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Cite This: J. Med. Chem. 2018, 61, 7767−7784 pubs.acs.org/jmc

Discovery of a Potent and Selective Steroidal 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* ORIC Pharmaceuticals, 240 East Grand Avenue, Fl2, South San Francisco, California 94080, United States

*S Supporting Information

ABSTRACT: The (GR) has been linked to therapy resistance across a wide range of types. Preclinical data suggest that antagonists of this may enhance the activity of anticancer therapy. The first- generation GR antagonist 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 codosed with chemotherapeutic agents readily metabolized by CYP2C8 such as . Furthermore, 28 demonstrated in vivo antitumor activity by enhancing response to in the GR+ OVCAR5 ovarian cancer xenograft model. Clinical evaluation of safety and therapeutic potential of 28 is underway.

■ INTRODUCTION enzalutamide.6 In addition, cumulative preclinical data over (GCs) such as are hormones the past decade have functionally linked GR activation with secreted by the in a circadian and stress- resistance to a variety of chemotherapeutic agents across an associated manner to regulate metabolism, cell growth, array of solid tumors that include ovarian cancer, triple- Downloaded via ORIC PHARMACEUTICALS INC on September 14, 2018 at 19:56:33 (UTC). ff fl negative breast cancer (TNBC), prostate cancer, pancreatic See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. apoptosis and di erentiation, in ammation, mood, and 7−13 cognitive function.1 GCs signal through the glucocorticoid cancer, nonsmall cell lung cancer, urological , etc. receptor (GR), a member of the superfamily of nuclear At the molecular level, GR drives resistance to therapy, at least in part, by driving transcriptional activation of mediators of cell receptors expressed across a wide variety of tissues. Upon 9,14−17 ligand binding, GR translocates to the nucleus where it triggers survival, antiapoptosis, cell adhesion, and invasion. transcription of a spectrum of genes that mediate its Collectively, these studies have established that genetic pleiotropic biological effects. Dysregulation of GR signaling ablation or pharmacologic inhibition of GR reverses the pro- ff has been associated with a variety of pathological states survival e ects mediated by this nuclear receptor and enhances including Cushing’s syndrome, psychotic depression, obesity, chemotherapy and antiandrogen activity in cancer mod- 6−9,14,18 and cancer.2 Therefore, there has been considerable interest to els. These data have also provided rationale to evaluate ffi target this nuclear receptor for therapeutic purposes. the e cacy of GR antagonists in combination with chemo- Several lines of evidence point to GR as a potential therapeutic agents as well as enzalutamide in the clinic. mechanism of therapy resistance in cancers of epithelial Furthermore, it is well established that GR is expressed in a 19 origin.3 Elevated levels of GR protein have been correlated variety of immune cells. Endogenous and synthetic with poor prognosis in ESR1− breast cancer4 and endometrial cancer patients5 as well as to poor outcome in castration- Received: May 8, 2018 resistant prostate cancer (CRPC) patients treated with Published: August 9, 2018

© 2018 American Chemical Society 7767 DOI: 10.1021/acs.jmedchem.8b00743 J. Med. Chem. 2018, 61, 7767−7784 Journal of Medicinal Chemistry Article glucocorticoids are potent regulators of inflammation.20,21 Glucocorticoid binding to GR activates or represses tran- scription of a variety of pro- and anti-inflammatory 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 a Figure 1. Mifepristone (1) and ORIC-101 (28). IC50 in GR and T cell exhaustion have been implicated in cancer immune luciferase antagonist assay. bEC [E ]* in AR luciferase 27 50 max escape as well poor response to immune therapy. assay. * = % mifepristone. Furthermore, disruption in cortisol secretion has been correlated with poor prognosis and across ■ RESULTS AND DISCUSSION a variety of advanced solid tumors including breast, ovarian, − and lung cancer.28 30 In addition, extra-adrenal production of With the goal of developing a potent, steroidal GR antagonist cortisol by colorectal cancer, squamous skin cancer, and with reduced AR agonism as compared to 1, an analysis of the melanoma cells has been associated with decreased CD8+ T binding mode of mifepristone to AR and GR was initiated. The cell proliferation and activity,31,32 suggesting that aberrant protein cocrystal structure of 1 bound to GR, but not to AR, has been solved (Figure 2).49,50 Because the two nuclear activation of GR could contribute to local tumor immunosup- receptors are closely related, a homology model of AR was pression. Altogether these observations point to the constructed based on the GR structure (pdb 3H52). Figure 2A immunomodulatory potential of GR and provide a rational shows a view of the mifepristone (1)/GR binding site basis for the clinical evaluation of GR antagonists in illustrating the key hydrogen bond interactions of the steroid combination with emerging immunotherapies. carbonyl with Arg611 and Gln570. Outside of the interaction The marketed drug mifepristone (RU-486) is a steroidal GR with these two polar residues, the majority of binding site antagonist, discovered in the 1980s as a potent 33 residues are hydrophobic in nature and form favorable van der receptor (PR) antagonist. Because of its potent GR Waals interactions with the predominately hydrophobic ligand. inhibitory activity, mifepristone has also been evaluated for fi 34 Figure 2B shows the complementary t between the ligand and conditions associated with alterations in GR activity. the binding site, with 1 almost completely buried in the steroid Mifepristone was approved in 2011 for the treatment of binding pocket. The helices that contain residues that interact Cushing’s syndrome and is currently undergoing clinical with mifepristone and the NCoR peptide are also labeled. testing in various solid tumors. The agonistic and antagonistic functions of GR and AR − Compared to recently discovered synthetic GR ultimately depend on the protein conformation,51 53 in − antagonists and allosteric inhibitors,35 40 mifepristone (1) particular, the positions of helices 11 and 12 and the ability stands out for its potency. However, mifepristone is a partial or inability of the receptor to bind coactivators or AR agonist, and its agonistic activity is sufficient to stimulate corepressors.54 To that end, structural hypotheses were the proliferation of CRPC LNCaP/AR-luc (LNAR) cells both developed along three distinct vectors with the goal of altering in vitro and in vivo. In addition, mifepristone has also been the GR and AR structures and thus affecting the agonist and shown to induce AR target gene expression in AR+ TNBC antagonist profiles of the ligands. Using the mifepristone MDA-MB-453 cells.41 Besides prostate cancer, previous studies scaffold as a foundation, modification of the C11 4- have shown the oncogenic roles of AR and androgen signaling dimethylaminophenyl group provided a vector to alter the − in TNBC and ovarian cancer.42 45 In fact, the therapeutic position of helix 12 and also to directly challenge copeptide binding. Modification of the C17 hydroxyl and introduction of potential of the antiandrogen enzalutamide is currently being ff clinically evaluated in AR+ TNBC and ovarian cancer C16 substituents a orded the opportunity to disrupt the − 55 fi patients.45 47 Clearly, partial AR agonism activity is an positions of both helices 11 and 12 directly, and modi cation unwanted feature for any GR antagonist with potential clinical of the C17 1-propynyl group provided a cascading structural effect where disruption of helix 7 in turn altered the position of application in AR-driven cancers. We envisioned that the helices 11 and 12. undesirable AR agonism of mifepristone could be eliminated Initial investigation of 1 began with replacement of the 4- while retaining its GR antagonistic potency by structure-based fi dimethylaminophenyl group at the C11 position in the steroid modi cation of mifepristone. We report here the results of our core with various substituents (Table 1, compounds 2− − − structure activity relationship (SAR) studies, which led to the 6).56 58 To measure antagonist and agonist activities of these discovery of ORIC-101 (28)(Figure 1), a highly potent GR fi compounds on GR and AR, luciferase reporter assays were antagonist with signi cantly reduced AR agonism and employed.59,60 Substitution of the 4-dimethylaminophenyl fi improved cytochrome P450 (CYP) inhibition pro les. These group with a small alkyl group, such as an ethyl, switched features of 28 make this novel compound an attractive the GR activity, converting antagonism to agonism (2 vs 1). + combination partner to antiandrogens and paclitaxel in AR Increase of the substituent’s size to cyclohexyl or unsubstituted cancers including CRPC as well as a subset of TNBC and phenyl group restored GR antagonism and attenuated GR ovarian cancers.14,44,46,48 In addition, the favorable CYP agonism (3 and 4 vs 2). These results imply that a phenyl or a inhibition profile of 28 makes this compound combinable similar-sized cyclohexyl moiety at the C11 position is an with therapeutic agents that are metabolized by CYP2C8 and important feature for GR antagonism. Moving the dimethyla- CYP2C9. mino group from the para-position to the meta-position

7768 DOI: 10.1021/acs.jmedchem.8b00743 J. Med. Chem. 2018, 61, 7767−7784 Journal of Medicinal Chemistry Article

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. reduced AR agonism dramatically (5.8% of 1) but decreased in a shift in the positions of helices 11 and 12 as well. In fact, GR antagonism by 20-fold as well (5 vs 1). The 4- the helix 12 position prevents the binding of corepressor or isopropylphenyl derivative elicited improvement in GR coactivator peptides (Supporting Information, Figure S4).62 fi antagonism (IC50 = 1.93 nM for 6). However, it also induced While mifepristone does not show signi cant agonism toward more AR agonism (200% of 1). GR (Emax relative to dexamethasone of 3.1%), 11 further fi Attention was then focused on the modi cation of the 1- reduces the relative Emax by more than 50%. propynyl group at the C17 position (Table 1, compounds 7− As mentioned, the addition of t-butyl to the alkyne has a 11).56,61 Changing the 1-propynyl group to a saturated n-alkyl significant effect on AR selectivity. A more than 7-fold fi group ampli ed AR agonism while diminishing GR antagonism reduction in AR agonism (Emax) is observed for 11 versus (7 vs 1), suggesting that a C−C triple bond at the C17 mifepristone. To date, efforts to obtain a cocrystal structure of position is crucial for minimized AR agonism and enhanced AR with 11 have not been successful. However, given the close GR antagonism. The introduction of an electron withdrawing structural relationship between AR and GR, a similar structural group by replacing the 1-propynyl group with a 3,3,3-trifluoro- hypothesis can be developed through molecular modeling to 1-propynyl group maintained the same level of AR agonism (8 explain the reduced AR agonism of 11. The orientation of 11 vs 1). Interestingly, gradually increasing alkyl substituent size in the binding site of AR is expected to be similar to the one in the 1-propynyl group led to the 3,3-dimethyl-1-butynyl observed for GR, and thus a similar disruption of the positions derivative 11, which possessed substantially reduced partial of helices 6, 7, 11, and 12 is expected. These large agonism in the AR luciferase assay (13% of 1). However, 11 conformational changes in turn disrupt the copeptide binding also exhibited a 4.5-fold decrease in GR antagonism potency site further reducing the AR agonism for 11 versus mifepri- compared to 1 (compounds 9−11 vs 1). stone. As part of the lead optimization process, a cocrystal structure A single-crystal small- X-ray structure of 11 was of 11 bound to human GR was determined at a resolution of also obtained (Figure 4). This structure allowed for 3.05 Å (Figure 3). The ketone moiety of 11 makes key confirmation of the absolute stereochemistry of all of the hydrogen bond interactions with Arg611 and Gln570, and chiral centers in the molecule and was also used to evaluate the overall, the ligand sits in a similar position as that of cocrystal structure binding mode. The steroid scaffold is rigid, mifepristone in the binding site. There is, however, a significant and overall 11 has few rotatable bonds. Nevertheless, structural difference in the protein structure in the region interacting distortion of the ligand is still possible and could result in a with the C17 substituent. As can be observed in the high energy binding conformation. The ligand conformation of mifepristone cocrystal structure, the 1-propynyl group sits in 11 from the GR protein cocrystal structure is shown in B, while a hydrophobic region between helices 3, 6, and 7 (Figure 2). C shows the overlay of the two and illustrates that the The orientation of these helices in turn has an effect on the conformations are almost identical. There is no significant position of helices 11 and 12. The resulting overall change in ligand conformation upon binding to GR, and thus conformation of GR is favorable for binding of the corepressor no additional strain is introduced into the molecule. peptide, NCoR. This is consistent with the antagonistic nature Having identified the 3,3-dimethyl-1-butynyl group as the of mifepristone toward GR. The replacement of the alkynyl optimized 1-alkynyl group at the C17 position with respect to methyl with a t-butyl group in 11 results in significant changes AR selectivity, attention was turned to substituent modification in the shape and size of the binding site and the overall of the aryl ring at the C11 position (Table 2). Replacing the 4- conformation of GR. Figure 3 illustrates a significant increase dimethylamino group in 11 by small alkyl groups was tolerated in the size and depth of this part of the binding pocket that is in terms of GR antagonism potency, but those derivatives necessary to accommodate 11. Consequently, significant displayed greater AR agonism as the size of the alkyl group changes occur in the orientations of helices 6 and 7, resulting increased (12, 13,and14). Replacement of the 4-

7769 DOI: 10.1021/acs.jmedchem.8b00743 J. Med. Chem. 2018, 61, 7767−7784 Journal of Medicinal Chemistry Article

c Table 1. Exploration of Substituents at C11 and C17 Positions in Mifepristone

a b c Percent dexamethasone. Percent mifepristone. Potency and Emax data with SD are reported as the average of at least two determinations. dimethlyamino group with a methoxy or methyloxetanyl group with an isopropyl group, was lower in GR antagonism activity was also well tolerated for GR antagonism (15 and 25). compared to 11 (IC50 = 43 nM for 21 vs IC50 = 14 nM for 11) Shifting the dimethylamino substituent from the para-position but improved AR agonism selectivity (Emax = 8.2% of 1 for 21 to the meta-position led to a drop in GR antagonism potency vs Emax = 13% of 1 for 11). Introduction of various cyclic (16) as observed in the previous SAR with compound 5. tertiary amines was also tolerated with regard to GR antagonist Additional substitutions at the meta-and ortho-positions were potency (22, 23, and 24). Overall, no significant improvement then evaluated while preserving the dimethylamino group at in GR antagonist potency was observed from the aryl group the para position. The 2,5-difluorophenyl analogue 18 showed modification at the C11 position. GR antagonism potency similar to that of 11, whereas potency The X-ray cocrystal structure of 11 bound to GR protein of all other analogues was diminished (17, 19, and 20). The (Figure 3) shows that the A ring carbonyl oxygen forms direct optimization of the N,N-dialkyl group within 11 was attempted hydrogen bonds to the guanidinium of Arg611 and to the γ- to improve GR antagonism potency and to address the amide of Gln570. The A-ring carbonyl oxygen in compound 11 inherent metabolic instability of the N,N-dimethylamino is α,β−γ,δ-diene carbonyl oxygen with extended conjugation, group. Compound 21, which exchanged one N-methyl group which is known to be less electronegative than α,β-unsaturated

7770 DOI: 10.1021/acs.jmedchem.8b00743 J. Med. Chem. 2018, 61, 7767−7784 Journal of Medicinal Chemistry Article

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.

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 MDA-MB-453 cell lines at about 100 nM. These expression levels are similar to those achievable by 0.1 nM Figure 4. Comparison of small-molecule single-crystal X-ray structure R1881, the potent synthetic androgen in the same cell lines. of 11 (A, orange) with the GR cocrystal X-ray structure 11 (B, dark On the other hand, 1 μM enzalutamide completely reverted gray, pdb 6DXK). The overlay of the two structures is shown in C. 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 carbonyl oxygen. Therefore, we hypothesized that reducing the (Figures 6 and 7). These data suggest that compound 28 C9 C−C double bond in the B ring could enhance hydrogen exhibits no AR agonism and might have an advantage over 1 in bond acceptor ability of the A ring carbonyl oxygen. treating AR-driven cancers. Consequently, an effort was made to explore modification of The single-crystal small-molecule X-ray structure of 28 was the steroid core in 11 (Table 3). solved. Figure 8 shows the structure of 28 as compared to the Of significance was the observation that 26, the correspond- protein cocrystal structure of 11. Because the overlay shows ing C9 C−C double bond reduced analogue of 11, had a that the overall shape of both compounds is very similar, 28 marked 3-fold improvement in GR antagonist potency (IC50 = would be expected to have a very similar binding mode to that 5.6 nM for 26 vs IC50 = 14 nM for 11). However, 26 showed a of 11. This is consistent with the binding mode of 28,as slight increase in AR agonist activity (Emax = 22% of 1) predicted via docking calculation with GR. compared to 11 (Emax = 13% of 1). This increase is not To identify a molecule suitable for preclinical development, negligible. For example, the comparable partial agonistic several selected compounds were evaluated for their rat fi activity of bicalutamide (Emax = 23% of 1) compromises its pharmacokinetic (PK) pro le (Table 4). The results showed therapeutic efficacy as an antiandrogen relative to full that 13 and 23 had low clearance but poor oral antagonists such as enzalutamide (Figure 5) and apalutamide. (CL = 0.41 and 0.28 L/kg/h; %F = 0 and 0.7%, respectively). The 3,3-dimethyl-1-butynyl group in 26 was further Compounds 11 and 28 had moderate clearance (CL = 1.4 and modified by replacing one methyl of the t-butyl group with a 1.9 L/kg/h, respectively) and acceptable oral bioavailability (% methoxy moiety. This was attempted to reduce AR agonism F = 13 and 24%, respectively). The rest of the compounds 21, and improve solubility, but it also resulted in lower GR 25, and 27 showed high CL (CL = 3.0−5.0 L/kg/h), although antagonism (27). On the other hand, similar to previous 27 exhibited good oral bioavailability (%F = 54%). observations, the replacement of one N-methyl group in 26 Given the GR antagonist potency, low AR agonism, and with an isopropyl group provided 28 with significantly reduced favorable rat PK profile, compound 28 was selected for further AR agonism (Emax = 11% of 1), which is comparable to AR evaluation. The preclinical of 28 were agonism of enzalutamide (Emax = 8.7% of 1)(Figure 5). evaluated in mouse and minipig following single-dose intra- Overall, compound 28 is a weak full AR antagonist with IC50 of venous (iv) or . The molecule was found to 484 ± 110 nM in the AR luciferase assay. Furthermore, have low clearance and reasonable oral bioavailability in mouse compound 28 is equipotent to 26 and only 2-fold less potent and minipig (Table 5).63 ff than mifepristone in the GR luciferase antagonism assay (IC50 Species di erences in the unbound fraction of compound 28 = 5.6 nM). were minimal (<2−3-fold). The unbound fraction (0.14) of

7771 DOI: 10.1021/acs.jmedchem.8b00743 J. Med. Chem. 2018, 61, 7767−7784 Journal of Medicinal Chemistry Article

c Table 2. Exploration of Aryl Substituents at C11 Position in Compound 11

a b c Percent dexamethasone. Percent mifepristone. Potency and Emax data with SD are reported as the average of at least two determinations. compound 28 is 10-fold higher than that of compound 1 in that both 11 and 28 have low potential of CYP2C8 and human plasma. As compound 1 is reported to have high CYP2C9 inhibition compared to 1, although the IC50 values of potential for inhibition of CYP2C8, we next examined the 11 and 28 for CYP3A4 are lower than that of 1 (Table 6). In liability of 11 and 28 in the CYP inhibition assay. It was found general, the replacement of the C17 alkynyl methyl in 1 with a

7772 DOI: 10.1021/acs.jmedchem.8b00743 J. Med. Chem. 2018, 61, 7767−7784 Journal of Medicinal Chemistry Article

c Table 3. Modification of the Steroid Core of 11

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

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 drug−drug 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. 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; Figure 5. Relative AR agonism of 11 and 28 compared to 1, ± IC50 = 0.4 0.2 nM for 1). The PR/GR ratio of 28 is 3.8, enzalutamide, and bicalutamide. while that of 1 is 0.12. Compound 28 does not show any agonism in the PR luciferase reporter assay. Unlike the receptor (ER) ligand 17β-estradiol and the ER antagonist tamoxifen, 28 does not physically bind to ER up to 1 μM (Supporting Information, Figure S1). Similarly, 28 neither induces nor inhibits aldosterone-induced interaction between MR and its coactivator SRC at the concentration of 5 μM (Supporting Information, Figures S2 and S3), suggesting 28 is not a 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 Figure 6. Induction of endogenous AR target genes by synthetic androgen (R1881) and 1 in LNAR cells. Endogenous AR target evaluated using human, rat, dog, monkey, and minipig genes, PSA and TMPRSS2, are induced by R1881 and 1 in LNAR peripheral blood mononuclear cells (PBMCs) treated ex vivo cells while those genes are not induced by 28. Enz (enzalutamide) with dexamethasone in the presence or absence of 1 or 28. inhibits R1881-induced expression but does not fully inhibit the Figure 10 shows an example of quantitative polymerase chain expression induced by 1. reaction (RT-qPCR) results measuring the dose-dependent expression levels of two established GR target genes, GILZ and t-butyl group improves the CYP inhibition profile, especially FKBP5,64 in human PBMCs treated with dexamethasone and against CYP2C8 and CYP2C9. 28. Treatment with 28 significantly inhibited ligand-induced

7773 DOI: 10.1021/acs.jmedchem.8b00743 J. Med. Chem. 2018, 61, 7767−7784 Journal of Medicinal Chemistry Article

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.

Table 6. CYP Inhibition of Compounds 1, 11, and 28 μ IC50 ( M) P450 isoform substrate 11128 3A4 midazolam 9.5 2.9 1.6 2C8 amodiaquine 1.5 >10 >10 2C9 diclofenac 4.9 >10 >10

target gene expression with potencies comparable to 1 in all Figure 8. Comparison of the small-molecule single-crystal X-ray species tested (Table 7). structure of 28 (A, blue) with the GR cocrystal X-ray structure 11 (B, Next, we evaluated whether 28 could inhibit endogenous dark gray, pdb 6DXK). The overlay of the two structures is shown in GR target gene expression and compared its relative potency C. with 1 in cancer cells. To this end, the GR+ human ovarian cancer cell line OVCAR5, which has no detectable AR and low Table 4. Rat Pharmacokinetic Data for Selected a PR expression levels, was stimulated with dexamethasone in Compounds the presence or absence of 28 or 1 and mRNA levels of GR iv (0.5 mg/kg)b po (5 mg/kg)c target genes were measured by RT-qPCR. Compound 28 reduced expression of FKBP5 and GILZ in a dose-dependent CL Vss t1/2 Cmax AUC compd (L/kg/h) (L/kg) (h) F (%) (μg/L) (μg·h/L) manner (IC50 = 17.2 and 21.2 nM, respectively) with similar 1 3.7 4.1 1.6 6.4 24 85 potency as 1 (IC50 = 10.9 and 13.0 nM, respectively) (Figure 11 4.0 4.9 1.9 37 72 471 11). 13 0.41 1.7 3.4 0 0 0 The ability of 28 to inhibit GR transcriptional activity was further evaluated in vivo. The major murine glucocorticoid, 21 4.0 4.7 1.5 23 59 290 65 23 0.28 1.5 3.6 0.7 21 167 , is a weak agonist of human GR. Therefore, 25 3.0 6.7 4.4 7.2 59 123 when human cancer cells are grown as tumor xenografts in mice, it is necessary to provide an exogenous ligand such as 27 5.0 7.2 2.4 54 295 544 ff 28 1.9 4.6 4.4 24 149 635 cortisol or the synthetic ligand dexamethasone to e ectively a activate human GR. Expression of the GR target gene FKBP5 n = 3 mice for both iv and po. iv time points: one predose and 10 postdose. po time points: one predose and 8 postdose. bFormulated was analyzed in OVCAR5 tumors isolated from animals 6 h in 10% DMSO, 70% PEG 400, and 20% water. cFormulated in 5% after administration of vehicle or dexamethasone (0.5 mg/kg DMSO, 95% 0.2% Tween 80 in 0.25% CMC. results in a plasma glucocorticoid concentration equivalent to a human cortisol dose of 0.100−0.467 μM corrected for Table 5. Pharmacokinetic Data for 28 in Mouse and Minipig potency) in the presence or absence of a single oral dose of 28 at 3 different concentrations. In the presence of 28 at 25, po (5 mg/kg for mouse and 10 iv (0.5 mg/kg)a mg/kg for minipig)b 50, or 75 mg/kg, dexamethasone-induced FKBP5 expression was reduced in a dose dependent manner (Figure 12A). CL Vss F Cmax AUC species (L/kg/h) (L/kg) t (h) (%) (μg/L) (μg·h/L) Expression of FKBP5 in the 75 mg/kg group was not 1/2 significantly different than expression in the vehicle group. mouse 1.63 5.01 4.83 21 274 644 The unbound plasma concentration of 28 in circulating mouse minipig 1.1 7.8 12.2 20 153 1721 plasma ranged from 0.02 to 0.12 μM across the three groups a Formulation: 28 was in 5% DMA, 10% EtOH, 40% PEG400, and (Figure 12B). The plasma exposures of dexamethasone were 45% D5W for mouse iv study; and in 5% DMA, 10% EtOH, 40% PEG b similar in all animals that received the dose of glucocorticoid 400, 43.8% D5W, pH 5, for minipig iv study. Formulation: 28 was in (data not shown). These data suggest that 28 is effective at 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 inhibiting dexamethasone-induced GR target gene expression oral study. in OVCAR5 xenograft tumors. 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

7774 DOI: 10.1021/acs.jmedchem.8b00743 J. Med. Chem. 2018, 61, 7767−7784 Journal of Medicinal Chemistry Article

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

Figure 11. Inhibitory effects of 28 and 1 on GR-mediated target gene expression in ovarian cancer OVCAR5 cells. 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 h. ■ CHEMISTRY Compounds 2−23 were prepared in a straightforward manner, through the route outlined in Scheme 1.61 Their synthesis was previously mentioned, corticosterone is the prevailing circulat- initiated by incorporation of C17 substituent in the commercially available ethylene deltanone 29.Grignard ing glucocorticoid in rodents and is a weak activator of human reaction of ketone 29 with alkynylmagnesium bromide or GR. Therefore, to achieve proper activation of GR in the alkynyllithium provided 17β-hydroxy ketal 30 exclusively. human xenograft transplant model, cortisol was administered Treatment of 30 with 30% and chronically in the drinking water at 100 mg/L before tumor 1,1,1,3,3,3-hexafluoropropan-2-one trihydrate in the presence implantation and for the duration of the study in the groups of sodium hydrogen phosphate buffer provided epoxide 31 as a indicated. The resulting plasma cortisol levels are consistent major diastereomer (dr >3:1). Copper-catalyzed addition of an with reported mean plasma cortisol levels in cancer patients alkyl or aryl Grignard reagent to the vinylic epoxide 31, throughout the day, which range from 0.100 to 0.496 μM.66,67 followed by hydrolysis with 70% at 55 °C, gave Daily treatment with 28 did not affect the growth of the compounds 2−23 in moderate to high yields. tumors grown under chronic cortisol. Gemcitabine and Synthesis of compounds 24 and 25 was completed in a carboplatin induced tumor growth inhibition in groups with similar manner but required extra steps as illustrated in Scheme or without 28 compared to vehicle alone in the presence of 2. The primary aniline derivative 33a was prepared from epoxide 31f via copper catalyzed 1,4-addition of the cortisol. However, addition of 28 significantly enhanced the ff commercially available (4-(bis(trimethylsilyl)amino)phenyl) antitumor e ect of chemotherapy. Tumor volumes on day 21 magnesium bromide 34, followed by acetic acid-mediated in mice receiving gemcitabine + carboplatin + cortisol were hydrolysis. The double Michael addition of aniline 33a to fi signi cantly larger than the tumor volumes in mice receiving divinyl sulfone afforded 24. For synthesis of 25, a commercially gemcitabine + carboplatin + cortisol + 28. These data available diol 35 was converted to cyclic ketal 36 with 2,2- indicated that 28 was effective in reversing the GR mediated dimethoxypropane. 1,4-Addition of in situ generated Grignard resistance to chemotherapy driven by cortisol in vivo. reagent of 36 to epoxide 31f, followed by hydrolysis with 70%

Table 7. Cross-Species Evaluation of the Effect of 1 and 28 on Endogenous Target Gene Expression in PBMCs

human rat dog monkey minipig GILZ FKBP5 GILZ FKBP5 GILZ FKBP5 GILZ FKBP5 GILZ FKBP5 a a IC50 of 1 (nM) 17.8 14.8 ND ND 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 aNot determined.

7775 DOI: 10.1021/acs.jmedchem.8b00743 J. Med. Chem. 2018, 61, 7767−7784 Journal of Medicinal Chemistry Article

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 h 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 concentrations of 28 in the three treated groups 6 h 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 Dunnett’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).

a Scheme 1

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 two groups indicated, with p < 0.05. 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 = a  Reagents and conditions: (a) for 30a,CH3C CMgBr, THF, 0 to 25 4:1). Copper-catalyzed 1,4-addition of the Grignard reagent to ° − 2 − ° − C; for 30b 30f,RLi, 78 to 25 C, 24 87%; (b) 35% H2O2, − 1 − epoxide 38 followed by acetic acid-mediated hydrolysis CF3COCF3,Na2HPO4, DCM, 42 94%; (c) R MgBr, CuI, THF, 0 resulted in dienone 39. Birch reduction of conjugated α,β- 25 °C, 59−83%; (d) 70% AcOH, 55 °C, 1−2h,46−96%. 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 was oxidized to ketone through Oppenauer oxidation. Alkynyl without double bond migration.70 The hydroxyl group in 42 lithium addition to the ketone 43 followed by deprotection of

7776 DOI: 10.1021/acs.jmedchem.8b00743 J. Med. Chem. 2018, 61, 7767−7784 Journal of Medicinal Chemistry Article

a Scheme 2 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 5 μ C18 110A column (3.5 μm, 150 × mm 4.6 mm i.d.); mobile phase, A = H2O with 0.1% TFA, B = − − fl CH3CN with 0.1% TFA; gradient: 5 95% B (0.0 15.0 min); ow 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- 1,2,4,6,7,8,12,13,14,15,16,17-dodecahydrospiro[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 n-butyllithium (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 fi − puri ed by silica gel chromatography (SiO2,510% EtOAc in hexanes, gradient elution) to provide 30f (7.7 g, 18.9 mmol, 74% 1 δ yield) as a white solid. H NMR (400 MHz, CDCl3) 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 = a − ° Reagents and conditions: (a) (i) 34, CuI, THF, 0 25 C, (ii) 70% 18.0 Hz), 2.53 (1 H, m), 2.29 (2 H, m), 1.90−2.11 (4 H, m), 1.62− ° AcOH, 55 C, 1 h, 23% for two steps; (b) divinyl sulfone, IPA/H2O, 1.87 (5 H, m), 1.70−1.75 (1 H, m), 1.40−1.50 (1 H, 1m), 1.20−1.30 ° 115 C, 5 h, 26%; (c) 2,2-dimethoxypropane, PTSA, DMF, 77%; (d) (1 H, m), 1.20 (9 H, s), 0.83 (3 H, s). m/z (ESI, +ve ion) 397.3 (M + ° ° (i) 36, Mg, 60 C, 30 min, CuI, THF, 0 to 25 C, 98%, (ii) 70% H)+. ° AcOH, 60 C, 1.5 h, 73%; (e) PPh3, DEAD, Ziram, THF, toluene, (5′R,8′S,10′R,13′S,14′S,17′S)-17′-(3,3-Dimethylbut-1-yn-1-yl)- 69%. 13′-methyl-1′,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]- fi 17′-ol (31f). To a solution of 30f (7.5 g, 18.9 mmol) in DCM (120 the thioketal protecting group in 44 resulted in the nal ° compounds 26, 27, and 28. 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% ° ■ CONCLUSION aq, 4.36 mL, 56.7 mmol). The mixture was stirred at 0 C for 10 min and then at rt for 2 h. Aqueous Na2S2O3 solution was added, and the In summary, structural modifications of mifepristone (1) at the mixture was stirred at rt for 30 min and extracted with EtOAc. The C11 and C17 positions followed by reduction of the C9 C−C organic phase was washed with brine and dried over anhydrous double bond led to the discovery of 28 that features substantial Na2SO4. The mixture was then concentrated under reduced pressure, fi reduction in AR agonism as compared to 1, with minimized and the crude residue was puri ed by silica gel chromatography (SiO2, loss of GR antagonism potency and low potential of CYP2C8 10−15% EtOAc in hexanes, gradient elution) to provide 31f (6.9 g, 1 16.7 mmol, 88% yield) as a white solid. H NMR (400 MHz, CDCl3) and 2C9 inhibition. Compound 28 demonstrated antitumor δ − − − activity by overcoming GR-mediated chemoresistance in the 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− OVCAR5 ovarian cancer xenograft model and is currently − − 71 1.80 (5 H, m), 1.46 1.57 (1 H, m), 1.20 1.45 (2 H, m), 1.19 (9 H, undergoing clinical testing. s), 0.83 (3 H, s). (5R,8S,11R,13S,14S,17S)-11-(4-(Dimethylamino)phenyl)-17-(3,3- ■ EXPERIMENTAL SECTION dimethylbut-1-yn-1-yl)-13-methyl-1,2,6,7,8,11,12,13,14,15,16,17- General Chemistry. All reactions were conducted under an inert dodecahydrospiro[cyclopenta[a]phenanthrene-3,2′-[1,3]- dioxolane]-5,17(4H)-diol (32j). gas atmosphere (nitrogen or argon) using a Teflon-coated magnetic A mixture of 31f (80 mg, 0.19 mmol) stir bar at the temperature indicated. Commercial reagents and and copper(I) iodide (36.9 mg, 0.19 mmol) in THF (3 mL) was anhydrous solvents were used without further purification. Analytical degassed with Ar. After cooling in an ice bath, (4-(dimethylamino) thin layer chromatography (TLC) and flash chromatography were phenyl) magnesium bromide (0.5 M in THF, 3.1 mL, 1.55 mmol) performed on Teledyne RediSep Rf Flash silica gel columns. Removal was added dropwise. The resulting solution was maintained at rt for 1 of solvents was conducted by using a rotary evaporator, and residual h, then quenched with NH4Cl solution and extracted with EtOAc. solvent was removed from nonvolatile compounds using a vacuum The organic phase was washed with brine and dried over anhydrous manifold maintained at approximately 1 Torr. All yields reported are Na2SO4. The mixture was then concentrated under reduced pressure isolated yields. Preparative reversed-phase high pressure liquid and the crude residue was purified by silica gel chromatography to chromatography (RP-HPLC) was performed using an Agilent 1100 provide 32j (90 mg, 0.17 mmol, 89% yield) as a white solid. 1H NMR δ − series HPLC and Phenomenex Gemini C18 column (5 μm, 100 mm (400 MHz, CDCl3) ppm 7.07 (2 H, d, J = 8.5 Hz), 6.63 6.67 (2 H, × 21.2 mm i.d.), eluting with a binary solvent system A and B using a m), 4.41 (1 H, d, J = 1.2 Hz), 4.13 (1 H, d, J = 7.2 Hz), 3.90−4.05 (4 fl − − gradient elusion [A, H2O with 0.1% tri uoroacetic acid (TFA); B, H, m), 2.91 (6 H, s), 2.45 (1 H, m), 2.10 2.40 (4 H, m), 2.00 2.05 fi − − − CH3CN with 0.1% TFA] with UV detection at 220 nm. All nal (2 H, m), 1.85 2.00 (2 H, m), 1.75 1.80 (1H, m), 1.60 1.75 (4 H,

7777 DOI: 10.1021/acs.jmedchem.8b00743 J. Med. Chem. 2018, 61, 7767−7784 Journal of Medicinal Chemistry Article

a Scheme 3

a − ° Reagents and conditions: (a) NaBH4, THF, MeOH, 0 25 C, 100%; (b) 35% H2O2,CF3COCF3,Na2HPO4, DCM, 100%; (c) (i) ArMgBr, CuI, − ° − ° − − ° − fl THF, 0 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, re ux, ° − ° − 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 to 25 °C, 59−97%; (i) 4 N HCl, glyoxylic acid, AcOH, 38−43%. m), 1.50 (1 H, m), 1.30−1.40 (3 H, m), 1.24 (9 H, s), 0.49 (3 H, s). (8S,11R,13S,14S,17S)-11-Cyclohexyl-17-hydroxy-13-methyl-17- m/z (ESI, +ve ion) 534.3 (M + H)+. (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). 1 (8S,11R,13S,14S,17S)-11-(4-(Dimethylamino)phenyl)-17-(3,3-di- H NMR (400 MHz, CDCl3) methylbut-1-yn-1-yl)-17-hydroxy-13-methyl- δ ppm 5.70 (1 H, s), 3.74−3.77 (1 H, m), 2.55−2.83 (3 H, m), 2.21− 1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]- 2.49 (6 H, m), 1.75−2.25 (7 H, m), 1.50−1.75 (3 H, m), 1.05−1.50 phenanthren-3-one (11). A solution of 32j (90 mg, 0.17 mmol) in (11 H, m), 0.71−0.92 (3 H, m). 1:2 acetic acid/water (16.5 mL) was stirred at 55 °C for 90 min. The (8S,11R,13S,14S,17S)-17-Hydroxy-13-methyl-11-phenyl-17- reaction mixture was cooled to rt and slowly added aq NaHCO (prop-1-yn-1-yl)-1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3H- 3 cyclopenta[a]phenanthren-3-one (4). 1 solution. After extraction with EtOAc, the organic phase was washed H NMR (400 MHz, CDCl3) δ − − with brine, dried over anhydrous Na SO , and concentrated under ppm 7.22 7.35 (2 H, m), 7.13 7.22 (3 H, m), 5.77 (1 H, s), 4.44 2 4 − − reduced pressure. The crude residue was purified by silica gel (1 H, br d, J = 4.0 Hz), 2.65 2.85 (1 H, m), 2.50 2.63 (2 H, m), − − − chromatography to provide 11 (54 mg, 0.12 mmol, 70% yield) as a 2.13 2.50 (7 H, m), 1.90 2.15 (2 H, m), 1.90 (3 H, s), 1.62 1.88 (3 − pale-yellow solid. 1H NMR (400 MHz, CDCl ) δ ppm 7.03 (2 H, d, J H, m), 1.28 1.62 (2 H, m), 0.50 (3 H, s). 3 (8S,11R,13S,14S,17S)-11-(3-(Dimethylamino)phenyl)-17-hy- = 8.6 Hz), 6.67 (2 H, d, J = 8.6 Hz), 5.77 (1 H, s), 4.36 (1 H, br d, J = droxy-13-methyl-17-(prop-1-yn-1-yl)- 6.3 Hz), 2.93 (6 H, s), 2.76 (1 H, s), 2.59 (2 H, dd, J = 7.4, 3.1 Hz), 1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]- − − − − 2.27 2.49 (6 H, m), 2.17 2.25 (1 H, m), 1.86 2.06 (2 H, m), 1.64 phenanthren-3-one (5). 1 δ H NMR (400 MHz, CDCl3) ppm 7.12 (1 1.78 (3 H, m), 1.47 (1 H, br s), 1.34 (1 H, br dd, J = 11.7, 5.6 Hz), H, m), 6.51−6.57 (3 H, m), 5.76 (1 H, s), 4.39 (1 H, br d, J = 4.0 − 13 δ 1.20 1.29 (9 H, s), 0.56 (3 H, s). C NMR (101 MHz, CD2Cl2) Hz), 2.91 (6 H, s), 2.70−2.85 (1 H, m), 2.18−2.65 (9 H, m), 1.91− ppm 199.5, 157.5, 149.2, 147.3, 132.9, 129.6, 128.1 (2 C), 122.9, 2.18 (2 H, m), 1.90 (3 H, s), 1.70−1.85 (2 H, m), 1.28−1.65 (2 H, 113.1 (2 C), 95.5, 82.4, 80.2, 50.5, 47.5, 41.0 (2 C), 40.1, 39.8, 39.6, m), 0.58 (3 H, s); m/z (ESI, +ve ion) 430.4 (M + H)+. 39.4, 37.5, 31.6, 31.4 (3 C), 28.1, 27.9, 26.4, 23.8, 14.1. m/z (ESI, +ve (8S,11R,13S,14S,17S)-17-Hydroxy-11-(4-isopropylphenyl)-13- ion) 472.4 (M + H)+. methyl-17-(prop-1-yn-1-yl)-1,2,6,7,8,11,12,13,14,15,16,17-dodeca- 2−10 and 12−23 were prepared from 29 according to a similar hydro-3H-cyclopenta[a]phenanthren-3-one (6). 1H NMR (400 δ − procedure described for the synthesis of 11. MHz, CDCl3) ppm 7.05 7.18 (4 H, m), 5.73 (1 H, s), 4.37 (1 (8S,11S,13S,14S,17S)-11-Ethyl-17-hydroxy-13-methyl-17-(prop- H, br d, J = 4.0 Hz), 2.71−2.89 (2 H, m), 2.15−2.68 (9 H, m), 1.88− 1-yn-1-yl)-1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3H- 2.15 (2 H, m), 1.88 (3 H, s), 1.20−1.81 (5 H, m), 1.25 (6 H, d, J = cyclopenta[a]phenanthren-3-one (2). 1 H NMR (400 MHz, CDCl3) 4.0 Hz), 0.58 (3 H, s). m/z (ESI, +ve ion) 429.4 (M + H)+. δ ppm 5.70 (1 H, s), 2.87−2.98 (2 H, m), 2.54−2.62 (1 H, m), 2.33− (8S,11R,13S,14S,17S)-11-(4-(Dimethylamino)phenyl)-17-(hex-1- 2.51 (5 H, m), 2.25 (1 H, ddd, J = 13.9, 9.5, 5.8 Hz), 1.78−2.06 (7 H, yn-1-yl)-17-hydroxy-13-methyl-1,2,6,7,8,11,12,13,14,15,16,17-do- m), 1.45−1.72 (4 H, m), 1.20−1.43 (2 H, m) 1.05 (3 H, s), 0.95 (3 decahydro-3H-cyclopenta[a]phenanthren-3-one (7). 1HNMR + δ H, t, J = 7.3 Hz). m/z (ESI, +ve ion) 339.1 (M + H) . (400 MHz, CDCl3) ppm 7.02 (2 H, d, J = 8.2 Hz), 6.66 (2 H, d,

7778 DOI: 10.1021/acs.jmedchem.8b00743 J. Med. Chem. 2018, 61, 7767−7784 Journal of Medicinal Chemistry Article

J = 8.9 Hz), 5.76 (1 H, s), 4.35 (1 H, br d, J = 6.6 Hz), 2.92 (6 H, s), (6 H, m), 2.18−2.26 (1 H, m), 1.92−2.06 (2 H, m), 1.60−1.79 (3 H, 2.70−2.80 (1H, m), 2.45−2.60 (3 H, m), 2.30−2.48 (2 H, m), 2.12− m), 1.49 (1 H, br d, J = 6.3 Hz), 1.31−1.44 (1 H, m), 1.25 (9 H, s), 2.28 (1 H, m), 1.95−2.10 (3 H, m), 1.50−1.62 (2 H, m), 1.32−1.46 0.59 (3 H, s). m/z (ESI, +ve ion) 472.4 (M + H)+. (5 H, m), 1.22−1.29 (3 H, m), 0.95−1.05 (2 H, m), 0.58 (3 H, s). m/ (8S,11R,13S,14S,17S)-11-(4-(Dimethylamino)-3,5-difluorophen- z (ESI, +ve ion) 448.4 (M + H)+. yl)-17-(3,3-dimethylbut-1-yn-1-yl)-17-hydroxy-13-methyl- (8S,11R,13S,14S,17S)-11-(4-(Dimethylamino)phenyl)-17-hy- 1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]- phenanthren-3-one (17). 1 δ droxy-13-methyl-17-(3,3,3-trifl uoroprop-1-yn-1-yl)- H NMR (400 MHz, CDCl3) ppm 6.68 1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]- (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 phenanthren-3-one (8). 1 δ − H NMR (400 MHz, CDCl3) ppm 7.02 (2 (6 H, t, J = 1.6 Hz), 2.75 (1 H, br d, J = 14.3 Hz), 2.56 2.62 (2 H, H, m, J = 8.6 Hz), 6.67 (2 H, m, J = 8.4 Hz), 5.78 (1 H, s), 4.40 (1 H, m), 2.29−2.48 (5 H, m), 2.15−2.26 (2 H, m), 1.89−2.08 (2 H, m), br d, J = 6.8 Hz), 2.93 (6 H, s), 2.77 (1 H, m), 2.55−2.62 (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 2.44−2.54 (2 H, m), 2.29−2.40 (4 H, m), 2.23 (1 H, br d, J = 7.2 (9 H, s), 0.55 (3 H, s). m/z (ESI, +ve ion) 508.4 (M + H)+. Hz), 1.83−2.11 (4 H, m), 1.35−1.57 (3 H, m), 0.59 (3 H, s). m/z (8S,11S,13S,14S,17S)-11-(4-(Dimethylamino)-2,6-difluorophen- (ESI, +ve ion) 484.3 (M + H)+. yl)-17-(3,3-dimethylbut-1-yn-1-yl)-17-hydroxy-13-methyl- (8S,11R,13S,14S,17S)-17-(But-1-yn-1-yl)-11-(4-(dimethylamino)- 1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]- phenanthren-3-one (18). 1 δ − phenyl)-17-hydroxy-13-methyl-1,2,6,7,8,11,12,13,14,15,16,17-do- H NMR (400 MHz, CDCl3) ppm 6.01 decahydro-3H-cyclopenta[a]phenanthren-3-one (9). 1HNMR 6.24 (2 H, m), 5.70 (1 H, s), 4.52 (1 H, d, J = 8.4 Hz), 2.92 (6 H, s), δ − − − (400 MHz, CDCl3) ppm 7.15 7.22 (1 H, m), 7.31 (1 H, m), 2.69 (2 H, dt, J = 15.0, 5.3 Hz), 2.39 2.59 (3 H, m), 2.16 2.38 (4 H, 6.75−6.80 (1 H, m), 6.65 (1 H, m), 5.77 (1 H, s), 4.37 (1 H, m), m), 1.87−2.06 (3 H, m), 1.70−1.84 (1 H, m), 1.33−1.57 (3 H, m), 3.05−3.15 (4 H, m), 3.0 (1 H, m), 2.93 (6 H, s), 2.55−2.62 (1 H, m), 1.24 (9 H, s), 1 0.84 (3 H, s). m/z (ESI, +ve ion) 508.4 (M + H)+. 2.30−2.45 (4 H, m), 2.00−2.10 (1 H, m), 1.90−2.00 (1 H, m), 1.65− (8S,11R,13S,14S,17S)-11-(4-(Dimethylamino)-3-methoxyphen- 1.75 (3 H, m), 1.30−1.40 (2 H, m), 1.25 (1 H, m), 1.17 (3 H, t, J = yl)-17-(3,3-dimethylbut-1-yn-1-yl)-17-hydroxy-13-methyl- 1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]- 7.5 Hz), 0.56 (3 H, s). m/z (ESI, +ve ion) 444.4 (M + H)+. phenanthren-3-one (19). 1 δ (8S,11R,13S,14S,17S)-11-(4-(Dimethylamino)phenyl)-17-hy- H NMR (400 MHz, CDCl3) ppm 6.81 droxy-13-methyl-17-(3-methylbut-1-yn-1-yl)- (1 H, d, J = 8.2 Hz), 6.73 (1 H, s), 6.64 (1 H, d, J = 7.6 Hz), 5.78 (1 1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]- H, s), 4.39 (1 H, br d, J = 6.4 Hz), 3.86 (3 H, s), 2.71−2.81 (7 H, m), phenanthren-3-one (10). 1 δ − − − − H NMR (400 MHz, CDCl3) ppm 7.02 2.53 2.64 (2 H, m), 2.30 2.50 (6 H, m), 2.18 2.28 (1 H, m), 1.88 (2 H, m, J = 8.0 Hz), 6.66 (2 H, m, J = 8.0 Hz), 5.57 (1 H, s), 4.35 (1 2.12 (2 H, m), 1.61−1.79 (4 H, m), 1.31−1.56 (2 H, m), 1.20−1.28 H, br d, J = 8.0 Hz), 2.92 (6 H, s), 2.70−2.80 (1 H, m), 2.50−2.70 (2 (9 H, s), 0.56 (3 H, s). m/z (ESI, +ve ion) 502.4 (M + H)+. H, m), 2.15−2.50 (7 H, m), 1.85−2.15 (2 H, m), 1.65−1.85 (4 H, (8S,11R,13S,14S,17S)-11-(4-(Dimethylamino)-3-methylphenyl)- m), 1.20−1.60 (2 H, m), 1.17 (6 H, d, J = 4.0 Hz), 0.56 (3 H, s); m/z 17-(3,3-dimethylbut-1-yn-1-yl)-17-hydroxy-13-methyl- 1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]- (ESI, +ve ion) 458.3 (M + H)+. phenanthren-3-one (20). 1 δ (8S,11R,13S,14S,17S)-17-(3,3-Dimethylbut-1-yn-1-yl)-17-hy- H NMR (400 MHz, CDCl3) ppm 6.99 droxy-13-methyl-11-(p-tolyl)-1,2,6,7,8,11,12,13,14,15,16,17-dodec- (1 H, s), 6.89 (2 H, m), 5.78 (1 H, s), 4.33−4.38 (1 H, m), 2.75 (1 H, ahydro-3H-cyclopenta[a]phenanthren-3-one (12). 1H NMR (400 m), 2.67 (6 H, s), 2.54−2.64 (2 H, m), 2.26−2.47 (8 H, m), 2.18− δ − − − MHz, CDCl3) ppm 7.01 7.15 (4 H, m), 5.78 (1 H, s), 4.40 (1 H, 2.24 (1 H, m), 1.88 2.07 (2 H, m), 1.60 1.77 (3 H, m), 1.47 (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), br dd, J = 12.9, 9.4 Hz), 1.31−1.40 (1 H, m), 1.24 (9 H, s), 0.54 (3 H, 2.40−2.49 (1 H, m), 2.18−2.38 (9 H, m), 1.87−2.12 (2 H, m), 1.61− s). m/z (ESI, +ve ion) 486.4 (M + H)+. 1.80 (3 H, m), 1.42−1.56 (1 H, m), 1.35 (1 H, br dd, J = 11.7, 5.9 (8S,11R,13S,14S,17S)-17-(3,3-Dimethylbut-1-yn-1-yl)-17-hy- Hz), 1.25 (9 H, s), 0.53 (3 H, s). m/z (ESI, +ve ion) 443.5 (M + H)+. droxy-11-(4-(isopropyl(methyl)amino)phenyl)-13-methyl- (8S,11R,13S,14S,17S)-17-(3,3-Dimethylbut-1-yn-1-yl)-11-(4-eth- 1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]- phenanthren-3-one (21). 1 δ − ylphenyl)-17-hydroxy-13-methyl-1,2,6,7,8,11,12,13,14,15,16,17-do- H NMR (400 MHz, CDCl3) ppm 6.95 decahydro-3H-cyclopenta[a]phenanthren-3-one (13). 1HNMR 7.06 (2 H, m), 6.71 (2 H, m, J = 8.8 Hz), 5.77 (1 H, s), 4.35 (1 H, br δ − (400 MHz, CDCl3) ppm 7.10 (4 H, s), 5.78 (1 H, s), 4.41 (1 H, d, J = 5.7 Hz), 3.98 4.09 (1 H, m), 2.76 (1 H, m), 2.70 (3 H, s), br d, J = 6.3 Hz), 2.77 (1 H, br d, J = 14.5 Hz), 2.56−2.66 (4 H, m), 2.53−2.61 (2 H, m), 2.33−2.49 (4 H, m), 2.27−2.30 (1 H, m), 2.17− 2.28−2.50 (6 H, m), 2.18−2.26 (1 H, m), 1.89−2.12 (2 H, m), 1.60− 2.25 (1 H, m), 1.84−2.09 (2 H, m), 1.54−1.80 (3 H, m), 1.30−1.54 1.79 (3 H, m), 1.43−1.57 (1 H, m), 1.29−1.42 (1 H, m), 1.14−1.29 (2 H, m), 1.24 (9 H, s), 1.10−1.38 6 H, m), 0.56 (3 H, s). m/z (ESI, (12 H, m), 0.52 (3 H, s); m/z (ESI, +ve ion) 457.4 (M + H)+. +ve ion) 500.5 (M + H)+. (8S,11R,13S,14S,17S)-17-(3,3-Dimethylbut-1-yn-1-yl)-17-hy- (8S,11R,13S,14S,17S)-17-(3,3-Dimethylbut-1-yn-1-yl)-17-hy- droxy-11-(4-isopropylphenyl)-13-methyl- droxy-13-methyl-11-(4-morpholinophenyl)- 1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]- 1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]- phenanthren-3-one (14). 1 δ − phenanthren-3-one (22). 1 δ − H NMR (400 MHz, CDCl3) ppm 7.04 H NMR (400 MHz, CDCl3) ppm 7.03 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, 7.13 (2 H, m), 6.75−6.91 (2 H, m), 5.78 (1 H, s), 4.34−4.40 (1 H, J = 6.9 Hz), 2.75 (1 H, m), 2.54−2.62 (2 H, m), 2.27−2.50 (6 H, m), m), 3.79−3.90 (4 H, m), 3.08−3.18 (4 H, m), 2.76 (1 H, br d, J = 2.21 (1 H, m), 2.01 (1 H, m), 1.95 (1 H, m), 1.59−1.82 (3 H, m), 15.0 Hz), 2.54−2.62 (2 H, m), 2.27−2.49 (5 H, m), 2.18−2.25 (1 H, 1.28−1.45 (2 H, m), 1.17−1.27 (15 H, m), 0.52 (3 H, s). m/z (ESI, m), 1.90−2.07 (2 H, m), 1.64−1.78 (3 H, m), 1.48 (2 H, br d, J = 9.8 +ve ion) 471.4 (M + H)+. 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-hy- (8S,11R,13S,14S,17S)-17-(3,3-Dimethylbut-1-yn-1-yl)-17-hy- droxy-11-(4-methoxyphenyl)-13-methyl- droxy-13-methyl-11-(4-(pyrrolidin-1-yl)phenyl)- 1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]- 1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]- phenanthren-3-one (15). 1 δ phenanthren-3-one (23). 1 δ − H NMR (400 MHz, CDCl3) ppm 7.09 H NMR (400 MHz, CDCl3) ppm 6.98 (2 H, d, J = 8.4 Hz), 7.00 (1 H, d, J = 8.8 Hz), 5.78 (1 H, s), 4.39 (1 7.06 (2 H, m), 6.44−6.54 (2 H, m) 5.76 (1 H, s), 4.36 (1 H, br dd, J H, br d, J = 7.8 Hz), 3.79 (3 H, s) 2.77 (1 H, br d, J = 14.6 Hz), 2.54− = 5.6, 2.4 Hz), 3.27 (4 H, br t, J = 5.2 Hz), 2.73−2.81 (1 H, m), 2.67 (2 H, m) 2.27−2.53 (6 H, m), 2.18−2.25 (1 H, m),1.95 (1 H, br 2.53−2.61 (2 H, m), 2.26−2.48 (5 H, m), 2.17−2.24 (1 H, m), 1.91− d, J = 1.0 Hz) 1.59−1.85 (3 H, m) 1.30−1.56 (2 H, m), 1.25 (9 H, s), 2.06 (6 H, m), 1.63−1.77 (3 H, m), 1.42−1.56 (1 H, m), 1.30−1.41 0.53 (3 H, s). m/z (ESI, +ve ion) 459.4 (M + H)+. (1 H, m), 1.24 (9 H, s), 0.57 (3 H, s). m/z (ESI, +ve ion) 498.4 (M + (8S,11R,13S,14S,17S)-11-(3-(Dimethylamino)phenyl)-17-(3,3-di- H)+. methylbut-1-yn-1-yl)-17-hydroxy-13-methyl- (8S,11R,13S,14S,17S)-11-(4-Aminophenyl)-17-(3,3-dimethylbut- 1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]- 1-yn-1-yl)-17-hydroxy-13-methyl-1,2,6,7,8,11,12,13,14,15,16,17-do- phenanthren-3-one (16). 1 δ decahydro-3H-cyclopenta[a]phenanthren-3-one (33a). H NMR (400 MHz, CDCl3) ppm 7.13 Under Ar, (1 H, t, J = 7.9 Hz), 6.53−6.60 (3 H, m), 5.77 (1 H, s), 4.40 (1 H, br 31f (3 g, 7.27 mmol) and copper iodide (1.38 g, 7.27 mmol) in THF s), 2.93 (6 H, s), 2.73−2.82 (1 H, m), 2.53−2.62 (2 H, m), 2.31−2.52 (40 mL) was cooled to 0 °C. (4-(Bis(trimethylsilyl)amino)phenyl)

7779 DOI: 10.1021/acs.jmedchem.8b00743 J. Med. Chem. 2018, 61, 7767−7784 Journal of Medicinal Chemistry Article fi fl − magnesium bromide (34) (0.5 M in THF, 58.2 mL, 29.1 mmol) was residue was puri ed by Combi- ash (SiO2,0 80% EtOAc in hexanes, added dropwise. The reaction mixture was slowly brought to rt and gradient elution) to give 33b (32 mg, 0.06 mmol, 70% yield). 1H δ − stirred for 2 h. The reaction was quenched (satd NH4Cl), and the NMR (400 MHz, CDCl3 ppm 7.28 7.37 (2 H, m), 7.19 (2 H, d, J reaction mixture exposed to the air and stirred for 10 min. The = 8.2 Hz), 5.78 (1 H, s), 4.43 (1 H, br d, J = 6.6 Hz), 3.96 (2 H, d, J = mixture was extracted (EtOAc) and washed (brine). The organics 11.0 Hz), 3.83 (2 H, dd, J = 11.0, 1.2 Hz), 2.69−2.87 (1 H, m), 2.59 − − were separated and dried (MgSO4) before concentration to dryness. (2 H, br dd, J = 7.1, 3.7 Hz), 2.23 2.50 (7 H, m), 1.85 2.11 (2 H, The crude was used in next step without further purification. m), 1.75−1.84 (2 H, m), 1.45−1.55 (1 H, m), 1.25−1.40 (1 H, m), A solution of the crude material (641 mg g, 0.99 mmol) in 70% 1.30 (3 H, s), 1.25 (9 H, s), 0.50 (3 H, s). m/z (ESI, +ve ion) 517.4 acetic acid in water (5 mL) was stirred at 55 °C for 1 h. The reaction (M + H)+. × (8S,11R,13S,14S,17S)-17-(3,3-Dimethylbut-1-yn-1-yl)-17-hy- was quenched (satd aq NaHCO3), extracted (2 EtOAc), and washed (brine). The combined organic layers were dried (MgSO ) droxy-13-methyl-11-(4-(3-methyloxetan-3-yl)phenyl)- 4 1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]- and concentrated under reduced pressure. Purification of the residue − phenanthren-3-one (25). A flask was charged with 33b (33 mg, 0.07 by column chromatography (SiO2,5070% EtOAc in hexanes, gradient elution) gave 33a (100 mg, 0.23 mmol, 22% yield). 1H NMR mmol) and azeotroped with toluene. Triphenylphosphine (50.3 mg, δ 0.19 mmol), toluene (1.5 mL), THF (0.5 mL), and Ziram (39.1 mg, (400 MHz, CDCl3) 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, 0.13 mmol) were added successively. Upon adding ethyl N,N- ethoxycarbonyliminocarbamate (0.03 mL, 0.19 mmol), the solution m), 2.52−2.65 (2 H, m), 2.28−2.49 (5 H, m), 2.17−2.26 (2 H, m), became clear. The resulting solution was stirred overnight. The 1.85−2.09 (2 H, m), 1.62−1.85 (3 H, m), 1.25−1.51 (2 H, m), 1.24 + mixture was filtered through Celite and rinsed with EtOAc. The (9 H, s), 0.506 (3 H, s). m/z (ESI, +ve ion) 444.4 [M + H] . fi fi fl (8S,11R,13S,14S,17S)-17-(3,3-Dimethylbut-1-yn-1-yl)-11-(4-(1,1- ltrate was concentrated, and the residue was puri ed by Combi- ash − dioxidothiomorpholino)phenyl)-17-hydroxy-13-methyl- (SiO2,0 30% EtOAc in hexanes, gradient elution) to give the crude 1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]- product. Purification by RP-HPLC (30−70% A/B, gradient elution) 1 phenanthren-3-one (24). Divinyl sulfone (0.20 mL, 1.4 mmol) was provided 25 (17 mg, 0.034 mmol, 53% yield). H NMR (400 MHz, δ − added to a solution of 33a (60 mg, 0.14 mmol) in isopropyl CDCl3) ppm 7.13 7.19 (4 H, m), 5.79 (1 H, s), 4.94 (2 H, dd, J = (2 mL) and water (2 mL) and the mixture was refluxed at 115 °C for 5.6, 3.4 Hz), 4.63 (2 H, d, J = 5.6 Hz), 4.43 (1 H, br d, J = 6.7 Hz), − 5 h. The reaction was quenched (satd aq NH Cl), extracted (2 × 2.77 (1 H, br d, J = 14.6 Hz), 2.60 (2 H, dd, J = 8.0, 4.1 Hz), 2.26 4 − − − EtOAc), and washed (brine). The combined organic layers were dried 2.51 (6 H, m), 2.15 2.25 (1 H, m), 1.87 2.09 (2 H, m), 1.65 1.79 fi (5 H, m), 1.43−1.56 (2 H, m), 1.25 (9 H, s), 0.52 (3 H, s). m/z (ESI, (MgSO4) and concentrated under reduced pressure. Puri cation of + the crude product by column chromatography (SiO ,20−60% EtOAc +ve ion) 499.4 (M + H) . 2 (8S,13S,14S,17S)-13-Methyl-1,2,4,6,7,8,12,13,14,15,16,17- in hexanes, gradient elution) gave 24 (20 mg, 0.036 mmol, 26% dodecahydrospiro[cyclopenta[a]phenanthrene-3,2′-[1,3]- yield). 1H NMR (400 MHz, CDCl ) δ ppm 7.11 (2 H, d, J = 8.5 Hz), 3 dioxolan]-17-ol (37). To a solution of 29 (100 g, 318 mmol) in THF 6.78−6.93 (2 H, m), 5.79 (1 H, s), 4.38 (1 H, br d, J = 7.3 Hz), 3.76− (500 mL) and MeOH (50 mL) was added sodium borohydride (6.14 3.93 (4 H, m), 3.05−3.20 (4 H, m), 2.72−2.83 (1 H, m), 2.54−2.66 g, 159 mmol) in portions at 0 °C. The mixture was stirred at 0 °C for (2 H, m), 2.18−2.48 (7 H, m), 1.89−2.11 (3 H, m), 1.57−1.83 (1 H, − 30 min and then rt for 1 h. The reaction was quenched with 2 mL of m), 1.43 1.56 (2 H, m), 1.24 (9 H, s), 0.53 (3 H, s). m/z (ESI, +ve satd NaHCO solution and concentrated to remove MeOH. The ion) 562.4 [M + H]+. 3 5-(4-Bromophenyl)-2,2,5-trimethyl-1,3-dioxane (36). residue was dissolved in EtOAc (1 L), the organic phase was washed To a sol- with satd NaHCO solution and then brine and dried over anhydrous ution of 2-(4-bromophenyl)-2-methylpropane-1,3-diol (35) (688 mg, 3 Na2SO4. The organic solution was concentrated under reduced 2.81 mmol) and 4-methylbenzenesulfonic acid (24 mg, 0.14 mmol) in pressure to give 37 (99.5 g, 100% yield) as a colorless oil. 1H NMR DMF (12 mL) was added 2,2-dimethoxypropane (0.52 mL, 4.2 δ (400 MHz, CDCl3) ppm 5.61 (1 H, m), 4.01 (4 H, s), 3.78 (1 H, mmol). The solution was stirred at rt overnight and then was poured m), 2.48−2.64 (1 H, m), 1.75−2.36 (14 H, m), 1.12−1.58 (3 H, m), into saturated aq NaHCO3 and extracted with EtOAc. The combined + fi − 0.78 (3 H, s). m/z (ESI, +ve ion) 317.3 [M + H] . organic extracts were puri ed by column chromatography (SiO2,0 (5′ R,8′ S,10′ R,13′ S,14′ S,17′ S)-13′ -Methyl- 30% EtOAc in hexanes, gradient elution) to give 36 (618 mg, 2.22 1′,2′,7′,8′,12′,13′,14′,15′,16′,17′-decahydro-4′H,6′H-spiro[[1,3]- 1 δ mmol, 77% yield). H NMR (400 MHz, CDCl3) ppm 7.49 (2 H, d, dioxolane-2,3′-[5,10]epoxycyclopenta[a]phenanthren]-17′-ol (38). J = 7.42 Hz) 7.26−7.30 (2 H, m) 4.08 (2 H, d, J = 11.7 Hz) 3.80 (2 To a solution of 37 (43.0 g, 136 mmol) in DCM (320 mL) at 0 °C H, d, J = 11.7 Hz) 1.41 (3 H, s) 1.34 (3 H, s), 1.28 (3 H, s); m/z was added Na2HPO4 (9.65 g, 67.9 mmol), followed by addition of (ESI, +ve ion) 280.3 [M + H]+. 1,1,1,3,3,3-hexafluoropropan-2-one trihydrate (11.2 mL, 81.5 mmol) (8S,11R,13S,14S,17S)-11-(4-(1,3-Dihydroxy-2-methylpropan-2- and H2O2 (35% aq, 34.2 mL, 408 mmol). The mixture was stirred at 0 yl)phenyl)-17-(3,3-dimethylbut-1-yn-1-yl)-17-hydroxy-13-methyl- °C for 10 min and then at rt for 3 h. Sodium thiosulfate solution (10% 1,2,6,7,8,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]- ° phenanthren-3-one (33b). fl aq, 200 mL) was slowly added at 0 C, and the mixture was stirred at Adried ask was charged with rt for 30 min and extracted with DCM (300 mL). The organic phase magnesium chips (40 mg, 1.5 mmol) and a crystal of iodine. The was washed with brine and dried over anhydrous Na SO . The flask was flushed with Ar, heated briefly with a heat gun, and then 2 4 ° fi organic solution was then concentrated to provide 38 (43 g. 130 heated at 60 C. At rst, a small portion (0.5 mL) of a solution of 36 mmol, 95% yield) as a 4:1 mixture of epimers (the desired isomer is (azeotroped with toluene beforehand, 373 mg, 1.31 mmol) in THF (2 1 δ − major). H NMR (400 MHz, CDCl3) ppm 5.97 6.07 (1 H, m), mL) was added. After iodine color disappeared, the remaining 3.84−4.00 (4 H, m), 3.65−3.79 (1 H, m), 2.38−2.57 (1 H, m), 1.75− solution was added, and the mixture was continued stirring for 30 min 2.36 (14 H, m), 1.12−1.58 (3 H, m), 0.74 (3 H, s). at 60 °C. The mixture was then cooled down and added to a solution (8S,11R,13S,14S,17S)-17-Hydroxy-11-(4-(isopropyl(methyl)- of 31f (45 mg, 0.11 mmol) and copper(I) iodide (16.6 mg, 0.09 amino)phenyl)-13-methyl-1,2,6,7,8,11,12,13,14,15,16,17-dodeca- mmol) in THF (1 mL) at 0 °C. The mixture was kept at 0 °C for 10 hydro-3H-cyclopenta[a]phenanthren-3-one (39b). A dried flask was min and then raised to rt for 2 h. After the reaction was cooled to rt, it charged with magnesium chips (2.83 g, 117 mmol) and a crystal of was quenched with NH4Cl and extracted with EtOAc. The organics iodine. The flask was flushed with Ar and heated briefly with a heat were washed, dried, and concentrated. The residue was purified by gun and then put into a 60 °C of bath. A 10 mL of 4-bromo-N- fl − Combi- ash (SiO2,0 40% EtOAc in hexanes, gradient elution) to isopropyl-N-methyl-aniline (26.1 g, 115 mmol, azeotroped with give the Grignard addition product (55 mg, 81%). toluene beforehand) in THF (120 mL) was added. After the iodine To a flask charged the Grignard addition product (55 mg, 0.09 color disappeared, the remaining solution was added dropwise for 20 mmol) was added 70% acetic acid (2 mL). After the mixture was min and the mixture was continued to stir at 6 °C for 2 h. ° heated at 60 C for 2 h, it was quenched with NaHCO3 and extracted The generated Grignard reagent prepared above was cooled down with EtOAc. The organics were washed, dried, and concentrated. The and added to a solution of 38 (12.7 g, 38.2 mmol, azeotroped with

7780 DOI: 10.1021/acs.jmedchem.8b00743 J. Med. Chem. 2018, 61, 7767−7784 Journal of Medicinal Chemistry Article toluene) and copper(I) iodide (3.64 mg, 19.1 mmol) in THF (100 monohydrate (2.45 g, 12.9 mmol) in acetic acid (50 mL) was added mL) at 0 °C. The resulting mixture was allowed to warm up to rt and ethane-1,2-dithiol (5.4 mL, 64 mmol). After the resulting solution was stir for 1 h. The reaction was quenched (satd aq NH4Cl), extracted (2 stirred at rt under Ar for 2 h, the reaction was quenched (ice-cold 2.5 × EtOAc), and washed (brine). The combined organic layer was N aq NaOH) and extracted (2 × EtOAc). The combined organic dried (Na2SO4) and concentrated under reduced pressure. layer was washed (brine), dried (MgSO4), and concentrated under fi fl − fi fl Puri cation of the residue by Combi- ash (SiO2,45 65% EtOAc/ reduced pressure. Puri cation of the residue by Combi- ash (SiO2, hexanes, gradient elution) provided the intermediate (9.7 g, 20.1 30−60% EtOAc in hexanes, gradient elution) provided 42b (1.17 g, 1 mmol, 53% yield) as an off-white foam. 2.35 mmol, 36% yield) as a white solid. H NMR (400 MHz, CDCl3) A solution of the intermediate (9.7 g, 20.1 mmol) in 70% acetic δ ppm 7.23 (2 H, d, J = 8.8 Hz), 6.69 (2 H, d, J = 8.9 Hz), 5.61 (1 H, acid in water (80 mL, 1.33 mol) was stirred at 55 °C for 1 h. The d, J = 1.3 Hz), 4.01−4.11 (1 H, m), 3.26−3.41 (3 H, m), 3.15−3.24 reaction was concentrated under reduced pressure to remove most (2 H, m), 2.71 (3 H, s), 2.42−2.53 (1 H, m), 2.24−2.34 (1 H, m), AcOH. Then the residue was diluted (EtOAc), basified (satd aq 2.07−2.18 (2 H, m), 1.76−2.04 (5 H, m), 1.58−1.72 (3 H, m), 1.30− × − − − NaHCO3), extracted (2 EtOAc), and washed (brine). The 1.42 (2 H, m), 1.21 1.24 (1 H, m), 1.07 1.18 (8 H, m), 0.91 1.04 + combined organic layer was dried (Na2SO4) and concentrated (2 H, m), 0.54 (3 H, s). m/z (ESI, +ve ion) 498.3 [M + H] . under reduced pressure. Purification of the residue by Combi-flash (8R,9S,10R,11S,13S,14S)-11-(4-(Isopropyl(methyl)amino)phenyl)- − 13-methyl-1,6,7,8,9,10,11,12,13,14,15,16-dodecahydrospiro- (SiO2,50 100% EtOAc in hexanes, gradient elution) gave 39b (6.80 [cyclopenta[a]phenanthrene-3,2′-[1,3]dithiolan]-17(2H)-one (43b). g, 16.2 mmol, 80% yield) as a white solid. 1H NMR (400 MHz, δ To a solution of 42b (1.17 g, 2.35 mmol) in toluene (19.6 mL) was CDCl3) 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 added cyclohexanone (7.00 mL, 6.77 mmol) and aluminum propan-2- Hz), 3.62−3.73 (1 H, m), 2.73 (1 H, br s), 2.71 (3 H, s), 2.32−2.61 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 (7 H, m), 2.07−2.17 (1 H, m), 2.02 (1 H, dq, J = 12.9, 4.4 Hz), 1.63− Rochelle’s salt solution. The mixture was stirred for 10 min and 1.76 (2 H, m), 1.28−1.49 (5 H, m), 1.15 (3 H, d, J = 6.6 Hz), 1.14 (3 extracted (2 × EtOAc). The combined organic layer was washed H, d, J = 6.6 Hz) 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)- (brine), dried (MgSO4), and concentrated under reduced pressure. fi fl − amino)phenyl)-13-methyl-1,2,4,6,7,8,9,11,12,13,14,15,16,17-tetra- Puri cation of the residue by Combi- ash (SiO2,15 30% EtOAc in decahydro-3H-cyclopenta[a]phenanthren-3-one (40b). Under Ar, hexanes, gradient elution) provided 43b (695 mg, 1.40 mmol, 60% 1 δ lithium (2.03 g, 292 mmol) in pieces was added to liquid ammonia yield) as a white solid. H NMR (400 MHz, CDCl3) 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), (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 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 (kept the internal temperature below 60 C). The mixture was − − − stirred at −78 °C for 5 min before the reaction was quenched by 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 adding 10 mL of H2O, then the solution was poured into 500 mL + − × (ESI, +ve ion) 496.4 [M + H] . ice water, extracted by EtOAc (500 mL 2), and the combined (8R,9S,10R,11S,13S,14S,17S)-17-(3,3-Dimethylbut-1-yn-1-yl)-11- organic layer was dried and concentrated under reduced pressure. (4-(isopropyl(methyl)amino)phenyl)-13-methyl- fi fl Puri cation of the residue by Combi- ash (SiO2, EtOAc/DCM/hex = 1,2,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydrospiro- 1:1:10 to1:1:5, gradient elution) provided 40b (37.1 g, 74% yield) as [cyclopenta[a]phenanthrene-3,2′-[1,3]dithiolan]-17-ol (44c). To a 1 δ white foam. H NMR (400 MHz, CDCl3) ppm 7.17 (2 H, d, J = 8.6 solution of 1.6 M n-butyllithium in hexanes (8.80 mL, 14.2 mmol) in − Hz), 6.65 (2 H, d, J = 8.9 Hz), 4.04 (1 H, dt, J = 13.2, 6.6 Hz), 3.60 THF (7 mL) was added 3,3-dimethylbut-1-yne (2.10 mL, 17.3 mmol) 3.71 (1 H, m), 3.44 (1 H, br s), 2.81 (2 H, br s), 2.68 (3 H, s), 2.07− at −78 °C. After 30 min, a solution of 43b (390 mg, 0.79 mmol) in 2.45 (8 H, m), 1.98 (1 H, br d, J = 17.5 Hz), 1.82−1.91 (1 H, m), THF (8 mL) was added slowly. After the reaction was stirred at −78 1.60−1.71 (2 H, m), 1.20−1.45 (5 H, m), 1.14 (3 H, d, J = 6.6 Hz), °C for 1 h, it was allowed to warm to rt and stirred for 3 h. The × 1.13 (3 H, d, J = 6.6 Hz), 0.36 (3 H, s). m/z (ESI, +ve ion) 422.4 [M reaction was quenched (satd aq NH4Cl) and extracted (2 EtOAc). + +H]. The combined organic layers were washed (brine), dried (MgSO4), (8R,9S,10R,11S,13S,14S,17S)-17-Hydroxy-11-(4-(isopropyl- and concentrated under reduced pressure. Purification of the residue (methyl)amino)phenyl)-13-methyl- fl − 1,2,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3H-cyclopenta- by Combi- ash (SiO2,15 30% EtOAc in hexanes, gradient elution) provided 44c (440 mg, 0.76 mmol, 97% yield) as a white foam. 1H [a]phenanthren-3-one (41b). Under Ar, to a solution of 40b (11.3 g, δ NMR (400 MHz, CDCl3) ppm 7.24 (2 H, br d, J = 8.6 Hz), 6.68 (2 26.8 mmol) in anhydrous EtOH (68 mL) was added conc H, br d, J = 8.6 Hz), 5.61 (1 H, s), 4.06 (1 H, dt, J = 13.0, 6.6 Hz), hydrochloric acid (90 mL), and the resulting solution was stirred at − − − ° ∼ 3.32 3.41 (3 H, m), 3.27 3.31 (1 H, m), 3.17 3.25 (1 H, m), 2.71 65 C until most starting material disappeared ( 16 h). The reaction (3 H, s), 2.45−2.54 (1 H, m), 2.29 (1 H, br d, J = 13.7 Hz), 2.06− was cooled to rt and concentrated under reduced pressure to remove 2.24 (4 H, m), 1.77−2.00 (5 H, m), 1.69 (2 H, br d, J = 10.5 Hz), most HCl. Then the residue was diluted with DCM, basified with ice- − − × 1.41 1.50 (1 H, m), 1.29 1.38 (2 H, m), 1.25 (9 H, s), 1.16 (3 H, d, cold 2.5 M NaOH, and extracted (2 DCM). The combined organic J = 6.5 Hz), 1.16 (3 H, d, J = 6.5 Hz), 1.03−1.11 (2 H, m), 0.99 (1 H, layer was washed with brine, dried over Na2SO4, and concentrated br d, J = 12.0 Hz), 0.62 (3 H, s). m/z (ESI, +ve ion) 578.5 [M + H]+. under reduced pressure. Purification of the residue by Combi-flash (8R,9S,10R,11S,13S,14S,17S)-17-(3,3-Dimethylbut-1-yn-1-yl)-17- − (SiO2,25 50% EtOAc in hexanes, gradient elution) provided 41b hydroxy-11-(4-(isopropyl(methyl)amino)phenyl)-13-methyl- (8.90 g, 21.1 mmol, 78% yield) as an off-white foam. 1H NMR (400 1,2,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3H-cyclopenta- δ [a]phenanthren-3-one (28). MHz, CDCl3) ppm 7.24 (2 H, br d, J = 8.6 Hz), 6.71 (2 H, br d, J = To a solution of 44c (114 mg, 0.20 8.5 Hz), 5.85 (1 H, s), 4.02−4.12 (1 H, m), 3.53−3.63 (1 H, m), 3.25 mmol) in acetic acid (1.7 mL) was added glyoxylic acid monohydrate (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, (908 mg, 9.86 mmol) at rt, and the mixture was stirred at rt for 10 J = 14.3 Hz), 2.37 (1 H, td, J = 14.1, 5.0 Hz), 2.26 (1 H, dt, J = 16.3, min. Then 4 N HCl in water (0.89 mL, 3.6 mmol) was added and the 3.9 Hz), 2.18 (1 H, dd, J = 13.1, 1.4 Hz), 2.07−2.14 (2 H, m), 2.01− resulting solution was stirred at rt for 20 h. The reaction was × 2.05 (1 H, m), 1.86−1.96 (1 H, m), 1.65−1.74 (1 H, m), 1.60−1.65 quenched (satd aq NaHCO3), extracted (2 EtOAc), and washed (1 H, m), 1.27−1.51 (1 H, m), 1.27−1.51 (5 H, m), 1.18 (3 H, d, J = (brine). The combined organic layers were dried (Na2SO4) and 6.4 Hz), 1.16 (3 H, d, J = 6.4 Hz), 0.98−1.12 (2 H, m), 0.59 (3 H, s). concentrated under reduced pressure. Purification of the residue with + fl − m/z (ESI, +ve ion) 422.3 [M + H] . Combi- ash (SiO2,20 40% EtOAc in hexanes, gradient elution) (8R,9S,10R,11S,13S,14S)-11-(4-(Isopropyl(methyl)amino)phenyl)- provided 28 (38 mg, 0.076 mmol, 38% yield) as a white foam. 1H δ 13-methyl-1,2,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydrospiro- NMR (400 MHz, CDCl3) ppm 7.23 (2 H, br d, J = 8.3 Hz), 6.71 (2 [cyclopenta[a]phenanthrene-3,2′-[1,3]dithiolan]-17-ol (42b). To a H, br d, J = 8.3 Hz), 5.86 (1 H, s), 4.04−4.11 (1 H, m), 3.33 (1 H, br solution of 41b (2.80 g, 6.64 mmol) and p-toluenesulfonic acid s), 2.86 (1 H, br s), 2.72 (3 H, s), 2.53 (1 H, br d, J = 14.3 Hz), 2.33−

7781 DOI: 10.1021/acs.jmedchem.8b00743 J. Med. Chem. 2018, 61, 7767−7784 Journal of Medicinal Chemistry Article

2.43 (1 H, m), 2.07−2.31 (5 H, m), 2.03 (1 H, br s), 1.88−1.97 (3 H, GmbH for protein production, crystallography, and structure m), 1.68−1.78 (1 H, m), 1.63 (1 H, s), 1.46−1.55 (2 H, m), 1.34− determination, and Dennis Yamashita for critical reading of − 1.42 (1 H, m), 1.26 1.30 (1 H, m), 1.24 (9 H, s), 1.17 (3 H, d, J = manuscript. 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, CDCl ) δ ppm 200.2, 167.7, 147.8, 131.3, 130.5 3 ■ ABBREVIATIONS USED (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, AcCN, acetonitrile; AcOH, acetic acid; AR, androgen receptor; 22.9, 19.3, 19.2, 14.8. m/z (ESI, +ve ion) 502.4 [M + H]+. CL, clearance; CMC, carboxymethyl cellulose; CRPC, (8R,9S,10R,11S,13S,14S,17S)-11-(4-(Dimethylamino)phenyl)-17- (3,3-dimethylbut-1-yn-1-yl)-17-hydroxy-13-methyl- castration-resistant prostate cancer; CYP3A4, cytochrome 1,2,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3H-cyclopenta- P450 3A4; CYP 2C8, cytochrome P450 2C8; CYP2C9, [a]phenanthren-3-one (26). 26 was prepared from 29 according to a cytochrome P450 2C9; DCM, dichloromethane; DMA, similar procedure described for the synthesis of 28. 1H NMR (400 dimethylacetamide; DEAD, diethyl azodicarboxylate; DMSO, δ MHz, CDCl3) ppm 7.28 (2 H, br d, J = 8.4 Hz), 6.67 (2 H, br d, J = dimethyl sulfoxide; ER, estrogen receptor; EtOAc, ethyl 8.4 Hz), 5.87 (1 H, s), 3.34 (1 H, br t, J = 6.0 Hz), 2.95 (6 H, s), acetate; FKBP5, FK506 binding protein 5; GILZ, glucocorti- 2.80−2.90 (1 H, m), 2.49−2.57 (1 H, m), 2.33−2.43 (1 H, m), 2.22− coid-induced leucine zipper; GCs, glucocorticoids; GR, 2.30 (2 H, m), 2.02−2.22 (4 H, m), 1.87−1.97 (3 H, m), 1.68−1.79 − − glucocorticoid receptor; HPMC, hydroxypropyl methylcellu- (1 H, m), 1.60 1.67 (1 H, m), 1.47 1.54 (2 H, m), 1.38 (1 H, ddd J lose; IPA, isopropyl alcohol; LNAR, LNCaP/AR-luc; NCoR, = 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)+. nuclear receptor corepressor; MR, mineralocorticoid receptor; (8R,9S,10R,11S,13S,14S,17S)-11-(4-(Dimethylamino)phenyl)-17- OVCAR5, human ovarian carcinoma; PBMC, peripheral blood hydroxy-17-(3-methoxy-3-methylbut-1-yn-1-yl)-13-methyl- mononuclear cell; PEG, polyethylene glycol; PR, progesterone 1,2,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3H-cyclopenta- receptor; PTSA, p-toluenesulfonic acid; QD, once a day [a]phenanthren-3-one (27). 27 was prepared from 29 according to a dosing; RT-qPCR, quantitative reverse transcription polymer- similar procedure described for the synthesis of 28. 1H NMR (400 δ − − ase chain reaction; SEM, standard error of mean; TNBC, MHz, CDCl3) ppm 7.26 7.31 (2 H, m), 6.61 6.74 (2 H, m), 5.86 fl (1 H, s), 3.37 (3 H, s), 3.32−3.36 (1 H, m), 2.96 (3 H, br s), 2.96 (3 triple-negative breast cancer; TFA, tri uoroacetic acid; THF, 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 ■ REFERENCES − − (3 H, s), 1.48 (3 H, s), 1.37 1.45 (2 H, m), 1.22 1.32 (1 H, m), (1) Azher, S.; Azami, O.; Amato, C.; McCullough, M.; Celentano, 1.05−1.15 (1 H, m), 0.68 (3 H, s). m/z (ESI, +ve ion) 490.4 [M + + A.; Cirillo, N. The non-conventional effects of glucocorticoids in H] . cancer. J. Cell. Physiol. 2016, 231, 2368−2373. (2) Clark, R. D. Glucocorticoid receptor antagonists. Curr. Top. Med. ■ ASSOCIATED CONTENT Chem. 2008, 8, 813−838. *S Supporting Information (3) Volden, P. A.; Conzen, S. D. The influence of glucocorticoid The Supporting Information is available free of charge on the signaling on tumor progression. Brain, Behav., Immun. 2013, 30, S26− ACS Publications website at DOI: 10.1021/acs.jmed- S31. chem.8b00743. (4) Pan, D.; Kocherginsky, M.; Conzen, S. D. Activation of the glucocorticoid receptor is associated with poor prognosis in estrogen In vitro biological assays, in vivo study protocols, HPLC receptor-negative breast cancer. Cancer Res. 2011, 71, 6360−6370. profiles of compounds 11 and 28, ribbon view of GR (5) Tangen, I. L.; Veneris, J. T.; Halle, M. K.; Werner, H. M.; Trovik, cocrystal X-ray structures of 1 and 11, determination of J.; Akslen, L. A.; Salvesen, H. B.; Conzen, S. D.; Fleming, G. F.; the cocrystal structure of 11 with GR, and determination Krakstad, C. Expression of glucocorticoid receptor is associated with aggressive primary endometrial cancer and increases from primary to of the single crystal structures of 11 and 28 (PDF) − Molecular formula strings (CSV) metastatic lesions. Gynecol. Oncol. 2017, 147, 672 677. (6) Arora, V. K.; Schenkein, E.; Murali, R.; Subudhi, S. K.; Accession Codes Wongvipat, J.; Balbas, M. D.; Shah, N.; Cai, L.; Efstathiou, E.; The coordinates of 11 with GR have been deposited in the Logothetis, C.; Zheng, D.; Sawyers, C. L. Glucocorticoid receptor confers resistance to antiandrogens by bypassing androgen receptor Protein Data Bank with accession code 6DXK. Authors will − release the atomic coordinates and experimental data upon blockade. Cell 2013, 155, 1309 1322. (7) Kroon, J.; Puhr, M.; Buijs, J. T.; van der Horst, G.; Hemmer, D. article publication. M.; Marijt, K. A.; Hwang, M. 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7782 DOI: 10.1021/acs.jmedchem.8b00743 J. Med. Chem. 2018, 61, 7767−7784 Journal of Medicinal Chemistry Article

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