Tetrahydrofluorenones with conformationally restricted side chains as selective estrogen receptor beta ligands

Article abstract of DOI:10.1016/j.bmcl.2006.06.043

A series of 2-9a bridged tetrahydrofluorenone derivatives were prepared which exhibited significant binding affinity for ERβ and were highly selective.

Full text of DOI:10.1016/j.bmcl.2006.06.043

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4462–4466
Tetrahydrofluorenones with conformationally restricted side chains
as selective estrogen receptor beta ligands
Kenneth J. Wildonger,a,* Ronald W. Ratcliffe,a Ralph T. Mosley,a
Milton L. Hammond,a Elizabeth T. Birzinb and Susan P. Rohrerb
aDepartment of Medicinal Chemistry, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
bDepartment of Atherosclerosis & Endocrinology, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
Received 20 April 2006; accepted 12 June 2006
Available online 11 July 2006
Abstract—A series of 2–9a bridged tetrahydrofluorenone derivatives were prepared which exhibited significant binding affinity for
ERb and were highly selective.
Ó 2006 Elsevier Ltd. All rights reserved.
The estrogen receptor (ER) is a member of the super-
family of nuclear hormone receptors which act as ligand
activated transcriptional factors.1 The estrogen receptor
was first cloned in 1986 and, at the time, was thought to
be the exclusive receptor mediating physiological
responses to estradiol.2,3 In 1996 a second estrogen
receptor was discovered and identified as ERb,4,5 the
original cloned receptor being named ERa. It was found
that ERb is widely expressed in a variety of tissues,
including lung, prostate, ovarian granulosa cells, and
brain, but is not the dominant ER expressed in the uter-
us or breast.6,7 The discovery of ERb and its differential
tissue expression stimulated efforts both to define the rel-
ative physiological roles of ERa and ERb and to identi-
fy subtype selective ligands.
Several groups have disclosed efforts to design ERb sub-
type selective ligands working from a variety of diverse
structural platforms including tetrahydrochrysenes,9
6H-benzo[c]chromen-6-ones,10 androstenediols,11 diaryl-
propionitriles,12 benzimidazoles,13 arylbenzothiophenes,14
arylbenzoxazines,15 triazines,16 biphenyls,17,18 aryl diphen-
olic azoles,19 and 2-phenyl-benzofurans.20
O
O
O
Me
Me
R
2
9a
R1
HO
HO
HO
H
R2
R1
R1
1 R=Me, R1=Me
2 R=Me, R1=Et
3a R=Me, R1=Pr
3b R=Br, R1=Pr
6 R1=H, R2=H
7a R1=Et, R2=H
7b R1=H, R2=Et
8a R1=Pr, R2=H
8b R1=H, R2=Pr
4 R1=H
5 R1=Pr
Both estrogen receptors (human) show substantial
homology in the DNA binding domain (96%) and, to
a lesser extent (58%), in the ligand binding domain
(LBD).5 The ligand binding pocket differs in only two
amino acids; the ERb binding pocket has a Met336
replacing a Leu384 in ERa and an Ile373 replacing a
Met421.8 Given the subtle differences in the LBD it is
not surprising that estradiol binds equally well to both
receptors.
Our effort in this area has focused on the tetrahydro-
fluorenone platform. We recently disclosed a new class
of tetrahydrofluorenone compounds as potent ERb
selective agonists (e.g. 1–3).21 Within this series the 9a
alkyl substituent of these compounds was shown to be
a key determinant of binding potency and selectivity.
In addition, the 9a-(S) configuration of this substituent
was found to be essential for good activity. In this re-
port, we have explored the effect of conformationally
restricting this key binding element by incorporating it
into a constrained ring system. The derivatives prepared
include the simple ethylene and propylene bridged com-
pounds 4 and 6. These can be viewed as conformation-
Keywords: ERb; ERa; Tetrahydrofluorenones.
*
Corresponding author. Tel.: +1 732 594 7937; fax: +1 732 594
9556; e-mail: ken_wildonger@merck.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.06.043

K. J. Wildonger et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4462–4466
4463
O
O
ally restricted extensions of compounds 1 and 2.
Building upon these two derivatives, pendant alkyl
chains were incorporated in analogs 5, 7a, and 8a. The
comparison of compounds 4 and 6 to the crystallo-
graphically determined conformation of 3b in complex
with hERb21 (Fig. 1) indicated that the pendant alkyl
chains would have to be attached exo to the bridged
ring platform in order to map the substituents to the
butyl of 3b.
a or b
c,d,e
CH2(CH2)nOH
MeO
MeO
10a n=1
10b n=2
9
O
O
Me
Me
f,g,h
CH2(CH2)nOH
(CH2)n
MeO
MeO
The synthetic routes to racemic bridged analogs 4 and 6
are described in Scheme 1. With the exception of the first
step, the routes to the two analogs are similar. The route
to 4 started with the reductive alkylation of 5-methoxy
indanone 9 with commercially available glycoaldehyde
dimer to give the 2-alkylated indanone 10a. The route
to 6 began from the alkylation of 9 with 3-bromotriethy-
lsilyloxypropane22 to give 10b. Robinson annulation
conditions employing ethyl vinyl ketone (EVK) with
10a and 10b, followed by cyclization under acidic condi-
tions, and deacetylation of the resulting acetates gave
the alcohols 11a and 11b. Mesylation of 11a and 11b,
followed by their conversion to iodides and cyclization
with LDA gave the products 12a and 12b. The final step
involved demethylation of the aromatic methyl ethers
and was accomplished with aluminum chloride and eth-
anethiol to give phenols 4 and 6.
12a n=1
12b n=2
11a n=1
11b n=2
O
Me
i
(CH2)n
HO
4 n=1
6 n=2
Scheme 1. Reagents and conditions: (a) glycoaldehyde dimer,
NaOMe, H2, 10% Pd/C, MeOH, rt; (b) 1-bromo-3-triethylsilyloxypro-
pane, NaH, DMF, rt; (c) EVK, NaOMe, MeOH, rt; (d) 6 N HCl,
HOAc, 80 °C; (e) NaOMe, MeOH, rt; (f) MsCl, Et3N, CH2Cl2, 0 °C;
(g) NaI, acetone, reflux; (h) LDA, THF, À78 °C to rt; (i) AlCl3, EtSH,
CH2Cl2, rt.
O
As mentioned above, the preparation of the chiral ethyl-
ene bridged compound 5 bearing a pendant propyl chain
was guided by the need to establish the two contiguous
stereochemical centers. Thus, the stereochemistry of the
pendant propyl chain was established at the start with
the alkylation of (S)-3-propylbutyrolactone23 with
2-methoxy benzylbromide to give 13 (see Scheme 2).
Internal Friedel–Crafts alkylation of 13 in polyphos-
phoric acid gave indanone 14 in low yield. Michael addi-
tion of EVK in the Robinson annulation sequence gave
diastereomers 15a (21%) and 15b (57%). The desired dia-
stereomer 15a24 was processed following chemistry pre-
viously described to give 16. Demethylation of 16
afforded the desired product 5.
O
O
O
b
OH
H
Pr
a
O
MeO
MeO
H
Pr
c
H
Pr
13
14
O
O
O
O
Et
Et
+
MeO
MeO
OH
OH
H
H
Pr
Pr
15a
15b
O
O
Me
Me
i
d,e,f,g,h
15a
MeO
HO
H
H
Pr
Pr
16
5
Scheme 2. Reagents and conditions: (a) LDA, 3-methoxybenzyl
bromide, THF, À78 °C; (b) PPA, 100 °C; (c) EVK, NaOMe, MeOH,
60 °C; (d) 6 N HCl, HOAc, 80 °C; (e) 6 N HCl, MeOH, rt; (f) MsCl,
Et3N, CH2Cl2, 0 °C; (g) NaI, acetone, reflux; (h) LDA, THF, À78 °C
to rt; (i) AlCl3, EtSH, CH2Cl2, rt.
The relative stereochemical assignment of 5 was
1
made on the basis of H NMR25 utilizing COSY and
NOESY experiments (see Fig. 2 for key NOESY
interactions).26
The racemic propylene analogs 7a, 7b, 8a, and 8b were
prepared as described in Scheme 3 following chemistry
similar to that previously described. Michael addition
of the appropriate acrylate to indanone 9 gave the alkyl-
ated indanones 16a and 16b. Reduction of the esters
Figure 1. Superposition of the non-bridged 3b (green) as determined in
a crystallographic complex with hERb and modeled bridged com-
pounds 4 (yellow) and 6 (purple).

4464
K. J. Wildonger et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4462–4466
Table 1. Binding affinities27
O
H7
H8a
H8b
H11b
H3C
H10b
Compound
Human ERa
IC50 (nM)
Human ERb
IC50 (nM)
Selectivity
ERa/ERb
H4
H1
H11a
H3
HO
1
1210
455
567
793
97
28
14
19
12
1
43
33
30
66
97
37
29
7
H9
Hb
2
3aa
Hd
H10a
Ha
4
Hc
5a
6
CH3
414
146
310
84
11
5
Figure 2. Key 1H NOESY interactions observed.
7a
7b
8a
8b
45
5
16
4
O
OH
O
605
141
CO2R'
c,d
a or b
a Chiral, all others racemates.
9
R
R
MeO
MeO
MeO
16a R=Et, R'=Me
16b R=Pr, R'=Et
17a R=Et
17b R=Pr
Analog 5, which incorporates a pendant propyl chain in
the ethylene bridge, shows significantly more potency in
ERb binding and more selectivity than its parent 4.
Moreover, compound 5 with its conformationally
restricted side chain shows a greater than 10-fold
enhancement in ERb potency and more than a doubling
of selectivity relative to its unconstrained analog 3a.
O
O
Me
O
Et
f,g
e
OH
MeO
OH
R1
R2
R2 R1
18a R1=Et, R2=H
18b R1=H, R2=Et
19a R1=Pr, R2=H
19b R1=H, R2=Pr
20a R1=Et, R2=H
20b R1=H R2=Et
21a R1=Pr, R2=H
21b R1=H R2=Pr
The binding results shown in Table 1 for the pairs of
compounds 7a, 7b, and 8a, 8b clearly confirm the mod-
eling prediction of a preference for exo substitution on
the bridge and show the significant effect that orienta-
tion of the pendant chain has on binding affinity
towards both ERa and ERb. The more active propylene
bridged analogs 7a and 8a, incorporating either a pen-
dant ethyl or propyl chain in the exo orientation,
showed equal ERb binding, although the selectivity of
7a was approximately twice that of 8a. Both of these
analogs were less potent in ERb binding and significant-
ly less selective than the ethylene analog 5 with a pen-
dant propyl chain.
O
Me
h,i,j,k
HO
R2
R1
7a R1=Et, R2=H
7b R1=H R2=Et
8a R1=Pr, R2=H
8b R1=H, R2=Pr
Scheme 3. Reagents and conditions: (a) LDA, methyl 2-pentenoate,
THF, À78 °C; (b) LDA, ethyl 2-hexenoate, THF, À78 °C; (c) LAH,
THF, 0 °C; (d) Jones reagent, acetone, À78 °C; (e) EVK, NaOMe,
MeOH, 60 °C; (f) 6 N HCl, HOAc, 80 °C; (g) 6 N HCl, MeOH, rt; (h)
MsCl, Et3N, CH2Cl2, 0 °C; (i) NaI, acetone, reflux; (j) LDA, THF,
À78 °C; (k) AlCl3, EtSH, CH2Cl2, rt.
As has been described previously for the tetrahydro-
fluorenone lead class,21 we believe that the ERb selectiv-
ity for the bridged variant arises principally from two
sources. The first is the planar nature of the tetrahydro-
fluorenone core which is maintained in the bridged sys-
tem. This putative stabilizing interaction between the
planar/aromatic surface of the tricyclic platform and
Met336 of hERb is not possible with the analogous
Leu384 in hERa (see Fig. 3). This feature appears to
be common to other planar ERb selective molecules
including some phytoestrogens such as genistein28 or
other synthetic molecules including some benzisoxaz-
oles,19 for example.
16a and 16b also resulted in the reduction of the inda-
none ketone to give diols which were reoxidized to pro-
vide the requisite indanones 17a and 17b. Robinson
annulation conditions using EVK with indanone 17a
gave diastereomers 18a (31%) and 18b (44%). Similar
reaction of 17b gave diastereomers 19a (21%) and 19b
(52%). Cyclization of the alcohols 18a, 18b, 19a, and
19b gave the tetrahydrofluorenone acetates which were
deacetylated to give the alcohols 20a, 20b, 21a, and
21b, respectively. Following established chemistry
these intermediates were converted to 7a and 7b, and
8a and 8b.
The second selectivity determinant postulated for the
tetrahydrofluorenone class is a favorable hydrophobic
interaction of the 9a-alkyl substituent which protrudes
orthogonally from the plane of the tricyclic core
toward Ile373 in hERb as depicted in Figure 3. We
speculate that Ile373 in hERb can nicely accommodate
the presence of the alkyl substituent into space which
is not available in hERa because the side chain of the
analogous Met421 fills this space. Fixing the 9a-alkyl
substituent into the proper orientation as in com-
pound 5 may enhance this favorable hydrophobic
The ER binding results of analogs prepared are shown
in Table 1. Comparison of the ethylene bridged analog
4 to the unbridged ethyl analog 1 shows at least equiva-
lent or slightly better ERb binding with an improvement
in selectivity. Comparison of the propylene bridged ana-
log 6 to its unbridged equivalent 2 shows equal ERb
binding and selectivity. Both the ethylene 4 and propyl-
ene 6 bridged analogs have similar ERb binding,
although 4 shows better selectivity.

K. J. Wildonger et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4462–4466
4465
9. Meyers, M. J.; Sun, J.; Carlson, K. E.; Katzenellenbogen,
B. S.; Katzenellenbogen, J. A. J. Med. Chem. 1999, 42,
2456.
10. Sun, W.; Cama, L. D.; Birzin, E. T.; Warrier, S.; Locco,
L.; Mosley, R.; Hammond, M. L.; Rohrer, S. P. Bioorg.
Med. Chem. Lett. 2006, 16, 1468.
11. Blizzard, T. A.; Gude, C.; Morgan, J. D.; Chan, W.;
Birzin, E. T.; Mojena, M.; Tudela, C.; Chen, F.; Knecht,
K.; Su, Q.; Kraker, B.; Mosley, R. T.; Holmes, M. A.;
Sharma, N.; Fitzgerald, P. M. D.; Rohrer, S. P.;
Hammond, M. L. Bioorg. Med. Chem. Lett. 2006, 16, 834.
12. Meyers, M. J.; Sun, J.; Carlson, K. E.; Gwendolyn, M. A.;
Katzenellenbogen, B. S.; Katzenellenbogen, J. A. J. Med.
Chem. 2001, 44, 4230.
13. Chesworth, R.; Wessel, M. D.; Heyden, L.; Mangano, F.
M.; Zawistoski, M.; Gegnas, L.; Galluzzo, D.; Lefker, B.;
Cameron, K. O.; Tickner, J.; Lu, L.; Castleberry, T. A.;
Petersen, D. N.; Brault, A.; Perry, P.; Ng, O.; Owen, T. A.;
Pan, L.; Ke, H-Z.; Brown, T. A.; Thompson, D. D.;
DaSilva-Jardine, P. Biorg. Med. Chem. Lett. 2005, 15,
5562.
14. Schopfer, U.; Schoeffter, P.; Bischoff, S. F.; Nozulak, J.;
Feuerbach, D.; Floersheim, P. J. Med. Chem. 2002, 45,
1399.
Figure 3. Superposition of the docked model of compound 5 (cyan)
with the crystallographic complexes of compound 3b (white) with
hERb (purple) and hERa (green) (pdb entry: 1ERE). Unless otherwise
indicated, residue numbering is that of hERb.
15. Yang, W.; Wang, Y.; Ma, Z.; Golla, R.; Stouch, T.;
Seethala, R.; Johnson, S.; Zhou, R.; Gungor, T.; Feyen, J.
¨
H. M.; Dickson, J. K., Jr. Bioorg. Med. Chem. Lett. 2004,
14, 2327.
interaction and thus account for its increased ERb
potency and selectivity relative to the corresponding
unconstrained analog 3a. The reduced potency and
selectivity of the propylene bridged analog 8a may
be a consequence of its greater conformational flexibil-
ity and/or a less favorable orientation of the pendant
propyl group.
16. Henke, B. R.; Consler, T. G.; Go, N.; Hale, R. L.;
Hohman, D. R.; Jones, S. A.; Lu, A. T.; Moore, L. B.;
Moore, J. T.; Orband-Miller, L. A.; Robinett, R. G.;
Shearin, J.; Spearing, P. K.; Stewart, E. L.; Turnbull, P. S.;
Weaver, S. L.; Williams, S. P.; Wisely, G. B.; Lambert, M.
H. J. Med. Chem. 2002, 45, 5492.
17. Edsall, R. J., Jr.; Harris, H. A.; Manas, E. S.; Mewshaw,
R. E. Bioorg. Med. Chem. 2003, 11, 3457.
In summary, we have designed a series of conformation-
ally constrained 2–9a bridged tetrahydrofluorenones
and incorporated pendant alkyl chains to optimize
ERb binding affinity and selectivity. The ethylene
bridged analog 5, which conformationally constrains
the butyl side chain of 3a, exhibits improved ERb affin-
ity by an order of magnitude while increasing subtype
selectivity.
18. Yang, C.; Edsall, R., Jr.; Harris, H. A.; Zhang, X.; Manas,
E. S.; Mewshaw, R. A. Bioorg. Med. Chem. 2004, 12, 2553.
19. Malamas, M. S.; Manas, E. S.; McDevitt, R. E.; Guna-
wan, I.; Xu, Z. B.; Collini, M. D.; Miller, C. P.; Dinh, T.;
Henderson, R. A.; Keith, J. C., Jr.; Harris, H. A. J. Med.
Chem. 2004, 47, 5021.
20. Collini, M. D.; Kaufman, D. H.; Manas, E. S.; Harris, H.
A.; Henderson, R. A.; Xu, Z. B.; Unwalla, R. J.; Miller, C.
P. Bioorg. Med. Chem. Lett. 2004, 14, 4925.
21. Wilkening, R. R.; Ratcliffe, R. W.; Tynebor, E. C.;
Wildonger, K. J.; Fried, A. K.; Hammond, M. L.; Mosley,
R. T.; Fitzgerald, P. M. D.; Sharma, N.; McKeever, B. M.;
Nilsson, S.; Carlquist, M.; Thorsell, A.; Locco, L.; Katz,
R.; Frisch, K.; Birzin, E. T.; Wilkinson, H. A.; Mitra, S.;
Cai, S.; Hayes, E. C.; Schaeffer, J. M.; Rohrer, S. P.
Bioorg. Med. Chem. Lett. 2006, 16, 3489.
22. Prepared from the reaction of 3-bromopropanol with
chlorotriethylsilane.
23. Prepared according to the method of Bode, J. W.; Doyle,
M. P.; Protopopova, M. N.; Zhou, Q-L. J. Org. Chem.
1996, 61, 9146.
24. Stereochemistry determined retrospectively from 5.
25. 1H NMR (benzene-d6): d 0.65–0.72 (1H, m, Hb), 0.70 (3H,
t, J = 7.1 Hz, Me), 0.80 (1H, m, Hc), 1.05 (1H, m, Hd),
1.21 (1H, m, Ha), 1.40 (1H, ddd, J = 4.8, 7.1, 13.3 Hz,
H8b), 1.48 (1H, dd, J = 4.3, 11.1 Hz, H11b), 1.55–1.60 (1H,
m, H9), 1.59 (1H, d, J = 11.1 Hz, H11a), 1.77 (1H, dd,
J = 9.7, 13.3 Hz, H8a), 2.18 (3H, s, Me), 2.49 (1H, d,
J = 17.0 Hz, H10b), 2.79 (1H, d, J = 17.0 Hz, H10a), 3.00
(1H, dd, J = 4.3, 7.1 Hz, H7), 6.54 (1H, d, J = 8.4 Hz, H3),
6.59 (1H, s, H1), 7.43 (1H, d, J = 8.4 Hz, H4).
References and notes
1. Mangelsdorf, D. J.; Thummel, C.; Beato, M.; Herrlich, P.;
Schutz, G.; Umesono, K.; Blumberg, B.; Kastner, P.;
¨
Mark, M.; Chambon, P.; Evans, R. M. Cell 1995, 83, 835.
2. Green, S.; Walter, P.; Kumar, V.; Krust, A.; Bornert, J.
M.; Argos, P.; Chambon, P. Nature 1986, 320, 134.
3. Green, S.; Gilna, P.; Waterfield, M.; Baker, A.; Hort, Y.;
Shine, J. Science 1986, 231, 1150.
4. Kuiper, G. G. J. M.; Gustafsson, J.-A. Proc. Natl. Acad.
Sci. U.S.A. 1996, 93, 5925.
5. Mosselman, S.; Polman, J.; Dijkema, R. FEBS Lett. 1996,
393, 49.
6. Harris, H. A.; Katzenellenbogen, J. A.; Katzenellenbogen,
B. S. Endocrinology 2002, 143, 4172.
7. Enmark, E.; Pelto-Huikko, M.; Grandien, K.; Lagerc-
rantz, S.; Lagercrantz, J.; Fried, G.; Nordenskjo¨ld, M.;
Gustafsson, J-A. J. Clin. Endocrinol. Metab. 1997, 82,
4258.
8. Pike, A. C. W.; Brzozowski, A. M.; Hubbard, R. E.;
Bonn, T.; Thorsell, A.; Engstrom, O.; Ljunggren, J.;
Gustafsson, J.; Carlquist, M. EMBO. J. 1999, 18, 4608.
26. Compound 15b was also converted by Scheme 2 to give
the diastereomer shown below:

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K. J. Wildonger et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4462–4466
27. The IC50 values were generated in an estrogen receptor
O
Me
ligand binding assay. This scintillation proximity assay
was conducted in NEN basic flash plates using tritiated
estradiol and full length recombinant human ERa and
ERb proteins, with incubation times of 3–22 h. Most
compounds are single point determinations.
HO
Pr
H
28. Kuiper, G. G. J. M.; Lemmen, J. G.; Carlsson, B.;
Corton, J. C.; Safe, S. H.; van der Saag, P. T.; van der
Burg, B.; Gustafsson, J.-A. Endocrinology 1998, 139,
4252.
1
A H NOESY experiment similar to that done for 5 showed
the propyl function to be endo.