Novel (2E,4E,6Z)-7-(2-alkoxy-3,5-dialkylbenzene)-3-methylocta-2,4,6-trienoic acid retinoid X receptor modulators are active in models of type 2 diabetes.

Previous data have shown that RXR-selective agonists (e.g., 3 and 4) are insulin sensitizers in rodent models of non-insulin-dependent diabetes mellitus (NIDDM). Unfortunately, they also produce dramatic increases in triglycerides and profound suppression of the thyroid hormone axis. Here we describe the design and synthesis of new RXR modulators that retain the insulin-sensitizing activity of RXR agonists but produce substantially reduced side effects. These molecules bind selectively and with high affinity to RXR and, unlike RXR agonists, do not activate RXR homodimers. To further evaluate the antidiabetic activity of these RXR modulators, we have designed a concise and systematic structure-activity relationship around the 2E,4E,6Z-7-aryl-3-methylocta-2,4,6-trienoic acid scaffold. Selected compounds have been evaluated using insulin-resistant rodents (db/db mice) to characterize effects on glucose homeostasis. Our studies demonstrate the effectiveness of RXR modulators in lowering plasma glucose in the db/db mouse model.

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.

Effects of isosteric pyridone replacements in androgen receptor antagonists based on 1,2-dihydro- and 1,2,3,4-tetrahydro-2,2-dimethyl-6-trifluoromethyl-8-pyridono[5,6-g]quin olines.

A series of nonsteroidal human androgen receptor (hAR) antagonists based on 8-substituted 1,2-dihydro- and 1,2,3,4-tetrahydro-2,2-dimethyl-6-trifluoromethylpyrido[3,2-g]quin olines was synthesized. Compounds in this series were tested for the ability to bind to hAR and inhibit hAR-dependent transcription in a mammalian cellular background.

Nonsteroidal progesterone receptor antagonists based on 6-thiophenehydroquinolines.

Synthesis and biological evaluation of 6-thiophene 1,2-dihydro or 1,2,3,4-tetrahydroquinoline derivatives resulted in a number of potent nonsteroidal antiprogestins.

4-Alkyl- and 3,4-dialkyl-1,2,3,4-tetrahydro-8-pyridono[5,6-g]quinolines: potent, nonsteroidal androgen receptor agonists.

A series of human androgen receptor (hAR) agonists based on 4-alkyl-; 4,4-dialkyl-; and 3,4-dialkyl-1,2,3,4-tetrahydro-8-pyridono[5,6-g]quinoline was synthesized and evaluated in competitive receptor binding assays and an androgen receptor cotransfection assay in a mammalian cell background. A number of compounds in this series demonstrated activity equal to or better than dihydrotestosterone in both assays and represent a novel class of compounds for use in androgen replacement therapy.

Nonsteroidal androgen receptor agonists based on 4-(trifluoromethyl)-2H-pyrano[3,2-g]quinolin-2-one.

A series of 2H-pyrano[3,2-g]quinolin-2-ones was prepared and tested for the ability to modulate the transcriptional activity of the human androgen receptor (hAR). The parent compound, 4-(trifluoromethyl)-2H-pyrano[3,2-g]quinolin-2-one, displayed moderate interaction with hAR, but substituted analogues were potent hAR modulators in vitro as measured by an hAR cotransfection assay in CV-1 cells and bound to hAR with high affinity in a whole cell assay. Several analogues were able to activate hAR-mediated gene transcription more potently and efficaciously than dihydrotestosterone.

Switching androgen receptor antagonists to agonists by modifying C-ring substituents on piperidino[3,2-g]quinolinone.

New nonsteroidal human androgen receptor (hAR) agonists were developed from an hAR antagonist pharmacophore, 2(1H)-piperidino[3,2-g]quinolinone. (+/-)-trans-7,8-Diethyl-4-trifluoromethyl-2(H)-piperidino-[3,2-g]quinoli none was synthesized and demonstrated potent hAR agonist activity (EC50=3 nM) in the cell-based cotransfection assay and high binding affinity (Ki=16 nM) in the competitive receptor binding assay.

5-Aryl-1,2,3,4-tetrahydrochromeno[3,4-f]quinolin-3-ones as a novel class of nonsteroidal progesterone receptor agonists: effect of A-ring modification.

Optimization of the 1,2-dihydroquinoline A-ring of a nonsteroidal human progesterone receptor (hPR) agonist pharmacophore (1) was performed by using the cotransfection and receptor binding assays as guides. The 3-keto group was discovered to regain the potent agonist activity which was lost upon removal of the 3,4-olefin, and it led to a novel hPR agonist series, 5-aryl-1,2,3,4-tetrahydrochromeno[3, 4-f]quinolin-3-ones. The new progestins demonstrated potent hPR agonist activity in the cotransfection assay and high binding affinity similar to progesterone. T47D human breast cancer cell line was employed for further characterization of the new progestins and a number of reference analogues. It was found that the new 3-keto analogues showed full agonist activity in the T47D assay, while the reference compounds from other related nonsteroidal hPR agonist series exhibited only partial agonist activity.

Estrogen receptor beta activates the human retinoic acid receptor alpha-1 promoter in response to tamoxifen and other estrogen receptor antagonists, but not in response to estrogen.

Human estrogen receptor-alpha (hERalpha) or -beta (hERbeta) transfected into Hep G2 or COS1 cells each responded to estrogen to increase transcription from an estrogen-responsive element (ERE)-driven reporter vector with similar fold induction through a classical mechanism involving direct receptor binding to DNA. ER antagonists inhibited this estrogen induction through both hERalpha and hERbeta, although raloxifene was more potent through ERalpha than ERbeta, and tamoxifen was more potent via ERbeta than ERalpha. We have shown previously that estrogen stimulated the human retinoic acid receptor-alpha-1 (hRARalpha-1) promoter through nonclassical EREs by a mechanism that was ERalpha dependent, but that did not involve direct receptor binding to DNA. We show here that in contrast to hERalpha, hERbeta did not induce reporter activity driven by the hRARalpha-1 promoter in the presence of estrogen. While hERbeta did not confer estrogen responsiveness on this promoter, it did elicit transcriptional activation in the presence of 4-hydroxytamoxifen (4-OH-Tam). Additionally, this 4-OH-Tam agonist activity via ERbeta was completely blocked by estrogen. Like ERalpha, transcriptional activation of this promoter by ERbeta was not mediated by direct receptor binding to DNA. While hERalpha was shown to act through two estrogen-responsive sequences within the promoter, hERbeta acted only at the 3'-region, through two Sp1 sites, in response to 4-OH-Tam. Other ER antagonists including raloxifene, ICI-164,384 and ICI-182,780 also acted as agonists through ERbeta via the hRARalpha-1 promoter. Through the use of mutant and chimeric receptors, it was shown that the 4-OH-Tam activity via ERbeta from the hRARalpha-1 promoter in Hep G2 cells required the amino-terminal region of ERbeta, a region that was not necessary for estrogen-induced ERbeta activity from an ERE in Hep G2 cells. Additionally, the progesterone receptor (PR) antagonist RU486 acted as a weak (IC50 >1 microM) antagonist via hERalpha and as a fairly potent (IC50 approximately 200 nM) antagonist via hERbeta from an ERE-driven reporter in cells that do not express PR. Although RU486 bound only weakly to ERalpha or ERbeta in vitro, it did bind to ERbeta in whole-cell binding assays, and therefore, it is likely metabolized to an ERbeta-interacting compound in the cell. Interestingly, RU486 acted as an agonist through ERbeta to stimulate the hRARalpha-1 promoter in Hep G2 cells. These findings may have ramifications in breast cancer treatment regimens utilizing tamoxifen or other ER antagonists and may explain some of the known estrogenic or antiestrogenic biological actions of RU486.

5-Benzylidene 1,2-dihydrochromeno[3,4-f]quinolines, a novel class of nonsteroidal human progesterone receptor agonists.

A novel series of nonsteroidal progestins, 5-benzylidene-1, 2-dihydrochromeno[3,4-f]quinolines (2), was discovered, and a preliminary structure-activity relationship study around the 5-benzylidene ring generated several potent human progesterone receptor agonists (compounds 8, 16). These new progestins showed biological activities (EC50 = 5.7 and 7.6 nM) similar to progesterone (EC50 = 2.9 nM) in the cotransfection assay with high efficacy (132% and 166%) and binding affinity (Ki = 0.66 and 0.83 nM) similar to medroxyprogesterone acetate (MPA) (Ki = 0.34 nM). A representative analogue, 8, demonstrated similar oral potency to MPA in the uterine wet weight/mammary gland morphology assay in ovariectomized rats.

Discovery and preliminary SAR studies of a novel, nonsteroidal progesterone receptor antagonist pharmacophore.

A series of 6-aryl-1,2-dihydro-2,2,4-trimethylquinolines was synthesized and tested for functional activity on the human progesterone receptor isoform B (hPR-B) in mammalian (CV-1) cells. The lead compound LG001447 (1,2-dihydro-2,2, 4-trimethyl-6-phenylquinoline) was discovered via directed high throughput screening of a defined chemical library utilizing an hPR-B cotransfection assay. Electron-withdrawing substituents at the meta position of the C(6) aryl group afforded substantial improvements in hPR modulatory activity. Several analogues were able to potently block the effects of progesterone in vitro. Two compounds, 10 (LG120753) and 11 (LG120830) with potencies comparable or equal to the steroidal hPR antagonist onapristone (ZK98,299), were demonstrated to act as antiprogestins in vivo after oral administration to rodents. This is the first disclosure of orally active nonsteroidal antiprogestins.

Synthesis and biological activity of a novel series of nonsteroidal, peripherally selective androgen receptor antagonists derived from 1,2-dihydropyridono[5,6-g]quinolines.

A new nonsteroidal antiandrogenic pharmacophore has been discovered using cell-based cotransfection assays with human androgen receptor (hAR). This series of AR antagonists is structurally characterized by a linear tricyclic 1,2-dihydropyridono[5,6-g]quinoline core. Analogues inhibit AR-mediated reporter gene expression and bind to AR as potently as or better than any known AR antagonists. Several analogues also showed excellent in vivo activity in classic rodent models of AR antagonism, inhibiting growth of rat ventral prostate and seminal vesicles, without accompanying increases in serum gonadotropin and testosterone levels, as is seen with other AR antagonists. Investigations of structure-activity relationships surrounding this pharmacophore resulted in molecules with complete specificity for AR, antagonist activity on an AR mutant commonly observed in prostate cancer patients, and improved in vivo efficacy. Molecules based on this series of compounds have the potential to provide unique and effective clinical opportunities for treatment of prostate cancer and other androgen-dependent diseases.

5-Aryl-1,2-dihydrochromeno[3,4-f]quinolines: a novel class of nonsteroidal human progesterone receptor agonists.

The development of a novel class of nonsteroidal human progesterone receptor (hPR) agonists, 5-aryl-1,2-dihydro-5H-chromeno[3,4-f]quinolines 2, is described. The introduction of a 5-aryl group into the 1,2-dihydrocoumarino[3,4-f]quinoline core 1 is the key for progestational activities. The structure-activity relationship (SAR) studies of the 5-aryl substituents generated a series of potent hPR agonists, which exhibited similar biological activity (EC50 = 8-30 nM) to the natural hormone progesterone (EC50 = 2.9 nM) in cell-based assays with efficacies ranging from 28% to 96%. Most of the analogues displayed similar or greater binding affinity (Ki = 0.41-3.6 nM) than progesterone (Ki = 3.5 nM). Three representative analogues (13, 15, and 24) demonstrated in vivo activities in mammary gland morphology/uterine wet weight assay in ovariectomized rats.

5-Aryl-1,2-dihydro-5H-chromeno[3,4-f]quinolines as potent, orally active, nonsteroidal progesterone receptor agonists: the effect of D-ring substituents.

Several 5-(4-chlorophenyl)-1,2-dihydro-5H-chromeno[3,4-f]quinolines were prepared to determine the effects of substitution at C(8) and C(9) on the progestational activity of this pharmacophore. In combination with a halogen (F or Cl) at C(9), replacement of the C(5) aryl group with variously substituted aryl groups resulted in optimization of the progestational activity, affording compounds with in vitro activity greater than that of progesterone as measured by a cotransfection assay using human progesterone receptor subtype-B (hPR-B). Binding affinities (Ki) to hPR-A were subnanomolar in many cases. These in vitro effects were verified in vivo using a rodent model. Compound 10 (LG120794, 9-chloro-5-(4-chlorophenyl)-1,2-dihydro-2,2,4-trimethyl-5H-chromeno++ +[3,4-f] quinoline) was more potent than medroxyprogesterone acetate at counterpoising the effects of estradiol benzoate in the uterine wet weight assay using immature rats.

5-Alkyl 1,2-dihydrochromeno[3,4-f]quinolines: a novel class of nonsteroidal progesterone receptor modulators.

A series of nonsteroidal human progesterone receptor (hPR) agonists, 5-alkyl 1,2-dihydrochromeno[3,4-f]quinolines, was synthesized and evaluated in cotransfection and competitive receptor binding assays. The 5-alkyl substitution was shown to be responsible for the agonist activity and substitution at C9 dramatically enhanced the potency. A number of analogues in this series showed activities similar to or better than progesterone in the cotransfection and binding assays and analogue 15 exhibited similar in vivo activity as medroxyprogesterone acetate (MPA) in murine uterine wet weight/mammary gland morphology assays.

New nonsteroidal androgen receptor modulators based on 4-(trifluoromethyl)-2(1H)-pyrrolidino[3,2-g] quinolinone.

A series of 2(1H)-pyrrolidino[3,2-g]quinolinones was prepared and tested for the ability to modulate the transcriptional activity of the human androgen receptor (hAR). The parent compound, 4-(trifluoromethyl)-2(1H)-pyrrolidino[3,2-g]quinolinone, displayed moderate interaction with hAR, but more substituted analogues, particularly 6,7-disubstituted compounds, were potent hAR agonists in vitro.

Nonsteroidal progesterone receptor antagonists based on a conformationally-restricted subseries of 6-aryl-1,2-dihydro-2,2,4-trimethylquinolines.

A series of nonsteroidal human progesterone receptor (hPR) antagonists based on conformationally-restricted analogues of a 6-aryl-1,2-dihydro-2,2,4-trimethylquinoline pharmacophore were synthesized and evaluated for their ability to bind to the human progesterone receptor and inhibit progesterone-stimulated reporter gene expression in mammalian cells.

Androgen receptor-mediated antagonism of estrogen-dependent low density lipoprotein receptor transcription in cultured hepatocytes.

Postmenopausal women receiving hormone replacement therapy have a lower risk of coronary heart disease than women who do not receive hormone treatment. Multiple mechanisms are likely to underlie estrogen's cardioprotective action, including lowering of plasma low density lipoprotein (LDL) cholesterol. Using an in vitro system exhibiting normal regulation of LDL receptor (LDLR) gene transcription, we show that 17beta-estradiol activates the LDLR promoter in transiently transfected HepG2 cells. LDLR activation by estrogen in HepG2 cells is dependent on the presence of exogenous estrogen receptor, and the estrogen-responsive region of the LDLR promoter colocalizes with the sterol response element previously identified. The estrogen response is concentration dependent, saturable, and sensitive to antagonism by estrogen receptor antagonists. Further, we show that compounds with androgen receptor agonist activity attenuate the estrogen-induced up-regulation of LDLR in our model system. Progestins with androgen receptor agonist activity, such as medroxyprogesterone acetate, also suppress estrogen's effects on LDLR expression through their androgenic properties. Characterization of the interplay between these hormone receptors on the LDLR in vitro system may allow a better understanding of the actions of sex steroids on LDLR gene expression and their roles in cardiovascular disease.

Estrogen and estrogen receptor antagonists stimulate transcription from the human retinoic acid receptor-alpha 1 promoter via a novel sequence.

We and others previously reported that up-regulation of retinoic acid receptor-alpha (RAR alpha) RNA and protein levels is elicited by estrogen in human breast cancer cells. We set out to determine the mechanism by which estrogen up-regulates RAR alpha. Cloning of 500 bp of the human (h) RAR alpha 1 promoter has been reported previously; we obtained this 500-bp DNA sequence by PCR techniques from human genomic DNA and tested its activity in the context of a luciferase-containing reporter vector in Hep G2 cell contransactivation assays. Estradiol elicited a 6- to 8-fold increase in luciferase activity from the reporter vector driven by hRAR alpha promoter sequence between -491 and +36 bp that was dependent on the presence of contransfected estrogen receptor (ER). Analysis of various truncated versions of this promoter sequence indicated that two regions of the sequence are sensitive to estrogen stimulation. The first resides in the region -49 to -79 bp upstream from the transcription start site and conferred approximately 2-fold activation by estrogen. This region does not contain a consensus estrogen response element, and ER binding to this DNA sequence was not observed. The second responsive sequence lies at -455 to -491 bp and conferred in additional 4- to 6-fold activation by estrogen. This upstream sequence contains two A/TGGTCA half-sites; however, direct binding of ER to this sequence was not observed. Additionally, ER DNA-binding domain mutants that are not capable of binding to DNA were just as effective as wild type ER in their ability to confer estrogen responsiveness to the RAR alpha promoter, implying that ER DNA-binding ability is not required for the estrogen-induced increase in transcriptional activity. Mutation of either half-site or of an additional immediate downstream sequence in the context of the -491 to +36 bp construct reduced the luciferase activity induction by estrogen from 6-fold to 1.5- to 2-fold. Placement of the region between -455 to -491 bp upstream of an SV40 promoter-driven luciferase vector conferred approximately 20- to 30-fold stimulation of luciferase activity by estrogen in an ER-dependent manner. The ER antagonists, 4-hydroxy-tamoxifen, keoxifene, and ICI 164384, each acted as weak agonist via the hRAR alpha promoter in contransactivation assays, exhibiting 20-30% of the efficacy that was demonstrated by estradiol. Interestingly, upon treatment of MCF7 cells with estradiol or the ER antagonists, increased levels of RAR alpha RNA and protein were observed with the antagonists as well as with estrogen.