Estrogen receptor ligands. Part 13: Dihydrobenzoxathiin SERAMs with an optimized antagonist side chain

Article abstract of DOI:10.1016/j.bmcl.2005.05.089

An optimized side chain for dihydrobenzoxathiin SERAMs was discovered and attached to four dihydrobenzoxathiin platforms. The novel SERAMs show exceptional estrogen antagonist activity in uterine tissue and an MCF-7 breast cancer cell assay.

Full text of DOI:10.1016/j.bmcl.2005.05.089

Bioorganic & Medicinal Chemistry Letters 15 (2005) 3912–3916
Estrogen receptor ligands. Part 13: Dihydrobenzoxathiin
SERAMs with an optimized antagonist side chain
Timothy A. Blizzard,* Frank DiNinno, Helen Y. Chen, Seongkon Kim, Jane Y. Wu,
Wanda Chan, Elizabeth T. Birzin, Yi Tien Yang, Lee-Yuh Pai, Edward C. Hayes,
Carolyn A. DaSilva, Susan P. Rohrer, James M. Schaeffer and Milton L. Hammond
Merck Research Laboratories, RY800-B116, P.O. Box 2000, Rahway, NJ 07065, USA
Received 28 March 2005; revised 23 May 2005; accepted 25 May 2005
Available online 29 June 2005
Abstract—An optimized side chain for dihydrobenzoxathiin SERAMs was discovered and attached to four dihydrobenzoxathiin
platforms. The novel SERAMs show exceptional estrogen antagonist activity in uterine tissue and an MCF-7 breast cancer cell
assay.
ꢀ 2005 Elsevier Ltd. All rights reserved.
The importance of the selective estrogen receptor mod-
ulators (SERMs) is well-documented.1 The discovery2a,b
of a second estrogen receptor, ERb, has generated
considerable interest in the development of receptor
sub-type-selective SERMs.2c–m Previous reports from
this laboratory have reported the discovery of dihydro-
benzoxathiins (e.g., 1 and 2) as a novel class of selective
estrogen receptor alpha modulators (SERAMs).3a–e We
have also described studies on the side chain SAR of this
class of SERAMs.3d–f These studies resulted in the
discovery that addition of a methyl group to the side
chain at the appropriate position and with the right
stereochemistry, either on the pyrrolidine ring (as in 3)
or on the linker (as in 5), substantially increased
estrogen antagonist activity in uterine tissue. These
compounds were also more potent inhibitors in an
MCF-7 breast cancer cell assay.
Although our previous studies3f strongly suggested that
the optimum stereochemistry for these methyl groups
would be as in side chain 20 (compare compound 3 with
4 and 5 with 6), we also prepared the other three diaste-
reomers of 20 (21–23) to be sure that we had the best
combination and to probe the relative contribution of
the two side chain methyl groups to the uterine antago-
nist properties of the final compounds. Once evaluation
of the corresponding dihydrobenzoxathiins 7–10 con-
firmed that the orientation of the methyl groups in 20
was optimal, we proceeded to append this side chain
to three additional dihydrobenzoxathiin platforms,
affording analogs 11–13 for evaluation.
Side chains 20–23 were prepared by a significantly im-
proved modification of our previously reported synthe-
sis of mono-alkylated pyrrolidine side chains (Scheme
1).3f Condensation of 2-(R)-methylsuccinic acid 174 with
L-alaninol 184 in refluxing toluene with azeotropic
Compounds 3 and 5, had what we considered to be, an
excellent ER antagonist profile.3f However, we felt that
it might be possible to find a compound with an even
better profile. Since introduction of a single methyl
group at either the 10 or the 3 position of the side chain
of 2 resulted in such a significant increase in uterine
antagonism and MCF-7 activity, we prepared and
report herein the ÔcomboÕ compounds 7–10, which incor-
porate methyl groups at both positions in the side chain.
Keywords: SERMs; SERAMs; Estrogen.
*
Scheme 1. Reagents and conditions: (i) toluene, reflux, 24 h, 75–85%;
(ii) chiral HPLC; (iii) LiAlH4, ether, 63–69%.
Corresponding author. Tel.: +1 732 594 6212; fax: +1 732 594
9556; e-mail: tim_blizzard@merck.com
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.05.089

T. A. Blizzard et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3912–3916
3913
removal of water afforded imide 195 as a 9:1 mixture of
diastereomers in 75–85% yield on multigram scale. This
improved condensation procedure eliminated some re-
agents (no acetyl chloride or acetic anhydride) and affor-
ded the desired product in higher yields than the
previous procedure. Chiral HPLC6 removed the minor
diastereomer resulting from partial epimerization at C-
3. Reduction of 19 with LiAlH4 afforded side chain 20
in 63–69% yield on multigram scale. As we expected
based on our experience with an earlier sequence3f there
was no epimerization at C-3 in the reduction step.7
important than the methyl at C-3 in the ring as 9 (ÔrightÕ
stereochemistry at C-10, ÔwrongÕ stereochemistry at C-3)
has a much better uterine profile than 8 (ÔwrongÕ stereo-
chemistry at C-10, ÔrightÕ stereochemistry at C-3). Ana-
log 7, which has the ÔwrongÕ stereochemistry at both
sites, is clearly the worst of the four diastereomers.
We were encouraged by the finding that the beneficial ef-
fect of adding a methyl group to the pyrrolidine ring or
to the linker appeared to be additive so that the ÔcomboÕ
compound 10 did have an improved uterine profile and
enhanced MCF-7 activity relative to the mono-methyl-
ated compounds 3 and 5. Although the improvement
was small, we felt it to be sufficient to warrant additional
investigation. We therefore prepared and evaluated
three additional analogs wherein the optimized antago-
nist side chain 20 was appended to three modified dihy-
drobenzoxathiin platforms. All three of these new
analogs (11–13) exhibited a substantially improved uter-
ine profile, relative to the mono-methylated analogs 3
and 5, which were in turn significantly better than the
unmethylated analog 2. The uterine profiles of com-
pounds 10–13 were clearly superior to raloxifene (14)
and were comparable to the known pure antagonist ful-
vestrant (16).10 Note, however, that compounds 1–13
are orally active, whereas fulvestrant must be dosed par-
enterally. In addition to their excellent uterine activity,
all of the new analogs showed improved potency in an
MCF-7 assay.11 The fluorinated analogs 11 and 13 were
especially noteworthy in this regard. In contrast to the
analogous methylated chromane system,12 wherein, the
introduction of the fluorine atom does not effect selectiv-
ity, we speculate that a decrease in the selectivity of flu-
oro-dihydrobenzoxathiins may arise from a reduction of
the electron density on sulfur, and in turn, a reduction of
the electronic repulsion with the Met366 residue of ERb.
The diastereomeric side chains 21–23 were prepared by
substitution of 2-(S)-methylsuccinic acid4 for the 2-(R)-
acid and/or substitution of D-alaninol4 for L-alaninol,
as appropriate, in the synthetic scheme outlined above.
The dihydrobenzoxathiin core structures (e.g., 24) were
synthesized using the procedure previously reported by
Kim et al. (Scheme 2).3a Attachment of the side chain
to the core using a previously described procedure3a, fol-
lowed by deprotection, afforded the novel SERAMs 7–
13. As previously noted during the synthesis of 5 and
6, 3f the presence of a methyl group in the linker resulted
in the formation of varying amounts of a rearranged by-
product. However, 7–13 were the major products.
The novel bis-methylated pyrrolidine analogs 7–10 re-
tained an excellent ERa potency exhibited by previously
reported compounds 1–6 in an in vitro estrogen receptor
binding assay (Table 1).8 Although all of the novel ana-
logs were alpha selective, 7 and 8 were particularly selec-
tive (note that a longer incubation time was used in the
evaluation of these two analogs in the binding assay; we
do not believe that a shorter incubation time would re-
sult in lower selectivity but this has not been proven).
As we had anticipated, the 10-(S),3-(R) configuration
present in side chain 20 (analog 10) proved to be optimal
for uterine antagonism as measured in a highly sensitive
immature rat assay.9 Interestingly, the methyl group at
C-10 in the linker appears to be considerably more
Although it was not possible to obtain X-ray quality
crystals of the ligand-bound ER complexes of com-
pounds 10–13, we were able to obtain and have reported
X-ray data for 3 and 5.3f The antagonist activity of 3
and 5 can be rationalized as resulting from the positive
interaction of the 3-(R)-configured ring methyl of 3
and the 10-(S)-linker methyl group of 5 with residues
in the antagonist-arm region of the ligand binding do-
main of ERa.3f The apparent additivity of the uterine
antagonist effects of 3 and 5 observed for the combo
compounds 10–13 prompts us to speculate that the
exceptional antagonism exhibited by 10–13 arises from
a sum of positive interactions previously demonstrated
for 3 and 5, thereby further enhancing the stabilization
of the antagonist configuration.
Because of their excellent activity in the MCF-7 and rat
uterine assays, compounds 10–13 were evaluated for
anti-tumor activity against MCF-7 human breast carci-
noma xenografts in athymic mice. All of the compounds
were found to be superior to the clinically approved
therapies, tamoxifen and fulvestrant, in that both tumor
growth rates and final tumor burdens were significantly
lower.13 In this regard, compound 13 was most effica-
cious. In a mode similar to the known SERD (selective
estrogen receptor down-regulator) fulvestrant,14 the
Scheme 2. Reagents: (i) 20, DIAD, PPh3, THF; (ii) Pd, HCO2NH4,
EtOH; (iii) nBu4NF, AcOH, THF.

3914
T. A. Blizzard et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3912–3916
Table 1. ER ligand binding data, rat uterine growth data, and MCF-7 data
Compound
X
R
Human ER binding
(IC50, nM)8
Immature rat uterine growth assay9
MCF-710
hERa
hERb
b/a
% Antagonism
0.3 mpk 1 mpk
% Agonism
IC50 (nM)
0.3 mpk
1 mpk
1
2
H
H
H
H
H
H
H
H
H
H
F
0.9
2.6
1.6
1.0
1.0
1.7
0.8
0.6
2.5
1.3
0.8
1.2
0.7
43
64
87
50
45
71
87
90
45
52
5.3
69
9.1
48
25
54
50
45
42
109
150
18
40
7
80
55
99
73
11
38
9
34
3.0
3.3
0.3
1.0
0.3
0.5
2.1
0.9
0.4
0.1
0.03
0.1
0.03
3
98
109
85
ꢀ1
14
ꢀ14
8
4
70
5
85
102
18
9
ꢀ8
71
6
40
59
7
34
30
54
69
8
57
72
33
27
9
100
109
108
93
118
123
118
118
122
ꢀ7
ꢀ18
ꢀ12
ꢀ8
ꢀ19
ꢀ9
ꢀ29
ꢀ17
ꢀ17
ꢀ20
10
11
12
13
H
F
58
13
102
14
15
16
Raloxifene
Estradiol
Fulvestrant
0.7
1.3
8.0
1.9
1.1
8.4
3
1
1
39
—
81
—
28
—
24
100
—
0.8
—
104
—
ꢀ24
0.1
exceptional activity of 10–13 against breast cancer cells
may be due, in part, to their ability to down-regulate
the estrogen receptor.15
covery of an optimized antagonist side chain for this
platform. This side chain has been attached to four dihy-
drobenzoxathiin platforms to prepare four novel a-se-
lective estrogen receptor ligands (10–13) with
exceptional antagonist activity. These orally active opti-
mized SERAMs are comparable or superior to previous-
ly reported SERMs with regard to estrogen antagonism
in uterine tissue and breast cancer cells. A future com-
munication from this laboratory will discuss the applica-
bility of the optimized antagonist side chain 20 to
known SERM platforms.
To our knowledge, compounds 10–13 represent the first
reported examples wherein a relatively small structural
modification of an existing SERM (i.e., addition of
two methyl groups as in 2 vs 10) results in conversion
of the SERM to a SERD. By analogy to our previous
classification of the ERa sub-type-selective SERMs as
SERAMs, the ERa sub-type-selective SERDs 10–13,
especially the more selective analogs 10 and 12, may rea-
sonably be described as SERADs (selective estrogen
receptor alpha down-regulators).
Acknowledgments
In conclusion, our effort to delineate the side-chain SAR
of the dihydrobenzoxathiin SERAMs has led to the dis-
The authors thank Dr. Derek Von Langen and Ms. Judy
Pisano for assistance in the large-scale preparation of

T. A. Blizzard et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3912–3916
3915
Morgan, J. D.; Wu, J. Y.; Chen, H. Y.; Kim, S.; Chan, W.;
Birzin, E. T.; Yang, Y.; Pai, L.; Zhang, Z.; Hayes, E. C.;
DaSilva, C. A.; Tang, W.; Rohrer, S. P.; Schaeffer, J. M.;
Hammond, M. L. Bioorg. Med. Chem. Lett. 2004, 14,
3865; (f) Blizzard, T. A.; DiNinno, F.; Morgan, J. D., II;
Chen, H. Y.; Wu, J. Y.; Kim, S.; Chan, W.; Birzin, E. T.;
Yang, Y.; Pai, L.; Li, Y.; Zhang, Z.; Hayes, E. C.;
DaSilva, C. A.; Fitzgerald, P. M. D.; Sharma, N.; Tang,
W.; Rohrer, S. P.; Schaeffer, J. M.; Hammond, M. L.
Bioorg. Med. Chem. Lett. 2005, 15, 107.
the dihydrobenzoxathiin cores. We also thank Dr. Der-
ek Von Langen, Mr. Daniel Kim, Mr. Steve Young, and
Mr. Jerry Morgan for assistance in the large-scale prep-
aration of side chain 20. We thank Dr. Sudha Warrier
Mitra and Mr. Joel Yudkovitz for the estrogen receptor
down-regulation experiments.
References and notes
4. Aldrich Chemical Company.
1. (a) Jordan, V. C. J. Med. Chem. 2003, 46, 883; (b) Jordan,
V. C. J. Med. Chem. 2003, 46, 1081.
5. All new compounds were characterized by LC–MS and
400, 500, or 600 MHz 1H NMR. Selected compounds were
characterized further by elemental analysis.
2. (a) Mosselman, S.; Polman, J.; Dijkema, R. FEBS Lett.
1996, 392, 49; (b) Kuiper, G. G.; Enmark, E.; Pelto-
Huikko, M.; Nilsson, S.; Gustafsson, J. A. Proc. Natl.
Acad. Sci. U.S.A. 1996, 93, 5925; (c) Malamas, M. S.;
Manas, E. S.; McDevitt, R. E.; Gunawan, I.; Xu, Z.;
Collini, M. D.; Miller, C. P.; Dinh, T.; Henderson, R. A.;
Keith, J. C.; Harris, H. A. J. Med. Chem. 2004, 47, 5021;
(d) 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; (e) 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. Bioorg. Med. Chem. Lett. 2004, 14, 2327; (f)
Yang, C.; Edsall, R. J.; Harris, H. A.; Zhang, X.; Manas,
E. S.; Mewshaw, R. E. Bioorg. Med. Chem. 2004, 12, 2553;
(g) Chesworth, R.; Zawistoski, M. P.; Lefker, B. A.;
Cameron, K. O.; Day, R. F.; Mangano, F. M.; Rosati, R.
L.; Colella, S.; Petersen, D. N.; Brault, A.; Lu, B.; Pan, L.
C.; Perry, P.; Ng, O.; Castleberry, T. A.; Owen, T. A.;
Brown, T. A.; Thompson, D. D.; DaSilva-Jardine, P.
Bioorg. Med. Chem. Lett. 2004, 14, 2729; (h) Meyers, M.
J.; Sun, J.; Carlson, K. E.; Marriner, G. A.; Katzenel-
lenbogen, B. S.; Katzenellenbogen, J. A. J. Med. Chem.
2001, 44, 4230; (i) Mortensen, D. S.; Rodriguez, A. L.;
Carlson, K. E.; Sun, J.; Katzenellenbogen, B. S.; Katzen-
ellenbogen, J. A. J. Med. Chem. 2001, 44, 3838; (j)
Muthyala, R. S.; Carlson, K. E.; Katzenellenbogen, J. A.
Bioorg. Med. Chem. Lett. 2003, 13, 4485; (k) 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; (l) Schopfer, U.; Schoefter, P.;
Bischoff, S. F.; Nozulak, J.; Feuerbach, D.; Floersheim, P.
J. Med. Chem. 2002, 45, 1399; (m) Manas, E. S.; Unwalla,
R. J.; Xu, Z. B.; Malamas, M. S.; Miller, C. P.; Harris, H.
A.; Hsiao, C.; Akopian, T.; Hum, W. T.; Malakian, K.;
Wolfrom, S.; Bapat, A.; Bhat, R. A.; Stahl, M. L.; Somers,
W. S.; Alvarez, J. C. J. Am. Chem. Soc. 2004, 126, 15106.
3. (a) Kim, S.; Wu, J. Y.; Birzin, E. T.; Frisch, K.; Chan, W.;
Pai, L.; Yang, Y. T.; Mosley, R. T.; Fitzgerald, P. M. D.;
Sharma, N.; DiNinno, F.; Rohrer, S.; Schaeffer, J. M.;
Hammond, M. L. J. Med. Chem. 2004, 47, 2171; (b) Chen,
H. Y.; Kim, S.; Wu, J. Y.; Birzin, E. T.; Chan, W.; Yang,
Y. T.; DiNinno, F.; Rohrer, S.; Schaeffer, J. M.;
Hammond, M. L. Bioorg. Med. Chem. Lett. 2004, 14,
2551; (c) Kim, S.; Wu, J.; Chen, H. Y.; Birzin, E. T.; Chan,
W.; Yang, Y. T.; Colwell, L.; Li, S.; DiNinno, F.; Rohrer,
S.; Schaeffer, J. M.; Hammond, M. L. Bioorg. Med. Chem.
Lett. 2004, 14, 2741; (d) Blizzard, T. A.; DiNinno, F.;
Morgan, J. D., II; Chen, H. Y.; Wu, J. Y.; Gude, C.; Kim,
S.; Chan, W.; Birzin, E. T.; Yang, Y.; Pai, L.; Zhang, Z.;
Hayes, E. C.; DaSilva, C. A.; Tang, W.; Rohrer, S. P.;
Schaeffer, J. M.; Hammond, M. L. Bioorg. Med. Chem.
Lett. 2004, 14, 3861; (e) Blizzard, T. A.; DiNinno, F.;
6. Analytical chiral HPLC analysis was performed on a
Chiralpak AD 4.6 · 250 mm 10 lm column eluted with 3:1
heptane/isopropanol at 0.4 mL/min. Under these condi-
tions, the major (R,S)-diastereomer of 19 has a retention
time of 23.7 min, while the minor (S,S)-diastereomer
elutes at 15.1 min.
7. The optical purity of 20 was confirmed by NMR analysis of
the corresponding Mosher ester. The 600 MHz 1H NMR of
the Mosher ester clearly showed the presence of only one
diastereomer. By contrast, the corresponding NMR spec-
trum of the Mosher ester of the 1:1 diastereomeric mixture
generated using racemic acid as the starting material clearly
showed both diastereomers. Similarly, when the reaction
sequence was run with racemic acid and racemic amine as
starting materials, the NMR spectrum of the Mosher ester
of the product showed P3 diastereomers.
8. (a) The IC50 values were generated in an estrogen receptor
ligand binding assay. This scintillation proximity assay
was conducted in NEN Basic Flashplates using tritiated
estradiol and full-length recombinant human ERa and
ERb proteins. Compounds were evaluated in duplicate in
a single assay. In our experience, this assay provided IC50
values that are reproducible to within a factor of 2–3.
Dihydrobenzoxathiin 1 (n = 36) and estradiol (n > 100)
were tested in multiple assays; data reported in Table 1 are
an average of all determinations; (b) Data for 7 and 8
reflect a 20 h incubation prior to radioactive quantifica-
tion; all other data obtained with 3 h incubation.
9. (a) The uterine weight assay is an in vivo assay based on a
published procedure9c that measures estrogen agonism
and antagonism in rat uterine tissue. Compounds are
dosed orally at the indicated doses, except for fulvestrant,
which was dosed subcutaneously. Agonism is reported as
percentage of estradiol control; antagonism reported as
percentage antagonism of estradiol; (b) Estradiol exhibited
100% agonism at 4 lg/kg; (c) Wakeling, A. E.; Valcaccia,
B.; Newboult, E.; Green, L. R. J. Steroid Biochem. 1984,
20, 111.
10. This is an in vitro MCF-7 breast cancer cell proliferation
assay adapted to a 96-well format. Cells are grown in
estrogen-depleted media for 6 days and then treated with
the test compound for 7 days. To evaluate the antagonist
activity of a test compound, this treatment is given in the
presence of 0.003 nM (the EC70) estradiol. The amount of
cell growth is determined by measuring the protein content
of living cells and an IC50 for the test compound is
determined.
11. Levin, M.; DÕSouza, N.; Castaner, J. Drugs Future 2001,
26, 841.
12. Tan, Q.; Blizzard, T. A.; Morgan, J. D., II; Birzin, E. T.;
Chan, W.; Yang, Y.; Pai, L.; Hayes, E. C.; DaSilva, C. A.;
Mitra, S. W.; Yudkovitz, J.; Wilkinson, H. A.; Sharma,
N.; Fitzgerald, P. M. D.; Li, S.; Colwell, L.; Fisher, J. E.;
Adamski, S.; Reszka, A. A.; Kimmel, D.; DiNinno, F.;
Rohrer, S. P.; Freedman, L. P.; Schaeffer, J. M.;

3916
T. A. Blizzard et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3912–3916
Hammond, M. L. Bioorg. Med. Chem. Lett. 2005, 15,
1675.
13. Hayes, E. C. et al., manuscript in preparation.
14. Osborne, C. K.; Wakeling, A.; Nicholson, R. I. Br. J.
Cancer 2004, 90, S2.
15. Warrier, S. et al., manuscript in preparation.