Estrogen receptor ligands. Part 8: Dihydrobenzoxathiin SERAMs with heteroatom-substituted side chains

Article abstract of DOI:10.1016/j.bmcl.2004.05.073

A series of benzoxathiin SERAMs with heteroatom-substituted amine side chains was prepared. Minor modifications in the side chain resulted in significant effects on biological activity, especially in uterine tissue.

Full text of DOI:10.1016/j.bmcl.2004.05.073

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3865–3868
Estrogen receptor ligands. Part 8: Dihydrobenzoxathiin
SERAMs with heteroatom-substituted side chains
Timothy A. Blizzard,* Frank DiNinno, Jerry D. Morgan, II, Jane Y. Wu,
Helen Y. Chen, Seongkon Kim, Wanda Chan, Elizabeth T. Birzin, Yi Tien Yang,
Lee-Yuh Pai, Zhoupeng Zhang, Edward C. Hayes, Carolyn A. DaSilva, Wei Tang,
Susan P. Rohrer, James M. Schaeffer and Milton L. Hammond
Merck Research Laboratories, Medicinal Chemistry, RY800-B116, PO Box 2000, Rahway, NJ 07065, USA
Received 2 April 2004; revised 25 May 2004; accepted 27 May 2004
Available online 19 June 2004
Abstract—A series of benzoxathiin SERAMs with heteroatom-substituted amine side chains was prepared. Minor modifications in
the side chain resulted in significant effects on biological activity, especially in uterine tissue.
Ó 2004 Elsevier Ltd. All rights reserved.
The clinical significance of the selective estrogen recep-
tor modulators (SERMs) is well documented.1 The
recent discovery of a second estrogen receptor subtype2
prompted interest in the development of receptor sub-
type-selective SERMS.3 Previous reports from this lab-
oratory have reported the discovery of benzoxathiins
(e.g., 1) as a novel class of selective estrogen receptor
alpha modulators (SERAMs).4a;b;c
of covalent adducts with biological proteins. We there-
fore examined alternative side chains for 1 with the goal
of finding a piperidine replacement that would maintain
potency and selectivity for ERa while reducing the for-
mation of covalent protein adducts.
To date, there have been few reports on the systematic
exploration of SERM side chains.5 In our previous
communication, we described benzoxathiin analogs with
bicyclic amine side chains that should be less readily
oxidized than the piperidine residue of 1 due to steric
constraints.4d Amine side chains with heteroatoms such
as those present in compounds 2–15 should also be less
oxidizable than the piperidine of 1 due to both steric and
electronic constraints. In addition, side chains 8a–10a
and 14a–15a also contain functional groups that could
act as internal iminium ion traps. The latter targets were
especially attractive since the internal traps provided a
second layer of defense against adduct formation. Al-
though internal trapping would be reversible, the cyclic
form should predominate thereby significantly reducing
the amount of iminium ion present. We therefore tar-
geted analogs 2–15 for synthesis. The requisite amino-
alcohol side chain synthons 2a–15a were prepared by a
variety of methods as summarized below (Table 1 and
Schemes 1–6).6
OH
HO
S
O
N
O
1
More recently, we have also described our initial studies
on the side chain SAR of 1, which were aimed at
maintaining the potency and selectivity of 1 while
reducing oxidative metabolism of the side chain.4d We
hypothesized that an iminium ion resulting from oxi-
dation of the piperidine residue present in the side chain
of 1 might be a significant contributor to the formation
Keywords: SERMs; SERAMs; Estrogen.
* Corresponding author. Tel.: +1-732-594-6212; fax: +1-732-594-9556;
e-mail: tim_blizzard@merck.com
The first method, illustrated by the preparation of thio-
morpholine analog 2a, involved alkylation of the
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.05.073

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T. A. Blizzard et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3865–3868
Table 1. Side chain preparation summary and biodata
#
R
ER binding
(IC50, nM)8
Cyanide
adduct?10
Uterine activity11
% Antag. % Ag.
Side chain
Starting
material6
Scheme
(yield)6
MCF-79
hERa hERb b=a
IC50 (nM)
OH
N
1
2
0.8
1.5
45
72
56
48
Yes
Yes
2.8
99
30
9
Commercial (Aldrich)
N
1a
S
S
OH
OH
S
15.6
17
N
1 (73%)
N
N
H
2a
16
SO2
SO2
3
4
2.0
2.5
247
116
124
46
No
––
26.3
––
1
0
See Ref. 6
N
3a
N
OH
F
F
OH
56
47
4 (32%)
5 (15%)
1 (71%)
1 (54%)
2 (49%)
N
N
N
H
4a
17
F
F
O
F
Ph
OH
F
5
6
7
8
16
275
267
331
436
17
134
184
198
No
No
––
42
9
28
33
20
2
38
29
9
N
N
N
18
5a
O
O
O
OH
OH
O
O
2.0
1.8
2.2
42.3
7.5
O
N
N
N
N
H
H
19
6a
OH
OH
OH
N
N
7a
17
TIPSO
OH
OH
OH
OH
Yes
11.3
N
N
N
N
H
H
8a
20
HO
TIPSO
HO
9
7.3 1113
143
No
42.5
––
––
2 (65%)
1 (61%)
N
N
9a
21
H2NOC
CONH2
H2N
O
10
11
2.2
2.6
366
64
166
25
No
No
21
0
5
N
N
OH
OH
N
N
10a
22
H
N
3.3
72
34
Commercial (Aldrich)
11a
OH
OH
F
F
N
N
12
13
0.5
1.7
59
81
118
48
––
––
3.8
0.6
58
15
35
32
6 (41%)
3 (10%)
F
N
N
H
H
23
12a
OR
OH
N
N
OH
24
13a R = TIPS
TIPSO
N
HO
HO
HO
14
15
2.0
3.9
134
67
21
No
No
3.2
1.9
22
49
78
61
2 (67%)
2 (75%)
N
25
N
N
H
HO
14a
TIPSO
HO
81
12
N
26
N
HO
H
15a
––
––
Raloxifene
17b-Estradiol
1.8
1.3
7
1
No
––
0.8
––
81
––
24
10011b
N/A
N/A
N/A
N/A
N/A
N/A
1.1

T. A. Blizzard et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3865–3868
3867
to protect the secondary hydroxyl in 7a since this group
did not interfere in the subsequent attachment of the
side chain to the benzoxathiin nucleus.
S
S
i
OH
N
N
2a
H
16
Scheme 1. Reagents and conditions: (i) 2-bromoethanol, K2CO3,
MeCN, reflux.
Alternatively, a masked hydroxyethyl group could be
installed first by alkylation with bromoethyl acetate
(Scheme 3). Subsequent TIPS protection and reduction
completed the synthesis of the hydroxypyrrolidine 13a.
OH
OTIPS
The fluoropiperidine side chain 4a was synthesized in a
similar manner (Scheme 4). Alkylation of commercial 4-
hydroxypiperidine 17 with bromoethyl acetate followed
by fluorination and deacetylation afforded 4a.
i, ii
OH
N
N
H
20
8a
Scheme 2. Reagents and conditions: (i) TIPS-Cl, imidazole, DMAP,
CH2Cl2; (ii) 2-bromoethanol, K2CO3, MeCN, reflux.
The difluoropiperidine side chain 5a was prepared from
commercial N-benzylpiperidone 18 (Scheme 5).8 Di-
fluorination with DAST followed by a-chloroethyl
chloroformate mediated debenzylation7 afforded the
difluoro intermediate 29. Acylation of 29 with acetoxy-
acetyl chloride followed by LiAlH4 reduction gave 5a in
15% overall yield.
OAc
ii, iii
i
OH
OH
OH
N
N
OTIPS
N
H
24
27
13a
Scheme 3. Reagents and conditions: (i) 2-bromoethyl acetate, K2CO3,
MeCN, reflux; (ii) TIPS-Cl, imidazole, DMAP, CH2Cl2; (iii) LiAlH4,
Et2O.
The fluoropyrrolidine side chain 12a was easily prepared
by acylation of 3-(R)-fluoropyrrolidine (23)6 with acet-
oxyacetyl chloride followed by reduction with LiAlH4
(Scheme 6).
OH
OH
F
Synthesis of the final products proceeded via attachment
of the hydroxyethylamine side chains to the ben-
zoxathiin core (+)-30 using the previously reported
procedure4a;c followed by deprotection (Scheme 7).
OAc
OH
ii, iii
i
N
N
N
H
28
4a
17
Scheme 4. Reagents and conditions: (i) 2-bromoethyl acetate, K2CO3,
MeCN, reflux; (ii) DAST, CH2Cl2; (iii) CH3OH, 40 °C.
With the exception of the difluoropiperidine 5 and the
hydroxyethylpiperidine 9, all of the novel benzoxathiin
analogs (2–15) retained the excellent ERa potency
exhibited by the piperidine analog 1 (Table 1) in an
in vitro estrogen receptor binding assay.8 Although the
magnitude of receptor subtype selectivity (ERb/ERa
ratio) varied considerably (from 17X to 198X), all of the
novel analogs were alpha selective. Unfortunately, most
of the new piperidine analogs suffered a substantial loss
of activity in the MCF-7 assay.9 The pyrrolidine analogs
11–15, however, retained excellent MCF-7 activity. As
expected, analogs with strong electron withdrawing
substituents (e.g., 3, 5, and 6) were less prone to oxi-
dative metabolism, as measured by their failure to form
a detectable cyanide adduct.10 With the exception of the
hydroxymethyl piperidine 8, all of the analogs with
functional groups positioned to act as internal iminium
ion traps (8–10 and 14–15) were found to have no
detectable cyanide adducts, suggesting that a prop-
erly positioned internal trap is a viable method for
alleviating the detrimental effects of amine metabolism.
F
F
O
Ph
F
F
iv, v
OH
i, ii, iii
N
N
N
H
18
29
5a
Scheme 5. Reagents and conditions: (i) DAST, CH2Cl2; (ii) a-chlo-
roacetyl chloride, Et3N, CH2Cl2; (iii) MeOH; (iv) acetoxyacetyl chlo-
ride, Et3N, CH2Cl2; (v) LiAlH4, Et2O.
OH
i, ii
F
F
N
N
H
23
12a
Scheme 6. Reagents and conditions: (i) acetoxyacetyl chloride, Et3N,
CH2Cl2; (ii) LiAlH4, Et2O.
appropriate secondary amine, for example, 16, with
2-bromoethanol (Scheme 1). Amines 2a, 6a, 7a, and 10a
were prepared by this route.6
OTIPS
i, ii, iii
OH
The presence of a hydroxyl group in the side chain
necessitated a protecting group in some instances. For
example, side chain 8a was prepared by first introducing
a tri-isopropylsilyl (TIPS) protecting group onto the
primary hydroxyl group of 20, then introducing the
hydroxyethyl group (Scheme 2). Amines 8a–9a and 14a–
15a were prepared via this method. It was not necessary
BnO
S
HO
S
R
O
O
(+)-30
1-15
OH
O
Scheme 7. Reagents and conditions: (i) 1a–15a, DIAD, PPh3, THF;
(ii) Pd, HCO2NH4, EtOH, EtOAc, H2O; (iii) n-Bu4NF, AcOH, THF.

3868
T. A. Blizzard et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3865–3868
H. Y.; Kim, S.; Wu, J. Y.; Birzin, E. T.; Chan, W.; Yang,
Y. T.; DiNinno, F.; Rohrer, S.; Schaeffer, J. M.; Ham-
mond, 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. T.; Pai, L.-Y.;
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, preceding paper in this issue.
doi:10.1016/j.bmcl.2004.05.074.
Interestingly, none of the pyrrolidine analogs, even the
unsubstituted pyrrolidine 11, formed detectable cyanide
adducts. Unfortunately, with the possible exception of
11, all of the novel analogs exhibited a less favorable
uterine profile than 1.11
Overall, the heteroatom-substituted analogs exhibited a
less desirable SERM profile than 1 although many of the
novel analogs successfully avoided cyanide adduct for-
mation. However, in conjunction with the results de-
scribed in the preceding manuscript,4d the observations
described herein suggested an ultimately successful
direction for our piperidine replacement effort. Further
results in this area will be reported in future publications
from this laboratory.
5. (a) Grese, T. A.; Sluka, J. P.; Bryant, H. U.; Cullinan, G.
J.; Glasebrok, A. L.; Jones, C. D.; Matsumoto, K.;
Palkowitz, A. D.; Sato, M.; Termine, J. D.; Winter, M. A.;
Yang, N. N.; Dodge, J. A. Proc. Natl. Acad. Sci. U.S.A.
1997, 94, 14105; (b) Robertson, D. W.; Katzenellenbogen,
J. A.; Hayes, J. R.; Katzenellenbogen, B. S. J. Med. Chem.
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Minato, H.; Kohayakawa, C.; Tsuji, J. Bioorg. Med.
Chem. Lett. 2003, 13, 4317; (d) Miller, C. P.; Tran, B. D.;
Collini, M. D. EP0802183A1, 1997.
6. All new compounds were characterized by LC–MS and
400, 500, or 600 MHz 1H NMR. Side chains were prepared
using starting materials and methods indicated in Table 1
(yields are the overall yield of the side chain from the
indicated starting material). Starting material sources: 1a,
11a, 16–22, 25, and 26: Aldrich. 3a: (a) Ford-Moore, A.
H.; Lidstone, A. G.; Waters, W. A. J. Chem. Soc. 1946,
819. 23: (b) Caldwell, C. G.; Chen, P.; He, J.; Parmee, E.
R.; Leiting, B.; Marsilio, F.; Patel, R. A.; Wu, J. K.;
Eiermann, G. J.; Petrov, A.; He, H.; Lyons, K. A.;
Thornberry, N. A.; Weber, A. Bioorg. Med. Chem. Lett.
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Acknowledgements
The authors thank Dr. Derek Von Langen and Ms. Judy
Pisano for the preparation of (+)-30.
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8. 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 provides IC50
values that are reproducible to within a factor of 2–3.
Benzoxathiin 1 ðn ¼ 36Þ and estradiol ðn > 100Þ were
tested in multiple assays; data reported in Table 1 is an
average of all determinations.
9. 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 then treated with the test
compound for 7 days. To evaluate the antagonist activity
of a test compound, this treatment occurs in the presence
of low levels of estradiol. The protein content of living
cells is then measured and an IC50 determined.
10. The cyanide adduct assay was used as a surrogate measure
of protein adduct formation subsequent to microsomal
oxidation. Compounds were incubated with liver micro-
somes in the presence of cyanide ion then LC–MS was
used to analyze for the presence of cyanide adducts.
11. (a) The uterine weight assay is an in vivo assay that
measures estrogen agonism and antagonism in rat uterine
tissue. Compounds are dosed orally at 1 mpk. Agonism
results are reported as % of estradiol control; antagonism
results are reported as % antagonism of estradiol; (b)
Estradiol exhibited 100% agonism @ 4 lg/kg.
4. (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,