Discovery of a potent steroidal glucocorticoid receptor antagonist with enhanced selectivity against the progesterone and androgen receptors (OP-3633)

Article abstract of DOI:10.1021/acs.jmedchem.9b00711

Structure-based modification of mifepristone (1) led to the discovery of novel mifepristone derivatives with improved selectivity profile. Addition of a methyl group at the C10 position of the steroid has a significant impact on progesterone receptor (PR) and androgen receptor (AR) activity. Within this series, OP-3633 (15) emerged as a glucocorticoid receptor (GR) antagonist with increased selectivity against PR and AR, improved cytochrome P450 inhibition profile, and significantly improved pharmacokinetic properties compared to 1. Furthermore, 15 demonstrated substantial inhibition of GR transcriptional activity in the GR positive HCC1806 triple negative breast cancer xenograft model. Overall, compound 15 is a promising GR antagonist candidate to clinically evaluate the impact of GR inhibition in reversal or prevention of therapy resistance.

Full text of DOI:10.1021/acs.jmedchem.9b00711

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Article
Discovery of a Potent Steroidal Glucocorticoid Receptor
Antagonist with Enhanced Selectivity against the
Progesterone and Androgen Receptors (OP-3633)
Xiaohui Du, John Eksterowicz, Haiying Zhou, Yosup Rew, Liusheng Zhu, Xuelei Yan, Julio
C. Medina, Tom Huang, Xi Chen, Dena Sutimantanapi, Nadine Jahchan, Wayne Kong,
Jessica Sun, Tatiana Zavorotinskaya, Qiuping Ye, Valeria R. Fantin, and Daqing Sun
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00711 • Publication Date (Web): 25 Jun 2019
Downloaded from http://pubs.acs.org on June 26, 2019
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Discovery of a Potent Steroidal Glucocorticoid Receptor
Antagonist with Enhanced Selectivity against the
Progesterone and Androgen Receptors (OP-3633)
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Xiaohui Du*, John Eksterowicz, Haiying Zhou, Yosup Rew, Liusheng Zhu, Xuelei Yan, Julio C.
Medina, Tom Huang, Xi Chen, Dena Sutimantanapi, Nadine Jahchan, Wayne Kong, Jessica Sun,
Tatiana Zavorotinskaya, Qiuping Ye, Valeria R. Fantin, Daqing Sun*
ORIC Pharmaceuticals, 240 E Grand Avenue, Fl2, South San Francisco, CA 94080, USA.
KEYWORDS: Glucocorticoid receptor (GR), antagonist, androgen receptor (AR), progesterone
receptor (PR), selectivity.
ABSTRACT: Structure-based modification of mifepristone (1) led to the discovery of novel
mifepristone derivatives with improved selectivity profile. Addition of a methyl group at the C10
position of the steroid has a significant impact on progesterone receptor (PR) and androgen
receptor (AR) activity. Within this series, OP-3633 (15) emerged as a glucocorticoid receptor
(GR) antagonist with increased selectivity against PR and AR, improved cytochrome P450
inhibition profile, and significantly improved pharmacokinetic properties compared to 1.
Furthermore, 15 demonstrated substantial inhibition of GR transcriptional activity in the GR
positive HCC1806 triple negative breast cancer xenograft model. Overall, compound 15 is a
promising GR antagonist candidate to clinically evaluate the impact of GR inhibition in reversal
or prevention of therapy resistance.
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INTRODUCTION
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The glucocorticoid receptor (GR) is a member of the superfamily of nuclear hormone receptors
that is activated by both natural and synthetic glucocorticoids (GCs), such as cortisol and
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dexamethasone, respectively. The GR is expressed across a variety of tissues. Upon ligand
binding, GR translocates into the nucleus, where it binds to glucocorticoid response elements
(GREs) and other transcription factors such as NF-B, and AP1 to regulate the transcription of a
wide range of genes controlling metabolism, cell growth, differentiation, apoptosis, inflammation,
and nervous system activities, including cognition and mood.3
Given the plethora of biological processes regulated by GCs, dysregulation in receptor signaling
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has been implicated in a number of disease states including Cushing’s syndrome, diabetes,
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depression, cancer, and immunosuppression. Accordingly, there has been considerable interest
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in the development of GR antagonists for therapeutic purposes. For example, mifepristone (1), a
potent steroidal GR antagonist, was approved by the FDA in 2012 for the treatment of Cushing’s
syndrome.10 More recently, GR has been shown to play a role in mediating resistance to
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chemotherapy in a variety of solid tumors including ovarian cancer, triple-negative breast
1
3
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cancer (TNBC), pancreatic cancer, non-small cell lung cancer, and urological cancers. In
prostate cancer, GR has been shown to create a bypass to the related androgen receptor (AR) and
to drive resistance to antiandrogens such as enzalutamide and apalutamide.17-18 Therefore, the
ability of GR antagonists, including 1, to overcome resistance to numerous standard of care agents
is under clinical evaluation.
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For example, the combinations of 1 with enzalutamide and nab-paclitaxel are being evaluated in
castration-resistant prostate cancer (CRPC)19 and TNBC , respectively. However, 1 exhibits
partial AR agonistic activity and potent progesterone receptor (PR) antagonistic activity (IC =
20
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.4 nM, Figure 1).21-22 More specifically, the AR agonistic activity of 1 is sufficient to stimulate
the proliferation of CRPC LNCaP/AR-luc (LNAR) cells both in vitro and in vivo and induces AR
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target gene expression in AR TNBC MDA-MB-453 cells. In addition, because of its CYP450
inhibition profile, when co-dosed with paclitaxel, 1 increases the paclitaxel exposure due to
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CYP2C8-driven drug-drug interactions. These unwanted features of 1 limit its potential use in
certain settings such as AR-driven cancers or in combinations that include paclitaxel and related
chemotherapeutic agents, and highlight the need for selective and potent GR antagonists that do
not carry the AR, PR, and CYP2C8 liabilities of 1.
Recently, we reported the discovery of compound 2, a potent steroidal GR antagonist.24
Introducing a t-butyl group substitution onto the alkyne moiety attenuated the AR agonism
associated with 1. Here we report the continued investigation of 1, which led to the discovery of a
series of novel C10-methyl steroidal GR antagonists. Among them, OP-3633 (15) exhibits lower
AR agonism and excellent selectivity against GR over PR, as well as an improved CYP inhibition
profile compared to 1 (Figure 1).
N
O
N
O
N
O
OH
OH
OH
H
H
H
H
H
H
H
Mifepristone (1)
2
15
GR antagonism IC50 = 3.26 ± 0.62 nMa GR antagonism IC50 = 14.6 ± 4.5 nMa GR antagonism IC50 = 29.0 ± 11.0 nMa
AR agonism EC50 = 11.9 nM [100]b
PR antagonism IC50 = 0.4 ± 0.2 nMc
AR agonism EC50 = >2500 nM [13]b AR agonism EC50 = >2500 nM [13.3]b
PR antagonism IC50 = 14.4 ± 6.0 nMc PR antagonism IC50 = 1135 ± 202 nMc
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a
b
Figure 1. Mifepristone (1), compound 2 and 15. IC in GR luciferase antagonist assay. EC
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c
[
Emax]* in AR luciferase agonist assay. * = % mifepristone. IC in PR luciferase antagonist
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assay.
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RESULTS AND DISCUSSION
Starting from 2, reduction of the C9-C10 double bond in 2 led to the synthesis of 3, which was
more potent against GR, but also showed a slight increase of AR agonism compared to 2. Both 2
and 3 had lower affinity for PR compared to 1 (IC = 14.4 and 3.4 nM, respectively), resulting
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in a slightly better PR/GR ratio. Several more analogs of 3 with various aniline N-alkyl
substituents at the C11 position were synthesized with the goal of evaluating their interactions
with PR (Table 1, compounds 4-7), while taking advantage of the increased potency on GR
antagonism as a result of the reduced C9-C10 double bond. Luciferase (luc.) GR, AR and PR
reporter assays were employed to characterize agonism and antagonism of compounds. None of
the tested compounds showed meaningful GR and PR agonism, and data for these assays are not
reported in the tables below. Increase of the substituent’s size to N, N-diethyl group (4)
attenuated AR agonism (Emax = 9.4% of 1) and maintained high GR antagonism; however, it did
not improve selectivity against PR. Replacing N,N-dimethyl group with a more constrained
morpholine moiety (5) maintained GR antagonism and low AR agonism but had no impact on
PR selectivity. The 4-methylpiperazinyl substitution on compound 6 weakened PR affinity (IC50
=
25.4 nM) compared to 1 and 2. However, this modification also decreased GR antagonism
slightly, with no net change in PR selectivity. Replacement of the 4-dimethylamino group with
the electron-withdrawing 4-(methylsulfonyl)piperazinyl group led to 7, which was potent against
GR (IC = 12.2 nM) and PR (IC = 4.6 nM). Installing a 3,3-dimethylpentynyl group, slightly
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bulkier than t-butyl group at the C17 position (8), diminished the GR inhibition potency (IC =
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2.7 nM) to a lesser extent than the PR inhibition potency (IC = 21.1 nM) compared to 3, and
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resulted in a small improvement in the selectivity against PR compared to 1, 2 and 3. Overall, the
compounds shown in Table 1 have lower affinity for PR compared to 1, but have no significant
improvement in PR selectivity compared to 2. Compounds in Table 1 were also tested for their
AR antagonistic activity and their IC values are shown in the table. These compounds are
1
1
1
1
1
1
1
1
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2
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2
3
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3
3
4
4
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4
4
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4
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5
5
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5
5
5
5
6
0
1
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8
9
0
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moderate to weak AR antagonists, resulting in a 10-30-fold GR over AR selectivity.
Table 1. Exploration of analogs of 2a
GR luc.
antagonism
IC50 (nM)
AR luc. agonism
EC50 (nM)
AR luc.
antagonism
IC50 (nM)
PR luc.
antagonism
IC50 (nM)
Compound
Structure
b
[Emax]
N
O
OH
2
14.6±4.5
5.6±2.4
13.9±9.4
>2500 [13]
224±97 [22]
>2500 [9.4]
129±49
165±14
263±62
14.4±6.0
3.4±0.2
6.0±2.4
H
H
N
OH
3
4
H
H
H
H
O
N
OH
H
H
H
H
O
O
N
OH
5
6
7
10.3±6.5
35.8±2.8
12.2±1.1
>2500 [5.1]
>2500 [7.9]
>2500 [18.1]
208
4.8±0.6
25.4±11.1
4.6±2.6
H
H
H
H
O
N
N
OH
1005±407
359±47
H
H
H
H
O
O
O
S
N
N
O
OH
H
H
H
H
N
OH
8
12.7±9.9
>2500 [7.3]
175±9.9
21.1±11.4
H
H
H
H
O
aPotency and Emax data with SD are reported as the average of at least two determinations. % mifepristone.
b
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In the literature, it has been noted that the -C10-methyl group on the androstene steroid can
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weaken the affinity towards PR. The hypothesis is that the steric interaction between the -
C10-methyl group and the -C11-aryl moiety results in ring A bending downwards compared to
the corresponding analog with a C9-C10 double bond (when using C7, C11 and O17 as anchor
positions), and the displacement of the carbonyl functional group leads to the low affinity for PR.
We decided to introduce the -C10-methyl group into our existing GR antagonists to evaluate its
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impact on PR, GR, and AR interactions. Table 2 shows a number of -C10-methyl analogs with
various propynyl groups at the C17 position (compounds 9-15) while maintaining 4-
dimethylaminophenyl substitution at C11. With a methyl substitution at the C10 position,
compound 9 showed a reduction in AR agonism (Emax = 7.4% of 1 for 9 compared to Emax = 22%
of 1 for 3) and a 6-fold decrease in GR antagonism compared to its C10-hydrogen analog 3 (IC50
=
33.6 nM for 9 and IC = 5.6 nM for 3). Strikingly, compound 9 was found to have a much-
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weakened PR affinity (IC = 2.5 M), which was significantly lower than that of 3 (IC = 3.4
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nM) and led to a 70-fold GR over PR selectivity.
Encouraged by the increased PR selectivity of 9, we next explored whether small alkyne
substitutions at the C17 position would improve GR antagonism while maintaining PR
selectivity. Changing the t-butyl group to smaller alkyl groups maintained high selectivity
against PR (Table 2). The PR antagonist IC of 10-15 ranged from 0.91 M to 4.24 M.
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Interestingly, decrease of the substituent’s size from a t-butyl group to a methyl group triggered
no substantial AR agonism (9 vs 15). All of the C10-methyl analogs had remarkably decreased
AR agonism compared to their corresponding C9-C10 double bond analogs reported by us
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previously. For example, compound 10 with an isopropyl alkyne substitution at C17 showed
low AR agonism (Emax = 8.3% of 1), while the corresponding C9-C10 double bond analog had
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much higher AR agonism (Emax = 50% of 1). Similarly, compound 15 with a methyl group
substitution had low AR agonism (Emax = 13.3% of 1), while the corresponding C9-C10 double
bond analog 1 exhibited maximum AR agonism (Emax = 100%). These results imply that a
methyl group at the C10 position is crucial to minimize AR agonism and PR antagonism, and
that a large t-butyl group is not required to attenuate AR agonism of the C10-methyl analogs.
Furthermore, most of the C10-methyl analogs in Table 2 are much weaker AR antagonists
compared to those in Table 1. On the other hand, the C10-methyl compounds in Table 2 suffer
from loss of GR antagonism at various degrees. Among them, compound 9, 11, and 15 were
1
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1
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most potent (IC from 28 nM to 34 nM), within 2-fold difference compared to 2.
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Table 2. Exploration of the alkyne substituents at the C17 position in the -C10-methyl analogs
N
OH
R
21
12
22
13 17
11
16
1
9 H 14
2
5 H 8
6
15
10
3
H
7
O
4
GR luc.
antagonism
IC50 (nM)
AR luc.
agonism
AR luc.
antagonism
IC50 (nM)
PR luc.
antagonism
IC50 (nM)
Compound
R
EC50 (nM)
b
[Emax]
9
33.6±8.5
>2500 [7.4]
>5000
2488±195
1
0
62.0±12.2
28.5±6.3
>2500 [8.3]
>2500 [5.4]
>5000
2084±359
1487±508
11
1452±526
O
1
2
116±13.3
>2500 [17.5]
>5000
4242±411
13
14
15
73.7±32.4
47.2±11.7
29.0±11.0
>2500 [13.2]
>2500 [7.3]
>2500 [13.3]
2955±339
642±7
1546±170
910±242
1135±202
CF3
912±403
aPotency and Emax data with SD are reported as the average of at least two determinations. b % mifepristone.
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In addition to the GR IC values shown in Table 1 and 2, Figure 2 shows the dose response
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curve for the luciferase assay of several most potent GR antagonists in each Table using
compound 1 (mifepristone) as control. All of them can completely inhibit the activity of GR,
demonstrating that they are GR full antagonists.
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Figure 2. Dose response curve for the GR luciferase assay of selected compounds in Table 1 and
Table 2.
To elucidate the structural basis for the improved PR selectivity observed upon the addition of
a methyl group at C10, a variety of approaches were undertaken. Consistent with earlier
observations, modeling studies suggested that reduction of the C9-C10 double bond would
change the shape of the scaffold.25 In addition, ab initio calculations showed that the -C10-
methyl group has a significant effect on the rotational profile of the -C11 4-
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dimethylaminophenyl group, restricting the C -C -C -C rotation (Figure 3) such that the plane
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of the phenyl ring is essentially fixed, sandwiched between the C10 and C13 methyl groups.
N
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7
8
9
0
21
11
9
22
H
H
H
O
15
Figure 3. Illustration of C -C -C -C rotation.
9
11
21
22
Finally, small molecule x-ray structures of both 1 and 15 were obtained. As shown in Figure 4,
the data are consistent with the results from the ab initio calculations. (A) shows the structure of
1
(green), in which the A-B-C-D rings of the scaffold adopted a relatively flat orientation with a
slight twist of the C -C -C -C torsional angle of 15.4 degrees. In comparison, (B) shows the
9
11
21
22
structure of 15 (purple) in the same orientation. The puckering of the scaffold is evident and
may affect the ability of the A-ring carbonyl to make a key hydrogen bond interaction present in
PR, AR and GR. Furthermore, the addition of the -C10-methyl group causes rotation of the C9-
C -C -C bond to 38.4 degrees. This forced change in the conformation may result in
11
21
22
2
6
additional steric conflicts with PR, resulting in improved selectivity toward GR. (C) shows the
overlay of the two structures further highlighting the key differences.
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Journal of Medicinal Chemistry
Page 10 of 51
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Comparison of the small molecule X-ray structures of mifepristone (A, green) with 15
(B, purple) with key torsional angle highlighted. The overlay of the two structures is shown in
C.
Inspired by the discovery of the -C10-methyl analogs, we further investigated modification of
the aryl group at the C11 position to improve GR antagonism. Bulkier substitutions were
introduced to the aryl group and their functional impact assessed. Replacement of one of the N-
methyl groups on the 4-dimethylaminophenyl in 15 with an isopropyl group (16) improved GR
antagonism (IC = 20.6 nM) and also maintained good selectivity against PR. However,
5
0
compound 16 had an unexpected higher AR agonism (Emax = 55.9% of 1). Expanding the 4-
dimethylaminophenyl to an N-methyl-N-methoxyethylphenyl (17) restored low AR agonism and
maintained high selectivity against PR. However, compound 17 was a less potent GR antagonist
compared to 15. Replacing the 4-dimethylaminophenyl with a constrained substituent 4-
pyrrolidinylphenyl (18) did not improve its GR antagonistic potency. Similarly, changing the 4-
dimethylaminophenyl to a 4-pyrrolylphenyl (19) provided no improvement in GR antagonism. A
bigger change resulting from the replacement of the C11 aniline by a 4-methoxylphenyl (20) did
not improve the GR potency either.
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We noted that one GR antagonist recently reported in the literature carries a 4-
chlorobenzyloxy group at C11.27 To determine the impact of this group on our scaffold, 4-
chlorobenzyloxy group was introduced at C11. 21 had improved GR antagonism (IC = 17.6
5
0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
nM), but showed a significant decrease in selectivity against PR (IC = 8.2 nM) as well as a
5
0
significant increase in AR agonism (Emax = 47.8%) and AR antagonism (IC = 85 nM). These
50
results suggest that the reduced steric clash between the -C10-methyl group and the -C11 4-
chlorobenzyloxy moiety in 21 is not able to hold the conformation required for the reduced
affinity towards PR.
a
Table 3. Modification of the substituents at the C11 position
GR luc.
antagonismI
AR luc.
agonism
EC50 (nM)
AR luc.
antagonism
IC50 (nM)
PR luc.
antagonis
m
Compound
Structure
C
50 (nM)
b
[Emax]
IC50 (nM)
N
O
OH
16
20.6±6.6
52.1±3.5
1142 [55.9]
2246±217
1295±160
527±126
H
H
H
H
N
O
O
OH
17
>2500 [20.0]
1640±220
H
H
N
OH
1
8
57.9 ±24.5
52.2±16.4
>2500 [10.6]
>2500 [16.5]
3146±2622
354±87
4068±1614
523±257
H
H
H
O
N
OH
19
H
H
H
O
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
O
OH
2
0
58.3±27.0
17.6±12.8
>2500 [14.1]
561±93
85±21
1689±125
8.2±3.8
H
H
H
Cl
OH
O
2
1
1885 [47.8]
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H
H
H
O
aPotency and Emax data with SD are reported as the average of at least two determinations. % mifepristone.
b
Given the GR antagonistic potency, high selectivity against PR, and low AR agonism,
compounds 9, 11 and 15 were selected for evaluation in the CYP inhibition assays (Table 4). The
results from those studies showed that compound 15 had relative low potential for CYP3A4 and
CYP2C8 inhibition compared to 9 and 11 at the concentration of 10 M.
Table 4. CYP inhibition of compounds 9, 11, and 15
% inhibition (10 M)
P450
Substrate
Isoform
9
11
15
3A4
Midazolam
Paclitaxel
71
53
70
34
58
2
C8
84
Since compound 1 was reported to have high potential for CYP2C8 inhibition (IC =1.5 M), a
5
0
detailed CYP IC evaluation of 15 was performed in comparison to 1 and 2. As shown in Table
5
0
5, compound 2 had a much lower potential for CYP2C8 inhibition but elevated CYP3A4
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inhibition relative to compound 1. Compound 15 offered an improved overall profile with low
inhibition potential for both CYP3A4 and CYP2C8.
Table 5. CYP profiles of 1, 2, and 15a
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
IC50 (M)
P450
Substrate
Isoform
1
2
15
9.5b
2.9c
>10d
3
A4
Midazolam
Paclitaxel
2
C8e
1.5
>10
8
a
b
CYP2C8 and CYP3A4 data are reported as the average of at least two determinations. IC50 is between 8.1-13
C
d
e

M (n=8). IC50 is between 2.4-3.4 M (n=2). IC50 is >10 M each time (n=2). CYP2C8 IC50 for compounds 1,
2, 15 is within 10% of average value in each measurement.
Next, compound 15 was subjected to further in vitro profiling. In the PolarScreen
Glucocorticoid Receptor Competitor assay (Figure 5), the potency of compound 15 (IC = 8.5
5
0
nM was comparable to that of 2 (IC = 8.0 nM), and 3.5-fold lower than that of 1 (IC = 2.3
5
0
50
nM). In the GR-coactivator interaction assay (Figure 6), compound 15 was 2-fold less potent
than 2 (IC = 16 nM and IC = 7.5 nM, respectively) and approximately 4.5-fold less
50
50
potent than 1 (IC = 3.5 nM).
50
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
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4
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4
5
5
5
5
5
5
5
5
5
5
6
350
300
250
200
150
100
cortisol
1
2
1
5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
10-1
100
101
102
103
104
competitor(nM)
Figure 5. Binding affinities of 1, 2 and 15 to human GR in the PolarScreen GR Competitor
Assay.
10000
1
2
1
8000
5
6
4
2
000
000
000
0
1
0-1
100
101
102
103
104
Concentration (nM)
Figure 6. 2 and 15 block the interaction between GR and its coactivator in the GR-coactivator
protein-protein interaction assay.
To determine whether 15 would be suitable for preclinical development, its pharmacokinetic
properties were characterized in preclinical species, namely rat, dog, and minipig. Although
compound 15 had similar clearance to that of 1 and 2 in rat, it showed about 10-fold and 2-fold
higher oral exposure than that of 1 and 2, respectively (Table 6). Additionally, compound 15 had
moderate clearance and good oral exposure in both dog and mini-pig (Table 7). The favorable
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solubility of 15 (Table 8) across all pH levels might contribute to the high oral exposures across
species.
Table 6. Pharmacokinetic data of 1, 2 and 15 in rat
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
iv (0.5 mg/kg)a
po (5 mg/kg)
compound
CL
Cmax
AUC
V
ss (L/kg)
t
1/2 (iv, h)
F (%)
(
L/kg/h)
(g/L)
(g·h/L)
1b
3.7
4.0
4.1
1.6
1.9
6.4
37
24
72
85
2b
4.9
471
1
5c
3.1
4.7
1.8
62
518
1020
a
b
formulated in 10% DMSO, 70% PEG 400, and 20% water; formulated in 5% DMSO, 95% 0.2% Tween 80 in
c
0
.25% CMC; 5% DMSO/ 95% 0.2% Tween 80 in 0.25% CMC, pH 4
Table 7. Pharmacokinetic data of 15 in dog and mini-pig
iv (0.5 mg/kg)a
po (5 mg/kg )b
Species
CL (L/kg/h)
V
ss (L/kg)
t
1/2 (h)
F (%)
Cmax (g/L)
AUC (g·h/L)
Dog
0.61
0.75
8.32
1.45
16.2
2.71
67.6
35.9
1261
249
5927
2451
Mini-pig
a
b
Formulations: 15 was in 5% DMA, 10% EtOH, 40% PEG400, and 45% D5W for iv study; 15 was in 95% 0.2%
Tween 80, and 5% DMSO in 0.25% CMC, for dog oral study, and 15 was in 95% 0.2% Tween 80, and 5% DMSO
in 0.25% CMC, pH 4 for minipig oral study.
Table 8. Solubility of compound 1 and 15
Cmd #
Solubility (M)
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
FaSSGF
PH=1.2
FaSSIF
PH=6.5
PBS
PH=7.4
1
>1164
1130
5.4
3.0
1
5
59.5
20.6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Furthermore, we evaluated whether 15 could inhibit GR transcriptional activity in the
HCC1806 xenograft model. A bolus oral dose of cortisol (5 mg/kg) was administered to
effectively activate human GR in HCC1806 triple negative breast cancer cells. Expression of the
GR target gene FKBP5 was analyzed in HCC1806 tumors collected from mice at 3 and 6 h after
cortisol administration, receiving either a single oral dose of 15 or vehicle as a control. Cortisol
treatment resulted in 2.3- to 4.0-fold induction of FKBP5 expression compared to the vehicle
group, assessed by RT-qPCR. In the presence of 15 at 150 mg/kg, cortisol-mediated induction of
FKBP5 expression was reduced 2.8-fold and 2.3-fold, respectively. The levels of FKBP5 in
tumors from mice treated with 15 and cortisol at both 3 h and 6 h were comparable to those
detected in tumors from the vehicle treated mice (Figure 7A). The unbound plasma concentration
of 15 reached 400 nM at 1h and remained at that level during the study period (Figure 7B).
These data suggest that 15 is effective at inhibiting cortisol-induced GR target gene expression in
HCC1806 xenograft tumors, and the reduction of GR target gene expression could be correlated
with the plasma exposure of 15.
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#
#
5
4
3
2
1
0
#
*
**
*
*
1
0
1
5 at 150 mg/kg
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ns
1
ns
0.1
e
h
3
0.01
3
h
6h
l
6h
icl
eh
l
t
a
at
iso
ritso
V
rt
15
15
o
+
o
+
l
C
l
C
0.001
iso
iso
0
1
2
3
4
5
6
7
8
rt
rt
o
o
C
C
Time (hr)
(A)
(B)
Figure 7. In vivo pharmacodynamics of compound 15. (A) mRNA levels of GR target gene
FKBP5 in HCC1806 tumors relative to vehicle 3 and 6 h after treatment (n= 3 mice/group).
Cortisol was administered orally at 5 mg/kg. A single dose of 15 at 150 mg/kg was administered
orally by gavage. (B) Unbound plasma concentration-time profile of 15 up to 6 h in group treated
with cortisol at 5 mg/kg and 15 at 150 mg/kg (n = 3 mice per group). Significance of effects on
FKBP5 expression was determined by One-Way ANOVA using Dunnett’s test to correct for
multiple comparisons. ***, p < 0.001, and **, p<0.01 vs. vehicle group. ns, no significant
#
#
#
difference vs. vehicle group. , p < 0.01, and , p < 0.05 vs. corresponding cortisol group.
As anticipated from previous reports evaluating safety of GR antagonists such as 1, adrenal
gland enlargement and a change in uterine weight were noted.22, 28 Since compound 15 exhibited
enhanced selectivity for GR relative to PR in the luciferase assay and had high oral exposure in
rat, the safety profile of 15 was explored, and particular attention was paid to the impact on the
female reproductive system. We reasoned that the reduced inhibitory activity of 15 on PR could
help minimize potential risks associated with inhibition of this nuclear receptor in female
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Page 18 of 51
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
reproductive organs such as endometrial hypertrophy, irregular vaginal bleeding and pain. Based
on a 2-fold safety margin above the effective inhibition exposure of 15 (Figure 7B) on GR target
gene expression in HCC1806 xenograft tumors, oral (gavage) administration of 15 once daily to
rats for 14 days at the dose of 250 mg/kg/day was performed to determine its potential toxicity.
The study revealed that 15 was well tolerated in female rats across the entire study period. There
were no clinical observations and effects on mean body weights, or changes in food consumption
related to 15 during this study. Importantly, no noticeable 15-related effects were observed in
ovarian or uterine weight. The 15-related findings were considered non-adverse based on
minimal to mild severity and lack of clinical pathology correlates suggestive of organ
dysfunction.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CHEMISTRY
2
4
Compounds (3-8) were synthesized according to the procedures reported previously. Scheme
25
1
described the synthesis of 15 via a modified known synthetic route. In a similar fashion,
compounds 9-14, and 16-20 were prepared from the appropriate starting materials.
Our route toward 15 began with the conversion of adrenosterone 22 to bis-ketal 23 under acidic
conditions. The carbonyl group in 23 was converted to enol nonaflate 24 via lithiation with LDA
and subsequent treatment with 1,1,2,2,3,3,4,4,4-nonafluoro-1-butanesulfonyl fluoride. Suzuki
coupling of 24 with Boc-protected methylaminophenyl boronic acid afforded 25 exclusively. An
attempt to install the desired stereochemistry at C11 via the Birch reduction in a trial reaction
29
resulted in the undesired stereochemistry. Alternatively, selective epoxidation of the C5-C6
double bond with hydrogen peroxide activated by hexafluoroacetone furnished -substituted
epoxide 26, which was reduced to alcohol 27 with lithium aluminum hydride. Oxidation of the
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C11-C12 double bond in 27 with mCPBA provided a 1:1.7 α/β mixture of epoxides, with the -
epoxide 28 as the minor diastereomer. Birch reduction of 28 yielded desired -C11 aryl
compound 29. Fortuitously, the Boc group was removed during the reaction. Reductive
amination of 29 with formaldehyde enabled installation of the N-methyl group. Deprotection of
bis-ketal on 30 and elimination of the C5 hydroxyl group in the presence of HCl, gave diketone
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
1. Barton deoxygenation of the C12 hydroxyl group in 31 via an imidazole carbothioate
intermediate 32 provided 33, which was then selectively protected as ethoxy dienone 34 under
3
0
acidic conditions. Finally, addition of prop-1-yn-1-ylmagnesium bromide to 34 followed by in
situ deprotection of the ethoxy dienone afforded compound 15.
The synthesis of compound 21 is illustrated in Scheme 2. Reduction of intermediate 23 with
lithium aluminum hydride provided alcohol 35 as a single isomer. Alkylation of 35 with 4-
chlorobenzyl bromide followed by hydrolysis with 4 N hydrochloric acid, gave enone 37, which
was then protected as ethoxy dienone 38 under acidic conditions. Changing the solvent from
ethanol to a mixture of THF and ethanol (30:1) minimized the formation of bis-enol ether and
3
1
improved the yield of 38. Addition of prop-1-yn-1-ylmagnesium bromide to 38 followed by in
situ deprotection of the ethoxy dienone afforded the final compound 21.
a
Scheme 1 . Synthesis of 15
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1
1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
O
O
O
O
O
C F SO O
4
9
2
O
H
a
H
b
H
c
H
H
H
H
O
O
H
H
O
2
2
O
23
O
24
O
N
O
N
O
N
O
O
O
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
O
O
O
O
H
H
H
d
e
H
H
H
O
H
H
O
O
H
O
O
O
OH 27
O
N
25
26
O
O
HN
O
N
O
OH
H
O
OH
H
O
O
f
H
g
O
O
h
O
H
H
H
H
O
H
H
O
N
OH
28
O
OH
29
O
OH
30
N
N
N
OH
H
O
O
S
N
O
k
i
j
O
H
O
H
H
H
H
H
O
O
H
H
31
3
2
33
N
N
O
O
OH
l
H
H
H
H
H
H
EtO
3
4
15
a
Reagents and conditions: (a) CH(OMe) , ethylene glycol, TsOH, 40 °C, 18 h, 65%; (b) LDA,
3
THF, -78 °C, 30 min, then C F SO F, 2 d, 55%; (c) (4-((tert-
4
9
2
butoxycarbonyl)(methyl)amino)phenyl)boronic acid, LiCl, 2 M Na CO , toluene, ethanol,
2
3
reflux, 42 h, 92%; (d) H O , Na HPO , CF COCF , 2 d, 88%; (e) LiAlH , THF, 1 h, 88%; (f)
2
2
2
4
3
3
4
mCPBA, DCM, 20 h, 27%; (g) Li, NH , THF, 81%; (h) HCHO, HOAc, DCM, NaBH(OAc) , 1
3
3
h, 92%; (i) 4 N HCl, acetone, 2 h, 96%; (j) 1,1'-thiocarbonyldiimidazole, Et N, DCM, 4 d, 83%;
3
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(
k) Bu SnH, toluene, reflux, 3 h, 84%; (l) (i) TsOH, CH(OEt) , EtOH, 1 h, 29%; (ii) prop-1-yn-
3
3
1
-ylmagnesium bromide, THF, overnight, followed by 4 N HCl, 1 h, 75%.
a
Scheme 2 . Synthesis of compound 21
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
Cl
O
O
O
O
HO
O
O
O
H
a
c
H
b
H
O
H
H
H
H
O
O
H
H
O
O
23
35
O
36
Cl
Cl
Cl
O
O
OH
O
O
O
H
d
H
H
H
H
H
H
H
H
O
EtO
O
37
21
38
a
Reagents and conditions: (a) LiAlH , THF, 0 °C to RT, 3 h, 61%; (b) NaH, 4-Cl BnBr, 0 °C to
4
RT, overnight, 68%; (c) 4 N HCl, acetone, 2.5 h, 86%; (d) (i) TsOH, CH(OEt) , THF /EtOH, 3 h,
3
5
6%; (ii) prop-1-yn-1-ylmagnesium bromide, THF, 0 °C to RT, 15 h, followed by 1 N HCl, 30
min, 76%.
CONCLUSION
In summary, structural modification of 1 by incorporation of a methyl group at the C10 position
3
2
led to the discovery of 15, a potent, selective , and orally bioavailable C10-methyl GR
antagonist. The enhanced selectivity for GR over PR and AR, and excellent bioavailability of 15
can be rationalized by the conformation changes, which were triggered by the increased steric
interaction between the C10-methyl group and the C11-aniline moiety. Those changes were
observed by superimposition of the single crystal structures of 15 and 1. In addition, 15
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
6
demonstrated substantial inhibition of cortisol-induced GR target gene expression in HCC1806
TNBC xenograft tumors. In a 14-day rat exploratory toxicology study, oral administration of 15
was well tolerated with no signs of PR inhibition-related effects in uterus and ovaries. The
combination of GR antagonistic potency, enhanced selectivity and superior cytochrome P450
inhibition profile, as well as suitable pharmacokinetic properties, makes compound 15 a potential
candidate for the treatment of cancer in patients.
0
1
2
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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3
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5
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0
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9
0
Experimental Section
General Chemistry. All reactions were conducted under an inert gas atmosphere (nitrogen or
argon) with a Teflon-coated magnetic stirbar at the temperature indicated. Commercial reagents
and anhydrous solvents were used without further purification. Flash chromatography were
performed on Teledyne RediSep Rf Flash silica-gel columns. Removal of solvents was
conducted via a rotary evaporator, and residual solvent was removed from nonvolatile
compounds using a vacuum manifold maintained at approximately 1 Torr. All yields reported are
isolated yields. Preparative reversed-phase high pressure liquid chromatography (RP-HPLC) was
performed using an Agilent 1100 Series HPLC and Phenomenex Gemini C18 column (5 micron,
1
00 mm × 21.2 mm i.d.), eluting with a binary solvent system A and B using a gradient elusion
[
A: H O with 0.1% trifluoroacetic acid (TFA); B: CH CN with 0.1% TFA] with UV detection at
2
3
2
20 nm. All final compounds were purified to ≥95% purity as determined by a Agilent 1100
Series HPLC with UV detection at 220 nm using the following method: Phenomenex Gemini 5µ
C18 110A column (3.5 μm, 150mm × 4.6 mm i.d.); mobile phase, A = H O with 0.1% TFA, B =
2
CH CN with 0.1% TFA; gradient: 5−95% B (0.0–15.0 min); flow rate, 1.5 mL/min. Low-
3
resolution mass spectral (MS) data were determined on an Agilent 1100 Series LCMS with UV
1
detection at 254 nm and a low resolution electrospray mode (ESI). H NMR spectra were
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obtained on a Bruker 400 (400 MHz) spectrometer. Chemical shifts () are reported in parts per
million (ppm) relative to residual undeuterated solvent as an internal reference. The following
abbreviations were used to explain the multiplicities: s = single; d = doublet, t = triplet, q =
quartet, dd = doublet of doublets, dt = doublet of triplets, sep = septet, m=multiplet, br=broad.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compounds 3-8 were prepared by procedures similar to those described in Scheme 3 of our
previous publication.24
(8R,9S,10R,11S,13S,14S,17S)-11-(4-(Dimethylamino)phenyl)-17-(3,3-dimethylbut-1-yn-1-
yl)-17-hydroxy-13-methyl-1,2,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3H-
1
cyclopenta[a]phenanthren-3-one (3). H NMR (400 MHz, CDCl )  ppm 7.28 (br d, J = 8.4
3
Hz, 2 H), 6.67 (br d, J = 8.4 Hz, 2 H), 5.87 (s, 1 H), 3.34 (br t, J = 6.0 Hz, 1 H), 2.95 (s, 6 H),
2.80–2.90 (m, 1 H), 2.49–2.57 (m, 1 H), 2.33–2.43 (m, 1 H), 2.22–2.30 (m, 2 H), 2.02–2.22 (m,
4
H), 1.87–1.97 (m, 3 H), 1.68–1.79 (m, 1 H), 1.60–1.67 (m, 1 H), 1.47–1.54 (m, 2 H), 1.38
(ddd, J = 11.9, 5.7, 5.7 Hz, 1 H), 1.26–1.30 (m, 1 H), 1.24 (s, 9 H), 0.67 (s, 3 H). m/z (ESI, +ve
ion) = 474.4 (M+H)+.
(8R,9S,10R,11S,13S,14S,17S)-11-(4-(Diethylamino)phenyl)-17-(3,3-dimethylbut-1-yn-1-
yl)-17-hydroxy-13-methyl-1,2,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3H-
1
cyclopenta[a]phenanthren-3-one (4). H NMR (400 MHz, CDCl ) δ 7.23 (br d, J = 8.3 Hz, 2
3
H), 6.60 (br d, J = 8.8 Hz, 2 H), 5.86 (br s, 1 H), 3.27–3.39 (m, 5 H), 2.81–2.92 (m, 1 H), 2.47–
2
.58 (m, 1 H), 2.31–2.43 (m, 1 H), 2.01–2.32 (m, 6 H), 1.86–1.99 (m, 3 H), 1.67–1.77 (m, 1 H),
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
.64 (br s, 1 H), 1.45–1.54 (m, 2 H), 1.33–1.42 (m, 1 H), 1.26–1.31 (m, 1 H), 1.24 (s, 9 H), 1.17
br t, J = 6.94 Hz, 6 H), 1.04–1.11 (m, 1 H), 0.67 (s, 3 H). m/z (ESI, +ve ion) = 502.4 [M+H]+.
(
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
8R,9S,10R,11S,13S,14S,17S)-17-(3,3-Dimethylbut-1-yn-1-yl)-17-hydroxy-13-methyl-11-
(4-morpholinophenyl)-1,2,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3H-
1
cyclopenta[a]phenanthren-3-one (5). H NMR (400 MHz, CDCl ) δ ppm 7.33 (br d, J = 8.3
3
Hz, 2 H), 6.84 (br d, J = 7.5 Hz, 2 H), 5.87 (s, 1 H), 3.88 (br s, 4 H), 3.33–3.42 (m, 1 H), 3.17 (br
s, 4 H), 2.77–2.89 (m, 1 H), 2.50–2.59 (m, 1 H), 2.32–2.45 (m, 1 H), 2.03–2.31 (m, 5 H), 1.84–
1
9
.99 (m, 3 H), 1.68–1.79 (m,1 H), 1.63 (s, 1 H), 1.45–1.60 (m, 2 H), 1.25–1.42 (m, 3 H), 1.24 (s,
; H), 1.07–1.18 (m, 1 H), 0.64 (s, 3 H). m/z (ESI, +ve ion) = 516.3 [M+H]+.
(
8R,9S,10R,11S,13S,14S,17S)-17-(3,3-Dimethylbut-1-yn-1-yl)-17-hydroxy-13-methyl-11-
(4-(4-methylpiperazin-1-yl)phenyl)-1,2,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3H-
1
cyclopenta[a]phenanthren-3-one (6). H NMR (400 MHz, CDCl ) δ ppm 7.30 (d, J = 8.8 Hz, 2
3
H), 6.85 (d, J = 8.8 Hz, 2 H), 5.86 (s, 1 H), 3.35 (br t, J = 5.8 Hz, 1 H), 3.15–3.26 (m, 4 H), 2.80
–
2.87 (m, 1 H), 2.58–2.61 (m, 4 H), 2.52–2.56 (m, 1 H), 2.36(s, 3 H), 2.02–2.29 (m, 6 H), 1.85–
.98 (m, 3 H), 1.66–1.77 (m, 2 H), 1.33–1.54 (m, 3 H), 1.26–1.28 (m, 1 H), 1.24 (s, 9 H), 1.08–
1
1.17 (m, 1 H), 0.64 (s, 3 H). m/z (ESI, +ve ion)= 529.4 [M+H]+.
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(
8R,9S,10R,11S,13S,14S,17S)-17-(3,3-Dimethylbut-1-yn-1-yl)-17-hydroxy-13-methyl-11-
(
4-(4-(methylsulfonyl)piperazin-1-yl)phenyl)-1,2,6,7,8,9,10,11,12,13,14,15,16,17-
1
tetradecahydro-3H-cyclopenta[a]phenanthren-3-one (7). H NMR (400 MHz, CDCl ) δ ppm
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
.32–7.35 (m, 2 H), 6.86 (d, J = 8.9 Hz, 2 H), 5.87 (s, 1 H), 3.39–3.43 (m, 4 H), 3.35–3.37 (m, 1
H), 3.27–3.29 (m, 4 H), 2.84 (s, 3 H), 2.77–2.83 (m, 1 H), 2.51–2.58 (m, 1 H), 2.33–2.42 (m, 1
H), 2.24–2.30 (m, 2 H), 2.14–2.22 (m, 1 H), 2.03–2.12 (m, 3 H) 1.84–1.96 (m, 3 H), 1.71–1.78
(
m, 1 H), 1.62 (s, 3 H), 1.44–1.56 (m, 2 H), 1.34–1.42 (m, 1 H), 1.25–1.29 (m, 1 H), 1.24 (s, 9
H), 1.11–1.18 (m, 1 H), 0.63(s, 3 H). m/z (ESI, +ve ion) = 593.4 [M+H]+.
(8R,9S,10R,11S,13S,14S,17S)-11-(4-(Dimethylamino)phenyl)-17-(3,3-dimethylpent-1-yn-1-
yl)-17-hydroxy-13-methyl-1,2,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3H-
1
cyclopenta[a]phenanthren-3-one (8). H NMR (400 MHz, CDCl ) δ ppm 7.25–7.35 (m, 2 H),
3
6.67 (br d, J = 6.4 Hz, 2 H), 5.86 (s, 1 H), 3.31–3.39 (m, 1 H), 2.95 (br s, 6 H), 2.77–2.90 (m, 1
H), 2.49–2.57 (m, 1 H), 2.37 (td, J = 14.1, 4.3 Hz, 1 H), 2.27 (dt, J = 16.3, 3.9 Hz, 2 H), 2.19
(
ddd, J = 13.8, 9.6, 5.7 Hz, 1 H), 2.02–2.14 (m, 3 H), 1.87–1.99 (m, 3 H), 1.69–1.78 (m, 1 H),
1
.63 (s, 1 H), 1.48–1.54 (m, 2 H), 1.44 (q, J = 7.6 Hz, 2 H), 1.33–1.41 (m, 1 H), 1.22–1.31 (m, 1
H), 1.19 (s, 3 H), 1.19 (s, 3 H), 1.10 (br d, J = 12.0 Hz, 1 H), 0.99 (t, J = 7.6 Hz, 3 H), 0.66 (s, 3
H). m/z (ESI, +ve ion) = 488.5 [M+H]+.
(8S,9R,10R,11S,13S,14S,17S)-11-(4-(Dimethylamino)phenyl)-17-(3,3-dimethylbut-1-yn-1-
yl)-17-hydroxy-10,13-dimethyl-1,2,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3H-
cyclopenta[a]phenanthren-3-one (9). Compound 9 was prepared by procedures similar to those
described for the synthesis of 15, substituting prop-1-yn-1-ylmagnesium bromide in Step l with
(3,3-dimethylbut-1-yn-1-yl)lithium, which was synthesized from the reaction of n-butyl lithium
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2
2
2
2
2
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3
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3
3
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4
4
4
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4
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5
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5
5
5
5
5
5
5
6
1
with 3,3-dimethylbut-1-yne. H NMR (400 MHz, CDCl ) δ ppm 7.15–7.41 (br s, 2 H), 6.60 (br
3
d, J = 8.6 Hz, 2 H), 5.68 (d, J = 1.2 Hz, 1 H), 3.41 (t, J = 5.8 Hz, 1 H), 2.93 (s, 6 H), 2.46–2.57
(m, 1 H), 2.11–2.33 (m, 7 H), 1.82–2.04 (m, 3 H), 1.68–1.79 (m, 2 H), 1.35–1.53 (m, 3 H), 1.23
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
s, 9 H), 1.05–1.16 (m, 1 H), 1.01 (s, 3 H), 0.88 (s, 3 H). m/z (ESI, +ve ion) = 488.5 [M+H]+.
(
(
8S,9R,10R,11S,13S,14S,17S)-11-(4-(Dimethylamino)phenyl)-17-hydroxy-10,13-dimethyl-
17-(3-methylbut-1-yn-1-yl)-1,2,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3H-
cyclopenta[a]phenanthren-3-one (10). Compound 10 was prepared by procedures similar
to those described for the synthesis of 15, substituting prop-1-yn-1-ylmagnesium
bromide in Step l with (3-methylbut-1-yn-1-yl)lithium, which was synthesized from the
1
reaction of n-butyl lithium with 3-methylbut-1-yne. H NMR (400 MHz, CDCl ) δ ppm 7.25–
3
7
2
.38 (br s, 2 H), 6.61 (br dd, J = 6.8, 1.2 Hz, 2 H), 5.68 (d, J = 1.3 Hz, 1 H), 3.37–3.45 (m, 1 H),
.94 (s, 6 H), 2.61 (dt, J = 13.7, 6.9 Hz, 1 H), 2.47–2.56 (m, 1 H), 2.11–2.38 (m, 7 H), 1.83–2.04
(
m, 3 H), 1.69–1.78 (m, 2 H), 1.36–1.52 (m, 3 H), 1.18 (d, J = 6.8 Hz, 6 H), 1.07–1.15 (m, 1 H),
1
.02 (s, 3 H), 0.88 (m, 3 H). m/z (ESI, +ve ion) = 474.4 [M+H]+.
(
8S,9R,10R,11S,13S,14S,17S)-17-(Cyclopropylethynyl)-11-(4-(dimethylamino)phenyl)-17-
hydroxy-10,13-dimethyl-1,2,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3H-
cyclopenta[a]phenanthren-3-one (11). Compound 11 was prepared by procedures similar
to those described for the synthesis of 15, substituting prop-1-yn-1-ylmagnesium
bromide in Step l with cyclopropylethynyl)lithium, which was synthesized from the
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reaction of n-butyl lithium with ethynylcyclopropane. H NMR (400 MHz, CDCl ) δ ppm
3
7
2
1
.15–7.43 (br s, 2 H), 6.60 (br d, J = 7.3 Hz, 2 H), 5.68 (d, J = 1.0 Hz, 1 H), 3.37–3.46 (m, 1 H),
.93 (s, 6 H), 2.45–2.58 (m, 1 H), 2.09–2.35 (m, 7 H), 1.84–2.03 (m, 3 H), 1.66–1.81 (m, 2 H),
.62 (s, 1 H), 1.36–1.54 (m, 3 H), 1.29 (tt, J = 8.3, 5.0 Hz, 1 H), 1.09–1.22 (m, 1 H), 1.02 (s, 3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
3
4
5
6
7
8
9
0
H), 0.87 (s, 3 H), 0.77–0.84 (m, 2 H), 0.64–0.71 (m, 2 H). m/z (ESI, +ve ion) = 472.4 [M+H]+.
(8S,9R,10R,11S,13S,14S,17S)-11-(4-(Dimethylamino)phenyl)-17-hydroxy-17-(3-
methoxyprop-1-yn-1-yl)-10,13-dimethyl-1,2,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-
3H-cyclopenta[a]phenanthren-3-one (12). Compound 12 was prepared by procedures
similar to those described for the synthesis of 15, substituting prop-1-yn-1-ylmagnesium
bromide in Step l with (3-methoxyprop-1-yn-1-yl)lithium, which was synthesized from the
1
reaction of n-butyl lithium with 3-methoxyprop-1-yne. H NMR (400 MHz, CDCl ) δ ppm
3
7
.15–7.40 (br s, 2 H), 6.52–6.68 (m, 2 H), 5.68 (d, J = 1.2 Hz, 1 H), 4.18 (d, J = 0.88 Hz, 2H),
3.41–3.45 (m, 1 H), 3.40 (s, 3 H), 2.94 (s, 6 H), 2.42–2.58 (m, 1 H), 2.20–2.35 (m, 6 H), 2.08–
2
1
.17 (m, 1 H), 1.85–2.00 (m, 3 H), 1.72–1.80 (m, 2 H), 1.42–1.53 (m, 3 H), 1.11–1.20 (m, 1 H),
.02 (3 H, s), 0.90 (3 H, s). m/z (ESI, +ve ion) = 476.4 [M+H]+.
(
8S,9R,10R,11S,13S,14S,17S)-17-(But-1-yn-1-yl)-11-(4-(dimethylamino)phenyl)-17-
hydroxy-10,13-dimethyl-1,2,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3H-
cyclopenta[a]phenanthren-3-one (13). Compound 13 was prepared by procedures similar
to those described for the synthesis of 15, substituting prop-1-yn-1-ylmagnesium
bromide in Step l with but-1-yn-1-yllithium, which was synthesized from the reaction of n-
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butyl lithium with but-1-yne. H NMR (400 MHz, CDCl ) δ ppm 7.22–7.39 (br s, 2 H), 6.61 (br
3
dd, J = 7.0, 1.5 Hz, 2 H), 5.68 (d, J = 1.2 Hz, 1 H), 3.36–3.47 (m, 1 H), 2.94 (s, 6 H), 2.41–2.60
(
m, 1 H), 2.10–2.34 (m, 8 H), 1.82–2.05 (m, 3 H), 1.69–1.80 (m, 2 H), 1.60–1.67 (m, 1 H), 1.43–
0
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9
0
1
2
3
4
5
6
7
8
9
0
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6
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9
0
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.52 (m, 3 H), 1.05–1.20 (m, 4 H), 1.02 (s, 3 H), 0.88 (m, 3 H). m/z (ESI, +ve ion) = 460.4
[
M+H]+.
(8S,9R,10R,11S,13S,14S,17S)-11-(4-(Dimethylamino)phenyl)-17-hydroxy-10,13-dimethyl-
1
7-(3,3,3-trifluoroprop-1-yn-1-yl)-1,2,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3H-
cyclopenta[a]phenanthren-3-one (14). Compound 14 was prepared by procedures similar
to those described for the synthesis of 15, substituting prop-1-yn-1-ylmagnesium
bromide in Step l with (3,3,3-Trifluoroprop-1-yn-1-yl)lithium, which was synthesized from
1
the reaction of LDA with 2-bromo-3,3,3-trifluoro-prop-1-ene. H NMR (400 MHz, CDCl ) δ
3
ppm 7.15–7.40 (br s, 2 H), 6.61 (d, J = 8.8 Hz, 2 H), 5.69 (d, J = 1.3 Hz, 1 H), 3.45 (br t, J = 5.9
Hz, 1 H), 2.94 (s, 6 H), 2.45–2.58 (m, 1 H), 2.22–2.37 (m, 5 H), 2.09–2.19 (m, 2 H), 1.80–2.03
m, 6 H), 1.30–1.54 (m, 3 H), 1.03 (s, 3 H), 0.92 (s, 3 H). m/z (ESI, +ve ion) = 500.3 [M+1]+.
(
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(
8S,9R,10R,11S,13S,14S,17S)-11-(4-(Dimethylamino)phenyl)-17-hydroxy-10,13-dimethyl-
1
7 -(prop-1-yn-1-yl)-1,2,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3H-
cyclopenta[a]phenanthren-3-one (15). Step a: (8'S,9'S,10'R,13'S,14'S)-10',13'-Dimethyl-
',2',4',7',8',9',10',12',13',14',15',16'-dodecahydro-11'H-dispiro[[1,3]dioxolane-2,3'-
cyclopenta[a]phenanthrene-17',2''-[1,3]dioxolan]-11'-one (23). Adrenosterone (22) (50.2 g,
67.1 mmol) was dissolved in DCM (390 mL). Trimethylorthoformate (91 g, 857.5 mmol) and
ethylene glycol (119 g, 1.92 mol) were added successively. Then toluenesulfonic acid (1.9 g,
0.0 mmol) was added. The reaction mixture was heated to 40 °C for 18 h and was then added
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pyridine (4 mL). The solution was concentrated and the residue was extracted with DCM and
washed with water. The organic layer was dried with MgSO , filtered and concentrated to give
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7
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4.3 g of crude product, which was recrystallized from hot ethyl acetate to provide 23 (42.3 g,
1
5%). H NMR (400 MHz, CD Cl ) δ ppm 5.28–5.30 (m, 1 H), 3.76–3.93 (m, 8 H), 2.47–2.62
2
2
(m, 3 H), 2.08–2.15 (m, 2 H), 1.95–2.06 (m, 3 H), 1.77–1.93 (m, 6 H), 1.53–1.61 (m, 2 H), 1.31–
.41 (m, 1 H), 1.19 (s, 3 H), 0.78 (s, 3 H). m/z (ESI, +ve ion) 389.3 (M+H)+.
1
Step b: (8'S,9'S,10'R,13'R,14'S)-10',13'-Dimethyl-1',4',7',8',9',10',13',14',15',16'-
decahydro-2'H-dispiro[[1,3]dioxolane-2,3'-cyclopenta[a]phenanthrene-17',2''-
1,3]dioxolan]-11'-yl 1,1,2,2,3,3,4,4,4-nonafluorobutane-1-sulfonate (24). To an oven-dried
00 mL flask charged with THF (125 mL) and anhydrous diisopropylamine (5.77 mL, 40.9
[
5
mmol) was added n-butyl lithium (1.6 M in hexanes, 25.1 mL, 40.2 mmol) dropwise at –78 °C.
The resulting solution was stirred at the same temperature for 25 min. In a separate flask, bis-
ketal 23 (3.9 g, 10.0 mmol) was azeotroped from toluene, dried under vacuum, and flushed with
argon before it was dissolved in THF (40 mL). This solution was added slowly to the freshly
prepared lithium diisopropylamide solution at –78 °C. After the mixture was stirred for 30 min,
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