Estrogen receptor ligands. Part 7: Dihydrobenzoxathiin SERAMs with bicyclic amine side chains

Article abstract of DOI:10.1016/j.bmcl.2004.05.074

A series of benzoxathiin SERAMs with bicyclic 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.2004.05.074

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3861–3864
Estrogen receptor ligands. Part 7: Dihydrobenzoxathiin
SERAMs with bicyclic amine side chains
Timothy A. Blizzard,* Frank DiNinno, Jerry D. Morgan, II, Helen Y. Chen,
Jane Y. Wu, Candido Gude, 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
Abstract—A series of benzoxathiin SERAMs with bicyclic 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 re-
cent 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).4
significant contributor to the formation of covalent
adducts. We therefore 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 formation of covalent protein
adducts.
To date, there are few reports on the systematic explo-
ration of SERM side chain SAR.5 We decided to target
side chains that should be less susceptible to oxidation
for mechanistic reasons. Bicyclic amine side chains such
as those present in compounds 2–15 should be less
readily oxidized than the piperidine residue of 1 due to
steric constraints. Furthermore, the amines in 2–9 and
15, wherein the nitrogen atom is attached to a bridge-
head carbon or is part of an azetidine ring, should be
much less susceptible to iminium ion formation upon
oxidation. We therefore targeted analogs 2–15 for syn-
thesis. The requisite aminoalcohol side chain synthons
2a–15a were prepared by a variety of methods as sum-
marized below (Table 1 and Schemes 1–5).6
OH
HO
S
O
N
O
1
Although benzoxathiin 1 has excellent potency and
selectivity for ERa, it was judged to be unacceptably
prone to oxidative metabolism with subsequent forma-
tion of covalent protein adducts. We hypothesized that
an iminium ion resulting from oxidation of the piper-
idine residue present in the side chain of 1 might be a
The first method, illustrated by the preparation of
bicyclic amine 2a, involved a-chloroethyl chloroformate
mediated dealkylation7 of a tertiary amine, such as 16,
followed by acylation of the resulting secondary amine,
for example, 29, with acetoxyacetyl chloride and LiAlH4
reduction to afford the desired hydroxyethyl amine.
Amines 2a–4a were prepared by this route although only
Keywords: SERMs; SERAMs; Estrogen.
* Corresponding author. Tel.: +1-732-594-6212; fax: +1-732-594-9556;
e-mail: tim_blizzard@merck.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.05.074

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T. A. Blizzard et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3861–3864
Table 1. Side chain preparation summary and biodata
#
R
ER Binding (IC50, nM)10
MCF-711
IC50 (nM)
Uterine activity13
%Antag. %Ag.
Side chain
Starting
material
Scheme
(yield)6
Cyanide
adduct?12
hER
0.8
HERb
b/a
N
1
2
3
4
5
6
45
56
Yes
No
No
No
No
No
2.8
2.2
3.0
3.6
3.3
1.6
99
30
47
42
37
33
9
61
53
53
70
66
N/A
N/A
OH
N
1a
N
N
N
N
0.6
0.6
0.9
0.7
1.3
51
47
85
78
1 (28%)
1 (17%)
1 (47%)
2 (42%)
2 (33%)
HO
HO
2a
16
N
N
17
HN
18
3a
N
N
118
73
131
104
40
HO
OH
4a
O
O
O
N
HN
N
N
5a
19
OH
52
HN
N
6a
20
7
0.6
0.6
0.5
0.7
0.8
40
33
67
55
No
No
No
Yes
Yes
5.8
0.4
0.5
2.6
2.5
30
79
43
86
76
61
23
70
8
2 (71%)
3 (2%)
N
N
H
HO
OH
N
7a
21
HN
N
8
N
N
N
8a
22
OH
OH
HN
N
9
56
112
194
106
3 (2%)
22
9a
O
O
N
10
11
136
85
4 (41%)
4 (21%)
O
23
10a
O
OH
N
19
O
N
11a
O
24
O
OH
OH
12
13
0.3
1.0
18
33
60
33
Yes
Yes
1.3
7.7
––
––
––
4 (28%)
4 (8%)
N
O
O
N
N
12a
25
26
O
O
O
O
––
N
13a
14
1.2
39
26
33
No
No
29.8
0.3
17
––
1
4 (30%)
5 (2%)
O
OH
OH
N
N
N
27
14a
15a
O
H2N
HO
15
0.4
1.8
65
12
––
N
28
––
––
Raloxifene
12
No
––
0.8
––
81
––
24
10013b
N/A
N/A
N/A
N/A
N/A
N/A
17b-Estradiol 1.3
1.1
1.1
-
H
N+
Cl
i, ii
iii, iv
N
N
i
HO
H
N
N
16
29
2a
H
HO
20
6a
Scheme 1. Reagents and conditions: (i) a-chloroethyl chloroformate,
Et3N, CH2Cl2; (ii) MeOH; (iii) acetoxyacetyl chloride, Et3N, CH2Cl2;
(iv) LiAlH4, Et2O.
Scheme 2. Reagents and conditions: (i) 2-bromoethanol, K2CO3,
MeCN, reflux.

T. A. Blizzard et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3861–3864
3863
OH
OTIPS
O
N
iii, iv
i, ii
N
N
BnO
+
S
HO
S
N
i, ii, iii
H
AcO
R
OH
OH
22
30
8a
O
O
9a
(+)-34
1-15
OH
O
Scheme 3. Reagents and conditions: (i) acetoxyacetyl chloride, Et3N;
(ii) Pd(OAc)2, CH2N2; (iii) chiral HPLC (see Ref. 8); (iv) LiAlH4,
Et2O.
Scheme 6. Reagents and conditions: (i) 1a–15a, DIAD, PPh3, THF;
(ii) Pd, HCO2NH4, 7:2:1 EtOH/EtOAc/H2O; (iii) n-Bu4NF, AcOH,
THF.
the last two steps were used for 4a since the des-methyl
starting material 18 was prepared by a different route.6
Synthesis of the final products proceeded via attachment
of the hydroxyethylamine side chains to the ben-
zoxathiin core (þ)-34 using the previously reported
procedure4a;c followed by deprotection (Scheme 6).
An alternative synthesis involved alkylation of the
appropriate secondary amine, for example, 20, with 2-
bromoethanol (Scheme 2). Amines 5a–7a were prepared
via this method.
All of the novel benzoxathiin analogs (2–15) retained the
excellent ERa potency exhibited by the monocyclic
analog 1 (Table 1) in an in vitro ER binding assay.10
Although the magnitude of receptor subtype selectivity
(ERb/ERa ratio) varied considerably (from 33 Â to
194 Â), all of the novel analogs remained alpha selective.
In addition to excellent binding, most of the new ana-
logs (2–12) retained antagonist activity in the MCF-7
proliferation assay11 that matched or exceeded the
activity of 1. Only analog 14 showed a substantial de-
crease in MCF-7 antagonist activity. As expected, ana-
logs with bicyclic amine side chains wherein the nitrogen
atom is attached to a bridgehead carbon (2–9) were less
prone to oxidative metabolism, as measured by their
failure to form a detectable cyanide adduct.12 However,
analogs 2–7 were also surprisingly potent agonists in
uterine tissue,13 in contrast to the uterine antagonism
exhibited by 1. Apparently, the larger bicyclic side chain
does not allow the receptor–ligand complex to assume
an antagonist conformation. Interestingly, with the
exception of 9, all of the fused bicyclic analogs 8–13 that
were evaluated in the uterine assay were found to be
antagonists. Unfortunately, with the exception of
bridgehead nitrogen analogs 8 and 9, the fused bicyclic
analogs all formed cyanide adducts. The differential
uterine activity of diastereomers 8 and 9 was especially
noteworthy and allowed tentative assignment of their
absolute stereochemistry.9
The 2,3-fused cyclopropylpyrrolidine 30 was prepared
by acylation of 2,3-dihydropyrrole 226 with acetoxy-
acetyl chloride (Scheme 3) followed by cyclopropana-
tion. Chiral HPLC separation of the enantiomers of 308
followed by LiAlH4 reduction gave the desired enan-
tiomeric side chains 8a and 9a.9
Synthesis of several of the fused bicyclic amines began
with conversion of a commercially available anhydride,
for example, 23, to an imide, for example, 31, by
sequential reaction with ethanolamine and acetic anhy-
dride (Scheme 4). Subsequent reduction with LiAlH4
afforded the desired side chain, for example, 10a.
Alternatively, hydroxyethylimide formation could be
achieved by reaction of the anhydride and ethanolamine
with azeotropic removal of water. The resulting hy-
droxyethylimide was then reduced with LiAlH4 to afford
the side chain. Amines 10a–14a were prepared using this
method.
The spiroazetidine 15a was prepared from the known
amino-alcohol 286 (Scheme 5). Reaction of 28 with
p-toluenesulfonyl chloride to form the chloro-tosylate 32
followed by cyclization afforded spiroazetidine 33.
Deprotection with Red-Al, acylation with acetoxy-
acetyl chloride, and reduction completed the synthesis of
15a.
Overall, the fused cyclopropyl analog 10 was the
most interesting of the novel analogs but was not
pursued further due to its cyanide adduct forma-
tion. However, its excellent potency, subtype selectivity,
and uterine profile encouraged us to continue the search
for a piperidine replacement and also suggested a
direction for these efforts. Further results in this area
will be reported in future publications from this labo-
ratory.
O
O
i, ii
iii
N
N
O
AcO
HO
O
O
23
31
10a
Scheme 4. Reagents and conditions: (i) ethanolamine; (ii) acetic
anhydride; (iii) LiAlH4, Et2O.
H
Ts
NH2
N
iii, iv, v
HO
i
ii
Acknowledgements
N
N
HO
Cl
Ts
The authors thank Dr. Derek Von Langen and Ms. Judy
Pisano for the preparation of (þ)-34 and thank Mr.
Daniel Kim for performing the preparative chiral HPLC
separation of the enantiomers of 30.
32
28
33
15a
Scheme 5. Reagents and conditions: (i) pTsCl; (ii) NaH, DMF; (iii)
Red-Al; (iv) acetoxyacetyl chloride, Et3N; (v) LiAlH4, Et2O.

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T. A. Blizzard et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3861–3864
using method indicated in Table 1. Starting material
References and notes
sources: 1a, 16, 23, and 26–27: Aldrich. Compound 17: (a)
Nelsen, S. F.; Hollinsed, W. C.; Kessel, C. R.; Calabrese,
J. C. J. Am. Chem. Soc. 1978, 100, 7876; Compound 18:
(b) Fraser, R. R.; Swingle, R. B. Can. J. Chem. 1970, 48,
2065; Compound 19: (c) Portoghese, P. S.; Turcotte, J. G.
J. Med. Chem. 1971, 14, 288; Compound 20: (d) Werner,
L. H.; Ricca, S. J. Am. Chem. Soc. 1958, 80, 2733;
Compound 21: (e) St. Georgiev, V.; Saeva, G. A.;
Kinsolving, C. R. J. Heterocycl. Chem. 1986, 23, 1023;
Compound 22: (f) Karadogan, B.; Parsons, P. J. Tetrahe-
dron 2001, 57, 8699, Compound 24: Maybridge. Com-
pound 25: Rieke Metals Inc.; Compound 28: (g) Nayer,
H.; Giudicelli, R.; Sette, J.; Menin, J. Bull. Soc. Chim. Fr.
1965, 204.
1. (a) Jordan, V. C. J. Med. Chem. 2003, 46, 883; (b) Jordan,
V. C. J. Med. Chem. 2003, 46, 1081; (c) Lonard, D. M.;
Smith, C. L. Steroids 2002, 67, 15.
2. (a) Kuiper, G. G. J. M.; Enmark, E.; Pelto-Kuikko, M.;
Nilsson, S.; Gustafsson, J. A. Proc. Natl. Acad. Sci. U.S.A.
1996, 93, 5925; (b) Mosselman, S.; Polman, J.; Dijkema,
R. FEBS Lett. 1996, 392, 49.
3. (a) Ghosh, U.; Ganessunker, D.; Sattigeri, V. J.; Carlson,
K. E.; Mortensen, D. J.; Katzenellenbogen, B. S.;
Katzenellenbogen, J. A. Bioorg. Med. Chem. Lett. 2003,
11, 629; (b) 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; (c) Schopfer, U.;
Schoefter, P.; Bischoff, S. F.; Nozulak, J.; Feuerbach, D.;
Floersheim, P. J. Med. Chem. 2002, 45, 1399; (d) Muth-
yala, R. S.; Carlson, K. E.; Katzenellenbogen, J. A.
Bioorg. Med. Chem. Lett. 2003, 13, 4485; (e) Myers, M. J.;
Sun, J.; Carlson, K. E.; Marriner, G. A.; Katzenellenbo-
gen, B. S.; Katzenellenbogen, J. A. J. Med. Chem. 2001,
44, 4230; (f) Mortensen, D. J.; Rodriguez, A. L.; Carlson,
K. E.; Sun, J.; Katzenellenbogen, B. S.; Katzenellenbogen,
J. A. J. Med. Chem. 2001, 44, 3838; (g) Myers, M. J.; Sun,
J.; Carlson, K. E.; Katzenellenbogen, B. S.; Katzenellenb-
ogen, J. A. J. Med. Chem. 1999, 42, 2456; (h) Edsall, R. J.;
Harris, H. A.; Manas, E. S.; Mewshaw, R. E. Bioorg. Med.
Chem. Lett. 2003, 13, 3457; (i) Miller, C. P.; Collini, M.
D.; Harris, H. A. Bioorg. Med. Chem. Lett. 2003, 13,
2399.
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,
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.
7. Olofson, R. A.; Martz, J. T.; Senet, J. P.; Piteau, M.;
Malfroot, T. J. Org. Chem. 1984, 49, 2081.
8. Analytical chiral HPLC performed on a Chiralcel OD
4.6 mm  250 mm 10 lm column eluted with 60:40 hep-
tane–isopropanol at 0.5 mL/min. Retention times:
(À)enantiomer of 30 (converts to 8a) 16 min; (þ)enantio-
mer (converts to 9a) 20 min.
9. The absolute stereochemistry of diasteromers 8 and 9 (and
thus enantiomers 8a and 9a) was assigned by analogy with
compounds of known stereochemistry based on uterine
assay data. These related compounds will be disclosed in a
future publication.
10. 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.
11. 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.
12. 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.
13. (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
reported as % of estradiol control; antagonism reported as
% antagonism of estradiol; (b) Estradiol exhibited 100%
agonism @ 4 lg/kg.
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.
1982, 25, 167; (c) Watanabe, N.; Nakagawa, H.; Ikeno, A.;
Minato, H.; Kohayakawa, C.; Tsuji, J. Bioorg. Med.
Chem. Lett. 2003, 13, 4317.
6. All new compounds were characterized by LC–MS and
400, 500, or 600 MHz 1H NMR. Side chains were prepared