Benzopyrans as selective estrogen receptor β agonists (SERBAs). Part 2: Structure-activity relationship studies on the benzopyran scaffold

Article abstract of DOI:10.1016/j.bmcl.2007.04.051

Benzopyrans are selective estrogen receptor (ER) β agonists (SERBAs), which bind the ER subtypes α and β in opposite orientations. Here we describe structure-activity relationship studies that led to the discovery of bezopyran 5b. X-ray crystal structures

Full text of DOI:10.1016/j.bmcl.2007.04.051

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3570–3574
Benzopyrans as selective estrogen receptor b agonists
(SERBAs). Part 2: Structure–activity relationship
studies on the benzopyran scaffold
Timothy I. Richardson,* Bryan H. Norman, Charles W. Lugar, Scott A. Jones,
Yong Wang, Jim D. Durbin, Venkatesh Krishnan and Jeffrey A. Dodge
Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA
Received 19 March 2007; revised 17 April 2007; accepted 18 April 2007
Available online 25 April 2007
Abstract—Benzopyrans are selective estrogen receptor (ER) b agonists (SERBAs), which bind the ER subtypes a and b in opposite
orientations. Here we describe structure–activity relationship studies that led to the discovery of bezopyran 5b. X-ray crystal struc-
tures of 5b and a non-selective analog 5c in ERa help explain the observed selectivity of the benzopyran platform.
Ó 2007 Elsevier Ltd. All rights reserved.
Estrogen activity is mediated by two members of the ste-
roid nuclear hormone receptor family, ERa and ERb.
They function as ligand-activated gene transcription fac-
tors and mediate a number of important physiological
processes with the classical receptor, ERa, being most
important for development and regulation of the female
reproductive system as well as maintenance of the skel-
etal and cardiovascular systems. It is possible to develop
synthetic estrogens that have tissue type selectivity.1
These selective ER modulators (SERMs), such as
raloxifene, act as agonists in some tissues such as bone,
liver, and cardiovascular, while behaving as antagonists
in others such as uterus and breast. The origin of tissue
selectivity is complex and not completely understood,
but is most likely due to the interaction of ligand bound
receptor with a wide array of cofactors, coactivators and
corepressors, which in concert with the receptor mediate
gene transcription. ERb, which was discovered in 1996,
adds another layer of complexity to our understanding
of estrogen physiology.2 While ERa is expressed in
nearly all tissues of both sexes, ERb is expressed in the
ovaries, uterus, and oviduct of the female reproductive
tract but not in breast tissue; while in males, ERb is
expressed in the prostate and epididymis but not in the
testes.3
Over the last decade several groups have developed ERb
selective ligands using structure based design methods
along with inspiration from the well-known ERb-selec-
tive isoflavone phytoestrogens daidzein (1) and genistein
(2).4 All of these ligands have in common two phenols
which bind to the Glu-Arg-water triad at one end of
the ERb receptor binding pocket and His475 at the
other end (Fig. 1).5 These two critical features of the
ERb pharmacophore (3) require a separation of the phe-
˚
nol oxygen atoms by a distance of about 10–12 A
(roughly 10 atoms). The benzopyran scaffold (4) served
as an excellent starting point to build ERb selective
ligands because the benzopyran substructure provides
a rigid platform with a distance between the phenol
˚
oxygens of 11.9 A. This distance positions the phenol
˚
oxygen atoms between the genistein distance of 12.2 A
˚
and the estradiol distance of 10.9 A. Herein we describe
structure–activity relationship studies that led to the
discovery of benzopyran 5b as a selective estrogen
receptor b agonist (SERBA).6
The unadorned benzopyran 4a was prepared as
described in Scheme 1. Base catalyzed condensation of
4-(methoxymethoxy)-benzaldehyde (7) and 2-hydroxy-
5-(methoxymethoxy)-acetophenone (6) gave 40,6-
dihydroxyflavanone 8. The ketone of 8 was reduced by
catalytic hydrogenation over Pd/C to give the corre-
sponding MOM-protected 40,6-flavandiol 9, which was
then deprotected under acidic conditions to give the
desired benzopyran 4a.
Keywords: Estrogen receptor; ERb selective ligands; Benzopyrans.
*
Corresponding author. Tel.: +1 317 433 2373; fax: +1 317 433
0552; e-mail: t_richardson@lilly.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.04.051

T. I. Richardson et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3570–3574
3571
Benzopyran Platform (4)
His475
11.9 Å
4
12.2 Å
HO
HO
3
O
O
HO
O
X
OH
OH
OH
Glu
ER ligands (3)
X = H, Daidzein (1)
X = OH, Genistein (2)
H2O
Arg
Figure 1. ER ligands: genistein, daidzein, and the benzopyran platform.
O
O
O
O
RO
RO
a
RO
Me
H
+
67%
OH
OR
7
8
6
OR
OR
23%
b
HO
c
O
72%
O
OH
4a
9
Scheme 1. Synthesis of benzopyran 4a. R = MOM; Reagents and conditions: (a) NaOH, H2O, EtOH, 0 °C ! rt; (b) H2, Pd/C, AcOH, EtOAc;
(c) HCl, H2O, THF.
The synthesis of the cyclopentane analog 5b was
described in part 1 of this series.6 The key step of the
synthesis was a reductive cyclization, which provided
the corresponding racemate 4b with excellent diastere-
oselectivity for the all syn diastereomer (Reaction a,
Scheme 2). This same route was used to prepare analogs
of 4a with cycloalkyl rings fused at the C3 and C4 posi-
tions of the benzopyran scaffold. The synthesis of the 5-
OH analog 4c followed the same route and is described
in Scheme 2. MOM-protected resorcinol 10 was lithiat-
ed, followed by transmetallization to the corresponding
aryl zinc, which was subjected to Palladium-mediated
Negishi cross coupling with the cyclopentyl vinyl triflate
116 to give cyclopentene 12. Palladium catalyzed hydro-
genation of the olefin in 12 gave exclusively the cis-ste-
reochemistry in cyclopentane 13. The methyl ester of
13 was converted to the Weinreb amide 14. Addition
of aryllithium 15 to Weinreb amide 14 provided aryl
ketone 16. Deprotection and cyclization of 16 was
accomplished in a one pot sequence: treatment with tol-
uenesulfonic acid, followed by the addition of sodium
cyanoborohydride under acidic conditions (monitoring
pH with bromocresol green) favored benzopyran 4c
with the all cis-stereochemistry.
lithiated and then coupled to cyclopentyl vinyl triflate
11 to give cyclopentene 22. Cyclopentene 22 was then
carried through the reductive cyclization route to pro-
vide 40,7-flavandiol 4e. Phenyl lithium was added to
Weinreb amide 236 to give aryl ketone 24. In like man-
ner, lithium–halogen exchange on MOM-protected
3-bromophenol 26 followed by addition to Weinreb
amide 23 gave aryl ketone 27. Both of these aryl ketones
were then carried through the reductive cyclization route
to provide 6-flavanol 4f and 30,6-flavandiol 4g.
The 3,4-cyclohexane-fused benzopyran 4h and the 40-flav-
anol analog 4i were prepared using the Pechmann Con-
densation Route described in Scheme 4. Hydroquinone
(29) was condensed with ethyl 2-cyclohexanonecarboxy-
late (30) in 80% H2SO4 to give 6-hydroxycoumarin 31.
The hydroxyl group of 31 was protected as its TBS-ether
to give 32. The d-lactone of 32 was reduced with DIBAL-
H to the corresponding lactol, which was then opened
with aryllithium 33 to give the styryl-benzyl alcohol 34.
The TBS-protecting groups on 34 were removed using
TBAF. When this deprotection reaction was acidified
with TFA, the styryl-benzyl alcohol was displaced by
the proximal phenol to give 2H-benzopyran 35. The al-
kene of 35 was reduced with hydrogen over Pd/C to give
3,4-cyclohexane-fused benzopyran 4h. In like manner
methyl 2-oxocyclopentanecarboxylate (36) was con-
densed with hydroquinone (29) in 80% H2SO4 and then
carried through the same reaction sequence to give 40-flav-
anol analog 4i.
The reductive cyclization route described in Scheme 2
supported the syntheses of the 3,4-cycloheptane-fused
benzopyran 4d as well as several other phenol isomers
of 4b (Scheme 3). Enol triflate 19 was prepared from
commercially available methyl 2-oxo-1-cycloheptane-
carboxylate (18) and then carried through the reductive
cyclization route to provide cycloheptane-fused benzo-
pyran 4d. MOM-protected 4-bromoresorcinol 21 was
As shown in Tables 1 and 2, benzopyrans 4a–i were
evaluated for their ability to bind estrogen receptors

3572
T. I. Richardson et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3570–3574
OR
HO
a
RO
O
45%
O
OR
4b
OH
OR
OR
b
+
CO2Me
TfO
CO2Me
41%
OR
OR
11
OR
12
10
c
OR
OR
Me
58%
NOMe
d
CO2Me
O
OR
13
67%
14
OR
e
93%
15
Li
OH
OR
OR
a
O
78%
O
OR
OH
16
4c
Scheme 2. Reductive cyclization route to 3,4-cycloalkyl fused benzopyrans. R = MOM; Reagents: (a) i—p-TsOH, MeOH, 50 °C; ii—HCl,
Na(CN)BH3, rt; (b) i—10, t-BuLi, THF, ꢀ78 ! 0 °C; ii—ZnCl2, 0 °C ! rt; iii—11, Pd(PPh3)4, rt ! 50 °C; (c) 5% Pd/C, MeOH, 60 psi H2; (d)
HN(OMe)MeÆHCl, iPrMgCl, THF, ꢀ10 °C; (e) 15, THF, ꢀ0 °C.
a
b
HO
O
CO2Me
TfO
CO2Me
O
18
19
4d
OH
TfO
CO2Me
11
Br
d
O
b
RO
OR
OMe
HO
HO
O
RO
OR
20, R = H
c
OH
22 (R = MOM)
4e
21, R = MOM
b
RO
O
RO
O
e
O
Ph
NOMe
OR
OR
Me
4f
24 (R = MOM)
23 (R = MOM)
f
HO
RO
O
b
OH
Br
OR
O
OR
25, R = H
c
4g
OR
26, R = MOM
27 (R = MOM)
Scheme 3. Benzopyrans prepared via the reductive cyclization route. Reagents and conditions: (a) Tf2O, i-Pr2EtN, CH2Cl2, ꢀ78 °C; (b) reductive
cyclization route, Scheme 2; (c) NaH, MOMCl, DMF, 0 °C; (d) i—s-BuLi, THF, 0 °C; ii—ZnCl2, 0 °C ! rt; iii—11, Pd(PPh3)4, rt ! 50 °C; (e) PhLi,
THF, 0 °C; (f) i—t-BuLi, THF, ꢀ78 °C; ii—23, THF, ꢀ78 °C.

T. I. Richardson et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3570–3574
3573
HO
a
HO
+
O
29%
OH
O
O
O
OEt
31
29
30
88%
b
OTBS
c, 67%
TBSO
TBSO
OH
OTBS
O
O
OH
34
32
Li
d, 85%
33
HO
HO
e
50%
OH
O
O
4h
35
OH
c, d, e
a
O
O
O
O
CO2Me
OH
36
37
4i
Scheme 4. Pechmann condensation route to 3,4-cycloalkyl fused benzopyrans. R = MOM; Reagents and conditions: (a) 80% H2SO4; (b) TBSCl,
imidazole, DMF; (c) i—DIBAL-H, THF, ꢀ78 °C; ii—35, THF, ꢀ78 °C; (d) TBAF, THF, TFA; (e) 5% Pd/C, MeOH, H2.
Table 1. Cycloalkyl ring SAR: ERa and ERb binding data
Table 2. Phenol isomer SAR: ERa and ERb binding data
4
5
HO
HO
6
n
3
R1
2'
7
O
O
O
R2
4'
4a
4b-d
4e-i
OH
OH
5'
Compound
n
ERbb (nM)
ERab (nM)
Ratio
Compound R1
R2
ERbb (nM) ERab (nM) Ratio
4a
4b
4h
4d
20.8 4.3
0.47 0.18
0.63 0.46
0.88 0.38
126 82
4.34 3.1
2.88 2.9
6.11 2.8
6
9
5
7
4b
4f
4g
4i
6-OH 40-OH 0.47 0.18 4.34 3.1
9
5
4
2
1
2
3
6-OH
6-OH 30-OH 18.5 7.4
40-OH 22.1 7.3
H
51 11
267 112
65 31
40 27
H
aAll compounds are racemic.
b Ki values are means of at least two determinations SD.
4c
4e
5-OH 40-OH 0.66 0.08 0.35 0.04 0.5
7-OH 40-OH 18.9 1.6
41 17
2
aAll compounds are racemic.
b Ki values are means of at least two determinations SD.
alpha and beta. As can be seen in Table 1, fusing a
cyclopentane ring to the 3,4-positions of benzopyran
4a dramatically improved binding affinity and afforded
a compound (4b) with 9-fold selectivity for ERb over
ERa. Increasing the ring size to cyclohexane (4h) or
cycloheptane (4d) did not improve ERb binding potency
over 4b. Since these two larger ring derivatives were
slightly less selective for ERb we chose to carry out phe-
nol isomer SAR studies with the cyclopentane ring in
place (Table 2). Removal of the hydoxyl group from
the pendant 40-aryl ring (4f) resulted in a 60-fold and a
100-fold loss in binding affinity for ERa and ERb,
respectively, demonstrating the importance of this phe-
nol in the ER pharmacophore.7 Compounds 4b and 4g
demonstrate that the 40-position is optimal because
when moved to the 30-position there is a 39-fold loss
in binding affinity for ERb. Removal of the hydroxyl
group from the 6-position on the benzopyran ring (4i)
also reduced binding affinity but not as dramatically
(47-fold vs 100-fold) as compared to the 40-position.
Moving the benzopyran hydroxyl group from the 6-po-
sition (4b) to the 5-position (4c) maintained potency at
ERb but increased potency at ERa, resulting in com-
plete loss of selectivity. Placing the hydroxyl group in
the 7-position resulted in a loss of affinity at both recep-
tors and gave a compound (4e) that was only 2-fold
selective for ERb.

3574
T. I. Richardson et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3570–3574
OH
have been deposited in the Protein Data Bank with
accession code 2POG.
C
C
__Ki (nM)__
ERα = 2.60
ERβ = 0.19
__Ki (nM)__
ERα = 0.29
ERβ = 0.47
HO
A
B
A
B
O
O
D
D
5b
In conclusion, we were able to increase the binding affin-
ity and selectivity of the benzopyran scaffold by fusing a
fourth ring at the 3,4-carbon positions of 4a. A cyclo-
pentane ring was marginally optimal compared to the
cyclohexane and cycloheptane rings. We demonstrated
that both phenols were critical for affinity. The position-
ing of the phenols was important for both affinity and
selectivity. The 5-OH isomer 4c was equipotent com-
pared to the 6-OH isomer 4b at ERb but was non-selec-
tive. Cocrystal structures of 5b and 5c, the more potent
enantiomers of 4b and 4c, revealed that moving the hy-
droxyl to the 5-position compensates for the shift in the
position of the A-ring with respect to His-524 and allows
the formation of a better hydrogen bond. Attempts to
increase the selectivity of the benzopyran scaffold will
be disclosed in subsequent papers.
5c
OH
OH
Better H-bond
His 524
Figure 2. Diagram of the X-ray structure of SERBA-1 (5b in yellow)
and the non-selective benzopyran 5e (green) bound to ERa.
References and notes
In order to better understand the basis for binding selec-
tivity we investigated the binding interactions that the
non-selective benzopyran 4c forms with ERa (Fig. 2).
We have already reported the cocrystal structures of
SERBA-1 (5b), the more potent enantiomer of benzopy-
ran 4b, with both ERa and ERb.6 The data indicate that
SERBA-1 binds to both receptors with its D-ring phenol
interacting with the hydrogen bond network of the Glu-
Arg-H2O triad, while the A-ring phenol interacts with
His-524 in ERa or the corresponding His-475 in ERb.
Although the phenols bind in the same places within
the binding pocket, the orientation of the benzopyran
scaffold is rotated by 180° on the bisphenol axis. The
positions of the A-ring phenol are also shifted with
respect to the histidines. The rotated binding orienta-
tions and shifted phenol positions result in slightly dis-
rupted binding interactions within the ERa pocket
compared to ERb and thus conferring selectivity for
ERb. The enantiomers of 4c were separated by chiral
chromatography (Chiralpak AD, 80% heptane-isopro-
panol). The more potent enantiomer 5c was cocrystal-
lized with ERa. Figure 2 shows an overlay of this
structure with that of 5b and ERa. As can be seen, mov-
ing the phenol from the 6-position (5b) to the 5-position
(5c) compensates for the shift in position of the A-ring
phenol with respect to His-524. As a result benzopyran
5c is able to form a better hydrogen bond to the histidine
compared to benzopyran 5b. Thus, the affinity to ERa is
increased and selectivity for ERb is diminished. Atomic
coordinates for ERa complexed with benzopyran 5c
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