5-Aryl indanones and derivatives as non-steroidal progesterone receptor modulators

Novel 5-aryl indanones, inden-1-one oximes, and inden-1-ols were synthesized and evaluated as progesterone receptor (PR) modulators using the T47D cell alkaline phosphatase assay. Both PR agonists and antagonists were achieved with appropriate 3- and 5-substitution from indanones and inden-1-ols while inden-1-one oximes provided only PR antagonists. Several compounds such as 10 and 11 demonstrated potent in vitro PR agonist potency similar to that of steroidal progesterone (1). In addition, a number of compounds (e.g., 12, 13, 17, 18) showed potent PR antagonist activity indicating the indanones and derivatives are promising PR modulator templates.     

1-Methyl-1H-pyrrole-2-carbonitrile containing tetrahydronaphthalene derivatives as non-steroidal progesterone receptor antagonists

Non-steroidal 1-methyl-1H-pyrrole-2-carbonitrile containing tetrahydronaphthalenes and acyclic derivatives were evaluated as novel series of progesterone receptor (PR) antagonists using the T47D cell alkaline phosphatase assay. Moderate to potent PR antagonists were achieved with these scaffolds. Several compounds (e.g., 15 and 20) demonstrated low nanomolar PR antagonist potency and good selectivity versus other steroid receptors.     

Novel 6-aryl-1,4-dihydrobenzo[d]oxazine-2-thiones as potent,selective, and orally active nonsteroidal progesterone receptor agonists

The functional activity of 6-aryl benzoxazinone-based progesterone (PR) antagonists changed to PR agonism when the 2-carbonyl group was replaced by a 2-thiocarbonyl moiety. Based on this finding novel 6-aryl benzoxazine-2-thiones were synthesized and evaluated as PR agonists in various in vitro and in vivo assays. Several analogues had sub-nanomolar in vitro potency and showed excellent oral activities in rats. Compounds 15 and 29 had similar potencies to medroxyprogesterone acetate (MPA) in the in vitro T47D alkaline phosphatase assay and in vivo rat decidualization model. In contrast to MPA, 29 was highly selective (>500-fold) for PR over glucocorticoid and androgen receptors.     

7-aryl 1,5-dihydro-benzo[e][1,4]oxazepin-2-ones and analogs as non-steroidal progesterone receptor antagonists

Novel 7-aryl benzo[1,4]oxazepin-2-ones were synthesized and evaluated as non-steroidal progesterone receptor (PR) modulators. The structure activity relationship of 7-aryl benzo[1,4]oxazepinones was examined using the T47D cell alkaline phosphatase assay. A number of 7-aryl benzo[1,4]oxazepinones such as 10j and 10v demonstrated good in vitro potency (IC(50) of 10-30 nM) and selectivity (over 100-fold) at PR over other steroidal receptors such as glucocorticoid and androgen receptors (GR and AR). Several 7-aryl benzo[1,4]oxazepinones were active in the rat uterine decidualization model. In this in vivo model, compounds 10j and 10u were active at 3 mg/kg when dosed orally.     

Parallel synthesis and SAR study of novel oxa-steroids as potent and selective progesterone receptor antagonists

Efficient parallel synthesis of novel 7-oxa-steroids 4 has been achieved from the key intermediate 3 via a one-pot four-step sequence. oxa-Steroids 4 with various ortho-, meta-, and para-monosubstituents on the phenyl ring, as well as disubstituted phenyl and heterocycles, were evaluated for progesterone receptor (PR) and glucocorticoid receptor (GR) antagonist activities. SAR study demonstrated that the para-fluorinated substituents on the phenyl ring not only increased the potency for PR in a T47D cell functional assay, but also improved the selectivity over GR in an A549 cell functional assay. The para-fluorophenyl oxa-steroid 4l and the para-trifluoromethylphenyl oxa-steroid 4p were found to be remarkably more potent and more selective PR antagonists than mifepristone, with subnanomolar potency and about 140-fold selectivity over GR. Molecular modeling of the oxa-steroid bound to PR provided meaningful insight for the SAR study. oxa-Steroids 4a and 4b were found to be more efficacious than mifepristone in vivo in a rat uterine complement C3 assay via the oral route, although they were less than or equally potent to mifepristone in the T47D assay.     

1,5-Dihydro-benzo[e][1,4]oxazepin-2(1H)-ones containing a 7-(5'-cyanopyrrol-2-yl) group as nonsteroidal progesterone receptor modulators

A series of novel 7-(5'-cyanopyrrol-2-yl) substituted benzo[1,4]oxazepin-2-ones were prepared and tested for their progesterone receptor (PR) agonist or antagonist activity in the alkaline phosphatase assay using the human T47D breast carcinoma cell line. Both PR agonists and antagonists were achieved with an appropriate choice of 5-substitution. Several analogs were potent PR agonists (e.g., 12 and 13) or PR antagonists (e.g., 18) with good selectivity over other steroid receptors.     

SAR studies of 6-aryl-1,3-dihydrobenzimidazol-2-ones as progesterone receptor antagonists

We have previously reported that the aryl substituted benzimidazolones, benzoxazinones, and oxindoles (e.g., 1-3) are progesterone receptor (PR) antagonists and have recently disclosed that the nature of 5- and 6-aryl moieties played a critical role in PR functional activity in the oxindole and benzoxazinone templates. For example, replacing the phenyl group of PR antagonists 2 and 3 with a 5'-cyanopyrrol-2'-yl moiety switched their functional activity to PR agonist activity (2a and 3a). These findings prompted us to examine if there is a similar effect of the 6-aryl moieties on the PR functional activity for the benzimidazolone template. Numerous analogs, such as 5, showed potent PR antagonist activity with about a 10-fold increase in potency as compared to those reported earlier in the same series. More interestingly, pyrrole-containing benzimidazolones 24-27 remained as PR antagonists in contrast to the PR agonist activity switch for oxindole and benzoxazinone scaffolds when a 5'-cyanopyrrol-2'-yl group was installed as a pendant aryl group.     

New progesterone receptor antagonists: 3,3-disubstituted-5-aryloxindoles

A new series of 3,3-disubstituted-5-aryloxindoles has been synthesized and evaluated for progesterone receptor antagonist (PR) activity in a T47D cell alkaline phosphatase assay and for their ability to bind PR in competition binding studies. In this communication, the synthesis and structure-activity relationships (SARs) of various 3,3-substituents are discussed where it is clear that small alkyl and spiroalkyl groups are required to achieve better PR antagonist activity.     

5-(3-Cyclopentyl-2-thioxo-2,3-dihydro-1H-benzimidazol-5-yl)-1-methyl-1H-pyrrole-2-carbonitrile: A novel, highly potent, selective, and orally active non-steroidal progesterone receptor agonist

We have recently discovered 5-(3-cyclopentyl-2-thioxo-2,3-dihydro-1H-benzimidazol-5-yl)-1-methyl-1H-pyrrole-2-carbonitrile (14) as a potent, selective, and orally active non-steroidal progesterone receptor (PR) agonist. Compound 14 and its analog 13 possessed sub-nanomolar in vitro potency (EC(50) 0.1-0.5nM) in the T47D alkaline phosphatase assay, similar to that of the steroidal PR agonist medroxyprogesterone acetate (MPA). In contrast to MPA, 14 was highly selective (>500-fold) for the PR over both glucocorticoid and androgen receptors. In the rat uterine decidualization and complement component C3 models, 14 had oral ED(50) values of 0.02 and 0.003mg/kg, respectively, and was from 6- to 20-fold more potent than MPA. In the monkey ovulation inhibition model, compound 14 was also highly efficacious and potent with an oral ED(100) of 0.03mg/kg.     

Discovery of substituted 4-anilino-2-(2-pyridyl)pyrimidines as a new series of apoptosis inducers using a cell- and caspase-based high throughput screening assay. Part 1: structure-activity relationships of the 4-anilino group

A series of 4-anilino-2-(2-pyridyl)pyrimidines has been discovered as a new class of potent inducers of apoptosis using a cell-based HTS assay. Compound 5a was found to arrest T47D cells in G2/M and induced apoptosis. SAR studies showed that a small and electron-donating group at the meta-position of the anilino ring is important for activity. A 20-fold increase in potency, from hit compound 4-(3-methoxyanilino)-2-(2-pyridinyl)-6-(trifluoromethyl)pyrimidine (5a) to lead compound 4-(2,5-dimethoxyanilino)-2-(2-pyridinyl)-6-(trifluoromethyl)pyrimidine (5l), was obtained through the SAR studies. Compound 5l is highly active with an EC50 value of 18 nM in the caspase activation assay in T47D breast cells. Interestingly, 5a and other meta-mono-substituted compounds were active against T47D cells but were not active against H1299 and HT29 cells, while 5l and other 2,5-disubstituted compounds were active against all the three cells. In a tubulin polymerization assay, compound 5l inhibited tubulin polymerization with an IC50 value of < 0.5 microM, while 5a was not active up to 50 microM.     

Characterization of a new class of selective nonsteroidal progesterone receptor agonists

The identification of a new series of selective nonsteroidal progesterone receptor (PR) agonists is reported. Using a high-throughput screening assay based on the measurement of transactivation of a mouse mammary tumor virus promoter-driven luciferase reporter (MMTV-Luc) in human breast cancer T47D cells, a benzimidazole-2-thione analog was identified. Compound 1 showed an apparent EC50 of 53 nM and efficacy of 93% with respect to progesterone. It binds to PR with high affinity (Ki nM), but had no or very low affinity for other steroid hormone receptors. Structure-activity relationship studies of a series of benzimidazole-2-thione analogs revealed critical positions for high PR binding affinity and transactivation potency as well as receptor selectivity, as exemplified by 25. Compound 25 binds to human PR with high affinity (Ki nM) and had at least > 1000-fold selectivity for PR versus other steroid receptors. Molecular modeling studies suggested that these agonists overlap favorably with progesterone in the ligand-binding domain of PR. In T47D cells, compound 25 acted as a full agonist in the MMTV-Luc reporter assay, as well as in the induction of endogenous alkaline phosphatase activity with apparent EC50 values of 4 and 9 nM, respectively. In the immature rat model, compound 25 provided a significant suppression of estrogen-induced endometrium hypertrophy as measured by luminal epithelial height. In contrast, compound 25 was inactive in the luteinizing hormone release assay in young ovariectomized rats. These benzimidazole-2-thione analogs constitute a new series of nonsteroidal PR agonists with an excellent steroid receptor selectivity profile. The differential activities observed in the in vivo progestogenic assays in rat models suggest that these analogs can act as selective PR modulators.     

Synthesis and antihormonal properties of novel 11β-benzoxazole-substituted steroids

Early studies led to the identification of 11β-aryl-4',5'-dihydrospiro[estra-4,9-diene-17β,4'-oxazole] analogs with potent and more selective antiprogestational activity compared to antiglucocorticoid activity than mifepristone. In the present study, we replaced the 4'-dimethylaminophenyl group of mifepristone with the benzoxazol group to give 5a-d. We also prepared the 17β-formamido analogs 6a,b using a new synthetic strategy via the intermediate epoxide 21. These compounds were evaluated for their antagonist hormonal properties using the T47D cell-based alkaline phosphatase assay and the A549 cell-based functional assay. Compound 5c showed potent antagonist activity at GR with better selectivity for GR versus PR than mifepristone and is a promising lead for further development.     

Synthesis and progesterone receptor antagonist activities of 6-aryl benzimidazolones and benzothiazolones

Novel 6-aryl benzimidazolones and benzothiazolones were prepared and examined as bioisosteres of the recently reported 6-aryl dihydroquinolines (1) for progesterone receptor (PR) antagonist activities. PR antagonist activities increased when compounds 9c-f possessed a more lipophilic group at position-1 and pendent aryl moiety para to NH moiety. Furthermore, conversion of carbonyl moiety of 9e,f to the thio-carbonyl led to benzoimidazolethiones 15a,b with significantly improved potency and binding affinity.     

8-alkylthio-6-thio-substituted theophylline analogues as selective noncompetitive progesterone receptor antagonists

The progesterone receptor (PR) plays a key role in reproduction and is important in cancers of the reproductive tract. Current PR antagonists usually compete for progestin binding in the PR ligand-binding pocket and often exhibit cross-binding with other members of the steroid receptor family. Using stably transfected cells expressing reporter genes, a set of ～150 theophylline analogues were screened for their ability to inhibit progesterone, estrogen, glucocorticoid and androgen signaling. The structure-activity studies presented here identify branched 8-alkylthio-6-thio-substitutions of theophylline as selective PR inhibitors. 6-Thio-8-(2-ethylbutyl)thiotheophylline (51), the most extensively studied derivative, does not act by competing with progestins for binding in the ligand-binding pocket of PR. It demonstrated the ability to inhibit the mouse mammary tumor virus (MMTV)-luciferase reporter and endogenous PR-regulated alkaline phosphatase activity in T47D breast cancer cells. Compound 51 is the lead member of a novel class of PR inhibitors that act outside the PR ligand-binding pocket, thus serving as a novel probe to investigate PR action and a lead for further development.     

Molecular and pharmacological properties of a potent and selective novel nonsteroidal progesterone receptor agonist tanaproget

Progesterone receptor (PR) agonists have several important applications in women's health, such as in oral contraception and post-menopausal hormone therapy. Currently, all PR agonists used clinically are steroids. Because of their interactions with other steroid receptors, steroid-metabolizing enzymes, or other steroid-signaling pathways, these drugs can pose significant side effects in some women. Efforts to discover novel nonsteroidal PR agonists with improved biological properties led to the discovery of tanaproget (TNPR). TNPR binds to the PR from various species with a higher relative affinity than reference steroidal progestins. In T47D cells, TNPR induces alkaline phosphatase activity with an EC(50) value of 0.1 nm, comparable with potent steroidal progestins such as medroxyprogesterone acetate (MPA) and trimegestone (TMG), albeit with a reduced efficacy ( approximately 60%). In a mammalian two-hybrid assay to measure PR agonist-induced interaction between steroid receptor co-activator-1 and PR, TNPR showed similar potency (EC(50) value of 0.02 nm) and efficacy to MPA and TMG. Importantly, in key animal models such as the rat ovulation inhibition assay, TNPR demonstrates full efficacy and an enhanced progestational potency (30-fold) when compared with MPA and TMG. Furthermore, TNPR has relatively weak interactions with other steroid receptors and binding proteins and little effect on cytochrome P450 metabolic pathways. Finally, the three-dimensional crystal structure of the PR ligand binding domain with TNPR has been delineated to demonstrate how this nonsteroidal ligand achieves its high binding affinity. Therefore, TNPR is a structurally novel and very selective PR agonist with an improved preclinical pharmacological profile.     

A structural and in vitro characterization of asoprisnil: a selective progesterone receptor modulator

Selective progesterone receptor modulators (SPRMs) have been suggested as therapeutic agents for treatment of gynecological disorders. One such SPRM, asoprisnil, was recently in clinical trials for treatment of uterine fibroids and endometriosis. We present the crystal structures of progesterone receptor (PR) ligand binding domain complexed with asoprisnil and the corepressors nuclear receptor corepressor (NCoR) and SMRT. This is the first report of steroid nuclear receptor crystal structures with ligand and corepressors. These structures show PR in a different conformation than PR complexed with progesterone (P4). We profiled asoprisnil in PR-dependent assays to understand further the PR-mediated mechanism of action. We confirmed previous findings that asoprisnil demonstrated antagonism, but not agonism, in a PR-B transfection assay and the T47D breast cancer cell alkaline phosphatase activity assay. Asoprisnil, but not RU486, weakly recruited the coactivators SRC-1 and AIB1. However, asoprisnil strongly recruited the corepressor NCoR in a manner similar to RU486. Unlike RU486, NCoR binding to asoprisnil-bound PR could be displaced with equal affinity by NCoR or TIF2 peptides. We further showed that it weakly activated T47D cell gene expression of Sgk-1 and PPL and antagonized P4-induced expression of both genes. In rat leiomyoma ELT3 cells, asoprisnil demonstrated partial P4-like inhibition of cyclooxygenase (COX) enzymatic activity and COX-2 gene expression. In the rat uterotrophic assay, asoprisnil demonstrated no P4-like ability to oppose estrogen. Our data suggest that asoprisnil differentially recruits coactivators and corepressors compared to RU486 or P4, and this specific cofactor interaction profile is apparently insufficient to oppose estrogenic activity in rat uterus.     

Potent nonsteroidal progesterone receptor agonists: synthesis and SAR study of 6-aryl benzoxazines

Novel 6-aryl benzoxazines were prepared and examined as progesterone receptor (PR) modulators. In contrast to the structurally related 6-aryl dihydroquinoline PR antagonists, the 6-aryl benzoxazines were potent PR agonists. Compounds 4e, 5b, and 6a with the 2,4,4-trimethyl-1,4-dihydro-2H-benzo[d][1,3]oxazine core were the most potent PR agonists in the series with sub-nanomolar activities (EC(50) 0.20-0.35nM). Compound 6a was more potent than progesterone (P4) in the in vivo decidualization assay in an ovariectomized female rat model by subcutaneous administration with an ED(50) of 1.5mg/kg (vs 5.62mg/kg for P4).     

Discovery, characterization and SAR of gambogic acid as a potent apoptosis inducer by a HTS assay

Gambogic acid (2), a natural product isolated from the resin of Garcinia hurburyi tree, was discovered to be a potent apoptosis inducer using our cell- and caspase-based high-throughput screening assays. Gambogic acid was found to have an EC(50) of 0.78 microM in the caspase activation assay in T47D breast cancer cells. The apoptosis-inducing activity of gambogic acid was further characterized by a nuclear fragmentation assay and flow cytometry analysis in human breast tumor cells T47D. Gambogic acid was found to induce apoptosis independent of cell cycle, which is different from paclitaxel that arrests cells in the G2/M phase. To understand the structure-activity relationship (SAR) of gambogic acid, derivatives of 2 with modifications to different function groups were prepared. SAR studies of gambogic acid, as measured by the caspase activation assay, showed that the 9,10 carbon-carbon double bond of the alpha,beta-unsaturated ketone is important for biological activity, while the 6-hydroxy and 30-carboxy group can tolerate a variety of modifications. The importance of the 9,10 carbon-carbon double bond was confirmed by the traditional growth inhibition assay. The high potency of 2 as an inducer of apoptosis, its novel mechanism of action, easy isolation and abundant supply, as well as the fact that it is amenable to chemical modification, makes gambogic acid an attractive molecule for the development of anticancer agents.     

Discovery of 4-aryl-4H-chromenes as a new series of apoptosis inducers using a cell- and caspase-based high-throughput screening assay. 2. Structure-activity relationships of the 7- and 5-, 6-, 8-positions

As a continuation of our efforts to discover and develop the apoptosis inducing 4-aryl-4H-chromenes as novel anticancer agents, we explored the SAR of 4-aryl-4H-chromenes with modifications at the 7- and 5-, 6-, 8-positions. It was found that a small hydrophobic group, such as NMe2, NH2, NHEt, and OMe, is preferred at the 7-position. Di-substitution at either the 5,7-positions or the 6,7-positions generally led to a large decrease in potency. Di-substitution at the 7,8-positions, in general, was found to result in potent compounds. 7-NMe2, 7-NHEt, 7-OMe, and 7,8-di-NH2 analogs were found to have similar SAR for the 4-aryl group, and several 7-substituted and 7,8-di-substituted analogs were found to have similar potencies as the lead compound MX58151 (2a) both as caspase activators and inhibitors of cell proliferation.     

Synthesis and identification of novel 11beta-aryl-4',5'-dihydrospiro[estra-4,9-diene-17beta,4'-oxazole] analogs with dissociated antiprogesterone activities

A series of novel 11beta-aryl-4',5'-dihydrospiro[estra-4,9-diene-17beta,4'-oxazole] analogs have been evaluated for their antagonist hormonal properties using the T47D cell-based alkaline phosphatase assay and the A549 cell-based functional assay. Some of the compounds showed highly potent, and more selective antiprogestational activity against antiglucocorticoid activity than mifepristone (RU 486).