BC-spiro-estradiols. Synthesis and estrogen receptor binding affinity of four new estradiol isomers

Article abstract of DOI:10.1016/j.bmcl.2012.04.022

The synthesis of four new isomers of estradiol in which the ring A to ring C planes are perpendicular to each other as a result of a spiro BC ring junction is described. Heterocyclic analogs and carbocyclic homologs of these compounds are also reported. Estrogen receptor binding studies show that the spiro compounds with the natural stereochemistry at C9 bind almost as strongly as estradiol but with greater β to α selectivity. These studies show that the estrogen receptors can readily accommodate isomers of estrogen with substantially different fixed shapes than the native ligand.

Full text of DOI:10.1016/j.bmcl.2012.04.022

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3713–3717
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
BC-spiro-estradiols. Synthesis and estrogen receptor binding affinity of four
new estradiol isomers
Muhammad Asim a, Daria Klonowska a, Christine Choueiri a, Ilia Korobkov a, Kathryn E. Carlson b,
John A. Katzenellenbogen b, Tony Durst a,
⇑
a Department of Chemistry, University of Ottawa, Ottawa, Canada K1N 6N5
b Department of Chemistry, University of Illinois, Urbana, IL 6180, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of four new isomers of estradiol in which the ring A to ring C planes are perpendicular to
each other as a result of a spiro BC ring junction is described. Heterocyclic analogs and carbocyclic
homologs of these compounds are also reported. Estrogen receptor binding studies show that the spiro
compounds with the natural stereochemistry at C9 bind almost as strongly as estradiol but with greater
Received 4 March 2012
Revised 2 April 2012
Accepted 4 April 2012
Available online 11 April 2012
b to
a selectivity. These studies show that the estrogen receptors can readily accommodate isomers of
estrogen with substantially different fixed shapes than the native ligand
Keywords:
Ó 2012 Published by Elsevier Ltd.
Estradiol
BC-spiro-estrogen
ER binding affinity
Synthesis
A large number of analogs of estradiol, the natural ligand for the
estrogen receptor (ER), have been prepared and evaluated for their
prepared in enantiomerically pure form,3 all the compounds de-
scribed herein were prepared as single enantiomers. Spiro deriva-
tive 2, or BC-spiro-estradiol, is most closely related to estradiol
since it is a constitutional isomer that has the same configuration
as estradiol at all of its chiral centers. BC-spiro-CD-cis-estradiol,
3, also has the same configuration as estradiol at all of its chiral
centers, with the exception that the CD ring junction is cis com-
pared to trans in estradiol. The spiro estradiol isomers 4 and 5 have
the non-natural configuration at the spiro carbon designated as C9
to allow for a facile comparison with estradiol. In the case of 4, this
results in a bent shaped structure which was confirmed by an X-
ray crystal structure (Fig. 3). Compound 5 can exist in two confor-
mations since the cis CD junction allows for a ring C chair-chair
interconversion; the energetically preferred conformation is as
shown.
We have also prepared compounds 6 and 7 which are homologs
of the spiro estradiols having a six-membered ring B. With the B
ring enlarged, the preferred dihedral angle made by the planes of
ring A and C is in the 60–70° range. The availability of these iso-
meric estradiols with fixed conformations different from estradiol
(2–5), the homologs 6 and 7, and the A-CD steroids, structures, 8
and 9, previously reported by us,8,9 which can adopt a variety of
conformations due to rotation about the ring A to ring C bond,
has allowed us to probe further into how variations in the shape
of ligands closely related to estradiol can affect their binding affin-
binding to the ER, and more recently, to the two ER subtypes, ER
and ERb. These analogs of estradiol provide interesting probes of
the ligand binding pocket of ER and ERb, and some have potential
a
a
for estrogen pharmaceutical applications. This work was reviewed
in detail some time ago,1,2 and the available information showed
clearly that both ER
tional substituents on the steroidal structure, in particular on the
b-face at C11 and on the -face at C7 and C16. All of these deriva-
a and ERb can accommodate quite readily addi-
a
tives are based on the natural, essentially planar ABCD ring archi-
tecture of estradiol (1). Substantial additional work describing
many non-steroidal compounds SERMs also supports the conclu-
sion that the estrogen receptors are capable of accommodating a
great variety of structures with a varied core composition and
geometries, and peripheral substituents.1–3
We report herein four new isomers of estradiol, 2–5, in which
the plane of ring A is constitutionally constrained to be perpendic-
ular to that of ring C through a spiro BC ring junction (Fig. 1). To the
best of our knowledge, these compounds represent the first iso-
mers of estradiol that have been reported in which rings B and C
are connected via a spiro-ring as opposed to the usual fused-ring
junction, although certain spiro-nonsteroidal compounds are
known to bind to ER,4,5 to potentiate estrogen action,6 or to inhibit
stereoid reductases.7 Since the CD portion of these compounds was
ity for the estrogen receptors, ERa and ERb.
In a previous paper,9 we described modeling studies which indi-
cated that when bound to the estrogen receptors, the A-CD ligands
⇑
Corresponding author. Tel.: +1 613 562 5800x6072; fax: +1 613 562 5170.
E-mail address: tdurst@uottawa.ca (T. Durst).
0960-894X/$ - see front matter Ó 2012 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2012.04.022

3714
M. Asim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3713–3717
CH3
CH3
D
HO
HO
CH3
D
OH
1
OH
A
A
OH
C
C
D
9
B
C
16
A
HO
B
B
3
H
3, BC-spiro-CD-cis-estradiol
A to C dihedral angle = 90°
RBAα = 6.4 1.7
1, estradiol
2
, BC-spiro-estradiol
A to C dihedral angle ~10°
RBAα = 100
A to C dihedral angle = 90°
RBAα = 0.98 0.26
RBAβ = 40.5 10
RBAβ = 100
RBAβ = 2.43 0.71
CH3
OH
HO
CH3
H
OH
A
OH
D
C
C
D
D
HO
B
B
A
CH3
C
9
A
B
9
HO
4, BC-spiro-C9 inverted
A to C dihedral angle = 90°
RBAα = 0.12 0.03
5, BC-spiro-CD-cis-C9 inverted
A to C dihedral angle = 90°
RBAα = 0.34 0.09
6, homo-BC-CD-cis-estradiol
A to C dihedral angle ~60-70°
RBAα = 3.7 0.97
RBAβ = 0.23 0.03
RBAβ = 0.37 0.09
RBAβ = 39.7 1.4
H
OH
CH3
CH3
HO
CH3
HO
HO
OH
A
A
OH
9
H
H
H
8
9
, A-CD-cis
, A-CD-trans
7
, homo-BC-CD-cis-C9-inverted
A to C dihedral angle; variable
RBAα = 2.38 0.19
A to C dihedral angle; variable
RBAα = 1.5 0.26
A to C dihedral angle = 60-70°
RBAα = 0.05 0.001
RBAβ = 9.97 1.3
RBAβ = 21.5 4.6
RBAβ = 0.16 0.02
Figure 1. Structures of estradiol (1), BC spiro estrogens (2–7), and A-CD estrogens (8, 9). Relative binding affinity (RBA) values are given for ER
a
(RBA
a
) and ERb (RBAb). The
and ERb
RBA value is the binding affinity relative to that of estradiol (RBA = 100), and are determined by a competitive radiometric binding assay using human, full length ER
a
and [3H]estradiol as tracer, as we have described.10 Values represent the mean standard deviation from 2–3 determinations. Ki values can be determined from the RBA
values (Kicompound = RBA ꢀ Kedstradiol/100; Kd = 0.2 nM (ER
a
); 0.5 nM (ERb)).
H
H
CH3
HO
CH3
OH
OH
OH
CH3
HO
OH
HO
HO
CH3
O
O
O
H
X
10, X=H, H;
14, X= O
12
11
13
Ring A to C dihedral angle= 90°
A to C dihedral angle= 90°
RBAa= 0.061 0.002
RBAb = 0.59 0.05
A to C dihedral angle = 90°
RBAa = 0.31 0.002
RBAb = 3.47 0.93
A to C dihedral angle= 90°
RBAa= 0.014 0.001
RBAb= 0.020 0.006
RBAa= 2.46 0.11 RBAa= 1.98 0.40
RBAb = 9.05 2.40 RBAb = 6.07 0.77
Figure 2. Structures of other spiro estrogens (10, 11, 13, 14) and an A-CD estrogen (12). See legend to Figure 1.
8 and 9 adjusted their conformation such that the dihedral angle
comparable to that of 3, while that of the oxa analog 11, with a
60–70° dihedral angle, is similar to 10, where the planes are at
90°. Interestingly, in contrast to estradiol, which binds at compara-
ble levels to ERa and ERb, these compounds show affinity prefer-
ence for ERb in the 6 to 15-fold range.
made by the aromatic and C rings was approximately 30°. During
the course of these studies, we realized that we could probe more
deeply into the shape of ligands capable of binding to the estrogen
receptors by preparing isomers of estrogen with fixed, or nearly
fixed, dihedral angles between these two planes by adding a
two-carbon bridge that created spiro ring compounds. We have
determined that 2 and 3, which are constitutional isomers of estra-
diol with a fixed dihedral angle of 90°, bind to ERb with a relative
binding affinity (RBA) value of 2.4 and 40.5 (RBA estradiol = 100)
We conclude that the estrogen receptors accommodate almost
equally well a series of isomeric or very similar ligands with dis-
tinctly different shapes: (a) the natural hormone estradiol, which
is essentially planar, (b) the spiro compounds 2 and 3, structural
isomers of estradiol in which the planes of the aromatic ring and
that of ring C are fixed at 90° to each other, and (c) the homolog
8, where the two planes are fixed at 60–70°. These findings are
consistent with the well-appreciated capacity of the estrogen
receptors to bind with good affinity to ligands having structurally
diverse core structures,11,12 as well as emerging evidence for the
‘plasticity’ of the ligand binding pocket13 and the concept that
the final shape of this pocket is only determined during ligand
binding, a process that enables the lower portion of the ligand
binding domain to complete its folding.3
and to ER
son, the conformationally mobile structures 8 and 9 gave RBAs of
10 and 21.5 for ERb, and 2.4 and 1.5 for ER , respectively. The 6-
a, with an RBA of 1.0 and 6.4, respectively. For compari-
a
oxo analog of 3, compound 10, also shows significant binding to
both estrogen receptors (Fig. 2). Thus, the quite rigid spiro estro-
gens have binding affinities comparable to those of the A-CD
ligands.
The homo analog 6, in which the ring B is expanded to a six
membered-ring, has a somewhat more flexible conformation. Nev-
ertheless, the A to C dihedral angle is restricted to a range no less
than about 60–70°. The binding of this compound to the ERs is
During the preparation of the above compounds, we also iso-
lated the spiro estrogen isomers with the opposite, non-natural

M. Asim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3713–3717
3715
sium bromide with the cis-ketone 16. When applied to the trans
fused CD-ketone 20,16 this same sequence gave an excellent yield
of the initial adduct 21. Acid-catalyzed cyclization gave only the
spiro derivative 22 resulting from attack of the aromatic ring on
the carbocation intermediate from the sterically less hindered bot-
tom side, opposite the methyl group. Demethylation afforded 4,
whose structure was secured by X-ray structure (Fig. 3) determina-
tion (Scheme 2).
Since the acid-catalyzed cyclization-deprotection sequence
starting with 21 resulted only in the isomer 4, having the unnatural
configuration at C9, an alternate approach to the estradiol isomer
2, which has the trans CD ring fusion and thus most closely resem-
bles estradiol was required. As shown in Scheme 3, addition of the
species 15 to the unsaturated enone 2316 afforded the expected
product 24. Acid-catalyzed dehydration gave, somewhat surpris-
ingly, the
D
14,15 alkene 25 as a single diastereomer, which was re-
acted with mCPBA to give exclusively the epoxide 26, whose
structure was secured by X-ray crystallography (Fig. 3). This not
only verified the configuration at C9 relative to the substituents
in ring D but also showed that the C ring existed in the chair con-
formation. Acid-catalyzed rearrangement of the epoxide resulted
in a hydride shift to form the ketone 27, which has the trans CD
ring junction, as again verified by an X-ray crystal structure.
The conversion of 27 into 2 was accomplished by the following
sequence: NaBH4 reduction gave a mixture of alcohols, with the
major compound resulting from the introduction of hydride from
the same side as the angular methyl group; the C–H of this alcohol
showed coupling of 11 Hz coupled with the adjacent axial C-14
methine hydrogen. Conversion of this alcohol to its thiocarbonate
28 was quite troublesome and occurred in less than 20% yield
due to its highly hindered nature. Finally, reaction of 28 with
Bu3SnH catalyzed by AIBN and subsequent removal of both the
methoxy and acetate groups gave the desired 2. The deoxygenation
of an alcohol by the above sequence has been described numerous
times as a preferred method for converting an alcohol into the cor-
responding hydrocarbon.17
The synthesis of the oxa spiro compounds followed our
previous syntheses of the A-CD compounds (Scheme 4). The lithio
derivative derived from di-MOM protected 3-hydroxymethyl-4-
bromophenol, 29, was added with the cis-CD moiety 16. The
mixture of benzylic alcohols thus obtained was treated with TsOH
in methanol to afford a separable mixture of 10 and 13. A similar
sequence when applied to 30 gave 11 and the homolog of 13;
the ER binding of the latter compound was not determined
(Scheme 4).
Figure 3. ORTEP of compound 4 and compound 26.
estrogen configuration at the spiro carbon. These compounds, 4, 5,
7 and 13, show considerably reduced binding to ERa and ERb rela-
tive to compounds 2, 3 and 6 but comparable to the A-CD com-
pound 12 having the non-natural configuration at C9 (Figs. 1 and
2). The reduced binding affinities of these compounds compared
to the analogs with the natural configuration at C9 is expected
since the relationship of the hydroxyl groups, the key pharmaco-
phore groups, in these compounds, especially in 4, is considerably
altered relative to estradiol and to the higher affinity ligands, 2, 3,
6, 11 and 14.
The synthesis of the spiro estrogens was relatively straightfor-
ward, with the exception of spiro-estradiol, 2, which has the same
absolute configuration at all of its chiral carbons as estradiol (its
synthesis is described later). The preparation of the spiro deriva-
tives 3 and 5 commenced with the addition of 2-(30-methoxy-
phenyl)-1-ethanemagnesium bromide 15 to the saturated CD
ring ketone 16,14 affording adduct 17, which was cyclized to a mix-
ture of the spiro derivatives 18 and 19 upon exposure to MeSO3H
in nitromethane at ꢁ10 °C. These compounds were separated via
preparative HPLC. Reaction with NaSC2H5 in DMF at 160 °C for
10 min afforded in essentially quantitative yield compounds 3
and 5,15 respectively (Scheme 1).
The preparation of the spiro lactone 14 involved metallation of
the bromo amide 31 and coupling with 16 to afford a mixture of
the adducts 17. Acid catalyzed hydrolysis of 17 afforded a mixture
of spiro lactones from which pure 14 was obtained via preparative
HPLC (Scheme 5).
The present study represents the first systematic probe of the
estrogen receptors using isomers and simple analogs of estradiol
having fixed shapes very different from that of the fused and planar
tetracyclic steroid. The spiro compounds 2, 3, 6, 10, 12 and 14,
The spiro-6 analogs 6 and 7 were also prepared following the
sequence shown in Scheme 1 starting with the addition of the
homolog of 15, namely 3-(30methoxyphenyl)-1-propanemagne-
OH
OMOM
OH
HO
a
b
H
MeO
MgBr
H
H
+
H3CO
H3CO
15
19
17
18
3
OCH3
c
c
5
Scheme 1. Reagents and conditions: (a) compound 16 (91%); (b) CH3SO3H/CH3NO2/ꢁ10 °C (85%); (c) NaSC2H5/DMF/160 °C (95%).

3716
M. Asim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3713–3717
H3C
OH
OH
OTBS
OTBS
H
H
22
a
c
HO
b
H
O
H3CO
H
4
HO
20
OCH3
21
Scheme 2. Reagents and conditions: (a) compound 15 (90%); (b) CH3SO3H/CH3NO2/ꢁ10 °C (80%); (c) NaSC2H5/DMF/160 °C (93%).
OTBS
b, c
H3C
CH3
O
CH
3 OAc
OTBS
a
H3CO
H3CO
OAc
HO
d
9
9
O
26
25
H3CO
23
24
CH3
CH3
HO
OH
CH3
H3CO
H3CO
OAc
OAc
f , g
h, i
e
9
O
O
H
H
H
S
2
27
28
OPh
Scheme 3. Reagents and conditions: (a) compound 15 (90%); (b) CH3SO3H/CH3NO2/ꢁ10 °C (80%); (c) Ac2O/py(99%); (d) mCPBA/DCM (82%); (e) BF3Et2O/DCM (88%); (f)
NaBH4/CH3OH (96%); (g) Ph-O-C(S)-Cl/DMAP (18%); (h) AIBN/Bu3SnH (82%); (i) NaSC2H5/DMF/160 °C (97%).
CH3
OH
Br
HO
OH
+
HO
OMOM
a, b, c
CH3
( )n
MOMO
O
( )n
O
10
11
29, n = 1
30, n = 2
, n = 1
( )n
, n = 2
13, n = 1
Scheme 4. Reagents and conditions: (a) nBuLi/THF/ꢁ78 °C; (b) compound 16 (15% & 14% n = 1 & n = 2); (c) H(+)
.
OMOM
CH3
HO
Br
O
HO
OH
a, b
MOMO
c, d
NEt2
MOMO
O
C(O)NEt2
O
31
17
14
Scheme 5. Reagents and conditions: (a) nBuLi/THF/ꢁ78 °C; (b) compound 16 (30%); (c) H(+) (97%); (d) HPLC spearation.
which have the natural estrogen configuration at C9, show good
binding affinity when compared to estradiol, but unlike estradiol
they exhibit modest to good selectivity (3 to 14-fold) for ERb over
ERa. These results verify the previous hypotheses that the overall
United States National Institutes of Health [PHS R01DK015556 to
J.A.K.] is gratefully acknowledged. C.C. would like to thank Dr.
Vijayaratnam Santhakumar for helpful discussions during her
internship at Astra Zeneca in Montreal.
shape of the estrogen molecule can be changed quite substantially
without significantly lowering the binding affinity of such mole-
cules to the estrogen receptors, and they are also consistent with
evidence that the ligand binding pocket is plastic and develops a
defined shape only upon ligand binding. The spiro compounds with
inverted configuration at C9 relative to estradiol, on the other
hand, show much weaker binding affinity. These results should en-
able us to model the estrogen receptor–ligand interactions with
greater confidence and allow us to generate additional compounds
with more predictable and potentially stronger binding to the
estrogen receptors.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.04.
022.
References and notes
1. Anstead, G. M.; Carlson, K. E.; Katzenellenbogen, J. A. Steroids 1997, 62,
268.
2. Minutolo, F.; Macchia, M.; Katzenellenbogen, B. S.; Katzenellenbogen, J. A. Med.
Res. Rev. 2011, 31, 364.
3. Katzenellenbogen, J. A. J. Med. Chem. 2011, 54, 5271.
4. Blizzard, T. A.; Morgan, J. D.; Mosley, R. T.; Birzin, E. T.; Frisch, K.; Rohrer, S. P.;
Hammond, M. L. Bioorg. Med. Chem. Lett. 2003, 13, 479.
5. Watanabe, N.; Ikeno, A.; Minato, H.; Nakagawa, H.; Kohayakawa, C.; Tsuji, J. J.
Med. Chem. 2003, 46, 3961.
Acknowledgments
The support of this project by the Canadian Breast Cancer Foun-
dation and NSERC Canada [Discovery Grant 1169 to T.D.] and the

M. Asim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3713–3717
3717
6. Bailey, D. J.; Doggett, N. S.; Ng, L. Y.; Qazi, T. J. Med. Chem. 1976, 19, 438.
7. Lin, S.-K.; Rasetti, V. Helv. Chim. Acta 1995, 78, 857.
8. Asim, M.; El-Salfiti, M.; Qian, Y.; Choueiri, C.; Salari, S.; Cheng, J.; Shadnia, H.;
Bal, M.; Christine Pratt, M. A.; Carlson, K. E.; Katzenellenbogen, J. A.; Wright, J.
S.; Durst, T. Bioorg. Med. Chem. Lett. 2009, 19, 1250.
9. Wright, J. S.; Shadnia, H.; Anderson, J. M.; Durst, T.; Asim, M.; El-Salfiti, M.;
Choueiri, C.; Pratt, M. A.; Ruddy, S. C.; Lau, R.; Carlson, K. E.; Katzenellenbogen,
J. A.; O’Brien, P. J.; Wan, L. J. Med. Chem. 2011, 54, 433.
10. Carlson, K. E.; Choi, I.; Gee, A.; Katzenellenbogen, B. S.; Katzenellenbogen, J. A.
Biochemistry 1997, 36, 14897.
12. Katzenellenbogen, J. A.; Muthyala, R. Pure Appl. Chem. 2003, 75, 1797.
13. Nettles, K. W.; Bruning, J. B.; Gil, G.; O’Neill, E. E.; Nowak, J.; Guo, Y.; Kim, Y.;
DeSombre, E. R.; Dilis, R.; Hanson, R. N.; Joachimiak, A.; Greene, G. L. EMBO Rep.
2007, 8, 563.
14. Hajos, Z. G.; Parrish, D. R. Org. Synth. 1985, 63, 26.
15. All numbered products were characterized by high field 1H and 13C NMR and
HRMS were obtained for all of the target structures.
16. Micheli, R. A.; Hajos, Z. G.; Cohen, N.; Parrish, D. R.; Portland, L. A.; Sciamanna,
W.; Scott, M. A.; Wehrli, P. A. J. Org. Chem. 1975, 40, 675.
17. Luzzio, F. A.; Fitch, R. W. J. Org. Chem. 1999, 64, 5485.
11. Katzenellenbogen, J. A. Environ. Health Perspect. 1995, 103, 99.