Benzopyrans as selective estrogen receptor β agonists (SERBAs). Part 4: Functionalization of the benzopyran A-ring

Article abstract of DOI:10.1016/j.bmcl.2007.07.009

Benzopyrans are selective estrogen receptor (ER) β agonists (SERBAs), which bind the ER receptor subtypes α and β in opposite orientations. We have used structure based drug design to show that this unique phenomena can be exploited via substitution at the 8-position of the benzopyran A-ring to disrupt binding to ERα, thus improving ERβ subtype selectivity. X-ray cocrystal structures with ERα and ERβ are supportive of this approach to improve selectivity in this structural class.

Full text of DOI:10.1016/j.bmcl.2007.07.009

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5082–5085
Benzopyrans as selective estrogen receptor b agonists
(SERBAs). Part 4: Functionalization of the benzopyran A-ring
Bryan H. Norman,a,* Timothy I. Richardson,a Jeffrey A. Dodge,a Lance A. Pfeifer,a
Gregory L. Durst,a Yong Wang,a Jim D. Durbin,a Venkatesh Krishnan,b Sean R. Dinn,c
Shengquan Liu,c John E. Reillyc and Kendal T. Ryterc
aDiscovery Chemistry Research, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA
bMusculoskeletal Research, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA
cAlbany Molecular Research, Inc., 21 Corporate Circle, PO Box 15098 Albany, New York, NY 12212, USA
Received 20 April 2007; revised 4 July 2007; accepted 6 July 2007
Available online 13 July 2007
Abstract—Benzopyrans are selective estrogen receptor (ER) b agonists (SERBAs), which bind the ER receptor subtypes a and b in
opposite orientations. We have used structure based drug design to show that this unique phenomena can be exploited via substi-
tution at the 8-position of the benzopyran A-ring to disrupt binding to ERa, thus improving ERb subtype selectivity. X-ray cocrys-
tal structures with ERa and ERb are supportive of this approach to improve selectivity in this structural class.
Ó 2007 Elsevier Ltd. All rights reserved.
Estrogen receptors (ERs, subtypes a and b) are nuclear
hormone receptors with diverse tissue distribution and
biological function.1 The understanding of ER ligands,
agonists and antagonists, is a fairly mature science, dating
back over the past several decades.2 More recently, the
development of selective estrogen receptor modulators
(SERMs) have impacted several disease targets, primarily
in women’s health.3 From a historical perspective, most of
the ER pharmacology to date has focused on ERa. How-
ever, since its discovery in 1996,4 much effort has gone into
understanding the relative importance of ERb.5,6 One
important ERb responsive tissue is the prostate.7 We have
recently described novel benzopyrans which are selective
ERb agonists (SERBAs) displaying striking effects on ro-
dent prostates in models of benign prostatic hyperplasia
(BPH).8 As part of this program, we have tried to improve
ERb selectivity through the use of structure based drug
design.9 In this paper, we describe our approaches to
selectivity improvements in the benzopyran A-ring.10
improving subtype specificity.11,12 Specifically, protein
crystallography has been instrumental in providing
information regarding the binding modes of ligands
within the ligand binding pockets (LBP) of ERa and
ERb. As we have already shown, benzopyrans, such as
1, are unique, in that they bind to ERa and ERb with
different orientations (a 180° rotation around the bis-
phenol axis). This is illustrated in Figure 1, which shows
an overlay of the X-ray cocrystal structures of 1/ERa
(blue) and 1/ERb (green). The residues lining the LBP
are also shown in the overlay, with phenol-binding res-
idues and a versus b residue changes highlighted. It
should be noted that the D-ring phenol binds within
the Arg–Glu network in both ERa (Arg394–Glu353)
and ERb (Arg346–Glu305). Similarly, the A-ring phenol
forms a hydrogen bond to the imidazole ring in His525
in ERa (His475 in ERb). We and others have shown
that these interactions are critical to the overall binding
affinity of ER ligands. Structure modifications which
compromise either interaction are expected to signifi-
cantly diminish ER binding affinity.
In recent years, we and others have utilized structure
based drug design (SBDD) tools to assist our under-
standing of ER-ligand binding, with the hopes of
The ‘ligand rotation’ phenomenon noted in Figure 1
provides a unique opportunity to improve ERb specific-
ity, which does not focus completely on exploiting
the conservative residue changes within the LBP
(M421(a) ! I373(b); L384(a) ! M336(b)). Since the
benzopyran scaffold already possesses very high affinity
Keywords: Estrogen receptor b; Prostate; Crystallography.
*
Corresponding author. Tel.: +1 317 277 2235; fax: +1 317 277
3652; e-mail: norman@lilly.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.07.009

B. H. Norman et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5082–5085
5083
shows that substitution at the 8 position of the benzo-
pyran should have minimal effect on ERb binding (note
the ‘open’ area), as compared to the 8 position of the
benzopyran in the ERa structure (crowded). When we
prepared the 8-methyl substituted analog of 1, we
observed no effect on binding affinity to either receptor
subtype. Therefore, steric interactions alone are insuffi-
cient and it is always possible that the binding modes
are altered when substitution at this position is made.
Here we describe the effect of substitutions at the 8
position that do disrupt ERa binding to a greater
extent than ERb binding, thereby improving ERb
selectivity.
The synthesis of the 8-methyl substituted analogs took
advantage of the reductive cyclization route reported
earlier and is described in Scheme 1.8,13 4-Methoxy
phenol was treated with benzyltrimethylammonium
tribromide to provide the dibromide. Demethylation
with trimethylsilyl iodide gave hydroquinone 5, which
was protected as the bis-methoxymethyl ether 6 using
sodium hydride and chloromethyl methyl ether. Ortho
mono-lithiation of protected hydroquinone 6 with sBu-
li for phenyllithium for 8 followed by quenching of the
anion with iodomethane or DMF provided 7a and 8,
respectively. 8 could be elaborated to 7b and 7c via
reduction to the benzyl alcohol 9, followed by alkyl-
ation with iodomethane (for 7b) or protection as the
TBS ether (for 7c). A second ortho lithiation followed
5
A
8
C
HO
4
B
2
O
D
OH
1
Figure 1. Overlay of 1 with ERa and ERb.
for ERb, the task of improving ERb selectivity becomes
an exercise in selectively disrupting ERa binding. Figure 1
MOMO
Br
PO
Br
MeO
d
a,b
g
OMOM
OP
TfO
95%
OH
7a, 81%
8, 70%
R
Br
CO2Me
MOMO
4
7a (R = Me)
8 (CHO)
5, P = H
10
h,
CO2Me
OMOM
95%
c
6, P = MOM
36-56%
R
MOMO
Br
MOMO
Br
11a-d
f
e
g
8
OMOM
OMOM
7b, 88%
7c,d, 95%
99%
R
CH2OH
7b (R = CH2OMe)
7c,d (R = CH2OTBS)
68-89%
i
9
Li
OMe
OMOM
OMOM
MOMO
N
MOMO
Me
MOMO
k, 14
CO2Me
OMOM
j
l
O
O
76-81%
OMOM
R
54-93%
OMOM
R
88-97%
R
12a-d
13a-d
15a-d
OMOM
OMOM
MOMO
HO
HO
MOMO
l
m
n
O
15c
(R = CH2OTBS)
O
OMOM
O
O
34%
95%
OMOM
OEt
R
(two steps)
R
EtO
CHO
OH
OH
2a- d
3a- d
17
16
Scheme 1. Reagents and conditions: (a) Benzyltrimethylammonium tribromide, CH2Cl2, MeOH; (b) TMSI, CH3CN; (c) NaH, DMF, MOMCl; (d)
sBuLi, THF; MeI for 7a or PhLi, THF; DMF for 8; (e) NaBH4, EtOH; (f) NaH, DMF, MeI for 7b or TBSCl, DMF, imidazole for 7c; (g) i—PhLi,
THF, ii—ZnCl2; (h) 10, Pd(PPh3)4; (i) H2, Pd/C, EtOH; (j) NH(OMe)Me, THF, i-PrMgCl; (k) 14, THF; (l) i—HCl, MeOH, NaBH3CN, ii—Chiral
separation; (m) i—TBAF, THF; ii—NaOCl, Bu4NBr; (n) H2, PtO2, EtOH.

5084
B. H. Norman et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5082–5085
by coupling with enol triflate 10 using Negeshi condi-
tions gave the unsaturated esters 11a–c. Unsaturated
esters 11a–c were hydrogenated over palladium on car-
bon to give 12a–c, and then transformed into racemic
Weinreb amides 13a–c. The Weinreb amides 13 were
reacted with lithiated p-bromophenyl methoxymethyl
ether 14 to give racemic ketones 15a–c. Deprotection
and cyclization of ketones 15 under acidic conditions
was followed by reduction with sodium cyanoborohy-
dride under acidic conditions to give racemic benzopy-
rans 2 and 3 (a, b, and d analogs), which were
separated into their individual enantiomers using chiral
preparative HPLC. Benzopyrans 2c and 3c were pre-
pared from 15c under slightly different conditions. Spe-
cifically, 15c was deprotected using TBAF and
oxidized with bleach to give benzaldehyde 16. This
material was subjected to hydrogenation conditions
in an attempt to selectively reduce the aldehyde in
the presence of the ketone. However, these conditions
did not result in reduction, but merely generated
diethyl acetal 17. Reductive cyclization of 17 using
conditions described above gave ethoxymethyl benzo-
pyrans 2c and 3c, which were separated into their indi-
vidual enantiomers using chiral preparative HPLC.
The 2R,3R,4R enantiomer (isomer 2, as drawn) was
found to have the higher binding affinity for all
analogs.
The A-ring, position 8 effects on ERa and ERb binding
are shown in Table 1. The addition of a methyl group at
the 8-position caused no change in binding affinity to
either receptor subtype; however, when larger, more po-
lar groups were added, the ERa binding affinity de-
creased significantly. We attribute this finding to the
limited space available (steric effect) and the nature of
the surrounding residues in this area of the LBP, which
are very hydrophobic (polarity effect). Perhaps ligands
such as 2a maintain good ERa binding due to the plas-
ticity of the ER LBPs,14 which may accommodate
hydrophobic substituents residing in the highly hydro-
phobic pocket. Alternatively, a larger, more polar
group, such as alkoxymethyl or hydroxymethyl is toler-
ated in the hydrophobic LBP only when adequate space
exists between the ligand polar groups and the hydro-
phobic residues. In this series, it appears that 2b (meth-
oxymethyl substitution) provides the best mix of
polarity and steric bulk to disrupt ERa binding, while
maintaining very good binding to ERb, thus improving
ERb selectivity to 43-fold.
With these results in hand, we sought to test our hypoth-
esis with X-ray crystal structures of 2b with both ERa
and ERb (Fig. 2).15 The similarity of binding modes, rel-
ative to 1, is demonstrated by overlaying the structures of
2b with the structures of 1 in both ERa and ERb. This
result clearly shows (b) that 2b binds to ERb in an iden-
tical manner, relative to 1/ERb. However, as shown in
Figure 2a, the methoxymethyl group in 2b forces the
A-ring out of its optimal position, thereby disrupting
the important A-ring phenol-His524 hydrogen bonding
interaction. We believe that this is responsible for the
ꢀ4-fold decrease in binding affinity of 2b, relative to 1.
Table 1. A-ring modifications: ERa and ERb binding data
HO
O
R
OH
In conclusion, we have shown that the introduction of
A-ring substitution to the 8 position of benzopyrans re-
sults in diminished ERa binding, while maintaining
strong ERb binding. We have shown that this is due
to the fact that compounds in this class bind to the
ER subtypes with different poses, and that substitution
at the 8 position is less tolerated in the ERa LBP, rela-
tive to ERb. Finally, the 8-methoxymethyl shows im-
proved binding selectivity (43-fold) in this class of
selective ERb agonists.
2a-e
Compound
R
ERa (nM) Kia ERb (nM) Kia Ratio
1
2a
2b
2c
2d
H
Me
2.29 ( 1.52)
2.82 ( 2.01)
0.17 ( 0.094)
0.26 ( 0.091)
0.28 ( 0.02)
0.745
14
11
43
36
25
CH2OMe 11.0 ( 5.64)
CH2OEt 27.2
CH2OH
147
5.97
a Standard errors shown in parentheses. Others not tested in duplicate.
Assay conditions described in Ref. 8.
Figure 2. Overlay of 1 and 2b with ERa (a) and ERb (b).

B. H. Norman et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5082–5085
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Osborne, H. E.; Amos, R. M.; Guo, S.; Boodhoo, A.;
Krishnan, V. J. Med. Chem. 2006, 49, 6155.
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