Synthesis and SAR study of novel pseudo-steroids as potent and selective progesterone receptor antagonists.

Synthesis of novel 7-pseudo-steroids 1c has been achieved from trenbolone 3 via an efficient 14 step sequence with overall yields of 10-15%. Various substitutions were incorporated at both the aromatic side chain as well as the D ring. The orientation of aromatic side chain at C10 plays a crucial role for progesterone receptor (PR) activity. Compound 2a (T47D=1nM) with -NMe(2) para to the aromatic group along with spirofurane groups in the D ring was the optimal substitution. All compounds were also evaluated for glucocorticoid receptor (GR) antagonist activities in vivo in a rat and found efficacious in uterine complement C3 assay via the oral route of administrations.

A selective androgen receptor modulator with minimal prostate hypertrophic activity restores lean body mass in aged orchidectomized male rats.

Androgens are required for the maintenance of normal sexual activity in adulthood and for enhancing muscle growth and lean body mass in adolescents and adults. Androgen receptor (AR) ligands with tissue selectivity (selective androgen receptor modulators, or SARMs) have potential for treating muscle wasting, hypogonadism of aging, osteoporosis, female sexual dysfunction, and other indications. JNJ-37654032 is a nonsteroidal AR ligand with mixed agonist and antagonist activity in androgen-responsive cell-based assays. It is an orally active SARM with muscle selectivity in orchidectomized rat models. It stimulated growth of the levator ani muscle with ED(50) 0.8 mg/kg, stimulating maximal growth at a dose of 3mg/kg. In contrast, it stimulated ventral prostate growth to 21% of its full size at 3mg/kg. At the same time, JNJ-37654032 reduced prostate weight in intact rats by 47% at 3mg/kg, while having no inhibitory effect on muscle. Using magnetic resonance imaging to monitor body composition, JNJ-37654032 restored about 20% of the lean body mass lost following orchidectomy in aged rats. JNJ-37654032 reduced follicle-stimulating hormone levels in orchidectomized rats and reduced testis size in intact rats. JNJ-37654032 is a potent prostate-sparing SARM with the potential for clinical benefit in muscle-wasting diseases.

Insight from molecular modeling into different conformation and SAR of natural steroids and unnatural 7-oxa-steroids.

Replacement of the 7-CH2 group of natural steroid with an oxygen atom led to identification of unnatural 7-oxa-steroids as potent and selective progesterone receptor antagonists. The unnatural 7-oxa-steroids exhibited a different structure-activity relationship (SAR) from natural steroids. Molecular modeling demonstrated that the switch of carbon to oxygen in the B-ring results in a subtle conformational change of the tetracyclic skeleton and induces a remarkable spatial shift at the terminal end of the side chain of the D-ring. This shift causes the phenyl ring on the D-ring to form a perfect parallel-displaced form of pi-pi interaction with the phenyl ring of Phe794. The unnatural 7-oxa-steroids were orally active in a rat complement C3 assay and showed comparable pharmacokinetic and metabolic profiles to those of the natural steroid, mifepristone.

Design, synthesis, and in vivo SAR of a novel series of pyrazolines as potent selective androgen receptor modulators.

A novel series of pyrazolines 2 have been designed, synthesized, and evaluated by in vivo screening as tissue-selective androgen receptor modulators (SARMs). Structure-activity relationships (SAR) were investigated at the R1 to R6 positions as well as the core pyrazoline ring and the anilide linker. Overall, strong electron-withdrawing groups at the R1 and R2 positions and a small group at the R5 and R6 position are optimal for AR agonist activity. The (S)-isomer of 7c exhibits more potent AR agonist activity than the corresponding (R)-isomer. (S)-7c exhibited an overall partial androgenic effect but full anabolic effect via oral administration in castrated rats. It demonstrated a noticeable antiandrogenic effect on prostate in intact rats with endogenous testosterone. Thus, (S)-7c is a tissue-selective nonsteroidal androgen receptor modulator with agonist activity on muscle and mixed agonist and antagonist activity on prostate.

A selective androgen receptor modulator that reduces prostate tumor size and prevents orchidectomy-induced bone loss in rats.

The pharmacological activity of JNJ-26146900 is described. JNJ-26146900 is a nonsteroidal androgen receptor (AR) ligand with tissue-selective activity in rats. The compound was evaluated in in vitro and in vivo models of AR activity. It binds to the rat AR with a K(i) of 400nM and acts as a pure androgen antagonist in an in vitro cell-based assay. Its in vitro profile is similar to the androgen antagonist bicalutamide (Casodex). In intact rats, JNJ-26146900 reduces ventral prostate weight with an oral potency (ED(50)) of 20-30mg/kg, again comparable to that of bicalutamide. JNJ-26146900 prevented prostate tumor growth in the Dunning rat model, maximally inhibiting growth at a dose of 10mg/kg. It slowed tumor growth significantly in a CWR22-LD1 mouse xenograft model of human prostate cancer. It was tested in aged male rats for its ability to prevent bone loss and loss of lean body mass following orchidectomy. After 6 weeks of dosing, bone volume decreased by 33% in orchidectomized versus intact vehicle-treated rats with a probability (P) of less than 0.05, as measured by micro-computerized tomography analysis. At a dose of 30mg/kg, JNJ-26146900 significantly reduced castration-induced tibial bone loss as indicated by the following parameters: bone volume, trabecular connectivity, trabecular number and spacing between trabeculae. Bone mineral density decreased from 229+/-34mg/cm(3) of hydroxyapatite to 166+/-26mg/cm(3) following orchidectomy, and was maintained at 194+/-20mg/cm(3) with JNJ-26146900 treatment (P<0.05 relative to orchidectomy alone). Using magnetic resonance imaging, the compound was found to partially prevent orchidectomy-induced loss of lean body mass. Our data show that selective androgen receptor modulators (SARMs) have the potential for anabolic effects on bone and muscle while maintaining therapeutic efficacy in prostate cancer.

Novel chromene-derived selective estrogen receptor modulators useful for alleviating hot flushes and vaginal dryness.

A novel SERM (selective estrogen receptor modulators), 1-(R), a chromene-derived bisbenzopyran, was discovered to alleviate hot flushes and effectively increase vaginal fluidity in rats. Moreover, 1-(R) was found to have beneficial effects on plasma cholesterol and bone metabolism while maintaining antiestrogenic activity in the uterus. The biological profile of its enantiomer 1-(S) was also evaluated.

Molecular properties and preclinical pharmacology of JNJ-1250132, a steroidal progesterone receptor modulator that inhibits binding of the receptor to DNA in vitro.

Progesterone receptor modulators have diverse potential therapeutic uses, including the treatment of endometriosis, uterine fibroids and breast cancer. Here we describe the molecular properties and preclinical pharmacology of a new steroidal progestin antagonist, JNJ-1250132. The compound is a high affinity ligand for the progesterone receptor, possessing cross-reactivity with other steroid receptors comparable to that of steroidal antagonists such as mifepristone. It inhibits progestin-inducible alkaline phosphatase gene expression in T47D human breast cancer cells, and also inhibits their in vitro proliferation. It inhibits gestation in rats and progesterone-dependent endometrial transformation in rabbits with efficacies comparable to mifepristone. Like mifepristone, it is a glucocorticoid antagonist in vivo. In cell-free DNA binding assays, the compound inhibits binding of the human progesterone receptor to a progesterone response element, and thus is similar to onapristone in this regard. In contrast, as judged by proteolytic analysis, JNJ-1250132 induces a receptor conformation more similar to that induced by mifepristone, which promotes receptor binding to DNA. Therefore, JNJ-1250132 has unique effects on the progesterone receptor that may translate into a novel clinical profile.

Novel phosphorus-containing 17beta-side chain mifepristone analogues as progesterone receptor antagonists.

A novel series of steroidal compounds were designed and synthesized with various phosphorus-containing groups on the 17beta-side chain as progesterone receptor antagonists. The structure-activity relationships of these compounds are discussed. Selected compounds were tested in an rat progesterone-sensitive assay. Some of these compounds are more potent than mifepristone, with a better selectivity profile in differentiating progesterone receptor from glucocorticoid receptor.

The putative tumor suppressor deleted in malignant brain tumors 1 is an estrogen-regulated gene in rodent and primate endometrial epithelium.

Deleted in malignant brain tumors 1 (DMBT1) is a candidate suppressor of malignancies of the brain, lung, gut, and breast. We have been studying gene expression in the uterus in the presence of estrogens and their antagonists. Here, we show that DMBT1 RNA levels are robustly increased by estrogen treatment in the uteri of ovariectomized monkeys and rats. In monkeys, the progestin antagonist mifepristone inhibits estrogen-dependent uterine proliferation. As determined by a microarray experiment and quantitative analysis of RNA levels, mifepristone inhibited estrogenic induction of DMBT1. DMBT1 was not expressed in intact monkeys that were treated with a gonadotropin agonist to suppress steroidogenesis. An in vitro transfection study with human DMBT1 promoter constructs showed that an Alu site approximately 3000 nucleotides upstream of the gene mediates estrogenic regulation. Surprisingly, the estrogen antagonists tamoxifen, raloxifene, and ICI 182,780 also induced gene expression via this Alu site. Rodents represent a more convenient model system for studying uterine biology than monkeys. In rats, uterine DMBT1 RNA levels were dramatically up-regulated by estrogen. Consistent with the transfection study, tamoxifen and raloxifene increased DMBT1 RNA levels in vivo, but ICI 182,780 inhibited an estrogen-induced increase. Immunohistochemical studies showed that DMBT1 is specifically induced in glandular and luminal epithelia of the rat endometrium. Our experiments establish that DMBT1 is an estrogen-responsive gene with a possible role in endometrial proliferation or differentiation, and they have implications for the putative tumor suppressive and mucosal protective functions of DMBT1 in the uterus.

Cell type-specific bidirectional regulation of the glucocorticoid-induced leucine zipper (GILZ) gene by estrogen.

Estrogen has numerous beneficial physiological actions; however, by acting as a mitogen, it plays a significant role in the induction and maintenance of breast cancer. Although the positive effects of estrogen on gene expression are well described, negative gene regulation is not. Using microarray analysis, we identified 27 genes that were up-regulated and 20 that were down-regulated by estrogen in MCF-7 human breast cancer cells. One gene encoding GILZ (glucocorticoid-induced leucine zipper protein), a putative apoptosis-regulating transcription factor, is rapidly down-regulated by estrogen in these cells. Estrogen antagonists block the down-regulation. The region of the GILZ promoter between nucleotides -104 and -69 mediates both basal activity and estrogen-dependent down-regulation in MCF-7 cells. This region contains a functional Oct-1 binding site and a cyclic AMP response element binding protein (CREB) binding site. The same DNA region mediates up-regulation by estrogen in HeLa and HEK293 cells, indicating that cell-specific factors are involved in estrogen regulation of this gene. The estrogen receptor (ER) is present in GILZ promoter protein complexes, but it does not bind directly to the promoter itself, as the DNA-binding domain of the estrogen receptor is not required for down-regulation. Elimination of the CREB binding site blocks both basal activity and estrogen regulation. Our results suggest that ER action at the CRE may mediate estrogen-dependent, cell-specific regulation of this gene.

Pharmacological characterization of an imidazolopyrazole as novel selective androgen receptor modulator.

Selective androgen receptor modulators (SARMs) are androgens with tissue-selective activity. SARMs that have anabolic activity on muscle while having minimal stimulatory activity on prostate are classified as SARM agonists. They can be used to prevent the loss of lean body mass that is associated with cancer, immunodeficiency, renal disease and aging. They may also have anabolic activity on bone; thus, unlike estrogens, they may reverse the loss of bone strength associated with aging or hypogonadism. Our in-house effort on SARM program discovers a nonsteroidal androgen receptor ligand with a unique imidazolopyrazole moiety in its structure. In vitro, this compound is a weak androgen receptor binder and a weak androgen agonist. Despite this, in orchidectomized mature rats it is an effective SARM agonist, with an ED(50) on levator ani muscle of 3.3mg/kg and an ED(50) on ventral prostate of >30mg/kg. It has its maximal effect on muscle at the dose of 10mg/kg. In addition, this compound has mixed agonistic and antagonistic activities on prostate, reducing the weight of that tissue in intact rats by 22% at 10mg/kg. The compound does not have significant effect on gonadotropin levels or testosterone levels in both orchidectomized and intact male rats. It does not have notable progestin, estrogen or glucocorticoid agonistic or antagonistic activity in rats. In a female sexual behavior model, it improves the sexual desire of ovariectomized female rats for sexually mature intact males over nonsexually ovariectomized females. Overall, the imidazolopyrazole is a potent prostate-sparing candidate for development as a SARM agonist with an appropriate pharmacological profile for clinical benefit in muscle-wasting conditions and female sexual function disorders.

Case study: Epidermoid cyst following percutaneous Topaz coblation for plantar fasciitis.

An epidermoid cyst is formed when there is proliferation of epidermal cells within an area of the dermis. They may be formed by the traumatic implantation of epidermal cells within the dermis as well as many other mechanisms. We present a case of epidermoid cyst formation following Topaz coblation for plantar fasciitis; a complication we believe is yet to be reported in the literature.

A rare case of tuberculosis of the midfoot.

Despite the increasing prevalence of tuberculosis in developed countries, tuberculous osteomyelitis of the foot remains an uncommon complication. The relative rarity of such a diagnosis means that delays are often incurred before clinicians can correctly identify the pathogen and therefore instigate appropriate chemotherapy. The authors present a case of midfoot pain secondary to tuberculous osteomyelitis.

Identification and structure-activity relationships of chromene-derived selective estrogen receptor modulators for treatment of postmenopausal symptoms.

As part of a program aimed at the development of selective estrogen receptor modulators (SERMs), novel chromene scaffolds, benzopyranobenzoxapanes, were discovered. Many compounds showed binding affinity as low as 1.6-200 nM, displayed antagonist behaviors in the MCF-7 human breast adenocarcinoma cell line as well in Ishikawa cell line with IC(50) values in the range 0.2-360 nM. On the basis of the side chain substitution, various compounds demonstrated strong inhibitory activity in anti-uterotropic assay. Compound 7-(R) and its major metabolites 5-(R) and 6-(R) were evaluated in several in vivo models of estrogen action. Relative to a full estrogen agonist (ethynyl estradiol) and the SERM raloxifene, 7-(R) was found to be a potent SERM that behaved as antagonist in the uterus and exhibited estrogen agonistic activity on bone, plasma lipids, hot flush, and vagina. The overall pharmacokinetic profile and stability were significantly improved compared to those of the phase 2 development compound 9-(R).

Serendipitous discovery of novel imidazolopyrazole scaffold as selective androgen receptor modulators.

A novel imidazolopyrazole derivative has been fortuitously discovered as potent selective androgen receptor modulator with in vivo efficacy.

Synthesis of potent and tissue-selective androgen receptor modulators (SARMs): 2-(2,2,2)-Trifluoroethyl-benzimidazole scaffold.

The synthesis and in vivo SAR of 2-(2,2,2)-trifluoroethyl-benzimidazoles are described. Prostate antagonism and/or levator ani agonism can be modulated by varying the substitution at the 2-position of 5,6-dichloro-benzimidazoles. Potent androgen agonists on the muscle were discovered that strongly bind to the androgen receptor (2-17 nM) and show potent in vivo efficacy (0.03-0.11 mg/day). True SARMs showing both prostate antagonism and levator ani agonism were revealed.

2-(2,2,2-Trifluoroethyl)-5,6-dichlorobenzimidazole derivatives as potent androgen receptor antagonists.

The synthesis and in vivo SAR of N-benzyl, N-aceto, and N-ethylene ether derivatives of 2-(2,2,2-trifluoroethyl)-5,6-dichloro-benzimidazole as novel androgen receptor antagonists are described. SAR studies led to the discovery of 4-bromo-benzyl benzimidazole 17 as a more potent androgen receptor antagonist in the rat prostate (ID(50)=0.13mg/day), compared with bicalutamide (ID(50)=0.23mg/day).

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.

beta-Alkylthio indolyl carbinols: potent nonsteroidal antiandrogens with oral efficacy in a prostate cancer model.

Through an in vivo screening model, we developed the in vivo SAR of beta-alkylthio indolyl carbinols. Through these efforts we identified a compound with potent oral in vivo efficacy in both immature and mature rat prostate weight reduction models and in a murine xenograft prostate cancer model.

Synthesis and SAR of potent and selective androgen receptor antagonists: 5,6-Dichloro-benzimidazole derivatives.

The synthesis and in vivo SAR of 5,6-dichloro-benzimidazole derivatives as novel selective androgen receptor antagonists are described. During screening of 2-alkyl benzimidazoles, it was found that a trifluoromethyl group greatly enhances antagonist activity in the prostate. Benzimidazole 1 is a potent AR antagonist in the rat prostate (ID50 = 0.15 mg/day).