, J = 8.1 Hz, 2H), 4.32 (q, J = 7.1 Hz,
2H), 2.36 (s, 3H), 1.33 (t, J = 7.1 Hz, 3H) ppm. HRMS (ESI)
calculated for C19H16O4Na 331.0946 [MNa]þ, found 331.0948.
Ethyl 6-(4-Methoxyphenyl)-2-oxo-2H-chromene-3-carboxy-
late (31). The title compound was prepared from 28 (2.00 g,
6.73 mmol), 4-methoxyphenylboronic acid (1.64 g, 10.77 mmol),
potassium carbonate (2.33 g, 16.83 mmol), and tetrakis-
(triphenylphosphine) palladium (90 mg, 101 μmol) in 50 mL
toluene, according to method E. The product was purified by
flash chromatography (ethyl acetate in hexane: 0-15%). Yield
1
69 mg (65%), white crystals. H NMR (300 MHz, CDCl3) δ:
7.74-7.79 (m, 2H), 7.67 (d, J = 2.0 Hz, 1H), 7.50 (d, J = 8.0 Hz,
2H), 7.41 (d, J = 8.6 Hz, 1H), 7.30 (d, J = 8.0 Hz, 2H), 6.48 (d,
J = 9.5 Hz, 1H), 2.43 (s, 3H) ppm. HRMS (ESI) calculated for
C16H13O2 237.0916 [MH]þ, found 237.0922.
6-(4-Methoxyphenyl)-2H-chromen-2-one (37). The title com-
pound was prepared from 34 (340 mg, 1.51 mmol), p-methoxy-
phenylboronic acid (370 mg, 2.43 mmol), potassium carbonate
(525 mg, 3.80 mmol), and tetrakis(triphenylphosphine) palla-
dium (15 mg, 17 μmol) in 17 mL toluene, according to method E.
The product was purified by flash chromatography (ethyl
acetate in hexane: 0-20%). Yield: 299 mg (78%), pale yellow
1
1.47 g (67%), yellow crystals. H NMR (300 MHz, DMSO) δ:
8.80 (s, 1H), 8.20 (d, J = 2.3 Hz, 1H), 8.00 (dd, J = 8.7 Hz, 2.3
Hz, 1H), 7.67 (d, J = 8.8 Hz, 2H), 7.49 (d, J = 8.7 Hz, 1H), 7.07
(d, J = 8.8 Hz, 2H), 4.32 (q, J = 7.1 Hz, 2H), 3.82 (s, 3H), 1.33 (t,
J = 7.1 Hz, 3H) ppm. HRMS (ESI) calculated for C19H16O5Na
347.0895 [MNa]þ, found 347.0891; anal. (C19H16O5) C, H, N.
1
crystals. H NMR (300 MHz, CDCl3) δ: 7.77 (d, J = 9.6 Hz,

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 259
1H), 7.73 (dd, J = 8.7 Hz, 2.0 Hz, 1H), 7.63 (d, J = 2.0 Hz, 1H),
7.53 (d, J = 8.7 Hz, 2H), 7.40 (d, J = 8.6 Hz, 1H), 7.02 (d, J =
8.7 Hz, 2H), 6.47 (d, J = 9.5 Hz, 1H), 3.88 (s, 3H) ppm. HRMS
(ESI) calculated for C16H13O3 253.0865 [MH]þ, found
253.0865.
6-(4-Hydroxyphenyl)-2H-chromen-2-one (37a). The title com-
pound was prepared from 37 (247 mg, 0.98 mmol) in toluene (12
mL), according to method B. The product was purified by flash
chromatography (0-1% MeOH in dichloromethane). Yield: 75
mg (32%), brown crystals. 1H NMR (300 MHz, DMSO) δ: 9.60
(s, 1H), 8.10 (d, J = 9.6 Hz, 1H), 7.94 (d, J = 1.9 Hz, 1H), 7.82
(dd, J = 8.6 Hz, 2.1 Hz, 1H), 7.54 (d, J = 8.5 Hz, 2H), 7.43 (d,
J = 8.6 Hz, 1H), 6.88 (d, J = 8.5 Hz, 2H), 6.52 (d, J = 9.5 Hz,
1H) ppm. HRMS (ESI) calculated for C15H11O3 [MH]þ
239.0708, found 281.0702.
radioflow detector (Raytest). The assays were performed in dupli-
cate (RSD was less than 15%) for determination of the percen-
tages of inhibition, and in triplicate (RSD was less than 5%) for
determination of the IC50 for 3, 4, 5, 6, and 6a. The conversion
was calculated according to the following formula %conversion
= (%E2/(%E2 þ %E1) ꢀ 100, and the inhibition was calculated
according to %inhibition = [(%conversion of control - %
conversion of sample)/%conversion of control] ꢀ 100.
Inhibition of 17β-HSD2. The 17β-HSD2 inhibition assay was
performed as described for 17β-HSD1 in terms of the percentage
of inhibition, but with the following modifications: sodium
phosphate buffer, pH 7.5, with E2 and [2,4,6,7-3H]-E2 (final
concentrations, 300 nM, 0.6 μCi), with the inhibitors present at
concentrations of 0.6 μM and 6 μM, or 6 μM and 60 μM, with
100 μM cofactor NADþ used and the reactions stopped after 5
min (time needed to convert ca. 20% of substrate in a control
assay without inhibitor).
6-(4-Chlorophenyl)-2H-chromen-2-one (38). The title com-
pound was prepared from 34 (100 mg, 0.44 mmol), p-chloro-
phenylboronic acid (111 mg, 0.71 mmol), potassium carbonate
(153 mg, 1.11 mmol), and tetrakis(triphenylphosphine) palla-
dium (4.5 mg, 5 μmol) in 5 mL toluene, according to method E.
The product was purified by flash chromatography (ethyl
acetate in hexane: 0-10%). Yield: 85 mg (74%), white crystals.
1H NMR (300 MHz, CDCl3) δ: 7.71-7.79 (m, 2H), 7.65 (d, J =
2.2 Hz, 1H), 7.42-7.55 (m, 5H), 6.50 (d, J = 9.5 Hz, 1H).
HRMS (ESI) calculated for C15H10O2Cl 257.0369 [MH]þ,
found 253.0377.
ER Affinity. The binding affinity of selected compounds to
ERR and ERβ was determined as described previously,61 with
minor modifications. Recombinant human ERR and ERβ
(0.9 nM) were incubated with 9 nM [2,4,6,7-3H]-E2 and the selected
inhibitor, for 18 h at 4 °C. The concentration range of the
inhibitors in the incubation mixtures was 10-9-10-5 M. Non-
specific binding was calculated using 6 μM E2 as the competing
ligand. After incubation, the ligand-receptor complex was
bound to 50% hydroxyapatite in Tris (pH 7.5) buffer and
washed four times. For radiodetection, the liquid scintillation
cocktail Emulsifier Safe (Perkin-Elmer) was added, and samples
were counted in a 1214 Rackbeta liquid scintillation counter
(LKB Wallac). Each compound was tested in triplicate (RSD
was less than 10%), and for determination of the RBAs, the
inhibitor and E2 concentrations required to displace 50% of the
receptor-bound 3H-E2 were determined. The RBA values were
calculated according to the following formula: RBA[%] =
(IC50(E2)/IC50(inhibitor) ꢀ 100. The RBA value for E2 was
arbitrarly set at 100%.
Biological Methods. [2,4,6,7-3H]-E1 and [2,4,6,7-3H]-E2 were
obtained from Perkin-Elmer, Boston (USA). Other chemicals
were purchased from Sigma, Merck, or Roth.
Preparation of 17β-HSD1 and 17β-HSD2 Enzymes for Activ-
ity Assays. Recombinant His-tagged human 17β-HSD1 was
overexpressed in the BL21-CodonPlus (DE3)-RIL strain of E.
coli containing the pQE30-17beta-HSD type 1 construct (pre-
pared at the Institute of Experimental Genetics, Neuherberg,
Germany). The bacteria were resuspended in PBS and soni-
cated; the resultant cell homogenate was used as the source of
the recombinant enzyme, and it was stored frozen until deter-
mination of enzymatic activity.
Molecular Modeling. The computational study was carried
out on a workstation with 4 dual-core AMD Opteron proces-
sors, 16 GB of RAM, a GeForce 7800 graphics card, and 1.2 TB
of hard disk space. For the docking experiments, FlexX 3.1.2
(BioSolveIT GmbH)63 installed on the 64-bit Fedora 7 was used.
For figure preparation, PyMol68 was used. We used the crystal
structure of 17β-HSD1 with co-crystallized E2 (PDB entry
Recombinant N-29 truncated 17β-HSD2 was overexpressed
in JM-107 E. coli containing the pGEXII-N-29 17beta-HSD
type 2 construct (prepared at the Institute of Experimental
Genetics, Neuherberg, Germany). The bacteria were resus-
pended in PBS and sonicated; the resultant cell homogenate
was used as the source of the recombinant enzyme, and it was
stored frozen until determination of enzymatic activity.
Inhibition of 17β-HSD1. The synthesized coumarins were
tested for their inhibition of 17β-HSD1, in terms of their percent-
ages of inhibition of E1 reduction, using E. coli cell homo-
genates.49 For 3, 4, 5, 6, and 6a, the IC50 was also determined.
Bacterial homogenate was incubated in 100 mM sodium phos-
phate buffer, pH 6.5, with E1 and [2,4,6,7-3H]-E1 (final con-
centrations, 55 nM, 0.6 μCi) at 37 °C, in the absence and
presence of potential inhibitors at concentrations of 0.6 μM
and 6 μM. For determination of IC50 values, the concentrations
of 3, 4, 5, and 6 ranged from 30 nM to 24 μM and concentrations
of 6a ranged from 10 nM to 6 μM. The concentration of the
cofactor NADPH was 100 μM. Inhibitor stock solutions were
prepared in DMSO and diluted with acetonitrile prior to use.
The final concentration of acetonitrile in the reactions was
adjusted to 1%. The reactions were stopped with addition of
ethyl acetate after 10 min (the time needed to convert ca. 25% of
the substrate in a control assay without inhibitor). The samples
were extracted with ethyl acetate, the solvent was evaporated,
and the steroids were dissolved in acetonitrile for separation on a
reverse-phase HPLC ODS Hypersil (Thermo Scientific) C18
column. Isocratic HPLC runs were performed at 25 °C and a
flow rate of 1 mL/min of acetonitrile/water (45:55) as mobile
phase. Detection and quantification of the radioactive steroids
were performed using Quickszint Flow 302 (Zinsser Analytic,
Frankfurt, Germany) as scintillation fluid, and a Ramona 2000
1A27).64 The active site was defined as the area within 6.5 A of
˚
the co-crystallized E2. Protonations and OH group orientations
of the active-site amino acid residues were manually assigned
with FlexX GUI. We validated the system by the redocking of
co-crystallized E2. The docking was carried out using the default
parameters of the program. For establishment of 3D pharma-
cophore model, we performed superposition of active com-
pounds with ROCS69 (OpenEye Scientific Software Inc.).
Visualization and detection of pharmacophoric parts was done
with LIQUID70 plugin for PyMol.
Acknowledgment. We thank Dr. Jerzy Adamski (Institute
of Experimental Genetics, Genome Analysis Center, Neuher-
berg, Germany) for providing the E. coli BL21-CodonPlus
(DE3)-RIL and JM-107 cells with pQE30-17beta-HSD type 1
and pGEXII-N-29 17beta-HSD type 2 constructs, respec-
ꢀ
tively; to Marjan Kuznik for help in performing the recep-
tor-binding assays, and to the Government of the Republic of
Slovenia, Ministry of Higher Education, Science and Technol-
ogy of the Republic of Slovenia for financial support. We also
thank Dr. Chris Berrie for critical reading of the manuscript.
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