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Discovery of a Potent and Selective Steroidal Glucocorticoid Receptor Antagonist (ORIC-101) Yosup Rew, Xiaohui Du, John Eksterowicz, Haiying Zhou, Nadine Jahchan, Liusheng Zhu, Xuelei Yan, Hiroyuki Kawai, Lawrence R. McGee, Julio C. Medina, Tom Huang, Chelsea Chen, Tatiana Zavorotinskaya, Dena Sutimantanapi, Joanna Waszczuk, Erica Jackson, Elizabeth Huang, Qiuping Ye, Valeria R. Fantin, and Daqing Sun J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00743 • Publication Date (Web): 09 Aug 2018 Downloaded from http://pubs.acs.org on August 9, 2018

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Journal of Medicinal Chemistry

Discovery of a Potent and Selective Steroidal Glucocorticoid Receptor Antagonist (ORIC-101) Yosup Rew,* Xiaohui Du, John Eksterowicz, Haiying Zhou, Nadine Jahchan, Liusheng Zhu, Xuelei Yan, Hiroyuki Kawai, Lawrence R. McGee, Julio C. Medina, Tom Huang, Chelsea Chen, Tatiana Zavorotinskaya, Dena Sutimantanapi, Joanna Waszczuk, Erica Jackson, Elizabeth Huang, 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), selectivity.

ABSTRACT: The glucocorticoid receptor (GR) has been linked to therapy resistance across a wide range of cancer types. Preclinical data suggest that antagonists of this nuclear receptor may enhance the activity of anti-cancer therapy. The first-generation GR antagonist mifepristone is currently undergoing clinical evaluation in various oncology settings. Structure-based modification of mifepristone led to the discovery of ORIC-101 (28), a highly potent steroidal GR antagonist with reduced androgen receptor (AR) agonistic activity amenable for dosing in androgen receptor positive tumors, and with improved CYP2C8 and CYP2C9 inhibition profile to minimize drug-drug interaction potential. Unlike mifepristone, 28 could be co-dosed with chemotherapeutic agents readily metabolized by CYP2C8 such as paclitaxel. Furthermore, 28

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demonstrated in vivo antitumor activity by enhancing response to chemotherapy in the GR+ OVCAR5 ovarian cancer xenograft model. Clinical evaluation of safety and therapeutic potential of 28 is underway.


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INTRODUCTION Glucocorticoids (GCs) such as cortisol are steroid hormones secreted by the adrenal gland in a circadian and stress-associated manner to regulate metabolism, cell growth, apoptosis and differentiation, inflammation, mood and cognitive function.1 GCs signal through the glucocorticoid receptor (GR), a member of the superfamily of nuclear receptors expressed across a wide variety of tissues. Upon ligand-binding, GR translocates to the nucleus where it triggers transcription of a spectrum of genes that mediate its pleiotropic biological effects. Dysregulation of GR signaling has been associated with a variety of pathological states including Cushing’s syndrome, psychotic depression, obesity and cancer.2 Therefore, there has been considerable interest to target this nuclear receptor for therapeutic purposes. Several lines of evidence point to GR as a potential mechanism of therapy resistance in cancers of epithelial origin.3 Elevated levels of GR protein have been correlated with poor prognosis in ESR1- breast cancer4 and endometrial cancer patients,5 as well as to poor outcome in castrationresistant prostate cancer (CRPC) patients treated with enzalutamide.6 In addition, cumulative preclinical data over the past decade have functionally linked GR activation with resistance to a variety of chemotherapeutic agents across an array of solid tumors that include ovarian cancer, triple-negative breast cancer (TNBC), prostate cancer, pancreatic cancer, non-small cell lung cancer, urological cancers, etc.7-13 At the molecular level, GR drives resistance to therapy at least in part, by driving transcriptional activation of mediators of cell survival, anti-apoptosis, cell adhesion and invasion.9, 14-17 Collectively, these studies have established that genetic ablation or pharmacologic inhibition of GR reverses the pro-survival effects mediated by this nuclear receptor and enhances chemotherapy and anti-androgen activity in cancer models.6-9, 14, 18


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These data have also provided rationale to evaluate the efficacy of GR antagonists in combination with chemotherapeutic agents as well as enzalutamide in the clinic. Furthermore, it is well established that GR is expressed in a variety of immune cells.19 Endogenous and synthetic glucocorticoids are potent regulators of inflammation.20-21 Glucocorticoid binding to GR activates or represses transcription of a variety of pro- and antiinflammatory genes, and the functional consequences of glucocorticoid action differ by cell type. GR activation has been shown to promote expansion of regulatory T cells and myeloid derived suppressor cells,22-24 stimulation of Th2 differentiation,25 as well as suppression of activated CD8+ T cells,26 and most of these effects were reversed by the GR antagonist mifepristone (also known as RU486). Importantly, promotion of immunosuppressive cells and T cell exhaustion have been implicated in cancer immune escape as well poor response to immune therapy.27 Furthermore, disruption in cortisol secretion has been correlated with poor prognosis and immunosuppression across a variety of advanced solid tumors including breast, ovarian and lung cancer.28-30 In addition, extra-adrenal production of cortisol by colorectal cancer, squamous skin cancer and melanoma cells has been associated with decreased CD8+ T cell proliferation and activity,31-32 suggesting that aberrant activation of GR could contribute to local tumor immunosuppression. Altogether these observations point to the immunomodulatory potential of GR, and provide a rational basis for the clinical evaluation of GR antagonists in combination with emerging immunotherapies. The marketed drug mifepristone (RU-486) is a steroidal GR antagonist, discovered in the 1980’s as a potent progesterone receptor (PR) antagonist.33 Due to its potent GR inhibitory activity, mifepristone has also been evaluated for conditions associated with alterations in GR


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activity.34 Mifepristone was approved in 2011 for the treatment of Cushing’s syndrome, and is currently undergoing clinical testing in various solid tumors. Compared to recently discovered synthetic non-steroidal GR antagonists and allosteric inhibitors,35-40 mifepristone (1) stands out for its potency. However, mifepristone is a partial AR agonist, and its agonistic activity is sufficient to stimulate the proliferation of CRPC LNCaP/ARluc (LNAR) cells both in vitro and in vivo. In addition, mifepristone has also been shown to induce AR target gene expression in AR+ TNBC MDA-MB-453 cells.41 Besides prostate cancer, previous studies have shown the oncogenic roles of AR and androgen signaling in TNBC and ovarian cancer.42-45 In fact, the therapeutic potential of the anti-androgen enzalutamide is currently being clinically evaluated in AR+ TNBC and ovarian cancer patients.45-47 Clearly, partial AR agonism activity is an unwanted feature for any GR antagonist with potential clinical application in AR-driven cancers. We envisioned that the undesirable AR agonism of mifepristone could be eliminated while retaining its GR antagonistic potency by structure-based modification of mifepristone. We report here the results of our structure-activity relationship (SAR) studies, which led to the discovery of ORIC-101 (28) (Figure 1), a highly potent GR antagonist with significantly reduced AR agonism and improved Cytochrome P450 (CYP) inhibition profiles. These features of 28 make this novel compound an attractive combination partner to anti-androgens and paclitaxel in AR+ cancers including CRPC, as well as a subset of TNBC and ovarian cancers.14, 44, 46, 48 In addition, the favorable CYP inhibition profile of 28 makes this compound combinable with therapeutic agents that are metabolized by CYP2C8 and CYP2C9.


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Figure 1. Mifepristone (1) and ORIC-101 (28). a IC50 in GR luciferase antagonist assay. b EC50 [Emax]* in AR luciferase agonist assay. * % mifepristone.


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RESULTS AND DISCUSSION With the goal of developing a potent, steroidal GR antagonist with reduced AR agonism as compared to 1, an analysis of the binding mode of mifepristone to AR and GR was initiated. The protein co-crystal structure of 1 bound to GR, but not to AR, has been solved (Figure 2).49-50 Since the two nuclear receptors are closely related, a homology model of AR was constructed based on the GR structure (pdb: 3H52). Figure 2A shows a view of the mifepristone (1)/GR binding site illustrating the key hydrogen bond interactions of the steroid carbonyl with Arg611 and Gln570. Outside of the interaction with these two polar residues, the majority of binding site residues are hydrophobic in nature and form favorable van der Waals interactions with the predominately hydrophobic ligand. Figure 2B shows the complementary fit between the ligand and the binding site, with 1 almost completely buried in the steroid binding pocket. The helices that contain residues that interact with mifepristone and the NCoR peptide are also labeled. The agonistic and antagonistic functions of GR and AR ultimately depend on the protein conformation,51-53 in particular, the positions of helices 11 and 12 and the ability or inability of the receptor to bind co-activators or co-repressors.54 To that end, structural hypotheses were developed along three distinct vectors with the goal of altering the GR and AR structures and thus affecting the agonist and antagonist profiles of the ligands. Using the mifepristone scaffold as a foundation, modification of the C11 4-dimethylaminophenyl group provided a vector to alter the position of helix 12 and also to directly challenge co-peptide binding. Modification of the C17 hydroxyl and introduction of C16 substituents afforded the opportunity to disrupt the positions of both helices 11 and 12 directly,55 and modification of the C17 1-propynyl group provided a cascading structural effect where disruption of helix 7 in turn altered the position of helices 11 and 12.


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(A)

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(B)

NCoR

H12 H3 H11

H5

H7

Figure 2. (A) 1 (gray) bound to GR (pdb: 3H52). GR active site residues shown in blue. (B) GR protein surface with helices labeled and NCoR peptide shown in purple. Initial investigation of 1 began with replacement of the 4-dimethylaminophenyl group at the C11 position in the steroid core with various substituents (Table 1, compounds 2-6).56-58 To measure antagonist and agonist activities of these compounds on GR and AR, luciferase reporter assays were employed.59-60 Substitution of the 4-dimethylaminophenyl group with a small alkyl group, such as an ethyl, switched the GR activity, converting antagonism to agonism (2 vs. 1). Increase of the substituent’s size to cyclohexyl or unsubstituted phenyl group restored GR antagonism and attenuated GR agonism (3 and 4 vs. 2). These results imply that a phenyl or a similar-sized cyclohexyl moiety at the C11 position is an important feature for GR antagonism. Moving the dimethylamino group from para-position to meta-position reduced AR agonism dramatically (5.8% of 1), but decreased GR antagonism by 20-fold as well (5 vs. 1). The 4-


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isopropylphenyl derivative elicited improvement in GR antagonism (IC50 = 1.93 nM for 6). However, it also induced more AR agonism (200% of 1). Attention was then focused on the modification of the 1-propynyl group at the C17 position (Table 1, compounds 7-11).56, 61 Changing the 1-propynyl group to a saturated n-alkyl group amplified AR agonism while diminishing GR antagonism (7 vs 1), suggesting that a C-C triple bond at the C17 position is crucial for minimized AR agonism and enhanced GR antagonism. The introduction of an electron withdrawing group by replacing the 1-propynyl group with a 3,3,3-trifluoro-1-propynyl group maintained the same level of AR agonism (8 vs 1). Interestingly, gradually increasing alkyl substituent size in the 1-propynyl group led to the 3,3dimethyl-1-butynyl derivative 11, which possessed substantially reduced partial agonism in the AR luciferase assay (13% of 1). However, 11 also exhibited a 4.5-fold decrease in GR antagonism potency compared to 1 (compounds 9-11 vs. 1).

Table 1. Exploration of substituents at C11 and C17 positions in mifepristone

GR luc. antagonism

GR luc. agonism

AR luc. agonism

IC50 (nM)

EC50 (nM) [Emax]a

EC50 (nM) [Emax]b

3.3±0.6

>2,500 [3.1±1.4]

11.9 ± 5.6 [100]

2

>5,000

57±35 [36±18]

>2,500 [47]

3

18.1±6.3

>2,500 [9.3±0.6]

>2,500 [26]

compound

R1

R2

1 (mifepristone)


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4

14.2±1.5

17.4±1.5 [7.0±3.4]

>2,500 [27]

5

64±20

>2,500 [1.5±0.5]

>2,500 [5.8]

6

1.93±0.20

>2,500 [3.1±2.0]

13±3 [198±14]

7

32±13

>2,500 [1.4±0.6]

1.5 [210]

8

11.2±2.8

>2,500 [2.4±1.2]

9

8.5±1.7

>2,500 [2.1±1.1]

10

26±12

>2,500 [1.8±0.9]

11

14.6±4.5

>2,500 [1.4±0.9]

54 [117]

30 [101]

>2,500 [50]

>2,500 [13.4±5.2]

Potency and Emax data with SD are reported as the average of at least two determinations. a % dexamethasone. b%

mifepristone.

As part of the lead optimization process, a co-crystal structure of 11 bound to human GR was determined at a resolution of 3.05 Å (Figure 3). The ketone moiety of 11 makes key hydrogen bond interactions with Arg611 and Gln570, and overall the ligand sits in a similar position as that of mifepristone in the binding site. There is however, a significant difference in the protein structure in the region interacting with the C17 substituent. As can be observed in the mifepristone co-crystal structure, the 1-propynyl group sits in a hydrophobic region between helices 3, 6 and 7 (Figure 2). The orientation of these helices in turn has an effect on the position


10

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of helices 11 and 12. The resulting overall conformation of GR is favorable for binding of the co-repressor peptide, NCoR. This is consistent with the antagonistic nature of mifepristone towards GR. The replacement of the alkynyl methyl with a t-butyl group in 11 results in significant changes in the shape and size of the binding site and the overall conformation of GR. Figure 3 illustrates a significant increase in the size and depth of this part of the binding pocket that is necessary to accommodate 11. Consequently, significant changes occur in the orientations of helices 6 and 7, resulting in a shift in the positions of helices 11 and 12 as well. In fact, the helix 12 position prevents the binding of co-repressor or co-activator peptides (Supplemental Figure S4).62 While mifepristone does not show significant agonism towards GR (Emax relative to dexamethasone of 3.1%), 11 further reduces the relative Emax by more than 50%.

(B)

(A)

H12

H3 H11

H5

H7

Figure 3. (A) 11 (dark gray) bound to GR (pdb: 6DXK). GR active site residues shown in olive. (B) GR protein surface with helices labeled.


11


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As mentioned, the addition of t-butyl to the alkyne has a significant effect on AR selectivity. A more than 7-fold reduction in AR agonism (Emax) is observed for 11 versus mifepristone. To date, efforts to obtain a co-crystal structure of AR with 11 have not been successful. However, given the close structural relationship between AR and GR, a similar structural hypothesis can be developed through molecular modeling to explain the reduced AR agonism of 11. The orientation of 11 in the binding site of AR is expected to be similar to the one observed for GR and thus a similar disruption of the positions of helices 6, 7, 11 and 12 is expected. These large conformational changes in turn disrupt the co-peptide binding site further reducing the AR agonism for 11 versus mifepristone. A single crystal small molecule x-ray structure of 11 was also obtained (Figure 4). This structure allowed for confirmation of the absolute stereochemistry of all of the chiral centers in the molecule and was also used to evaluate the co-crystal structure binding mode. The steroid scaffold is rigid and overall 11 has few rotatable bonds. Nevertheless, structural distortion of the ligand is still possible and could result in a high energy binding conformation. The ligand conformation of 11 from the GR protein co-crystal structure is shown in B, while C shows the overlay of the two and illustrates that the conformations are almost identical. There is no significant change in ligand conformation upon binding to GR and thus no additional strain is introduced into the molecule.


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(A)

(B)

(C)

Figure 4. Comparison of small molecule single crystal x-ray structure of 11 (A, orange) with the GR co-crystal x-ray structure 11 (B, dark gray, pdb: 6DXK). The overlay of the two structures is shown in C.

Having identified the 3,3-dimethyl-1-butynyl group as the optimized 1-alkynyl group at the C17 position with respect to AR selectivity, attention was turned to substituent modification of the aryl ring at the C11 position (Table 2). Replacing the 4-dimethylamino group in 11 by small alkyl groups was tolerated in terms of GR antagonism potency, but those derivatives displayed greater AR agonism as the size of the alkyl group increased (12, 13, and 14). Replacement of the 4-dimethlyamino group with a methoxy or methyloxetanyl group was also well tolerated for GR antagonism (15 and 25). Shifting the dimethylamino substituent from the para-position to metaposition led to a drop in GR antagonism potency (16) as observed in the previous SAR with compound 5. Additional substitutions at the meta-and ortho-positions were then evaluated while preserving the dimethylamino group at the para position. The 2,5-difluorophenyl analog 18 showed GR antagonism potency similar to that of 11, whereas potency of all other analogs was diminished (17, 19, and 20). The optimization of the N,N-dialkyl group within 11 was attempted to improve GR antagonism potency and to address the inherent metabolic instability of the N,Ndimethylamino group. Compound 21, which exchanged one N-methyl group with an isopropyl


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group, was lower in GR antagonism activity compared to 11 (IC50 = 43 nM for 21 vs. IC50 = 14 nM for 11) but improved AR agonism selectivity (Emax = 8.2% of 1 for 21 vs. Emax = 13% of 1 for 11). Introduction of various cyclic tertiary amines was also tolerated with regard to GR antagonist potency (22, 23, and 24). Overall, no significant improvement in GR antagonist potency was observed from the aryl group modification at the C11 position.

Table 2. Exploration of aryl substituents at C11 position in compound 11

GR luc. antagonism

GR luc. agonism

AR luc. agonism

IC50 (nM)

EC50 (nM) [Emax]a

EC50 (nM) [Emax]b

11

14.6±4.5

>2,500 [1.4±0.9]

12

16.1±7.8

>2,500 [1.5±0.8]

>2,500 [16±10]

13

17.5±9.3

>2,500 [1.5±0.7]

>2,500 [20±13]

14

16.0±7.0

>2,500 [1.5±0.8]

>2,500 [25]

15

16.6±3.5

>2,500 [1.1±0.6]

>2,500 [9.8±4.3]

16

205±112

>2,500 [1.3±0.6]

>2,500 [7.5]

17

43±19

>2,500 [1.3±0.6]

>2,500 [8.8±7.7]

compound

Ar

>2,500 [13.4±5.2]


14

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18

12.4±5.8

>2,500 [1.3±0.7]

>2,500 [11±8.4]

19

48±17

>2,500 [1.0±0.5]

>2,500 [5.7]

20

28±16

>2,500 [1.2±0.7]

>2,500 [17.0±1.7]

21

43±21

>2,500 [1.7±0.8]

>2,500 [8.2±4.4]

22

13.3±2.7

>2,500 [1.5±0.7]

>2,500 [18.7±3.2]

23

19.5±5.1

>2,500 [1.2±0.8]

>2,500 [13.3±4.1]

24

16.7±1.0

>2,500 [1.8±1.1]

>2,500 [23±10]

25

9.6±3.4

>2,500 [2.2±1.2]

>2,500 [11.0±4.3]

Potency and Emax data with SD are reported as the average of at least two determinations. a % dexamethasone. b%

mifepristone.

The X-ray co-crystal structure of 11 bound to GR protein (Figure 3) shows that the A ring carbonyl oxygen forms direct hydrogen bonds to the guanidinium of Arg611 and to the γ-amide of Gln570. The A-ring carbonyl oxygen in compound 11 is α,β-γ,δ-diene carbonyl oxygen with extended conjugation, which is known to be less electronegative than α,β-unsaturated carbonyl oxygen. Therefore, we hypothesized that reducing the C9 C-C double bond in the B ring could enhance hydrogen bond acceptor ability of the A ring carbonyl oxygen. Consequently, an effort was made to explore modification of the steroid core in 11 (Table 3).


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Table 3. Modification of the steroid core of 11 GR luc. antagonism

GR luc. agonism

AR luc. agonism

IC50 (nM)

EC50 (nM) [Emax]a

EC50 (nM) [Emax]b

3.3±0.6

>2,500 [3.1±1.4]

11.9±5.6 [100]

11

14.6±4.5

>2,500 [1.4±0.9]

26

5.6±2.4

>2,500 [2.2±1.0]

27

14.6±7.0

>2,500 [1.7±0.9]

5.6±2.6

>2,500 [2.3±1.1]

compound

Structure

1 (mifepristone)

>2,500 [13.4±5.2]

224±97 [22±11]

>2,500 [10.6±4.3]

28 >2,500 [11.0±5.8]

(ORIC-101) Potency and Emax data with SD are reported as the average of at least two determinations. a % dexamethasone. b%

mifepristone.

Of significance was the observation that 26, the corresponding C9 C-C double bond reduced analog of 11, had a marked 3-fold improvement in GR antagonist potency (IC50 = 5.6 nM for 26 vs. IC50 = 14 nM for 11). However, 26 showed a slight increase in AR agonist activity (Emax = 22% of 1) compared to 11 (Emax = 13% of 1). This increase is not negligible. For example, the comparable partial agonistic activity of bicalutamide (Emax = 23% of 1) compromises its


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therapeutic efficacy as an anti-androgen relative to full antagonists such as enzalutamide (Figure 5) and apalutamide.

Figure 5. Relative AR agonism of 11 and 28 compared to 1, enzalutamide, and bicalutamide.

The 3,3-dimethyl-1-butynyl group in 26 was further modified by replacing one methyl of the tbutyl group with a methoxy moiety. This was attempted to reduce AR agonism and improve solubility, but it also resulted in lower GR antagonism (27). On the other hand, similar to previous observations, the replacement of one N-methyl group in 26 with an isopropyl group provided 28 with significantly reduced AR agonism (Emax = 11% of 1), which is comparable to AR agonism of enzalutamide (Emax = 8.7% of 1) (Figure 5). Overall, compound 28 is a weak full AR antagonist with IC50 of 484 ± 110 nM in the AR luciferase agonism assay. Furthermore, compound 28 is equipotent to 26 and only 2-fold less potent than mifepristone in the GR luciferase antagonism assay (IC50 = 5.6 nM).


17


Figure 6. Induction of endogenous AR target genes by synthetic androgen (R1881) and 1 in LNAR cells. Endogenous AR target genes, PSA and TMPRSS2, are induced by R1881 and 1 in LNAR cells while those genes are not induced by 28. Enz (enzalutamide) inhibits R1881induced expression but does not fully inhibit the expression induced by 1.

CLDN8

1

28

Relative mRNA levels

SEC14L2


CYPGF8


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1

28

1

28

Figure 7. Stimulation of AR activity in AR+ TNBC cells by 1. Compound 1, but not 28, stimulates AR target gene expression in the AR+ MDA-MB-453 TNBC cell line.


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We also compared the AR agonism of compounds 1 and 28 in the LNAR cells, which exhibit high AR, but low GR, and low PR expression, as well as in the MDA-MB-453 cells with high AR, high GR, but no PR expression by RT-qPCR (Figures 6 and 7). The results showed that 1, but not 28, is able to stimulate endogenous AR target gene expression in both LNAR and MDAMB-453 cell lines at about 100 nM. These expression levels are similar to those achievable by 0.1 nM R1881, the potent synthetic androgen in the same cell lines. On the other hand, 1 µM enzalutamide completely reverted gene expression induced by R1881, but not 1, in the LNAR cell line (Figure 6). Notably, endogenous AR target gene induction by compound 28 was minimal in both cell lines (Figures 6 and 7). These data suggest that compound 28 exhibits no AR agonism and might have an advantage over 1 in treating AR-driven cancers. The single crystal small molecule x-ray structure of 28 was solved. Figure 8 shows the structure of 28 as compared to the protein co-crystal structure of 11. Since the overlay shows that the overall shape of both compounds is very similar, 28 would be expected to have a very similar binding mode to that of 11. This is consistent with the binding mode of 28, as predicted via docking calculation with GR.

(A)

(Β)

(C)

Figure 8. Comparison of the small molecule single crystal x-ray structure of 28 (A, blue) with the GR co-crystal x-ray structure 11 (B, dark gray, pdb: 6DXK). The overlay of the two structures is shown in C.


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To identify a molecule suitable for preclinical development, several selected compounds were evaluated for their rat pharmacokinetic (PK) profile (Table 4). The results showed that 13 and 23 had low clearance, but poor oral bioavailability (CL = 0.41 and 0.28 L/kg/h; %F = 0 and 0.7%, respectively). Compounds 11 and 28 had moderate clearance (CL =1.4 and 1.9 L/kg/h, respectively) and acceptable oral bioavailability (%F = 13 and 24%, respectively). The rest of the compounds 21, 25, and 27 showed high CL (CL = 3.0˗5.0 L/kg/h) although 27 exhibited good oral bioavailability (%F = 54%).

Table 4. Rat pharmacokinetic data for selected compoundsa po (5.0 mg/kg)c

iv (0.5 mg/kg)b compound

AUC

CL Vss (L/kg)

t1/2 (iv, h)

F (%)

Cmax (µg/L)

(L/kg/h)

(µgh/L)

1

3.7

4.1

1.6

6.4

11

4.0

4.9

1.9

37

13

0.41

1.7

3.4

0

21

4.0

4.7

1.5

23

23

0.28

1.5

3.6

25

3.0

6.7

27

5.0

28

1.9

24

85

72

471

0

0

59

290

0.7

21

167

4.4

7.2

59

123

7.2

2.4

54

295

544

4.6

4.4

24

149

635


20

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an

= 3 mice for both iv and po, iv time points: one pre-dose and 10 post-dose, po time points: one pre-dose and

8 post-dose; b formulated in 10% DMSO, 70% PEG 400, and 20% water; c formulated in 5% DMSO, 95% 0.2% Tween 80 in 0.25% CMC.

Given the GR antagonist potency, low AR agonism, and favorable rat PK profile, compound 28 was selected for further evaluation. The preclinical pharmacokinetics of 28 were evaluated in mouse and minipig following single-dose intravenous (iv) or oral administration. The molecule was found to have low clearance and reasonable oral bioavailability in mouse and minipig (Table 5).63

Table 5. Pharmacokinetic data for 28 in mouse and minipig po (5 mg/kg for mouse and 10 mg/kg for minipig)b

iv (0.5 mg/kg)a Species CL (L/kg/h)

Vss (L/kg)

t1/2 (h)

F (%)

Mouse

1.63

5.01

4.83

21

Minipig

1.1

7.8

12.2

20

Cmax (µg/L)

AUC (µgh/L)

274

644

153

1721

Formulations: a 28 was in 5% DMA, 10% EtOH, 40% PEG400, and 45% D5W for mouse iv study; and in 5% DMA, 10% EtOH, 40% PEG 400, 43.8% D5W, pH 5 for minipig iv study; b 28 was in 0.25% CMC, 0.2% Tween 80, and 5% DMSO for mouse oral study, and 28 HCl salt was in 2% HPMC and 0.2% Tween 80 for minipig-oral study.

Species differences in the unbound fraction of compound 28 were minimal (< 2-3 fold). The unbound fraction (0.14) of compound 28 is 10-fold higher than that of compound 1 in human plasma. As compound 1 is reported to have high potential for inhibition of CYP2C8, we next


21


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examined the liability of 11 and 28 in the CYP inhibition assay. It was found that both 11 and 28 have low potential of CYP2C8 and CYP2C9 inhibition compared to 1 although the IC50 values of 11 and 28 for CYP3A4 are lower than that of 1 (Table 6). In general, the replacement of the C17 alkynyl methyl in 1 with a t-butyl group improves the CYP inhibition profile, especially against CYP2C8 and CYP2C9.

Table 6. CYP inhibition of compounds 1, 11, and 28

IC50 (µM)

P450 Substrate Isoform

1

11

28

3A4

Midazolam

9.5

2.9

1.6

2C8

Amodiaquine

1.5

>10

>10

2C9

Diclofenac

4.9

>10

>10

Furthermore, the in vivo combination PK studies of 1 or 28 with paclitaxel, a CYP2C8 substrate, in female mice demonstrated that 28 showed less potential for CYP2C8 driven drugdrug interaction compared to 1 (Figure 9). The results showed that adding 1 to paclitaxel resulted in higher paclitaxel plasma exposure. Unlike 1, 28 did not result in the increase in paclitaxel concentration in plasma.


22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Plasma Concentration ( M)

Page 23 of 66

Figure 9. In vivo combination pharmacokinetics of 1 and 28 with paclitaxel in mice.

To evaluate the selectivity of compound 28 for GR over PR, both 28 and 1 were tested in the PR luciferase reporter assay. The results showed that the inhibitory activity of 28 was substantially lower than that of 1 (IC50 = 21 ± 10 nM for 28; IC50 = 0.4 ± 0.2 nM for 1). The PR/GR ratio of 28 is 3.8 while that of 1 is 0.12. Compound 28 does not show any agonism in the PR luciferase reporter assay. Unlike the estrogen receptor (ER) ligand 17β-estradiol and the ER antagonist tamoxifen, 28 does not physically bind to ER up to 1 µM (Supplemental Figure S1). Similarly, 28 neither induces nor inhibits aldosterone-induced interaction between MR and its coactivator SRC at the concentration of 5 µM (Supplemental Figures S2 and S3), suggesting 28 is not a mineralocorticoid receptor (MR) agonist or antagonist. To compare the activity of 1 and 28 on GR across multiple species, expression of endogenous GR target genes was evaluated using human, rat, dog, monkey, and minipig peripheral blood mononuclear cells (PBMCs) treated ex vivo with dexamethasone in the presence or absence of 1 or 28. Figure 10 shows an example of quantitative polymerase chain reaction (RT-qPCR) results measuring the dose-dependent expression levels of two established GR target genes, GILZ and


23


FKBP564 in human PBMCs treated with dexamethasone and 28. Treatment with 28 significantly inhibited ligand-induced target gene expression with potencies comparable to 1 in all species tested (Table 7).

1.2


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 66

GILZ FKBP5

0.9 0.6 0.3 0.0 0.1

1

10

100

1000

Concentration (nM)

Figure 10. GR target gene expression in PBMCs. Representative RT-qPCR measuring FKBP5 and GILZ in PBMCs from a healthy human donor treated with dexamethasone at 30 nM and 28 at dose ranging concentrations for 6 hours.

Table 7. Cross-species evaluation of the effect of 1 and 28 on endogenous target gene expression in PBMCs species

human

Target Gene

GILZ

FKBP5

GILZ

FKBP5

GILZ

FKBP5

GILZ

FKBP5

GILZ

FKBP5

IC50 of 1 (nM)

17.8

14.8

NDa

NDa

32.6

20.2

13.9

21.9

25.7

19.7

IC50 of 28 (nM)

24.9

19.5

59.4

45.6

34.3

24.5

14.9

11.1

181

39.3

a

Rat

dog

monkey

minipig

not determined.


24

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Next, we evaluated whether 28 could inhibit endogenous GR target gene expression and compared its relative potency with 1 in cancer cells. To this end, the GR+ human ovarian cancer cell line OVCAR-5, which has no detectable AR and low PR expression levels, was stimulated with dexamethasone in the presence or absence of 28 or 1 and mRNA levels of GR target genes were measured by RT-qPCR. Compound 28 reduced expression of FKBP5 and GILZ in a dosedependent manner (IC50 = 17.2 and 21.2 nM, respectively) with similar potency as 1 (IC50 = 10.9 and 13.0 nM, respectively) (Figure 11).

Figure 11. Inhibitory effects of 28 and 1 on GR-mediated target gene expression in ovarian cancer OVCAR-5 cells.

The ability of 28 to inhibit GR transcriptional activity was further evaluated in vivo. The major murine glucocorticoid, corticosterone, is a weak agonist of human GR.65 Therefore, when human cancer cells are grown as tumor xenografts in mice, it is necessary to provide an exogenous ligand such as cortisol or the synthetic ligand dexamethasone to effectively activate human GR. Expression of the GR target gene FKBP5 was analyzed in OVCAR5 tumors isolated from animals 6 hours after administration of vehicle or dexamethasone (0.5 mg/kg results in a


25


Page 26 of 66

plasma glucocorticoid concentration equivalent to a human cortisol dose of 0.100 to 0.467 µM corrected for potency) in the presence or absence of a single oral dose of 28 at 3 different concentrations. In the presence of 28 at 25, 50 or 75 mg/kg, dexamethasone-induced FKBP5 expression was reduced in a dose dependent manner (Figure 12A). Expression of FKBP5 in the 75 mg/kg group was not significantly different than expression in the vehicle group. The unbound plasma concentration of 28 in circulating mouse plasma ranged from 0.02 to 0.12 µM across the 3 groups (Figure 12B). The plasma exposures of dexamethasone were similar in all animals that received the dose of glucocorticoid (data not shown). These data suggest that 28 is effective at inhibiting dexamethasone-induced GR target gene expression in OVCAR-5 xenograft tumors.

(A)

(B)

Figure 12. Effect of 28 on dexamethasone-induced target gene expression in OVCAR5 xenograft tumors. (A) mRNA levels of the GR target gene, FKBP5, in OVCAR5 tumors relative to vehicle 6 hours after treatment (n = 4 mice per group). Dexamethasone (Dex) was administered by intraperitoneal injection at 0.5 mg/kg and a single dose of 28 was administrated orally by gavage at 25, 50, and 75 mg/kg. (B) Bar graph showing the mean unbound plasma


26

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concentrations of 28 in the 3 treated groups, 6 hours after treatment with Dex at 0.5 mg/kg (n = 4 mice per group). Significance of effects on FKBP5 expression was determined by one-Way ANOVA using Dunnet’s test to correct for multiple comparisons. The p-values compared to the vehicle group are 0.0004 (***) for the Dexamethasone group, 0.0036 (**) for the Dex + 28 at 25 mg/kg group, and not significant (ns) for the Dex + 28 at 50 mg/kg group (p = 0.1111) and the Dex + 28 at 75 mg/kg group (p = 0.7506).

An in vivo study was carried out to assess the efficacy of 28 in combination with gemcitabine and carboplatin in the OVCAR5 ovarian cancer xenograft model (Figure 13). As previously mentioned, corticosterone is the prevailing circulating glucocorticoid in rodents and is a weak activator of human GR. Therefore, to achieve proper activation of GR in the human xenograft transplant model, cortisol was administered chronically in the drinking water at 100 mg/L before tumor implantation and for the duration of the study in the groups indicated. The resulting plasma cortisol levels are consistent with reported mean plasma cortisol levels in cancer patients throughout the day, which range from 0.100 to 0.496 µM.66-67 Daily treatment with 28 did not affect the growth of the tumors grown under chronic cortisol. Gemcitabine and carboplatin induced tumor growth inhibition in groups with or without 28 compared to vehicle alone in the presence of cortisol. However, addition of 28 significantly enhanced the antitumor effect of chemotherapy. Tumor volumes on Day 21 in mice receiving gemcitabine + carboplatin + cortisol were significantly larger than the tumor volumes in mice receiving gemcitabine + carboplatin + cortisol + 28. These data indicated that 28 was effective in reversing the GR mediated resistance to chemotherapy driven by cortisol in vivo.


27


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*

Figure 13. Effects of 28 on gemcitabine and carboplatin response in OVCAR5 xenograft tumor in cortisol-treated mice. Tumor growth curves for mice with established OVCAR5 xenografts grown in the presence of cortisol at 100 mg/L (n = 10 mice per group). Animals were dosed with Gemcitabine 100 mg/kg and Carboplatin 60 mg/kg every 3 days for 4 doses. 28 was dosed at 75 mg/kg twice a day by oral gavage for the duration of the study. Data are displayed as mean ± SEM. The star (*) in both panels indicates statistical significance on day 21 between the 2 groups indicated, with p < 0.05.

CHEMISTRY Compounds 2–23 were prepared in a straightforward manner, through the route outlined in Scheme 1.61 Their synthesis was initiated by incorporation of C17 substituent in the commercially available ethylene deltanone 29. Grignard reaction of ketone 29 with alkynylmagnesium bromide or alkynyllithium provided 17β-hydroxy ketal 30 exclusively. Treatment of 30 with 30% hydrogen peroxide and 1,1,1,3,3,3-hexafluoropropan-2-one trihydrate in the presence of sodium hydrogen phosphate buffer provided epoxide 31 as a major


28

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diastereomer (dr > 3:1). Copper-catalyzed addition of an alkyl or aryl Grignard reagent to the vinylic epoxide 31, followed by hydrolysis with 70% acetic acid at 55 °C gave compounds 2–23 in moderate to high yields. Scheme 1a

a

Reagents and conditions: (a) for 30a: CH3C≡CMgBr, THF, 0 °C to 25 °C; for 30b–30f: R2Li,

˗78 °C to 25°C, 24–87%; (b) 35% H2O2, CF3COCF3, Na2HPO4, DCM, 42–94%; (c) R1MgBr, CuI, THF, 0 °C to 25°C, 59–83%; (d) 70% AcOH, 55 °C, 1–2 h, 46–96%.


29


Page 30 of 66

Scheme 2a

a

Reagents and conditions: (a) (i) 34, CuI, THF, 0 °C to 25 °C; (ii) 70% AcOH, 55 °C, 1 h, 23%

for two steps; (b) divinyl sulfone, IPA/H2O, 115 °C, 5 h, 26%; (c) 2,2-dimethoxypropane, PTSA, DMF, 77%; (d) (i) 36, Mg, 60 °C, 30 min; CuI, THF, 0 °C to 25 °C, 98%; (ii) 70% AcOH, 60 °C, 1.5 h, 73%; (e) PPh3, DEAD, Ziram, THF, toluene, 69%.

Synthesis of compounds 24 and 25 was completed in a similar manner but required extra steps as illustrated in Scheme 2. The primary aniline derivative 33a was prepared from epoxide 31f via copper catalyzed 1,4-addition of the commercially available (4-


30

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(bis(trimethylsilyl)amino)phenyl)magnesium bromide 34, followed by acetic acid-mediated hydrolysis. The double Michael addition of aniline 33a to divinyl sulfone afforded 24. For synthesis of 25, a commercially available diol 35 was converted to cyclic ketal 36 with 2,2dimethoxypropane. 1,4-Addition of in situ generated Grignard reagent of 36 to epoxide 31f, followed by hydrolysis with 70% acetic acid gave diol 33b. Finally, intramolecular Mitsunobu reaction of 33b yielded 25.68 Scheme 3 describes the synthesis of compounds 26, 27, and 28. The C17 ketone group in 29 was reduced by sodium borohydride to provide 17β-hydroxy ketal 37 as a single isomer, which was used in the next step without further purification. Oxidation of 37 with hydrogen peroxide led to a mixture of epoxides with the desired epoxide as major (dr = 4:1). Copper-catalyzed 1,4addition of the Grignard reagent to epoxide 38 followed by acetic acid-mediated hydrolysis resulted in dienone 39. Birch reduction of conjugated α,β-γ,δ-unsaturated ketone 39 into unconjugated β,γ-unsaturated ketone 40,69 followed by strong acid catalyzed double bond migration provided α,β-unsaturated ketone 41.70 Enone 41 was then protected as thioketal 42 under acidic conditions without double bond migration.70 The hydroxyl group in 42 was oxidized to ketone through Oppenauer oxidation. Alkynyl lithium addition to the ketone 43 followed by deprotection of the thioketal protecting group in 44 resulted in the final compounds 26, 27 and 28.


31


Page 32 of 66

Scheme 3a

29

a

b

H

O

O

O

38

O

R1 N

R1 N d

OH e

H H

H

OH f

H H

H

H

H

S

41

40 40a: R1 = Me 40b: R1 = i-Pr R1 N

H

H

R1 N

OH H

h

H H

S

42a: R1 = Me 42b: R1 = i-Pr

41a: R = Me 41b: R1 = i-Pr

O

R2

H

i

43 43a: R1 = Me 43b: R1 = i-Pr

H

R2

H H

H

O

S

S

OH

H

S

H

42

1

R1 N

H H

S

O

O

g

39 39a: R1 = Me 39b: R1 = i-Pr

R1 N

OH

44 44a: R1= Me, R2 = t-Bu 44b: R1 = Me, R2 = 1

2

44c: R = i-Pr, R = t-Bu

a

H

H

H

O

37

OH

c

H

H

O

R1 N

OH

OH

26: R1= Me, R2 = t-Bu O

27: R1 = Me, R2 =

O

28: R1 = i-Pr, R2 = t-Bu

Reagents and conditions: (a) NaBH4, THF, MeOH, 0 °C to 25 °C, 100%; (b) 35% H2O2,

CF3COCF3, Na2HPO4, DCM, 100%; (c) (i) ArMgBr, CuI, THF, 0 °C to 25 °C, 53–78%; (ii) 70% AcOH, 55 °C, 1 h, 77–80%; (d) liquid NH3, Li, THF, ˗78 °C, 74–100%; (e) for 41a: TsOH, benzene, reflux, 46%; for 41b: 37% HCl, MeOH, 65 °C, 73%; (f) HSCH2CH2SH, TsOH, AcOH, 36–61%; (g) Al(OiPr)3, cyclohexanone, toluene, 105 °C, 60–67%; (h) n-BuLi, alkyne, ˗78 °C to 25 °C, 59–97%; (i) 4 N HCl, glyoxylic acid, AcOH, 38–43%.


32

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CONCLUSION In summary, structural modifications of mifepristone (1) at the C11 and C17 positions followed by reduction of the C9 C-C double bond led to the discovery of 28 that features substantial reduction in AR agonism as compared to 1, with minimized loss of GR antagonism potency and low potential of CYP2C8 and 2C9 inhibition. Compound 28 demonstrated antitumor activity by overcoming GR-mediated chemo-resistance in the OVCAR5 ovarian cancer xenograft model, and is currently undergoing clinical testing.71

EXPERIMENTAL SECTION General Chemistry. All reactions were conducted under an inert gas atmosphere (nitrogen or argon) using a Teflon-coated magnetic stirbar at the temperature indicated. Commercial reagents and anhydrous solvents were used without further purification. Analytical thin layer chromatography (TLC) and flash chromatography were performed on Teledyne RediSep Rf Flash silica-gel columns. Removal of solvents was conducted by using 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, 100 mm × 21.2 mm i.d.), eluting with a binary solvent system A and B using a gradient elusion [A: H2O with 0.1% trifluoroacetic acid (TFA); B: CH3CN with 0.1% TFA] with UV detection at 220 nm. All final compounds were purified to ≥95% purity as determined by an 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


33


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i.d.); mobile phase, A = H2O with 0.1% TFA, B = CH3CN with 0.1% TFA; gradient: 5−95% B (0.0–15.0 min); flow rate, 1.5 mL/min. Low-resolution mass spectral (MS) data were determined on an Agilent 1100 Series LCMS with UV detection at 254 nm and a low resolution electrospray mode (ESI). NMR spectra were obtained on a Bruker 400 spectrometer. Chemical shifts (δ) 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, m=multiplet, br=broad.

(8S,13S,14S,17S)-17-(3,3-Dimethylbut-1-yn-1-yl)-13-methyl-1,2,4,6,7,8,12,13,14,15,16,17dodecahydrospiro[cyclopenta[a]phenanthrene-3,2'-[1,3]dioxolan]-17-ol (30f). To a solution of 3,3-dimethylbut-1-yne (3.34 g, 40.7 mmol) in THF (85 mL) at ˗78 °C was added dropwise nbutyllithium (1.6 M in hexanes, 24.6 mL, 39.4 mmol). After it was stirred at ˗78 °C for 25 min, a solution of 29 (8 g, 25.4 mmol) in THF (100 ml) was added slowly. The mixture was stirred at ˗78 °C for 17 min and then at rt for 40 min. The reaction mixture was quenched by slow addition of aq. NH4Cl solution and concentrated to remove the majority of THF. The residue was extracted with EtOAc (400 mL). The organic phase was washed with brine and dried over anhydrous Na2SO4. The mixture was then concentrated under reduced pressure and the crude residue was purified by silica-gel chromatography (SiO2, 5–10 % EtOAc in hexanes, gradient elution) to provide 30f (7.7 g, 18.9 mmol, 74% yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ ppm 7.27 (2 H, s), 5.51–5.69 (1 H, m), 3.92–4.09 (4 H, m), 2.64 (1 H, br dd, J = 18.0 Hz), 2.53 (1 H, m), 2.29 (2 H, m), 1.90–2.11 (4 H, m), 1.62–1.87 (5 H, m), 1.70–1.75 (1 H, m),


34

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1.40–1.50 (1 H, 1m), 1.20–1.30 (1 H, m), 1.20 (9 H, s), 0.83 (3 H, s); m/z (ESI, +ve ion) 397.3 (M+H)+. (5'R,8'S,10'R,13'S,14'S,17'S)-17'-(3,3-Dimethylbut-1-yn-1-yl)-13'-methyl1',2',7',8',12',13',14',15',16',17'-decahydro-4'H,6'H-spiro[[1,3]dioxolane-2,3'[5,10]epoxycyclopenta[a]phenanthren]-17'-ol (31f). To a solution of 30f (7.5 g, 18.9 mmol) in DCM (120 mL) at 0 °C was added Na2HPO4 (5.37 g, 37.8 mmol), followed by addition of CF3COCF3-3H2O (2.6 mL, 18.9 mmol) and H2O2 (35% aq., 4.36 mL, 56.7 mmol). The mixture was stirred at 0 °C for 10 min and then at rt for 2 h. Aq. Na2S2O3 solution was added, the mixture was stirred at rt for 30 min and extracted with EtOAc. The organic phase was washed with brine and dried over anhydrous Na2SO4. The mixture was then concentrated under reduced pressure and the crude residue was purified by silica-gel chromatography (SiO2, 10–15 % EtOAc in hexanes, gradient elution) to provide 31f (6.9 g, 16.7 mmol, 88% yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ ppm 6.07–6.11 (1 H, m), 3.87–3.99 (4 H, m), 2.45–2.65 (2 H, br s), 2.20–2.30 (2 H, m), 2.10 (1 H, m), 1.86–2.10 (4 H, m), 1.60–1.80 (5 H, m), 1.46–1.57 (1 H, m), 1.20–1.45 (2 H, m), 1.19 (9 H, s), 0.83 (3 H, s). (5R,8S,11R,13S,14S,17S)-11-(4-(Dimethylamino)phenyl)-17-(3,3-dimethylbut-1-yn-1-yl)-13methyl-1,2,6,7,8,11,12,13,14,15,16,17-dodecahydrospiro[cyclopenta[a]phenanthrene-3,2'[1,3]dioxolane]-5,17(4H)-diol (32j). A mixture of 31f (80 mg, 0.19 mmol) and copper(I) iodide (36.9 mg, 0.19 mmol) in THF (3 mL) was degassed with Ar. After cooling in an ice bath, (4(dimethylamino) phenyl) magnesium bromide (0.5 M in THF, 3.1 mL, 1.55 mmol) was added dropwise. The resulting solution was maintained at rt for 1 h, then quenched with NH4Cl solution and extracted with EtOAc. The organic phase was washed with brine and dried over anhydrous Na2SO4. The mixture was then concentrated under reduced pressure and the crude


35


Page 36 of 66

residue was purified by silica-gel chromatography to provide 32j (90 mg, 0.17 mmol, 89% yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ ppm 7.07 (2 H, d, J = 8.5 Hz), 6.63–6.67 (2 H, m), 4.41 (1 H, d, J = 1.2 Hz), 4.13 (1 H, d, J = 7.2 Hz), 3.90–4.05 (4 H, m), 2.91 (6 H, s), 2.45 (1 H, m), 2.10–2.40 (4 H, m), 2.00–2.05 (2 H, m), 1.85–2.00 (2 H, m), 1.75–1.80 (1H, m), 1.60– 1.75 (4 H, m), 1.50 (1 H, m), 1.30–1.40 (3 H, m), 1.24 (9 H, s), 0.49 (3 H, s); m/z (ESI, +ve ion) 534.3 (M+H)+. (8S,11R,13S,14S,17S)-11-(4-(Dimethylamino)phenyl)-17-(3,3-dimethylbut-1-yn-1-yl)-17hydroxy-13-methyl-1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3Hcyclopenta[a]phenanthren-3-one (11). A solution of 32j (90 mg, 0.17 mmol) in 1:2 acetic acid/water (16.5 mL) was stirred at 55 °C for 90 min. The reaction mixture was cooled to rt and slowly added aq. NaHCO3 solution. After extraction with EtOAc, the organic phase was washed with brine, dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude residue was purified by silica-gel chromatography to provide 11 (54 mg, 0.12 mmol, 70% yield) as a pale yellow solid. 1H NMR (400 MHz, CDCl3) δ ppm 7.03 (2 H, d, J = 8.6 Hz), 6.67 (2 H, d, J = 8.6 Hz), 5.77 (1 H, s), 4.36 (1 H, br d, J = 6.3 Hz), 2.93 (6 H, s), 2.76 (1 H, s), 2.59 (2 H, dd, J = 7.4, 3.1 Hz), 2.27–2.49 (6 H, m), 2.17–2.25 (1 H, m), 1.86–2.06 (2 H, m), 1.64–1.78 (3 H, m), 1.47 (1 H, br s), 1.34 (1 H, br dd, J = 11.7, 5.6 Hz), 1.20–1.29 (9 H, s), 0.56 (3 H, s); 13C NMR (101 MHz, CD2Cl2) δ ppm 199.5, 157.5, 149.2, 147.3, 132.9, 129.6, 128.1 (2 C), 122.9, 113.1 (2 C), 95.5, 82.4, 80.2, 50.5, 47.5, 41.0 (2 C), 40.1, 39.8, 39.6, 39.4, 37.5, 31.6, 31.4 (3 C), 28.1, 27.9, 26.4, 23.8, 14.1; m/z (ESI, +ve ion) 472.4 (M+H)+.

2-10 and 12-23 were prepared from 29 according to a similar procedure described for the synthesis of 11.


36

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(8S,11S,13S,14S,17S)-11-Ethyl-17-hydroxy-13-methyl-17-(prop-1-yn-1-yl)1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (2). 1H NMR (400 MHz, CDCl3) δ ppm 5.70 (1 H, s), 2.87–2.98 (2 H, m), 2.54–2.62 (1 H, m), 2.33– 2.51 (5 H, m), 2.25 (1 H, ddd, J = 13.9, 9.5, 5.8 Hz), 1.78–2.06 (7 H, m), 1.45–1.72 (4 H, m), 1.20–1.43 (2 H, m) 1.05 (3 H, s), 0.95 (3 H, t, J = 7.3 Hz); m/z (ESI, +ve ion) 339.1 (M+H)+.

(8S,11R,13S,14S,17S)-11-Cyclohexyl-17-hydroxy-13-methyl-17-(prop-1-yn-1-yl)1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (3). 1H NMR (400 MHz, CDCl3) δ ppm 5.70 (1 H, s), 3.74–3.77 (1 H, m), 2.55–2.83 (3 H, m), 2.21– 2.49 (6 H, m), 1.75–2.25 (7 H, m), 1.50–1.75 (3 H, m), 1.05–1.50 (11 H, m), 0.71–0.92 (3 H, m).

(8S,11R,13S,14S,17S)-17-Hydroxy-13-methyl-11-phenyl-17-(prop-1-yn-1-yl)1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (4). 1H NMR (400 MHz, CDCl3) δ ppm 7.22–7.35 (2 H, m), 7.13–7.22 (3 H, m), 5.77 (1 H, s), 4.44 (1 H, br d, J = 4.0 Hz), 2.65–2.85 (1 H, m), 2.50–2.63 (2 H, m), 2.13–2.50 (7 H, m), 1.90–2.15 (2 H, m), 1.90 (3 H, s), 1.62–1.88 (3 H, m), 1.28–1.62 (2 H, m), 0.50 (3 H, s).

(8S,11R,13S,14S,17S)-11-(3-(Dimethylamino)phenyl)-17-hydroxy-13-methyl-17-(prop-1-yn1-yl)-1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (5). 1

H NMR (400 MHz, CDCl3) δ ppm 7.12 (1 H, m), 6.51–6.57 (3 H, m), 5.76 (1 H, s), 4.39 (1 H,

br d, J = 4.0 Hz), 2.91 (6 H, s), 2.70–2.85 (1 H, m), 2.18–2.65 (9 H, m), 1.91–2.18 (2 H, m), 1.90 (3 H, s), 1.70–1.85 (2 H, m), 1.28–1.65 (2 H, m), 0.58 (3 H, s); m/z (ESI, +ve ion) 430.4 (M+H)+.


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(8S,11R,13S,14S,17S)-17-Hydroxy-11-(4-isopropylphenyl)-13-methyl-17-(prop-1-yn-1-yl)1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (6). 1H NMR (400 MHz, CDCl3) δ ppm 7.05–7.18 (4 H, m), 5.73 (1 H, s), 4.37 (1 H, br d, J = 4.0 Hz), 2.71–2.89 (2 H, m), 2.15–2.68 (9 H, m), 1.88–2.15 (2 H, m), 1.88 (3 H, s), 1.20–1.81 (5 H, m), 1.25 (6 H, d, J = 4.0 Hz), 0.58 (3 H, s); m/z (ESI, +ve ion) 429.4 (M+H)+.

(8S,11R,13S,14S,17S)-11-(4-(Dimethylamino)phenyl)-17-(hex-1-yn-1-yl)-17-hydroxy-13methyl-1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (7). 1

H NMR (400 MHz, CDCl3) δ ppm 7.02 (2 H, d, J = 8.2 Hz), 6.66 (2 H, d, J = 8.9 Hz), 5.76 (1

H, s), 4.35 (1 H, br d, J = 6.6 Hz), 2.92 (6 H, s), 2.70–2.80 (1H, m), 2.45–2.60 (3 H, m), 2.30– 2.48 (2 H, m), 2.12–2.28 (1 H, m), 1.95–2.10 (3 H, m), 1.50–1.62 (2 H, m), 1.32–1.46 (5 H, m), 1.22–1.29 (3 H, m), 0.95–1.05 (2 H, m), 0.58 (3 H, s); m/z (ESI, +ve ion) 448.4 (M+H)+.

(8S,11R,13S,14S,17S)-11-(4-(Dimethylamino)phenyl)-17-hydroxy-13-methyl-17-(3,3,3trifluoroprop-1-yn-1-yl)-1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3Hcyclopenta[a]phenanthren-3-one (8). 1H NMR (400 MHz, CDCl3) δ ppm 7.02 (2 H, m, J = 8.6 Hz), 6.67 (2 H, m, J= 8.4 Hz), 5.78 (1 H, s), 4.40 (1 H, br d, J= 6.8 Hz), 2.93 (6 H, s), 2.77 (1 H, m), 2.55–2.62 (2 H, m), 2.44–2.54 (2 H, m), 2.29–2.40 (4 H, m), 2.23 (1 H, br d, J = 7.2 Hz), 1.83–2.11 (4 H, m), 1.35–1.57 (3 H, m), 0.59 (3 H, s); m/z (ESI, +ve ion) 484.3 (M+H)+.

(8S,11R,13S,14S,17S)-17-(ut-1-yn-1-yl)-11-(4-(dimethylamino)phenyl)-17-hydroxy-13methyl-1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (9). 1

H NMR (400 MHz, CDCl3) δ ppm 7.15–7.22 (1 H, m), 7.31 (1 H, m), 6.75–6.80 (1 H, m), 6.65

(1 H, m), 5.77 (1 H, s), 4.37 (1 H, m), 3.05–3.15 (4 H, m), 3.0 (1 H, m), 2.93 (6 H, s), 2.55–2.62


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(1 H, m), 2.30–2.45 (4 H, m), 2.00–2.10 (1 H, m), 1.90–2.00 (1 H, m), 1.65–1.75 (3 H, m), 1.30– 1.40 (2 H, m), 1.25 (1 H, m), 1.17 (3 H, t, J = 7.5 Hz), 0.56 (3 H, s); m/z (ESI, +ve ion) 444.4 (M+H)+.

(8S,11R,13S,14S,17S)-11-(4-(Dimethylamino)phenyl)-17-hydroxy-13-methyl-17-(3methylbut-1-yn-1-yl)-1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3Hcyclopenta[a]phenanthren-3-one (10). 1H NMR (400 MHz, CDCl3) δ ppm 7.02 (2 H, m, J = 8.0 Hz), 6.66 (2 H, m, J = 8.0 Hz), 5.57 (1 H, s), 4.35 (1 H, br d, J = 8.0 Hz), 2.92 (6 H, s), 2.70– 2.80 (1 H, m), 2.50–2.70 (2 H, m), 2.15–2.50 (7 H, m), 1.85–2.15 (2 H, m), 1.65–1.85 (4 H, m), 1.20–1.60 (2 H, m), 1.17 (6 H, d, J = 4.0 Hz), 0.56 (3 H, s); m/z (ESI, +ve ion) 458.3 (M+H)+.

(8S,11R,13S,14S,17S)-17-(3,3-Dimethylbut-1-yn-1-yl)-17-hydroxy-13-methyl-11-(p-tolyl)1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (12). 1H NMR (400 MHz, CDCl3) δ ppm 7.01–7.15 (4 H, m), 5.78 (1 H, s), 4.40 (1 H, br d, J = 7.0 Hz), 2.76 (1 H, br d, J = 14.8 Hz), 2.56–2.63 (2 H, m), 2.40–2.49 (1 H, m), 2.18–2.38 (9 H, m), 1.87– 2.12 (2 H, m), 1.61–1.80 (3 H, m), 1.42–1.56 (1 H, m), 1.35 (1 H, br dd, J = 11.7, 5.9 Hz), 1.25 (9 H, s), 0.53 (3 H, s); m/z (ESI, +ve ion) 443.5 (M+H)+.

(8S,11R,13S,14S,17S)-17-(3,3-Dimethylbut-1-yn-1-yl)-11-(4-ethylphenyl)-17-hydroxy-13methyl-1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (13). 1H NMR (400 MHz, CDCl3) δ ppm 7.10 (4 H, s), 5.78 (1 H, s), 4.41 (1 H, br d, J = 6.3 Hz), 2.77 (1 H, br d, J = 14.5 Hz), 2.56–2.66 (4 H, m), 2.28–2.50 (6 H, m), 2.18–2.26 (1 H, m), 1.89– 2.12 (2 H, m), 1.60–1.79 (3 H, m), 1.43–1.57 (1 H, m), 1.29–1.42 (1 H, m), 1.14–1.29 (12 H, m), 0.52 (3 H, s); m/z (ESI, +ve ion) 457.4 (M+H)+.


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(8S,11R,13S,14S,17S)-17-(3,3-Dimethylbut-1-yn-1-yl)-17-hydroxy-11-(4-isopropylphenyl)13-methyl-1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (14). 1H NMR (400 MHz, CDCl3) δ ppm 7.04–7.20 (4 H, m), 5.78 (1 H, s), 4.41 (1 H, br d, J = 6.4 Hz), 2.87 (1 H, t, J = 6.9 Hz), 2.75 (1 H, m), 2.54–2.62 (2 H, m), 2.27–2.50 (6 H, m), 2.21 (1 H, m), 2.01 (1 H, m), 1.95 (1 H, m), 1.59–1.82 (3 H, m), 1.28–1.45 (2 H, m), 1.17–1.27 (15 H, m), 0.52 (3 H, s); m/z (ESI, +ve ion) 471.4 (M+H)+.

(8S,11R,13S,14S,17S)-17-(3,3-Dimethylbut-1-yn-1-yl)-17-hydroxy-11-(4-methoxyphenyl)13-methyl-1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (15). 1H NMR (400 MHz, CDCl3) δ ppm 7.09 (2 H, d, J = 8.4 Hz), 7.00 (1 H, d, J = 8.8 Hz), 5.78 (1 H, s), 4.39 (1 H, br d, J = 7.8 Hz), 3.79 (3 H, s) 2.77 (1 H, br d, J = 14.6 Hz), 2.54–2.67 (2 H, m) 2.27–2.53 (6 H, m), 2.18–2.25 (1 H, m),1.95 (1 H, br d, J = 1.0 Hz) 1.59–1.85 (3 H, m) 1.30–1.56 (2 H, m), 1.25 (9 H, s), 0.53 (3 H, s); m/z (ESI, +ve ion) 459.4 (M+H)+.

(8S,11R,13S,14S,17S)-11-(3-(Dimethylamino)phenyl)-17-(3,3-dimethylbut-1-yn-1-yl)-17hydroxy-13-methyl-1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3Hcyclopenta[a]phenanthren-3-one (16). 1H NMR (400 MHz, CDCl3) δ ppm 7.13 (1 H, t, J = 7.9 Hz), 6.53–6.60 (3 H, m), 5.77 (1 H, s), 4.40 (1 H, br s), 2.93 (6 H, s), 2.73–2.82 (1 H, m), 2.53– 2.62 (2 H, m), 2.31–2.52 (6 H, m), 2.18–2.26 (1 H, m), 1.92–2.06 (2 H, m), 1.60–1.79 (3 H, m), 1.49 (1 H, br d, J = 6.3 Hz), 1.31–1.44 (1 H, m), 1.25 (9 H, s), 0.59 (3 H, s); m/z (ESI, +ve ion) 472.4 (M+H)+.

(8S,11R,13S,14S,17S)-11-(4-(Dimethylamino)-3,5-difluorophenyl)-17-(3,3-dimethylbut-1yn-1-yl)-17-hydroxy-13-methyl-1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3H-


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cyclopenta[a]phenanthren-3-one (17). 1H NMR (400 MHz, CDCl3) δ ppm 6.68 (1 H, s), 6.65 (1 H, s), 5.81 (1 H, s), 4.32 (1 H, br d, J = 6.8 Hz), 2.87 (6 H, t, J = 1.6 Hz), 2.75 (1 H, br d, J = 14.3 Hz), 2.56–2.62 (2 H, m), 2.29–2.48 (5 H, m), 2.15–2.26 (2 H, m), 1.89–2.08 (2 H, m), 1.61– 1.80 (3 H, m), 1.43–1.57 (1 H, m), 1.29–1.43 (1 H, m), 1.24 (9 H, s), 0.55 (3 H, s); m/z (ESI, +ve ion) 508.4 (M+H)+.

(8S,11S,13S,14S,17S)-11-(4-(Dimethylamino)-2,6-difluorophenyl)-17-(3,3-dimethylbut-1-yn1-yl)-17-hydroxy-13-methyl-1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3Hcyclopenta[a]phenanthren-3-one (18). 1H NMR (400 MHz, CDCl3) δ ppm 6.01–6.24 (2 H, m), 5.70 (1 H, s), 4.52 (1 H, d, J = 8.4 Hz), 2.92 (6 H, s), 2.69 (2 H, dt, J = 15.0, 5.3 Hz), 2.39–2.59 (3 H, m), 2.16–2.38 (4 H, m), 1.87–2.06 (3 H, m), 1.70–1.84 (1 H, m), 1.33–1.57 (3 H, m), 1.24 (9 H, s), 1 0.84 (3 H, s); m/z (ESI, +ve ion) 508.4 (M+H)+.

(8S,11R,13S,14S,17S)-11-(4-(Dimethylamino)-3-methoxyphenyl)-17-(3,3-dimethylbut-1-yn1-yl)-17-hydroxy-13-methyl-1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3Hcyclopenta[a]phenanthren-3-one (19). 1H NMR (400 MHz, CDCl3) δ ppm 6.81 (1 H, d, J = 8.2 Hz), 6.73 (1 H, s), 6.64 (1 H, d, J = 7.6 Hz), 5.78 (1 H, s), 4.39 (1 H, br d, J = 6.4 Hz), 3.86 (3 H, s), 2.71–2.81 (7 H, m), 2.53–2.64 (2 H, m), 2.30–2.50 (6 H, m), 2.18–2.28 (1 H, m), 1.88–2.12 (2 H, m), 1.61–1.79 (4 H, m), 1.31–1.56 (2 H, m), 1.20–1.28 (9 H, s), 0.56 (3 H, s); m/z (ESI, +ve ion) 502.4 (M+H)+. (8S,11R,13S,14S,17S)-11-(4-(Dimethylamino)-3-methylphenyl)-17-(3,3-dimethylbut-1-yn-1yl)-17-hydroxy-13-methyl-1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3Hcyclopenta[a]phenanthren-3-one (20). 1H NMR (400 MHz, CDCl3) δ ppm 6.99 (1 H, s), 6.89


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(2 H, m), 5.78 (1 H, s), 4.33–4.38 (1 H, m), 2.75 (1 H, m), 2.67 (6 H, s), 2.54–2.64 (2 H, m), 2.26–2.47 (8 H, m), 2.18–2.24 (1 H, m), 1.88–2.07 (2 H, m), 1.60–1.77 (3 H, m), 1.47 (1 H, br dd, J = 12.9, 9.4 Hz), 1.31–1.40 (1 H, m), 1.24 (9 H, s), 0.54 (3 H, s); m/z (ESI, +ve ion) 486.4 (M+H)+. (8S,11R,13S,14S,17S)-17-(3,3-Dimethylbut-1-yn-1-yl)-17-hydroxy-11-(4(isopropyl(methyl)amino)phenyl)-13-methyl-1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro3H-cyclopenta[a]phenanthren-3-one (21). 1H NMR (400 MHz, CDCl3) δ ppm 6.95–7.06 (2 H, m), 6.71 (2 H, m, J = 8.8 Hz), 5.77 (1 H, s), 4.35 (1 H, br d, J = 5.7 Hz), 3.98–4.09 (1 H, m), 2.76 (1 H, m), 2.70 (3 H, s), 2.53–2.61 (2 H, m), 2.33–2.49 (4 H, m), 2.27–2.30 (1 H, m), 2.17– 2.25 (1 H, m), 1.84–2.09 (2 H, m), 1.54–1.80 (3 H, m), 1.30–1.54 (2 H, m), 1.24 (9 H, s), 1.10– 1.38 6 H, m), 0.56 (3 H, s); m/z (ESI, +ve ion) 500.5 (M+H)+.

(8S,11R,13S,14S,17S)-17-(3,3-Dimethylbut-1-yn-1-yl)-17-hydroxy-13-methyl-11-(4morpholinophenyl)-1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3Hcyclopenta[a]phenanthren-3-one (22). 1H NMR (400 MHz, CDCl3) δ ppm 7.03–7.13 (2 H, m), 6.75–6.91 (2 H, m), 5.78 (1 H, s), 4.34–4.40 (1 H, m), 3.79–3.90 (4 H, m), 3.08–3.18 (4 H, m), 2.76 (1 H, br d, J = 15.0 Hz), 2.54–2.62 (2 H, m), 2.27–2.49 (5 H, m), 2.18–2.25 (1 H, m), 1.90– 2.07 (2 H, m), 1.64–1.78 (3 H, m), 1.48 (2 H, br d, J = 9.8 Hz), 1.24 (9 H, s), 0.53 (3 H, s); m/z (ESI, +ve ion) 514.4 (M+H)+.

(8S,11R,13S,14S,17S)-17-(3,3-Dimethylbut-1-yn-1-yl)-17-hydroxy-13-methyl-11-(4(pyrrolidin-1-yl)phenyl)-1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3Hcyclopenta[a]phenanthren-3-one (23). 1H NMR (400 MHz, CDCl3) δ ppm 6.98–7.06 (2 H, m),


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6.44–6.54 (2 H, m) 5.76 (1 H, s), 4.36 (1 H, br dd, J = 5.6, 2.4 Hz), 3.27 (4 H, br t, J = 5.2 Hz), 2.73–2.81 (1 H, m), 2.53–2.61 (2 H, m), 2.26–2.48 (5 H, m), 2.17–2.24 (1 H, m), 1.91–2.06 (6 H, m), 1.63–1.77 (3 H, m), 1.42–1.56 (1 H, m), 1.30–1.41 (1 H, m), 1.24 (9 H, s), 0.57 (3 H, s); m/z (ESI, +ve ion) 498.4 (M+H)+.

(8S,11R,13S,14S,17S)-11-(4-Aminophenyl)-17-(3,3-dimethylbut-1-yn-1-yl)-17-hydroxy-13methyl-1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (33a). Under Ar, 31f (3 g, 7.27 mmol) and copper iodide (1.38 g, 7.27 mmol) in THF (40 mL) was cooled to 0 °C. (4-(Bis(trimethylsilyl)amino)phenyl)magnesium bromide (34) (0.5 M in THF, 58.2 mL, 29.1 mmol) was added dropwise. The reaction mixture was slowly brought to rt and stirred for 2 h. The reaction was quenched (sat. NH4Cl) and the reaction mixture exposed to the air and stirred for 10 min. The mixture was extracted (EtOAc) and washed (brine). The organics were separated and dried (MgSO4) before concentration to dryness. The crude was used in next step without further purification.

A solution of the crude material (641 mg g, 0.99 mmol) in 70% acetic acid in water (5mL) was stirred at 55 °C for 1 h. The reaction was quenched (sat. aq. NaHCO3), extracted (2 × EtOAc), and washed (brine). The combined organic layers were dried (MgSO4) and concentrated under reduced pressure. Purification of the residue by column chromatography (SiO2, 50–70 % EtOAc in hexanes, gradient elution) gave 33a (100 mg, 0.23 mmol, 22% yield). 1H NMR (400 MHz, CDCl3) δ ppm 6.96 (2 H, d, J = 8.5 Hz), 6.64 (2 H, br d, J = 8.2 Hz), 5.77 (1 H, s), 4.34 (1 H, br d, J = 6.9 Hz), 2.69–2.86 (1 H, m), 2.52–2.65 (2 H, m), 2.28–2.49 (5 H, m), 2.17–2.26 (2 H, m), 1.85–2.09 (2 H, m), 1.62–1.85 (3 H, m), 1.25–1.51 (2 H, m), 1.24 (9 H, s), 0.506 (3 H, s); m/z (ESI, +ve ion) 444.4 [M+H]+.


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(8S,11R,13S,14S,17S)-17-(3,3-Dimethylbut-1-yn-1-yl)-11-(4-(1,1dioxidothiomorpholino)phenyl)-17-hydroxy-13-methyl-1,2,6,7,8,11,12,13,14,15,16,17dodecahydro-3H-cyclopenta[a]phenanthren-3-one (24). Divinyl sulfone (0.20 mL, 1.4 mmol) was added to a solution of 33a (60 mg, 0.14 mmol) in isopropanol (2 mL) and water (2 mL) and the mixture was refluxed at 115 °C for 5 h. The reaction was quenched (sat. aq. NH4Cl), extracted (2 × EtOAc), and washed (brine). The combined organic layers were dried (MgSO4) and concentrated under reduced pressure. Purification of the crude product by column chromatography (SiO2, 20–60 % EtOAc in hexanes, gradient elution) gave 24 (20 mg, 0.036 mmol, 26% yield). 1H NMR (400 MHz, CDCl3) δ ppm 7.11 (2 H, d, J = 8.5 Hz), 6.78–6.93 (2 H, m), 5.79 (1 H, s), 4.38 (1 H, br d, J = 7.3 Hz), 3.76–3.93 (4 H, m), 3.05–3.20 (4 H, m), 2.72–2.83 (1 H, m), 2.54–2.66 (2 H, m), 2.18–2.48 (7 H, m), 1.89–2.11 (3 H, m), 1.57–1.83 (1 H, m), 1.43– 1.56 (2 H, m), 1.24 (9 H, s), 0.53 (3 H, s); m/z (ESI, +ve ion) 562.4 [M+H]+.

5-(4-Bromophenyl)-2,2,5-trimethyl-1,3-dioxane (36). To a solution of 2-(4-bromophenyl)-2methyl-propane-1,3-diol (35) (688 mg, 2.81 mmol) and 4-methylbenzenesulfonic acid (24 mg, 0.14 mmol) in DMF (12 mL) was added 2,2-dimethoxypropane (0.52 mL, 4.2 mmol). The solution was stirred at rt overnight and then was poured into saturated aq. NaHCO3 and extracted with EtOAc. The combined organic extracts were purified by column chromatography (SiO2, 0– 30 % EtOAc in hexanes, gradient elution) to give 36 (618 mg, 2.22 mmol, 77% yield). 1H NMR (400 MHz, CDCl3) δ ppm 7.49 (2 H, d, J = 7.42 Hz) 7.26–7.30 (2 H, m) 4.08 (2 H, d, J = 11.7 Hz) 3.80 (2 H, d, J = 11.7 Hz) 1.41 (3 H, s) 1.34 (3 H, s), 1.28 (3 H, s); m/z (ESI, +ve ion) 280.3 [M+H]+.


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(8S,11R,13S,14S,17S)-11-(4-(1,3-Dihydroxy-2-methylpropan-2-yl)phenyl)-17-(3,3dimethylbut-1-yn-1-yl)-17-hydroxy-13-methyl-1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro3H-cyclopenta[a]phenanthren-3-one (33b). A dried flask was charged with magnesium chips (40 mg, 1.5 mmol) and a crystal of iodine. The flask was flushed with Ar, heated briefly with a heat gun and then heated at 60 °C. At first, a small portion (0.5 mL) of a solution of 36 (azeotroped with toluene beforehand, 373 mg, 1.31 mmol) in THF (2 mL) was added. After iodine color disappeared, the remaining solution was added, and the mixture was continued stirring for 30 min at 60 °C. The mixture was then cooled down and added to a solution of 31f (45 mg, 0.11mmol) and copper (I) iodide (16.6 mg, 0.09 mmol) in THF (1 mL) at 0 °C. The mixture was kept at 0 °C for 10 min and then raised to rt for 2 h. After the reaction was cooled to rt, it was quenched with NH4Cl and extracted with EtOAc. The organics were washed, dried, and concentrated. The residue was purified by combi-flash (SiO2, 0–40 % EtOAc in hexanes, gradient elution) to give the Grignard addition product (55 mg, 81%).

To a flask charged the Grignard addition product (55 mg, 0.09 mmol) was added 70% acetic acid (2 mL). After the mixture was heated at 60 °C for 2 h, it was quenched with NaHCO3 and extracted with EtOAc. The organics were washed, dried and concentrated. The residue was purified by combi-flash (SiO2, 0–80 % EtOAc in hexanes, gradient elution) to give 33b (32 mg, 0.06 mmol, 70% yield). 1H NMR (400 MHz, CDCl3 δ ppm 7.28–7.37 (2 H, m), 7.19 (2 H, d, J = 8.2 Hz), 5.78 (1 H, s), 4.43 (1 H, br d, J = 6.6 Hz), 3.96 (2 H, d, J = 11.0 Hz), 3.83 (2 H, dd, J = 11.0, 1.2 Hz), 2.69–2.87 (1 H, m), 2.59 (2 H, br dd, J = 7.1, 3.7 Hz), 2.23–2.50 (7 H, m), 1.85– 2.11 (2 H, m), 1.75–1.84 (2 H, m), 1.45–1.55 (1 H, m), 1.25–1.40 (1 H, m), 1.30 (3 H, s), 1.25 (9 H, s), 0.50 (3 H, s); m/z (ESI, +ve ion) 517.4 (M+H)+.


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(8S,11R,13S,14S,17S)-17-(3,3-Dimethylbut-1-yn-1-yl)-17-hydroxy-13-methyl-11-(4-(3methyloxetan-3-yl)phenyl)-1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3Hcyclopenta[a]phenanthren-3-one (25). A flask was charged with 33b (33 mg, 0.07 mmol) and azeotroped with toluene. Triphenylphosphine (50.3 mg, 0.19 mmol), toluene (1.5 mL), THF (0.5 mL), and Ziram (39.1 mg, 0.13 mmol) were added successively. Upon adding ethyl N-Nethoxycarbonyliminocarbamate (0.03 mL, 0.19 mmol), the solution became clear. The resulting solution was stirred overnight. The mixture was filtered through celite and rinsed with EtOAc. The filtrate was concentrated, and the residue was purified by combi-flash (SiO2, 0–30 % EtOAc in hexanes, gradient elution) to give the crude product. Purification by RP-HPLC (30–70% A/B, gradient elution) provided 25 (17 mg, 0.034 mmol, 53% yield). 1H NMR (400 MHz, CDCl3) δ ppm 7.13–7.19 (4 H, m), 5.79 (1 H, s), 4.94 (2 H, dd, J = 5.6, 3.4 Hz), 4.63 (2 H, d, J = 5.6 Hz), 4.43 (1 H, br d, J = 6.7 Hz), 2.77 (1 H, br d, J = 14.6 Hz), 2.60 (2 H, dd, J = 8.0, 4.1 Hz), 2.26– 2.51 (6 H, m), 2.15–2.25 (1 H, m), 1.87–2.09 (2 H, m), 1.65–1.79 (5 H, m), 1.43–1.56 (2 H, m), 1.25 (9 H, s), 0.52 (3 H, s); m/z (ESI, +ve ion) 499.4 (M+H)+.

(8S,13S,14S,17S)-13-Methyl-1,2,4,6,7,8,12,13,14,15,16,17dodecahydrospiro[cyclopenta[a]phenanthrene-3,2'-[1,3]dioxolan]-17-ol (37). To a solution of 29 (100 g, 318 mmol) in THF (500 mL) and MeOH (50 mL) was added sodium borohydride (6.14 g, 159 mmol) in portions at 0 °C. The mixture was stirred at 0 °C for 30 min and then rt for 1 h. The reaction was quenched with 2 mL of sat. NaHCO3 solution and concentrated to remove MeOH. The residue was dissolved in EtOAc (1 L), the organic phase was washed with sat. NaHCO3 solution and then brine and dried over anhydrous Na2SO4. The organic solution was concentrated under reduced pressure to give 37 (99.5 g, 100% yield) as a colorless oil. 1H


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NMR (400 MHz, CDCl3) δ ppm 5.61 (1 H, m), 4.01 (4 H, s), 3.78 (1 H, m), 2.48–2.64 (1 H, m), 1.75–2.36 (14 H, m), 1.12–1.58 (3 H, m), 0.78 (3 H, s); m/z (ESI, +ve ion) 317.3 [M+H]+.

(5'R,8'S,10'R,13'S,14'S,17'S)-13'-Methyl-1',2',7',8',12',13',14',15',16',17'-decahydro4'H,6'H-spiro[[1,3]dioxolane-2,3'-[5,10]epoxycyclopenta[a]phenanthren]-17'-ol (38). To a solution of 37 (43.0 g, 136 mmol) in DCM (320 mL) at 0 °C was added Na2HPO4 (9.65 g, 67.9 mmol), followed by addition of 1,1,1,3,3,3-hexafluoropropan-2-one trihydrate (11.2 mL, 81.5 mmol) and H2O2 (35% aq, 34.2 mL, 408 mmol). The mixture was stirred at 0 °C for 10 min and then at rt for 3 h. Sodium thiosulfate solution (10% aq., 200 mL) was slowly added at 0 °C and the mixture was stirred at rt for 30 min and extracted with DCM (300 mL). The organic phase was washed with brine and dried over anhydrous Na2SO4. The organic solution was then concentrated to provide 38 (43 g. 130 mmol, 95% yield) as a 4:1 mixture of epimers (the desired isomer is major). 1H NMR (400 MHz, CDCl3) δ ppm 5.97–6.07 (1 H, m), 3.84–4.00 (4 H, m), 3.65–3.79 (1 H, m), 2.38–2.57 (1 H, m), 1.75–2.36 (14 H, m), 1.12–1.58 (3 H, m), 0.74 (3 H, s).

(8S,11R,13S,14S,17S)-17-Hydroxy-11-(4-(isopropyl(methyl)amino)phenyl)-13-methyl1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (39b). A dried flask was charged with magnesium chips (2.83 g, 117 mmol) and a crystal of iodine. The flask was flushed with Ar and heated briefly with a heat gun and then put into a 60 °C of bath. A 10 mL of 4-bromo-N-isopropyl-N-methyl-aniline (26.1 g, 115 mmol, azeotroped with toluene beforehand) in THF (120 mL) was added. After the iodine color disappeared, the remaining solution was added dropwise for 20 min and the mixture was continued to stir at 6 °C for 2 h.


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The generated Grignard reagent prepared above was cooled down and added to a solution of 38 (12.7 g, 38.2 mmol, azeotroped with toluene) and copper(I) iodide (3.64 mg, 19.1 mmol) in THF (100 mL) at 0 °C. The resulting mixture was allowed to warm up to rt and stir for 1 h. The reaction was quenched (sat. aq. NH4Cl), extracted (2 × EtOAc), and washed (brine). The combined organic layer was dried (Na2SO4) and concentrated under reduced pressure. Purification of the residue by combi-flash (SiO2, 45–65% EtOAc/hexanes, gradient elution) provided the intermediate (9.7 g, 20.1 mmol, 53% yield) as an off-white foam. A solution of the intermediate (9.7 g, 20.1 mmol) in 70% acetic acid in water (80 mL, 1.33 mol) was stirred at 55 °C for 1 h. The reaction was concentrated under reduced pressure to remove most AcOH. Then the residue was diluted (EtOAc), basified (sat. aq. NaHCO3), extracted (2 × EtOAc), and washed (brine). The combined organic layer was dried (Na2SO4) and concentrated under reduced pressure. Purification of the residue by combi-flash (SiO2, 50–100% EtOAc in hexanes, gradient elution) gave 39b (6.80 g, 16.2 mmol, 80% yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ ppm 7.01 (2 H, d, J = 8.5 Hz), 6.71 (2 H, d, J = 8.9 Hz), 5.76 (1 H, s), 4.29 (1 H, br d, J = 6.9 Hz), 4.06 (1 H, dt, J = 13.3, 6.6 Hz), 3.62–3.73 (1 H, m), 2.73 (1 H, br s), 2.71 (3 H, s), 2.32–2.61 (7 H, m), 2.07–2.17 (1 H, m), 2.02 (1 H, dq, J = 12.9, 4.4 Hz), 1.63–1.76 (2 H, m), 1.28–1.49 (5 H, m), 1.15 (3 H, d, J = 6.6Hz), 1.14 (3 H, d, J = 6.6Hz) 0.48 (3 H, s); m/z (ESI, +ve ion) 420.4 [M+H]+. (8S,9R,11S,13S,14S,17S)-17-Hydroxy-11-(4-(isopropyl(methyl)amino)phenyl)-13-methyl1,2,4,6,7,8,9,11,12,13,14,15,16,17-tetradecahydro-3H-cyclopenta[a]phenanthren-3-one (40b). Under Ar, lithium (2.03 g, 292 mmol) in pieces was added to liquid ammonia (500 mL) at ˗78 °C, when the color changed to dark blue, a solution of 39b (50.0 g, 119 mmol) in THF (500 mL) was added over 15 min (kept the internal temperature below ˗60 °C). The mixture was


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stirred at ˗78 °C for 5 min before the reaction was quenched by adding 10 mL H2O, then the solution was poured into 500 mL ice-water, extracted by EtOAc (500 mL × 2), the combined organic layer was dried, concentrated under reduced pressure. Purification of the residue by combi-flash (SiO2, EtOAc/DCM/hex = 1:1:10 to1:1:5, gradient elution) provided 40b (37.1 g, 74 % yield) as white foam. 1H NMR (400 MHz, CDCl3) δ ppm 7.17 (2 H, d, J = 8.6 Hz), 6.65 (2 H, d, J = 8.9 Hz), 4.04 (1 H, dt, J = 13.2, 6.6 Hz), 3.60–3.71 (1 H, m), 3.44 (1 H, br s), 2.81 (2 H, br s), 2.68 (3 H, s), 2.07–2.45 (8 H, m), 1.98 (1 H, br d, J = 17.5 Hz), 1.82–1.91 (1 H, m), 1.60–1.71 (2 H, m), 1.20–1.45 (5 H, m), 1.14 (3 H, d, J = 6.6 Hz), 1.13 (3 H, d, J = 6.6 Hz), 0.36 (3 H, s); m/z (ESI, +ve ion) 422.4 [M+H]+.

(8R,9S,10R,11S,13S,14S,17S)-17-Hydroxy-11-(4-(isopropyl(methyl)amino)phenyl)-13methyl-1,2,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3H-cyclopenta[a]phenanthren-3one (41b). Under Ar, to a solution of 40b (11.3 g, 26.8 mmol) in anhydrous EtOH (68 mL) was added conc. hydrochloric acid (90 mL) and the resulting solution was stirred at 65 °C until most starting material disappeared (~ 16 h). The reaction was cooled to rt, concentrated under reduced pressure to remove most HCl. Then the residue was diluted with DCM, basified with ice-cold 2.5 M NaOH, and extracted (2 × DCM). The combined organic layer was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. Purification of the residue by combiflash (SiO2, 25–50 % EtOAc in hexanes, gradient elution) provided 41b (8.90 g, 21.1 mmol, 78% yield) as an off-white foam. 1H NMR (400 MHz, CDCl3) δ ppm 7.24 (2 H, br d, J = 8.6 Hz), 6.71 (2 H, br d, J = 8.5 Hz), 5.85 (1 H, s), 4.02–4.12 (1 H, m), 3.53–3.63 (1 H, m), 3.25 (1 H, br t, J = 5.3 Hz), 2.83 (1 H, br s), 2.72 (3 H, s), 2.53 (1 H, br d, J = 14.3 Hz), 2.37 (1 H, td, J = 14.1, 5.0 Hz), 2.26 (1 H, dt, J = 16.3, 3.9 Hz), 2.18 (1 H, dd, J = 13.1, 1.4 Hz), 2.07–2.14 (2 H, m), 2.01–2.05 (1 H, m), 1.86–1.96 (1 H, m), 1.65–1.74 (1 H, m), 1.60–1.65 (1 H, m), 1.27–1.51


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(1 H, m), 1.27–1.51 (5 H, m), 1.18 (3 H, d, J = 6.4 Hz), 1.16 (3 H, d, J = 6.4 Hz), 0.98–1.12 (2 H, m), 0.59 (3 H, s); m/z (ESI, +ve ion) 422.3 [M+H]+.

(8R,9S,10R,11S,13S,14S)-11-(4-(Isopropyl(methyl)amino)phenyl)-13-methyl1,2,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydrospiro[cyclopenta[a]phenanthrene-3,2'[1,3]dithiolan]-17-ol (42b). To a solution of 41b (2.80 g, 6.64 mmol) and p-toluenesulfonic acid monohydrate (2.45 g, 12.9 mmol) in acetic acid (50mL) was added ethane-1,2-dithiol (5.4 mL, 64 mmol). After the resulting solution was stirred at rt under Ar for 2 h, the reaction was quenched (ice-cold 2.5 N aq. NaOH) and extracted (2 × EtOAc). The combined organic layer was washed (brine), dried (MgSO4), and concentrated under reduced pressure. Purification of the residue by combi-flash (SiO2, 30–60 % EtOAc in hexanes, gradient elution) provided 42b (1.17 g, 2.35 mmol, 36% yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ ppm 7.23 (2 H, d, J = 8.8 Hz), 6.69 (2 H, d, J = 8.9 Hz), 5.61 (1 H, d, J = 1.3 Hz), 4.01–4.11 (1 H, m), 3.26–3.41 (3 H, m), 3.15–3.24 (2 H, m), 2.71 (3 H, s), 2.42–2.53 (1 H, m), 2.24–2.34 (1 H, m), 2.07–2.18 (2 H, m), 1.76–2.04 (5 H, m), 1.58–1.72 (3 H, m), 1.30–1.42 (2 H, m), 1.21–1.24 (1 H, m), 1.07– 1.18 (8 H, m), 0.91–1.04 (2 H, m), 0.54 (3 H, s); m/z (ESI, +ve ion) 498.3 [M+H]+.

(8R,9S,10R,11S,13S,14S)-11-(4-(Isopropyl(methyl)amino)phenyl)-13-methyl1,6,7,8,9,10,11,12,13,14,15,16-dodecahydrospiro[cyclopenta[a]phenanthrene-3,2'[1,3]dithiolan]-17(2H)-one (43b). To a solution of 42b (1.17 g, 2.35 mmol) in toluene (19.6 mL) was added cyclohexanone (7.00 mL, 6.77 mmol) and aluminum propan-2-olate (648 mg, 3.17 mmol) successively. After the reaction was heated to 105 °C for 4 h, it was cooled to rt and quenched with saturated aq. Rochelle's salt solution. The mixture was stirred for 10 min and extracted (2 × EtOAc). The combined organic layer was washed (brine), dried (MgSO4), and


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concentrated under reduced pressure. Purification of the residue by combi-flash (SiO2, 15–30 % EtOAc in hexanes, gradient elution) provided 43b (695 mg, 1.40 mmol, 60% yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ ppm 7.22 (2 H, d, J = 8.8 Hz), 6.68 (2 H, d, J = 8.8 Hz), 5.64 (1 H, d, J=1.5 Hz), 4.06 (1 H, dt, J = 13.3, 6.6 Hz), 3.25–3.41 (3 H, m), 3.16–3.25 (2 H, m), 2.71 (3 H, s), 2.37–2.55 (2 H, m), 2.33 (1 H, dt, J = 13.3, 3.1 Hz), 1.94–2.21 (8 H, m), 1.62–1.80 (3 H, m), 1.25–1.37 (2 H, m), 1.16 (6 H, t, J=6.50 Hz), 1.01–1.13 (2 H, m), 0.66 (3 H, s); m/z (ESI, +ve ion) 496.4 [M+H]+.

(8R,9S,10R,11S,13S,14S,17S)-17-(3,3-Dimethylbut-1-yn-1-yl)-11-(4(isopropyl(methyl)amino)phenyl)-13-methyl-1,2,6,7,8,9,10,11,12,13,14,15,16,17tetradecahydrospiro[cyclopenta[a]phenanthrene-3,2'-[1,3]dithiolan]-17-ol (44c). To a solution of 1.6 M n-butyllithium in hexanes (8.80 mL, 14.2 mmol) in THF (7 mL) was added 3,3-dimethylbut-1-yne (2.10 mL, 17.3mmol) at ˗78 °C. After 30 min, a solution of 43b (390 mg, 0.79 mmol) in THF (8 mL) was added slowly. After the reaction was stirred at ˗78 °C for 1 h, it was allowed to warm to rt and stirred for 3 h. The reaction was quenched (sat. aq. NH4Cl) and extracted (2 × EtOAc). The combined organic layers were washed (brine), dried (MgSO4), and concentrated under reduced pressure. Purification of the residue by combi-flash (SiO2, 15–30% EtOAc in hexanes, gradient elution) provided 44c (440 mg, 0.76 mmol, 97% yield) as a white foam. 1H NMR (400 MHz, CDCl3) δ ppm 7.24 (2 H, br d, J = 8.6 Hz), 6.68 (2 H, br d, J = 8.6 Hz), 5.61 (1 H, s), 4.06 (1 H, dt, J = 13.0, 6.6 Hz), 3.32–3.41 (3 H, m), 3.27–3.31 (1 H, m), 3.17– 3.25 (1 H, m), 2.71 (3 H, s), 2.45–2.54 (1 H, m), 2.29 (1 H, br d, J = 13.7 Hz), 2.06–2.24 (4 H, m), 1.77–2.00 (5 H, m), 1.69 (2 H, br d, J = 10.5 Hz), 1.41–1.50 (1 H, m), 1.29–1.38 (2 H, m), 1.25 (9 H, s), 1.16 (3 H, d, J = 6.5 Hz), 1.16 (3 H, d, J = 6.5 Hz), 1.03–1.11 (2 H, m), 0.99 (1 H, br d, J = 12.0 Hz), 0.62 (3 H, s); m/z (ESI, +ve ion) 578.5 [M+H]+.


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(8R,9S,10R,11S,13S,14S,17S)-17-(3,3-Dimethylbut-1-yn-1-yl)-17-hydroxy-11-(4(isopropyl(methyl)amino)phenyl)-13-methyl-1,2,6,7,8,9,10,11,12,13,14,15,16,17tetradecahydro-3H-cyclopenta[a]phenanthren-3-one (28). To a solution of 44c (114 mg, 0.20 mmol) in acetic acid (1.7 mL) was added glyoxylic acid monohydrate (908 mg, 9.86 mmol) at rt and the mixture was stirred at rt for 10 min. Then 4 N HCl in water (0.89 mL, 3.6 mmol) was added and the resulting solution was stirred at rt for 20 h. The reaction was quenched (sat. aq. NaHCO3), extracted (2 × EtOAc), and washed (brine). The combined organic layers were dried (Na2SO4) and concentrated under reduced pressure. Purification of the residue with combi-flash (SiO2, 20˗40 % EtOAc in hexanes, gradient elution) provided 28 (38 mg, 0.076 mmol, 38% yield) as a white foam. 1H NMR (400 MHz, CDCl3) δ ppm 7.23 (2 H, br d, J = 8.3 Hz), 6.71 (2 H, br d, J = 8.3 Hz), 5.86 (1 H, s), 4.04 - 4.11 (1 H, m), 3.33 (1 H, br s), 2.86 (1 H, br s), 2.72 (3 H, s), 2.53 (1 H, br d, J = 14.3 Hz), 2.33–2.43 (1 H, m), 2.07–2.31 (5 H, m), 2.03 (1 H, br s), 1.88–1.97 (3 H, m), 1.68–1.78 (1 H, m), 1.63 (1 H, s), 1.46–1.55 (2 H, m), 1.34–1.42 (1 H, m), 1.26–1.30 (1 H, m), 1.24 (9 H, s), 1.17 (3 H, d, J = 5.6 Hz), 1.15 (3 H, d, J = 5.6 Hz), 1.06–1.12 (1 H, m), 0.67 (3 H, s); 13C NMR (101 MHz, CDCl3) δ ppm 200.2, 167.7, 147.8, 131.3, 130.5 (2 C), 124.4, 112.8 (2 C), 95.2, 81.9, 80.1, 52.7, 51.4, 48.9, 47.0, 42.0, 38.8, 38.6, 38.3, 37.8, 36.7, 35.5, 31.9, 31.1 (3 C), 29.7, 27.5, 26.8, 22.9, 19.3, 19.2, 14.8; m/z (ESI, +ve ion) 502.4 [M+H]+.

(8R,9S,10R,11S,13S,14S,17S)-11-(4-(Dimethylamino)phenyl)-17-(3,3-dimethylbut-1-yn-1yl)-17-hydroxy-13-methyl-1,2,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3Hcyclopenta[a]phenanthren-3-one (26). 26 was prepared from 29 according to a similar procedure described for the synthesis of 28. 1H NMR (400 MHz, CDCl3) δ ppm 7.28 (2 H, br d, J = 8.4 Hz), 6.67 (2 H, br d, J = 8.4 Hz), 5.87 (1 H, s), 3.34 (1 H, br t, J = 6.0 Hz), 2.95 (6 H, s), 2.80–2.90 (1 H, m), 2.49–2.57 (1 H, m), 2.33–2.43 (1 H, m), 2.22–2.30 (2 H, m), 2.02–2.22 (4 H,


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m), 1.87–1.97 (3 H, m), 1.68–1.79 (1 H, m), 1.60–1.67 (1 H, m), 1.47–1.54 (2 H, m), 1.38 (1 H, ddd J = 11.9, 5.7, 5.7 Hz), 1.26–1.30 (1 H, m), 1.24 (9 H, s), 0.67 (3 H, s); m/z (ESI, +ve ion) 474.4 (M+H)+. (8R,9S,10R,11S,13S,14S,17S)-11-(4-(dimethylamino)phenyl)-17-hydroxy-17-(3-methoxy-3methylbut-1-yn-1-yl)-13-methyl-1,2,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3Hcyclopenta[a]phenanthren-3-one (27). 27 was prepared from 29 according to a similar procedure described for the synthesis of 28. 1H NMR (400 MHz, CDCl3) δ ppm 7.26–7.31 (2 H, m), 6.61–6.74 (2 H, m), 5.86 (1 H, s), 3.37 (3 H, s), 3.32–3.36 (1 H, m), 2.96 (3 H, br s), 2.96 (3 H, br s), 2.80–2.89 (1 H, m), 2.50–2.58 (1 H, m), 2.33–2.43 (1 H, m), 2.19–2.31 (3 H, m), 2.01– 2.15 (3 H, m), 1.87–1.98 (3 H, m), 1.72–1.80 (1 H, m), 1.70 (1 H, s), 1.52 (1 H, br d, J = 7.6 Hz), 1.48 (3 H, s), 1.48 (3 H, s), 1.37 - 1.45 (2 H, m), 1.22–1.32 (1 H, m), 1.05–1.15 (1 H, m), 0.68 (3 H, s); m/z (ESI, +ve ion) 490.4 [M+H]+.


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ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. •

(i) In vitro biological assays, (ii) In vivo study protocols, (iii) HPLC profiles of compounds 11 and 28, (iv) Ribbon view of GR co-crystal x-ray structures of 1 and 11, (v) Determination of the co-crystal structure of 11 with GR, and (vi) Determination of the single crystal structures of 11 and 28 (PDF)

•

Molecular formula strings (CSV)

Accession Codes The coordinates of 11 with GR have been deposited in the pdb with accession code 6DXK. Authors will release the atomic coordinates and experimental data upon article publication. AUTHOR INFORMATION Corresponding Authors *Y.R.(phone, office) 650-388-5612; (e-mail) [email protected]. *D.S.(phone, office) 650-388-5607; (e-mail) [email protected]. ACKNOWLEDGMENT We thank Nigel Walker for his technical advice and guidance with protein crystallography efforts, Proteros Biostructures GmbH for protein production, crystallography, and structure determination, and Dennis Yamashita for critical reading of manuscript. ABBREVIATIONS


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AcCN, acetonitrile; AcOH, acetic acid; AR, androgen receptor; CL, clearance; CMC, carboxymethyl cellulose; CRPC, castration-resistant prostate cancer; CYP3A4, Cytochrome P450 3A4; CYP 2C8, Cytochrome P450 2C8; CYP2C9, Cytochrome P450 2C9; DCM, dichloromethane; DMA, dimethylacetamide; DEAD, diethyl azodicarboxylate; DMSO, dimethyl sulfoxide; ER, estrogen receptor; EtOAc, ethyl acetate; FKBP5, FK506 binding protein 5; GILZ, glucocorticoid-induced leucine zipper; GCs, glucocorticoids; GR, glucocorticoid receptor; HPMC, hydroxypropyl methylcellulose; IPA, isopropyl alcohol; LNAR, LNCaP/AR-luc; NCoR, nuclear receptor co-repressor; MR, mineralocorticoid receptor; OVCAR-5, human ovarian carcinoma; PBMC, peripheral blood mononuclear cell; PEG, Polyethylene glycol; PR, progesterone receptor; PTSA, p-toluenesulfonic acid; QD, once a day dosing; RT-qPCR, quantitative reverse transcription polymerase chain reaction; SEM, standard error of mean; TNBC, triple-negative breast cancer; TFA, trifluoroacetic acid; THF, tetrahydrofuran.

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Helices 11 and 12 are mobile and can be positioned to favorably bind co-activators

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Detailed SAR studies on the modification of the C17 hydroxyyl and introduction of the

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Several known mifepristone analogs (2, 4, 5, 6, and 10) were synthesized to test AR

agonism. Since its original discovery in 1981, the majority of medicinal chemistry research on mifepristone and its analogs has been focused on exploring the SAR around PR and GR selectivity.


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These large changes in the protein structure are localized to helices 6, 7, 11 and 12 while

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Metabolite profiles of compound 28 in human, rat, and minipig are similar in both in

vitro hepatocyte assay and in vivo PK study.


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