Estrogen receptor ligands. Part 9: Dihydrobenzoxathiin SERAMs with alkyl substituted pyrrolidine side chains and linkers

Article abstract of DOI:10.1016/j.bmcl.2004.10.036

A series of benzoxathiin SERAMs was prepared. Minor modifications in the side chain or linker resulted in significant effects on biological activity, especially in uterine tissue. A series of dihydrobenzoxathiin SERAMs with alkylated pyrrolidine side chains or alkylated linkers was prepared. Minor modifications in the side chain or linker resulted in significant effects on biological activity, especially in uterine tissue.

Full text of DOI:10.1016/j.bmcl.2004.10.036

Bioorganic & Medicinal Chemistry Letters 15 (2005) 107–113
Estrogen receptor ligands. Part 9: Dihydrobenzoxathiin
SERAMs with alkyl substituted pyrrolidine side chains and linkers
Timothy A. Blizzard,* Frank DiNinno, Jerry D. Morgan, II, Helen Y. Chen, Jane Y. Wu,
Seongkon Kim, Wanda Chan, Elizabeth T. Birzin, Yi Tien Yang, Lee-Yuh Pai,
Paula M. D. Fitzgerald, Nandini Sharma, Ying Li, Zhoupeng Zhang, Edward C. Hayes,
Carolyn A. DaSilva, Wei Tang, Susan P. Rohrer, James M. Schaeffer and
Milton L. Hammond
Merck Research Laboratories, RY800-B116, PO Box 2000, Rahway, NJ 07065, USA
Received 2 September 2004; revised 8 October 2004; accepted 8 October 2004
Available online 2 November 2004
Abstract—A series of dihydrobenzoxathiin SERAMs with alkylated pyrrolidine side chains or alkylated linkers was prepared.
Minor modifications in the side chain or linker resulted in significant effects on biological activity, especially in uterine tissue.
ꢀ 2004 Elsevier Ltd. All rights reserved.
The clinical significance of the selective estrogen recep-
tor modulators (SERMs) is well documented.1 Recently,
there has been much interest in the development of
receptor subtype-selective SERMS.2 Previous reports
from this laboratory have reported the discovery of
dihydrobenzoxathiins (e.g., 1) as a novel class of Selec-
tive Estrogen Receptor Alpha Modulators (SERAMs).3a–e
More recently, we have also described our initial
studies on the side chain SAR of 1, which were aimed
at maintaining potency and selectivity while reducing
oxidative metabolism of the side chain.3d,e
bicyclic analogs that we examined,3d 2 appeared to be
susceptible to metabolic oxidation of the side chain, as
measured by an in vitro liver microsome assay.4 Interest-
ingly, the unsubstituted pyrrolidine analog 3 did not
form a detectable cyanide adduct in this assay. How-
ever, 3 had a less favorable SERM profile than 1, parti-
cularly with regard to uterine agonism/antagonism.
We therefore targeted analogs 4–16 for synthesis with
the hope that we could find a pyrrolidine analog with
an improved SERM profile that was less susceptible
than 1 to oxidation. The requisite aminoalcohol side
chain synthons 4a–16a were prepared by a variety of
methods as summarized below (Table 1 and Schemes
1–3).5
OH
OH
HO
S
HO
S
O
O
The first method, illustrated by the preparation of the 3-
methyl analog 4a, involved conversion of a chiral diacid,
for example, 29, to the corresponding imide 45 by
sequential reaction with acetyl chloride (to form the
anhydride), ethanolamine, and acetic anhydride
(Scheme 1). As expected, some epimerization occurred
during this sequence; imide 45 was obtained in only
71% ee.6 For initial evaluation, this enantiomerically im-
pure material was converted to the final product, dihy-
drobenzoxathiin 4. However, it was possible to obtain
enantiomerically pure 4a by chiral HPLC separation
of the enantiomers of 456 followed by reduction of the
N
O
OR
1
1-28
During the course of these studies, we found that the
fused cyclopropyl analog 2 had excellent potency and
subtype selectivity while retaining a uterine profile com-
parable to that of 1 (Table 1). However, like all of the
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 ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.10.036

108
T. A. Blizzard et al. / Bioorg. Med. Chem. Lett. 15 (2005) 107–113
Table 1. Side chain preparation summary and biodata for alkylated pyrrolidine analogs
#
R
ER Binding
(IC50, nM)9
Cyanide MCF-710
adduct?4 IC50 (nM)
Uterine
activity11
Side chain
Starting material5 Scheme
(yield)5
hERa hERb b/a
%Ant. %Ag.
OH
N
1
2
0.8
0.7
45
56 Yes
2.8
2.6
99
86
9
8
Commercial (Aldrich)
See Ref. 4
N
N
1a
OH
136
194 Yes
25 No
54 No
50 No
30 No
60 No
27 ––
N
2a
OH
N
3
2.6
3.0
1.0
0.7
0.8
0.9
1.1
0.4
0.3
1.0
0.9
0.7
0.5
0.4
64
161
50
21
48
24
19
13
13
45
59
73
37
22
12
3.3
0.9
1.0
0.4
0.5
1.6
1.2
3.5
3.8
0.7
0.4
0.5
0.4
––
72
106
85
83
22
31
14
27
34
78
3
34
ꢀ19
8
Commercial (Aldrich)
N
N
3a
HO2C
OH
N
4
1 (50)
HO2C
4a
29
HO2C
OH
N
5
1 (48)
N
N
HO2C
5a
30
OH
N
6
12
65
63
77
36
46
2
2 (19)
2 (17)
1 (26)
1 (26)
1 (23)
1 (17)
1 (11)
3 (39)
1 (7)
HN
31
6a
OH
N
N
7
31
7a
OH
N
HO2C
8
N
N
N
N
HO2C
32
33
8a
OH
N
9
17 ––
32
9a
OH
N
HO2C
10
11
12
13
14
15
16
33 Yes
43 Yes
45 Yes
65 Yes
104 No
74 Weak
55 No
HO2C
10a
OH
N
33
11a
OH
N
HO2C
N
N
HO2C
12a
34
36
OH
N
N+
-
98
22
ꢀ10
39
24
O
13a
35
OH
N
HO2C
HO2C
75
103
52
N
N
N
14a
OH
N
36
36
1 (6)
15a
OH
N
1 (6)
16a
–– Raloxifene
–– 17b-Estradiol
1.8
1.3
7
1
No
––
0.8
––
81
––
n/a
10011b n/a
n/a
n/a
n/a
n/a
1.1

T. A. Blizzard et al. / Bioorg. Med. Chem. Lett. 15 (2005) 107–113
OTIPS
i, ii, iii
109
OH
i, ii, iii
iv
HO
OH
BnO
S
HO
S
O
O
N
N
O O
OAc
OH
O
O
29
45
4a
(+)-48
1-28
OH
OR
Scheme 1. Reagents and conditions: (i) AcCl (neat), reflux, 3h; (ii)
ethanolamine, Et3N, CH2Cl2, rt, 18h; (iii) ClCH2CH2Cl, Ac2O, reflux,
5h; (iv) chiral HPLC (see Ref. 6); (v) LiAlH4, ether, 15h.
Scheme 4. Reagents and conditions: (i) 1a–24a, DIAD, PPh3, THF;
(ii) Pd, HCO2NH4, EtOH, EtOAc, H2O; (iii) n-Bu4NF, AcOH, THF.
iii
All of the novel alkylated pyrrolidine analogs (4–16) re-
tained the excellent ERa potency exhibited by the initial
analogs 1–3 (Table 1) in an in vitro estrogen receptor
binding assay.9 Although the magnitude of receptor sub-
type selectivity (ERb/ERa ratio) varied considerably
(from 17 · to 104 ·), all of the novel analogs were a
selective. In addition, all of the new analogs that were
tested in the MCF-7 assay retained excellent potency.10
As we had hoped, the mono-substituted pyrrolidine ana-
logs more closely resembled the unsubstituted pyrrol-
idine 3 rather than the fused pyrrolidine 2 in the
cyanide adduct assay.4 Interestingly, there was a clear
trend toward increased metabolic activation as the size
of the pyrrolidine substituent increased. Fortunately,
the smaller substituents, which resulted in improved
metabolic stability also conferred an improved uterine
profile. In fact, several of the novel analogs exhibited a
substantially improved uterine profile relative to the
unsubstituted pyrrolidine 3.11 The 3-(R)-methyl pyrroli-
dine side chain present in 4 was especially noteworthy
in this regard. It is interesting that the uterine profile
appears to be highly dependent on the size and
stereochemistry of the pyrrolidine ring substituent.
Substituents that are larger than methyl are clearly det-
rimental (compare 4, 8, and 10) and the 3-(R) stereo-
chemistry is better than 3-(S) (compare 4 and 5).
N
N
OAc
OH
O
6a
46R
i
ii
N
H
N
OAc
O
iii
OAc
31
46
N
N
OH
O
7a
46S
Scheme 2. Reagents and conditions: (i) acetoxyacetyl chloride, Et3N,
CH2Cl2; (ii) chiral HPLC; (iii) LiAlH4, Et2O.
i, ii
iii
N+
N
H
N
-
O
OH
35
47
13a
Scheme 3. Reagents and conditions: (i) H2, Pd/C, EtOH; (ii) 1N HCl,
ether; (iii) 2-bromoethanol, K2CO3, MeCN, reflux.
enantiomerically pure imide. Mosher ester analysis of 4a
showed that no epimerization occurred during the imide
reduction.7 Amines 4a, 5a, and 12a were prepared by
this route.5 In a similar approach starting with a racemic
diacid, such as 32 or 33, chiral HPLC separation of the
imide provided both side chain enantiomers. Amines
8a–11a were prepared in this manner. Application of a
similar sequence to the diastereomeric diacid mixture
36 afforded the 2,3-dimethyl pyrrolidine side chains
14a–16a. Stereochemistry of diasteromers 15 and 16
was established by X-ray analysis of a complex of 15
bound in ERa (vide infra).
Similarly, for the 3,4-dimethyl pyrrolidine analogs 14–
16, the 3R,4R isomer 15 is clearly superior to the other
diastereomers 14 and 16, at least in terms of uterine
activity. Another striking stereochemical effect is seen
with compounds substituted at C-2 of the pyrrolidine
ring. The 2-(S) methyl analog 7 has a substantially infe-
rior uterine profile relative to the 2-(R) analog 6. This is
also illustrated by the large difference in uterine activity
between the 3,3-dimethyl analog 12 (a potent uterine
antagonist) and the corresponding 2,2-dimethyl analog
13 (a potent agonist in uterine tissue).
The enantiomeric 2-methyl pyrrolidine side chains 6a
and 7a were prepared by acylation of racemic 2-methyl
pyrrolidine 31 followed by chiral HPLC resolution of
46 and reduction of the enantiomeric amides 46R and
46S (Scheme 2).8
SERM side chains are generally connected to the core
structure by an unsubstituted ethylene linker. Encour-
aged by the superior uterine profiles of the methylated
pyrrolidines, we decided to examine methylated linkers
to see if a similar improvement in activity could be ob-
tained by adding a methyl group to the linker. To verify
that the two carbon linker was optimal we first prepared
the three carbon analog 17. The requisite side chain 17a
was readily prepared from pyrrolidine (Scheme 5) and
attached to the dihydrobenzoxathiin core using our
standard protocol (Scheme 4). The extended linker was
clearly detrimental with regard to uterine activity
(Table 2; compare 3 and 17). The alkylated-linker side
chains were easily synthesized by bis-alkylation of the
The 2,2-dimethylpyrrolidine side chain 13a was pre-
pared by alkylation of 2,2-dimethyl pyrrolidine 47,
which was, in turn, derived from the commercial
N-oxide 35 (Scheme 3).
Synthesis of the final products proceeded via attachment
of the hydroxyethylamine side chains to the ben-
zoxathiin core (+)-48 using the previously reported pro-
cedure3a,c followed by deprotection (Scheme 4).

from commercial L-alaninol 38 in one step (Scheme 6).
Side chains 18a–24a were prepared in this manner.
110
T. A. Blizzard et al. / Bioorg. Med. Chem. Lett. 15 (2005) 107–113
i
N
H
N
OH
With the alkylated linker, attachment of the side chain
to the dihydrobenzoxathiin core was complicated by
the formation of a by-product, such as 25, which results
from the presence of an unsymmetrical intermediate,
such as 49, in the Mitsunobu alkylation (Scheme 7).
Reaction of 49 with the phenol of (+)-48 via Ôpath aÕ
eventually leads to the ÔnormalÕ product 18 while alkyl-
ation via Ôpath bÕ results in the rearranged by-product
37
17a
Scheme 5. Reagents and conditions: (i) 3-bromopropanol, K2CO3,
MeCN, reflux.
appropriate amine precursor with 1,4-dibromobutane. For
example, the a-(S)-methyl side chain 18a was prepared
Table 2. Side chain preparation summary and biodata for linker alkylated analogs
#
R
ER Binding
(IC50, nM)9
Cyanide
adduct?4 IC50 (nM)
MCF-710
Uterine
activity11
Side chain
Starting material5
Scheme
(yield)5
hERa hERb b/a
%Ant. %Ag.
OH
N
3
2.6
1.7
64
81
25 No
3.3
4.1
72
26
34
69
Commercial (Aldrich)
N
N
3a
OH
N
HN
17
48 ––
5 (67)
6 (58)
6 (48)
6 (57)
N
37
17a
OH
OH
N
NH2
18
19
20
1.3
1.7
0.5
45
71
25
35 No
42 Weak
56 Yes
0.3
0.5
0.2
101
18
0
71
26
18a
38
OH
OH
N
NH2
N
N
19a
OH
N
39
OH
NH2
61
20a
40
OH
OH
N
N
N
N
N
NH2
21
22
23
0.7
0.7
0.5
38
46
15
51 Yes
66 Yes
30 Yes
0.4
2.0
0.4
2
44
20
78
55
79
6 (50)
6 (80)
21a
41
OH
OH
N
NH2
42
22a
OH
OH
N
NH2
6 (92)
6 (44)
43
23a
OH
N
OH
NH2
24
25
26
27
28
0.7
2.5
6.3
0.8
1.2
44
176
363
97
63 No
70 ––
0.5
2.3
9.1
1.3
3.1
31
68
21
30
28
71
29
26
28
40
44
24a
25 is a by-product from synthesis of 18
26 is a by-product from synthesis of 19
27 is a by-product from synthesis of 20
28 is a by-product from synthesis of 21
N
N
58 ––
120 Yes
95 Yes
N
N
114
–– Raloxifene
–– 17b-Estradiol
1.8
1.3
12
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. 15 (2005) 107–113
111
antagonism of 15 and 18, and the partial agonism of
16 and 19. As is illustrated in Figure 1, the disposition
of the methyl substituents of 15 allows a close hydro-
phobic interaction of the pyrrolidine ring with W383,
and with L536 and L539, two residues in helix 12. The
methyl substituents also allow a close approach of resi-
dues C530 and K531, in the loop that connects helix 11–
12. We suggest that all of these interactions may serve to
stabilize helix 12 in the antagonist conformation, leading
to the observed extreme uterine antagonism. In contrast,
the downward disposition of the methyl substituent on
the right in 16 (Fig. 1) forces the compound to the left
(in the view of that figure) to avoid steric clash with
W383, generating fewer hydrophobic interactions with
W383, L536, and L539. In addition, the upward dispo-
sition of the methyl on the left puts steric pressure on
K531, forcing it and the entire connecting loop away
from the ligand. We suggest that the less favorable inter-
actions in the complex with 16 do not provide added sta-
bilization for helix 12 in the antagonist conformation,
explaining the partial agonism of this compound.
i
NH2
N
HO
HO
18a
38
Scheme 6. Reagents and conditions: (i) 1,4-dibromobutane, K2CO3,
MeCN, reflux.
path a, ii, iii
18
25
i
N
N+
a
-
HO
path b, ii, iii
ArO
49
18a
b
Scheme 7. Reagents and conditions: (i) (+)-49, DIAD, PPh3, THF;
(ii) Pd, HCO2NH4, EtOH, EtOAc, H2O; (iii) n-Bu4NF, AcOH, THF.
25. Fortunately, the two products could be separated by
chromatography.
The biodata and preparation of the alkylated linker ana-
logs are summarized in Table 2. Introduction of a
methyl group at the a position of the ethylene linker
did result in an improved SERM profile provided that
the new stereocenter had the (S) stereochemistry. As
with the alkylated pyrrolidines, the size of the substitu-
ent had a strong impact on metabolic activation with
larger substituents resulting in increased oxidation as
measured by cyanide adduct formation. Similarly, uter-
ine activity was highly dependent on the stereochemistry
and site of alkylation as well as the size of the substitu-
ent. For example, while an (S)-methyl substituent at the
a position (next to nitrogen) of the linker improved the
SERM profile, an (R)-methyl at this position resulted in
a substantially less favorable profile (compare 18 and
19). Larger substituents at this position resulted in an
increase in uterine agonism. Alkylation at the beta
position (next to the oxygen) also resulted in a less desir-
able uterine profile (compounds 25–28).
There is a caveat to our proposal that the extreme
uterine antagonism of 15 is a result of the stabilization
of helix 12 in the antagonist conformation via direct
interactions with residues in helix 12 as well as stabiliz-
ing interactions with residues in the loop that connects
helices 11 and 12. We might expect that the residues in
the connecting loop (residues 526–532) would be signif-
icantly better ordered in 15 than in 16, but in fact the
loop is poorly ordered in both of these complexes. The
order is somewhat better for 15 than for 16, but it is still
very poor in absolute terms. We suspect that the poor
ordering of the connecting loop is an artifact induced
by the fact that the protein was carboxymethylated dur-
ing the purification process, to prevent aggregation via
the formation of interresidue disulfide bonds. C530 is
one of the modified residues, and the modification prob-
ably prevents the close approach of the connecting loop
to the antagonist arm of the ligand. Such density as is
observed for this loop is probably due to a subpopula-
tion of unmodified C530 residues. But even without
the full ordering of the connecting loop, 15 makes
X-ray analysis12 of complexes of 15, 16, 18, and 19
bound to the ligand-binding domain of ERa provides
a possible explanation for the extraordinary uterine
Figure 1. The structures of 15 (left) and 16 (right) bound to the ligand-binding domain of ERa. Nitrogen atoms are colored blue, oxygen red, and
sulfur gold. Carbon atoms in the ligand are colored yellow, and carbon atoms in the protein are colored gray. Protein residues that contain at least
˚
one atom within 5.0A of an atom in the ligand are show (residues in helix 3, which are on top in this view, have been removed for clarity). To
emphasize the stereochemistry of the methyl substituents on pyrrolidine ring, the labels ÔupÕ and ÔdownÕ have been added.

112
T. A. Blizzard et al. / Bioorg. Med. Chem. Lett. 15 (2005) 107–113
Figure 2. The structures of 18 (left) and 19 (right) bound to the ligand-binding domain of ERa. Atom coloring and residue labeling is as in Figure 1.
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significantly better direct contacts with residues in helix
12 than does 16, supporting our hypothesis.
Further support comes from a second pair of structures,
18 and 19, where the methyl substituents are now on the
linker, not the ring. As is illustrated in Figure 2, the
down disposition of the methyl substituent of 18 allows
a close approach of the ring to W383, and good interac-
tions with L536 and L539. In the structure of 19, the up
orientation of the methyl group generates a serious steric
clash with K531, causing the pyrrolidine ring to twist.
This reduces the interaction with W383, and loses the
interaction with L539 completely. Once again, 18, the
compound with better interactions with residues in helix
12 and with the connecting loop, is the full antagonist.
In conclusion, we have demonstrated that the SERM
profile of the dihydrobenzoxathiin SERAMs is highly
dependent on the size and location of side chain substit-
uents. Several of the novel compounds described herein
are clearly superior to our starting compound 1. Com-
pounds 4, 15, and 18 are especially noteworthy in this re-
gard. However, believing that further improvement
might be possible, we have continued our effort in this
area. The result of this ongoing effort will be reported
in future publications from this laboratory.
Acknowledgements
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.;
Morgan, J. D., II; 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.
The authors thank Dr. Derek Von Langen and Ms. Judy
Pisano for the preparation of (+)-48 and to Dr. Mats
Carlquist and his colleagues at Karo-Bio for providing
protein samples. Use of the Advanced Photon Source
beamline 17-ID was supported by the companies of
the Industrial Macromolecular Crystallography Associ-
ation through a contract with Illinois Institute of
Technology.
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2. (a) Malamas, M. S.; Manas, E. S.; McDevitt, R. E.;
Gunawan, I.; Xu, Z.; Collini, M. D.; Miller, C. P.; Dinh,

T. A. Blizzard et al. / Bioorg. Med. Chem. Lett. 15 (2005) 107–113
113
M.; Hammond, M. L. Bioorg. Med. Chem. Lett. 2004, 14,
3865.
values that are reproducible to within a factor of 2–3.
Benzoxathiin 1 (n = 36) and estradiol (n > 100) were tested
in multiple assays; data reported in Table 1 are an average
of all determinations.
4. Compounds were incubated with liver microsomes in the
presence of cyanide ion then LC–MS was used to analyze
for the presence of cyanide adducts. This cyanide adduct
assay was used as a surrogate measure of protein adduct
formation subsequent to microsomal oxidation. For more
details, see (a) Evans, D. C.; Watt, A. P.; Nicoll-Griffith,
D. A.; Baillie, T. A. Chem. Res. Toxicol. 2004, 17, 3.
5. All new compounds were characterized by LC–MS and
400, 500, or 600MHz 1H NMR. Side chains were prepared
using starting materials and methods indicated in Tables 1
and 2 (yields are the overall yield of the side chain from the
indicated starting material). Starting material sources: 29–
31 and 34–44: Aldrich; 32 and 33 were prepared from the
corresponding alkyl diethylmalonates (Aldrich) by a
procedure analogous to a literature synthesis of 2,3-
dimethyl succinic acid: Sutton, S. C.; Nantz, M. H.;
Hitchcock, S. R. Org. Prep. Proc. Int. 1992, 24, 39.
6. Enantiomeric excess of 45 was determined by chiral HPLC
analysis on a Chiralcel OD 4.6 · 250mm 10lm column
eluted with 4:1 heptane–isopropanol at 0.4mL/min. Under
these conditions, the (R)-enantiomer of 45 has a retention
time of 31.8min while the (S)-enantiomer elutes at
36.2min.
10. An in vitro MCF-7 breast cancer cell proliferation assay
adapted to a 96-well format. Cells were grown in estrogen-
depleted media for 6days then treated with the test
compound for 7days. 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.
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 1mpk. Agonism is
reported as % of estradiol control; antagonism reported as
%antagonism of estradiol; (b) Estradiol exhibited 100%
agonism at 4lg/kg.
12. Complexes of the ligand binding domain of ERa (residues
307–554) with the ligands 15, 16, 18, and 19 were
crystallized by vapor diffusion, using a precipitant con-
taining 100mM MgCl2, 6% PEG 3350, and 100mM
imidazole buffer, pH7.1. The space group of the crystals
was P6522, with cell dimensions a = b = 58.62, c = 276.79
(15), a = b = 58.57, c = 276.31 (16), a = b = 58.64,
c = 275.77 (18), and a = b = 58.69, c = 277.18 (19).
Diffraction data for all complexes were measured at
beamline 17-ID of the Advanced Photon Source. The data
were processed with program X-GEN, which yielded an
7. Enantiomeric purity of 4a was confirmed by analysis of
the 600MHz 1H NMR of the Mosher ester of 4a.
The Mosher ester of racemic 4a was also prepared for
comparison.
˚
R-merge of 0.073 for the data from 1 to 1.8A (15), 0.060
˚
8. The stereochemistry assignments of 6a and 7a were made
by comparison of their optical rotations to an authentic
sample of the (S)-enantiomer 7a, which was prepared from
the known (S)-N-benzoyl-2-methyl-pyrrolidine: (a) Ring-
dahl, B. Acta Chem. Scand. Ser. B 1984, 38, 141.
9. 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
for the data from 1 to 1.7A (16), 0.083 for the data from
˚
˚
1 to 1.8A (18), and 0.066 for the data from 1 to 1.6A
(19). The structures were refined using program SHELXL,
with final values for R-work and R-free of 0.181 and 0.269
˚
for the data from 10.0 to 1.80A resolution (15), 0.177 and
˚
0.267 for the data from 10.0 to 1.70A (16), 0.187 and
˚
0.299 for the data from 10.0 to 1.80A (18), and 0.184 and
˚
0.251 for the data from 10.0 to 1.60A (19). Coordinates
and structures factors have been deposited with the
Protein Data Bank (entries 1XP1, 1XP6, 1XP9, and
1XPC).