Synthesis and receptor binding in trans-CD ring-fused A-CD estrogens: Comparison with the cis-fused isomers

Article abstract of DOI:10.1016/j.bmcl.2014.06.066

Ligands which selectively activate only one of the estrogen receptors, ERα or ERβ, are current pharmaceutical targets. Previously, we have reported on substituted cis A-CD ligands in which the B-ring of the steroidal structure has been removed and cis refers the stereochemistry of the CD ring junction as compared to trans in estradiol. These compounds often showed good potency and selectivity for ERβ. Here we report the synthesis and binding affinities for a similar series of trans A-CD ligands, and compare them to the cis-series. Counterintuitively, trans A-CD ligands, which are structurally more closely related to the natural ligand estradiol, show weaker binding and less β-selectivity than their cis-counterparts.

Full text of DOI:10.1016/j.bmcl.2014.06.066

Bioorganic & Medicinal Chemistry Letters 24 (2014) 3841–3844
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and receptor binding in trans-CD ring-fused A-CD
estrogens: Comparison with the cis-fused isomers
Cristian Dabrota a, Muhammad Asim a, Christine Choueiri a, Ana Gargaun a, Ilia Korobkov a, Ammara Butt a,
Kathryn E. Carlson b, John A. Katzenellenbogen b, James S. Wright c, Tony Durst a,
⇑
a Department of Chemistry, University of Ottawa, D’Iorio Hall, 10 Marie Curie St., Ottawa K1N 6N5, Canada
b Department of Chemistry, University of Illinois, Urbana, IL 61801, USA
c Department of Chemistry, Carleton University, 1125 Colonel By Dr., Ottawa K1S 5B6, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Ligands which selectively activate only one of the estrogen receptors, ERa or ERb, are current pharmaceu-
Received 10 March 2014
Revised 19 June 2014
Accepted 20 June 2014
Available online 27 June 2014
tical targets. Previously, we have reported on substituted cis A-CD ligands in which the B-ring of the ste-
roidal structure has been removed and cis refers the stereochemistry of the CD ring junction as compared
to trans in estradiol. These compounds often showed good potency and selectivity for ERb. Here we report
the synthesis and binding affinities for a similar series of trans A-CD ligands, and compare them to the
cis-series. Counterintuitively, trans A-CD ligands, which are structurally more closely related to the
natural ligand estradiol, show weaker binding and less b-selectivity than their cis-counterparts.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Estrogen receptor
ER
a
ERb
b-Selectivity
Ligand synthesis
Current studies of the mechanism of disease initiation in breast
cancer and several other cancers have focused on the pathway
starting from the natural estrogen 17b-estradiol (E2), which by
P450 aromatic hydroxylation can form the catechol estrogen and
potentially the corresponding ortho-quinones.1 Of various possible
metabolites, the 3,4-quinone has been shown to intercalate into
DNA and undergo Michael addition with DNA bases, ultimately
forming depurinating adducts. DNA repair then leads to a high
mutation rate and tumor initiation.2 Once initiated, activation of
even stronger than the naturally occurring ligand E2, which has
the highest affinity of the natural ligands. We also observed some,
and at times considerable, enhancement of ERb- receptor binding
selectivity. In general, the compounds which showed this type of
binding selectivity also acted as strong b-selective agonists; such
compounds are denoted ‘‘super-agonists’’ when they exceed the
maximum level of transcription activation by E2.9 In our initial
report,6 we presented these compounds as having the general
structure 2 (see Fig. 1), where the CD ring junction is trans as in
E2. Subsequently,7,8 we corrected the structure assignment and
showed that the compounds we had prepared actually had the
cis-CD ring structure, as shown below in the parent A-CD com-
pound for series 1 (see Scheme 2).
We now present our work on the preparation of a series of com-
pounds of structural type 2 which have the trans-CD ring junction.
We compare their binding affinity to both estrogen receptors with
the corresponding cis-compounds in series 1. One might expect
that retaining the geometry of the natural ligand at the CD inter-
face would lead to optimum binding of the A-CD system. However,
we will show that the unintended synthesis of compounds with
structure 1 rather than 2 turns out to have been quite fortuitous,
since the A-CD compounds that most closely resemble E2 generally
bind less strongly and with lower selectivity to ERb than those in
the cis-series 1. As before, for convenience we have chosen the
the estrogen receptor ER
a by endogenous ligands such as E2 has
been demonstrated to promote proliferation of the cancer cells.3
On the other hand, in some experiments activation of the receptor
ERb may suppress cancer cell proliferation,4 hence the current
intense interest in developing ERb-selective ligands.5
We have recently reported the synthesis of a series of estrogen
agonists represented by structure 1 shown in Figure 1, in which the
A ring and C rings are connected by a single bond,6–8 which we
called A-CD estrogens. These compounds, especially those having
electronegative substituents at C5, showed high affinity binding
to the estrogen receptors (ERs), which for ERb was in some cases
Abbreviations: RBA, relative binding affinity; ER, estrogen receptor; E2,
17b-estradiol.
⇑
Corresponding author. Tel.: +1 (613)562 5800x6072; fax: +1 (613)562 5170.
E-mail address: tony.durst@uottawa.ca (T. Durst).
http://dx.doi.org/10.1016/j.bmcl.2014.06.066
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

3842
C. Dabrota et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3841–3844
OH
17
OH
17
OH
17
H3C
H3C
H3C
11
11
11
1
1
1
H
C
C
D
9
16
D
16
9
16
9
R2
R2
15
15
15
8
8
8
H
H
H
A
A
3
H
H
H
3
3
R5
HO
R5
HO
HO
R4
R4
trans 17β-estradiol, E2
cis A-CD, series 1
trans A-CD, series 2
Figure 1. Stereochemical relationship between the natural estrogen E2, the A-CD estrogens, cis-series 1 and the trans-series 2. The cis and trans designations refer to the
stereochemistry at the CD ring junction.
numbering in these compounds to correspond to the same num-
bering scheme as in E2 (see Fig. 1).
which was hydrogenated to give a mixture of compounds from
which 2i was obtained after reverse phase HPLC purification.
As pointed out above, the isomeric compounds 1 and 2 were
most readily distinguished by focusing on the quaternary methyl
group. In all of the compounds described these methyl hydrogens
absorbed in the 0.83–0.85 ppm range for the trans-compounds
but more downfield at 1.09–1.11 ppm for the cis-isomers. In the
13C spectrum this carbon was found near 12 ppm in the trans-com-
pounds but again considerably more downfield near 20 ppm for
the corresponding cis-isomers.10,11 Many of the trans compounds
were synthesized starting with the trans hydridanone 4, so the
trans CD stereochemistry is unambiguous. The others were pre-
pared by hydrogenation of the diene 10 and finally several includ-
ing the parent compound were prepared via both routes. In all
cases the trans ACD compounds showed clear and consistent pro-
ton and carbon spectra differences, as described above.12,13 For
structural details, see Supplementary data.
The trans-CD ring junction in the structures for series 2 was
installed in one of two ways.10 In the first approach (Scheme 1),
the CD ring ketone 4 having the trans-ring junction was prepared
following the method of Micheli et al.11 This ketone was then
reacted with the lithio-derivative of a suitably protected ring A
(moiety 5) to give the alcohol 6. Dehydration, with concomitant
removal of the 3-phenol and 17-alcohol protecting groups, gener-
ally MEM-ethers, gave exclusively the alkene 7. The typical overall
yield over these two steps was in the 80–90% range. Simple hydro-
genation with Pd/C as catalyst afforded quantitatively a mixture of
isomers at C9 from which the major desired isomer 2 was isolated
by preparative HPLC. The structure of the parent compound in the
trans-series was verified by an X-Ray structure determination,
shown in Figure 2 for 2a, the trans A-CD parent compound.
The 1H NMR spectrum of 2a showed the C13 methyl group at
0.83 ppm; this compared with 1.05 ppm for the cis-CD-fused deriv-
ative 1. This difference was consistent for all other pairs of com-
pounds described herein. 13C NMR also showed significant
differences for the C17, which helped subsequently to verify
whether either the trans- or the cis-CD ring juncture was present
in derivatives of 1 and 2.
Alternatively, reaction of 5 with the MEM-protected enone 8
yielded the allylic alcohol 9. Acid-catalyzed dehydration with con-
comitant deprotection afforded the diene 9 (Scheme 2) in excellent
yield. Hydrogenation with a variety of common catalysts, including
Pd/C, gave mainly the compound 2, as shown by 1H NMR examina-
tion of the crude reaction product. Small amounts of the three
other possible stereoisomers were also formed. The formation of
2 as the major product in the hydrogenation of 10 indicates that
the C14–C15 double bond is hydrogenated first, with preferential
delivery of hydrogen from the less hindered side, to give the
C8–C11 alkene 11 having the trans-CD ring junction. Subsequent
hydrogenation gives, as expected, mainly the isomer 2 with the
(S) configuration at C9. In contrast, hydrogenation of the cis
CD-junctioned alkene 12 gives a close to a 1:1 mixture of stereoiso-
mers at C9.
Binding affinities to the estrogen receptors ER
a and ERb are
given as relative binding affinities (RBAs), where the RBA for estra-
diol on both ERs is 100. The RBA value and selectivity ratio
RBA(ERb)/RBA(ERa) for a series of 10 cis A-CD (series 1) and 10
trans A-CD compounds (series 2) containing saturated C-rings are
given in Table 1. The binding affinity ratios RBA(trans)/RBA(cis)
for both receptors are given in Table 2.
The values of the RBAs shown in Table 1 are correlated via a sig-
moidal dependence with the transcription activation, or ‘potency’,
of each ligand in its receptor,8 so the RBAs are already useful pre-
dictors of potential drug activity. With four distinct data sets (cis-
and trans-ligands in two receptors, ER
ber of interesting comparisons that can be made. First, consider the
selectivity of trans versus cis ligands binding to ER and ERb. The
b-selectivity of the parent trans compound 2a, for example, is given
by the b/ ratio = 10/2.38 = 4.2 (see Table 1), whereas for the
cis compound 1a it is 21.5/1.47 = 14.6, over three times larger.
Looking at the selectivity ratios b/ for each series, the average
a and ERb) there are a num-
a
a
a
value for the ligands in the trans-series is 3.7, whereas for the
cis-series it is 9.1. Thus, the cis-series is much more b-selective
than the trans-series.
Either of the above sequences or slight variations thereof were
used to prepare a series of 10 derivatives carrying a variety of sub-
stituents in the A-ring.10 For example, the 5-hydroxy derivative 2i
was obtained in three steps by first converting 1,3-dibenzyloxy-4-
bromobenzene into its lithio-derivative and reaction of this species
with the enone 8. Purification of the reaction mixture via silica gel
chromatography (Scheme 3) resulted in formation of the diene 11,
It is also of interest to compare the magnitude of the RBAs of the
trans versus the cis compounds in Table 1. For example, comparing
the trans/cis RBA ratio for compound c (2c/1c) in ERa, the ratio is
4.22/27.3 = 0.15 (see Table 2); the cis-structure is much more
strongly bound. Continuing in this way, the average ratio for
trans/cis binding into ER
the binding of the cis-compounds into ER
a
for the 10 ligands is 0.48, showing that
is, generally speaking,
a
Scheme 1.

C. Dabrota et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3841–3844
OMEM
3843
CH3
OMEM
CH3
Li
THF
-78 0
H(+)
HO
+
C
MEMO
O
9
5
8
MEMO
OH
CH3
H3C
CH3
CH3
HO
OH
OH
H2
H2
OH
Ar
Ar
Pd/C
Pd/C
Ar
H
H
H
11
2
12
10
Scheme 2.
Table 2
Ratio of binding to ERa and ERb for the trans-compounds 2 versus the cis-compounds 1
Compound
ER
a
ERb
a(Parent, R = H)
b(4-F)
c(5-F)
d(5-CH3)
e(5-CF3)
f(4,5-DiF)
g(2,4,5-TriF)
h(2,4-DiF 5-CH3)
i(5-OH)
1.62
1.58
0.15
0.17
0.054
0.20
0.16
0.11
0.14
0.63
0.48
0.47
0.79
0.10
0.04
0.024
0.17
0.056
0.042
0.038
0.30
Figure 2. X-ray structure of compound 2a, the trans A-CD parent compound.
Cambridge Crystallographic Data Centre deposition no. is CCDC 1007460.
over twice as strong as that of the trans-compounds. Performing
the same operations on binding to the ERb receptor, the trans/cis
ratio for compound a (2a/1a) is 10/21.5 = 0.47. The average for
the whole series is only 0.20, so binding to the ERb receptor favors
the cis-structures by a factor of five.
j(5-OCH3)
Average b/
a
ratio
0.20
It should also be pointed out that in the cis series 1, the com-
pounds such as 1c (5-F) and 1e (5-CF3), which showed the highest
binding affinities, also had the lowest b/a selectivity, 5.0 and 2.3,
respectively. In contrast, some ligands with more modest binding
affinities to both receptors 1a (parent) and 1i (5-OH) showed the
these large differences by examining the interaction of these sub-
stituents with receptor residues, work that we have already begun.
Since the results presented above were so unexpected, we
looked at two more series of compounds with the trans-, cis-CD
ring fusion (Series 7 and Series 14, Table 3). These were prepared
during the course of our studies and are C9–C11 alkenes having the
trans CD ring fusion (Series 7) and the cis fusion (Series 14). The
compounds in Series 7 were obtained as intermediates in the syn-
thesis via Scheme 2; those in Series 14 were formed in the prepa-
ration of the cis A-CD compounds.10 As shown in Table 3, even
maximum selectivity for the ERb to ER
a ratio of ca. 15. The very
low binding affinity of the 5-OCH3 derivatives 1j relative to those
carrying CF3, CH3 and OH groups at this position was unexpected.
For comparison, the binding affinity to ERb of 1e (5-CF3) is about
1700 times greater than that of 1j. A similar comparison for 2e ver-
sus 2j gives a value of close to 130. It should be possible to explain
OH
OMEM
1.nBuLi
Br
H
H2/Pd
H
BnO
OBn
2. 8
2i
13
Scheme 3.
HO
OH
BnO
OBn
Table 1
Relative binding affinities for cis-series 1 and trans-series 2 to estrogen receptors ER
a
and ERb, selectivity ratio for ERb/ER
a
. Error bars in parentheses
Compound
cis A-CD structures (1)
RBA(b)
trans A-CD structures (2)
RBA(b)
RBA(
a)
b/
a
RBA(
a)
b/a
a(H)
b(4-F)
c(5-F)
d(5-CH3)
e(5-CF3)
f(4,5-diF)
g(2,4,5-triF)
h (2,4-diF 5-CH3)
i(5-OH)
j(5-OCH3)
Estradiol (E2)
1.47(0.26)
1.04(0.09)
27.3(0.70)
2.82(0.45)
89.7(13.8)
4.62(0.93)
0.186(0.01)
0.75(0.20)
0.26(0.07)
0.016(0.002)
21.5(4.6)
8.7(1.5)
135.5(7.3)
33.6(6.2)
205(23)
42.8(5.5)
1.73(0.02)
5.35(0.88)
3.97(0.08)
0.123(0.03)
14.6
8.4
5
11.9
2.3
9.3
9.3
7.1
15.3
7.7
2.38(0.19)
1.68(0.15)
4.22(0.06)
0.47(0.1)
4.8(1.1)
10(1.3)
4.2
4.1
3.2
2.8
1
7.9
3.3
2.8
4.1
3.7
1
6.84(0.41)
13.6(0.35)
1.3(0.3)
4.9(0.1)
7.3(1.8)
0.097(0.025)
0.226(0.04)
0.15(0.04)
0.037(0.008)
100
0.92(0.15)
0.029(0.004)
0.08(0.007)
0.037(0.003)
0.010(0.001)
100
Average b/
a
ratio*
9.1
3.7
*
E2 is not included in the averages.

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C. Dabrota et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3841–3844
Table 3
Relative binding affinities for cis-series 14 and trans-series 7 to estrogen receptors ER
a and ERb, and selectivity ratio for ERb/ERa
Compound
cis A-CD structures (14)
RBA(b)
trans A-CD structures (7)
RBA(
a
)
b/
a
RBA(
a)
RBA(b)
b/a
a(H)
b(5-F)
c(5-Cl)
d(5-CF3)
e(5-OCH3)
f(2,4,5-triF)
g (2,5-DiF)
h(4-F,5-Cl)
i(4,5-DiCl))
j(2,4-DiF 5-Cl)
0.39(0.04)
4.5*(0.8)
60(8)
122(11)
0.004(0.001)
—
0.44(0.02)
50(6)
3.0(0.8)
3.35(0.06)
6.8(0.6)
49*(7)
118(4)
174(8)
0.022(0.004)
—
2.7(0.5)
149(9)
55(9)
17
11*
2
1.4
5.5
—
6.1
3
18.3
1.8
6.6
2.38(0.19)
21.7(0.4)
9.7(2.2)
17.3(2.4)
38(11)
14.1(3.8)
—
0.029(0.002)
0.076(0.007)
4.7
1.7
1.5
—
4.8
0.8
—
0.006(0.001)
0.099(0.03)
6(2)
Average b/
a
ratio
2.7
*
Determined for a ꢀ1:1 mixture of 8–9 and 9–11 alkenes.
though relatively few pairs are currently available, the trends dis-
cussed above are also seen for these sets of isomers. Thus, the aver-
Supplementary data
age b/a selectivity ratio for the cis-series 7 (6.6) is higher than that
Supplementary data (spectra of the trans-compounds) associ-
ated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmcl.2014.06.066.
for the trans-series 14 (2.7) by a factor of 2.4; this is identical to the
ratio for the cis-series 1 and trans-series 2 (factor of 2.4). Since the
alkene series show no special advantages over the alkanes and is
more likely to be reactive in vivo, and since the cis-series 1 shows
improved b-selectivity and binding affinity compared to the trans-
series 2, future drug development using these compounds will be
focused on the cis-series 1.
References and notes
1. Cavalieri, E. L.; Rogan, E. G.; Chakravarti, D. Cell. Mol. Life Sci. 2002, 59, 665.
2. Li, K.-M.; Todorovic, R.; Devanesan, P.; Higginbotham, S.; Kofeler, H.;
Ramanathan, R.; Gross, M. L.; Rogan, E. G.; Cavalieri, E. L. Carcinogenesis 2004,
25, 289.
3. Zahid, M.; Kohli, E.; Saeed, M.; Rogan, E.; Cavalieri, E. Chem. Res. Toxicol. 2006,
19, 164.
4. Strom, A.; Hartman, J.; Foster, J. S.; Kietz, S.; Wimalasena, J.; Gustafsson, J.-A.
Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 1566.
5. Minutolo, F.; Macchia, M.; Katzenellenbogen, B. S.; Katzenellenbogen, J. A. Med.
Res. Rev. 2011, 31, 364.
H3C
H
H3C
H
OH
17
OH
11
9
11
9
HO
HO
6. Asim, M.; El-Salfiti, M.; Qian, Y.; Choueiri, C.; Salari, S.; Cheng, J.; Shadnia, H.;
Bal, M.; Pratt, M. A. C.; Carlson, K. E.; Katzenellenbogen, J. A.; Wright, J. S.; Durst,
T. Bioorg. Med. Chem. Lett. 2009, 19, 1250.
6
trans-series 7
cis-series 14
7. Erratum to Ref. in Bioorg. Med. Chem. Lett. 2010, 20, 6861.
8. Wright, J. S.; Shadnia, H.; Anderson, J. M.; Durst, T.; Asim, M.; El-Salfiti, M.;
Choueiri, C.; Pratt, M. A. C.; Ruddy, S. C.; Lau, R.; Carlson, K. E.;
Katzenellenbogen, J. A.; O’Brien, P. J.; Wan, L. J. Med. Chem. 2011, 54, 433.
9. Min, J.; Wang, P.; Srinivasan, S.; Nwachukwu, J. C.; Guo, P.; Huang, M.; Carlson,
K. E.; Katzenellenbogen, J. A.; Nettles, K. W.; Zhou, H.-B. J. Med. Chem. 2013, 56,
3346.
We have begun a series of modeling studies aimed at understanding
the trends in reactivity described above.8,14 In future work we will
focus on understanding the origins of the ERb-selectivity described
in this paper, as well as the preference for the cis- rather than the
10. Choueiri, C. (Ph.D thesis). University of Ottawa, 2013.
11. 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.
12. The preparation of the A ring precursor bromides is described in Ref.
trans-geometry for the binding affinity in the receptors ER
a
and
8
.
13. All new compounds were characterized by high field 1H and 13C NMR and
HREIMs. For example, for the 5-OH derivative, 2i: 1H NMR (400 MHz, acetone-
d6) d 8.11 (s, 1H, OH) 8.00 (s,1H, OH), 6.97 (d, J = 8.3 Hz, 1H), 6.36 (d, J = 2.4 Hz,
1H), 6.20 (dd, J = 8.3, 2.4 Hz, 1H) 3.69–3.61 (m, 2H, H17 and O-H)), 2.88–2.74
(m, 1H), 2.05–1.89 (m, 1H), 1.84 (dt, J = 12.5, 3.2 Hz, 1H), 1.74–1.27 (m, 8H),
1.17(dt, 12.7, 4.7 Hz, 1H), 0.84 (s, 3H). 13C NMR (100 MHz, acetone-d6 d 157.7,
157/1, 129.3, 126.3, 108.3, 104.4, 82.8, 47.5, 44.4, 39.4, 39.1, 34.0, 31.9, 30.2,
27.3, 11.9. HRMS calc’d for C16H22O3: 262.1569. Found: 262.1570.
ERb.
Acknowledgments
We thank the Canadian Breast Cancer Foundation (Ontario
Region) for grants to J.S.W. and T.D. which supported this research.
Support from the US National Institutes of Health (PHS 5R01
DK015556 to J. A.K.) is also gratefully acknowledged.
14. Wright, J. S.; Anderson, J. M.; Shadnia, H.; Durst, T.; Katzenellenbogen, J. A. J.
Comput. Aided Mol. Des. 2013, 27, 707.