Induction of Endoplasmic Reticulum Stress-Mediated Apoptosis by Aminosteroid RM-581 Efficiently Blocks the Growth of PC-3 Cancer Cells and Tumors Resistant or Not to Docetaxel.

Aminosteroid derivative RM-581 was previously identified as an endoplasmic-reticulum (ER) stress inducer with potent in vitro and in vivo anticancer activities. We report its evaluation in androgen-independent prostate cancer (PC-3) cells. RM-581 efficiently blocks PC-3 cell proliferation with stronger activity than that of a selection of known antineoplastic agents. This later also showed a synergistic effect with docetaxel, able to block the proliferation of docetaxel-resistant PC-3 cells and, contrary to docetaxel, did not induce cell resistance. RM-581 induced an increase in the expression level of ER stress-related markers of apoptosis, potentially triggered by the presence of RM-581 in the ER of PC-3 cells. These in vitro results were then successfully translated in vivo in a PC-3 xenograft tumor model in nude mice, showing superior blockade than that of docetaxel. RM-581 was also able to stop the progression of PC-3 cells when they had become resistant to docetaxel treatment. Concomitantly, we observed a decrease in gene markers of mevalonate and fatty acid pathways, and intratumoral levels of cholesterol by 19% and fatty acids by 22%. Overall, this work demonstrates the potential of an ER stress inducer as an anticancer agent for the treatment of prostate cancers that are refractory to commonly used chemotherapy treatments.

Minor chemical modifications of the aminosteroid derivative RM-581 lead to major impact on its anticancer activity, metabolic stability and aqueous solubility.

The aminosteroid (AM) RM-581 is built around a mestranol backbone and has recently emerged as this family's lead candidate, showing in vitro and in vivo potency over different types of cancer, including high fatality pancreatic cancer. To extend the structure-activity relationships (SAR) to other estrane analogs, we synthesized a focused series of RM-581 derivatives at position C3 or C2 of its steroidal core. These new AM derivatives were first tested on a large selection of prostate, breast, pancreatic and ovarian cancer cell lines. The impact of these modifications on metabolic stability (human liver microsomes) was also measured. A SAR study revealed a fine regulation of anticancer activity related to the nature of the substituent. Indeed, the addition of potential prodrug groups like acetate, sulfamate or phosphate (compounds 8, 9 and 10) at C3 of the phenolic counterpart provided better antiproliferative activities than RM-581 in breast and pancreatic cancer cell types while maintaining activity in other cancer cell lines. Also, the phosphate group was highly beneficial on water solubility. However, the bulkier carbamate prodrugs 6 (N,N-dimethyl) and 7 (N,N-diethyl) were less active. Otherwise, carbon homologation (CH2) at C2 (compound 33) was beneficial to metabolic stability and, in the meantime, this AM conserved the same anticancer activity as RM-581. However, the replacement of the hydroxy or methoxy at C3 by a hydrogen or an acetyl (compound 17 or 21b) was detrimental for anticancer activity, pointing to a crucial molecular interaction of the aromatic oxygen atom at this position. Overall, this work provided a better knowledge of the structural requirements to maintain RM-581's anticancer activity, and also identified minor structural modifications to increase both metabolic stability and water solubility, three important parameters of pharmacological development.

Induction of endoplasmic reticulum stress by aminosteroid derivative RM-581 leads to tumor regression in PANC-1 xenograft model.

The high fatality and morbidity of pancreatic cancer have remained almost unchanged over the last decades and new clinical therapeutic tools are urgently needed. We determined the cytotoxic activity of aminosteroid derivatives RM-133 (androstane) and RM-581 (estrane) in three human pancreatic cancer cell lines (BxPC3, Hs766T and PANC-1). In PANC-1, a similar level of antiproliferative activity was observed for RM-581 and RM-133 (IC50 = 3.9 and 4.3 µM, respectively), but RM-581 provided a higher selectivity index (SI = 12.8) for cancer cells over normal pancreatic cells than RM-133 (SI = 2.8). We also confirmed that RM-581 induces the same ER stress-apoptosis markers (BIP, CHOP and HERP) than RM-133 in PANC-1 cells, pointing out to a similar mechanism of action. Finally, these relevant in vitro results have been successfully translated in vivo by testing RM-581 using different doses (10-60 mg/kg/day) and modes of administration in PANC-1 xenograft models, which have led to tumor regression without any sign of toxicity in mice (animal weight, behavior and histology). Interestingly, RM-581 fully reduced the pancreatic tumor growth when administered orally in mice.

Chemical synthesis of C3-oxiranyl/oxiranylmethyl-estrane derivatives targeted by molecular modeling and tested as potential inhibitors of 17ß-hydroxysteroid dehydrogenase type 1.

17ß-Hydroxysteroid dehydrogenase type 1 (17ß-HSD1) is a promising therapeutic target known to play a pivotal role in the progression of estrogen-dependent diseases such as breast cancer, and endometriosis. This enzyme is responsible for the last step in the biosynthesis of the most potent estrogen, estradiol (E2) and its inhibition would prevent the growth of estrogen-sensitive tumors. Based on molecular modeling with docking experiments, we identified two promising C3-oxiranyl/oxiranylmethyl-estrane derivatives that would bind competitively and irreversibly in the catalytic site of 17ß-HSD1. They have been synthesized in a short and efficient route and their inhibitory activities over 17ß-HSD1 have been assessed by an enzymatic assay. Compound 15, with an oxiranylmethyl group at position C3, was more likely to bind the catalytic site and showed an interesting, but weak, inhibitory activity with an IC50 value of 1.3 µM (for the reduction of estrone into E2 in T-47D cells). Compound 11, with an oxiranyl at position C3, produced a lower inhibition rate, and the IC50 value cannot be determined. When tested in estrogen-sensitive T-47D cells, both compounds were also slightly estrogenic, although much less than the estrogenic hormone E2.

Targeting Cytochrome P450 (CYP) 1B1 Enzyme with Four Series of A-Ring Substituted Estrane Derivatives: Design, Synthesis, Inhibitory Activity, and Selectivity.

Cytochrome P450 (CYP) 1B1 is involved in the bioactivation of procarcinogens and drug resistance. To obtain selective CYP1B1 inhibitors over CYP1A1, we synthesized four series of estrane derivatives: (1) 12 estrone (E1)- and 17ß-estradiol (E2)-derivatives bearing a 3- or a 4-pyridinyl core at C2, C3, or C4, (2) eight estrane derivatives with different sulfur groups at C3, (3) 19 E1- and E2-derivatives bearing distinct aryls at C2, and (4) five D-ring derivatives. E2-derivatives were more active than oxidized E1-analogues, thus highlighting the key role of 17ß-OH for interaction with CYP1B1. 2-(4-Fluorophenyl)-E2 was the best CYP1B1 inhibitor (IC50 = 0.24 µM), with a selectivity index (SI) of 20 over CYP1A1. Furthermore, the addition of a C17α-ethynyl group as D-ring modification improved the selectivity index to 25 with only a slight loss of activity (IC50 = 0.37 µM). Our docking results showed that these compounds fit better into the CYP1B1 binding site than that of CYP1A1.

Synthesis of novel 17-(5'-iodo)triazolyl-3-methoxyestrane epimers via Cu(I)-catalyzed azide-alkyne cycloadditon, and an evaluation of their cytotoxic activity in vitro.

The regioselective Cu(I)-catalyzed 1,3-dipolar cycloaddition of 3-methoxyestrane 17α- and 17ß-azide epimers (3 and 5) with different terminal alkynes afforded novel 1,4-substituted triazolyl derivatives (8a-f and 11a-f). If the Ph3P in the classical CuAAC process was replaced by Et3N, the formation of small quantities of 5-iodotriazoles (9a-f and 11a-f) was observed. For the preparation of 5-iodo-1,2,3-triazoles (9a-f and 11a-f), an improved method was developed, directly from steroidal azides and terminal alkynes, in reactions mediated by CuI and ICl as iodinating agents. The antiproliferative activities of the structurally related triazoles were determined in vitro with the microculture tetrazolium assay on six malignant human cell lines of gynecological origin (HeLa, A2780, MCF7, MDA-MB-231, MDA-MB-361 and T47D). X-ray analysis revealed the presence of the iodo substituent on the 1,2,3-triazole ring.

Progestógenos: farmacologia e uso clínico / Progestagens: pharmacology and clinical use

Os progestógenos são esteroides que podem ser sintéticos ou naturais. A progesterona é o único progestágeno natural. Os progestógenos sintéticos tentam mimetizar o efeito da progesterona, e são chamados de progestinas. Cada progestina apresenta diferentes propriedades farmacológicas, dependendo da molécula da qual foi originada, usualmente testosterona e progesterona. Pequenas mudanças estruturais nas moléculas originais levam a diferenças consideráveis na atividade de cada uma das progestinas. O objetivo deste trabalho é revisar a origem dos progestógenos, as peculiaridades de cada grupo e seu uso clínico mais comum. As informações já levantadas sobre o efeito das progestinas em patologias importantes e prevalentes, como o câncer de mama e eventos tromboembólicos, também será abordado.

Progestagens are natural or synthetic steroids, and progesterone is the only natural one. Synthetic progestagens, called progestins, were created to mimic the effects of natural progesterone. The progestins have different pharmacological properties depending on the parent molecule, usually testosterone or progesterone, from which they are derived. Very small structural changes in the original molecule may induce considerable differences in the activity of the derivative. The aim of this paper is to review the origin of each progestin, the peculiarities of each group and its most common clinical use. The current knowledge about the effect of progestins on important and prevalent diseases, such as breast cancer and thromboembolic events, will also be addressed.

Synthesis, X-ray crystal structures and biological activity of 16-amino-17-substituted-D-homo steroid derivatives.

D-Homo derivatives in the androstane and estrane series, 12-19, were synthesized by a fragmentation-cyclization reaction of 16-oximino-17-hydroxy-17-substituted derivatives 3-9, or by cyclization of the corresponding D-seco derivatives 20-26. The structures were confirmed by X-ray analysis of compounds 12 and 16. Preliminary assessment of inhibitory effects of D-homo derivatives from androstane series towards aromatase, 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), 17 alpha-hydroxylase/C17-20 lyase (P450c17) and 17 beta-HSD indicated much lower inhibitory potential compared to previously tested activity of another type of D-modified steroids, namely D-seco derivatives. Also, assessment of potential antiestrogenic activity of derivatives from estrane series showed absence of such an activity.

The rationale for a wider range of progestogens.

Progestogens are commonly used in hormone replacement therapy, normally as opponents of estrogen to protect the endometrium from hyperplasia and cancer. While these benefits of endometrial protection are well recognized, the data related to the effect of progestogens on breast tissue and the cardiovascular system are conflicting. It has been demonstrated that, according to the type of progestogen used, and the dose and duration of its application, a predominant proliferative effect may be observed in human breast cells. As far as breast cancer is concerned, most epidemiological studies suggest no difference in risk between therapy with estrogens alone or estrogens combined with progestogens, but recent data do indicate an increased risk with combined therapy. When the cardiovascular risk factors are considered, some progestogen molecules with a higher androgenic potency than others attenuate the beneficial effects of estrogens on both the lipid profile and vasomotion. On the other hand, the epidemiological data on primary prevention do not suggest any negative effect of the progestogens administered together with estrogens on cardiovascular morbidity or mortality. Recent results have questioned the cardioprotective effect of hormone replacement therapy in women with established coronary heart disease. It has been suggested that the lack of a secondary preventive effect by hormone replacement therapy may be due to the progestogens selected. The effect on osteoporosis is also the subject of debate, with some progestogens having a neutral effect on bone mineral density and others producing a marked improvement. Awareness of the classic contraindications of hormone replacement therapy and selection of molecules devoid of estrogenic, androgenic or glucocorticoid effect should allow greater use of the progestogens without any major drawback.

Synthesis and structure-activity profiles of A-homoestranes, the estratropones.

2-Methoxyestradiol, a mammalian metabolite of estradiol, has reported antiangiogenic activity which has been proposed to be mediated through interaction at the colchicine binding site on the tubulin monomer. Subsequent structure-activity studies of 2-methoxyestradiol have yielded highly potent steroidal inhibitors of tubulin polymerization. In an effort to probe the scope of binding at the colchicine binding site and the nature of the relationship between 2-methoxyestradiol and colchicine, a series of colchicine/2-methoxyestradiol hybrids was synthesized. These A-homoestrane hybrid systems, collectively termed estratropones, possessed an A-ring tropone system with the keto functionality at either the C-2, C-3, or C-4 position of the steroid nucleus. The estratropones were evaluated for their ability to inhibit the polymerization of tubulin using an in vitro purified bovine brain assay. Most of these hybrids inhibit polymerization with greater potency than either of the natural products. The most potent of these congeners possessed an approximate 5-fold enhancement of the activity of colchicine for the inhibition of tubulin polymerization. alpha-Substituents on the tropone ring showed varied effects on the activities for the two classes of estratropones studied in this regard, the C-3 oxo and the C-4 oxo species. The 3-substituted 4-oxoestratropones exhibited antitubulin activity according to Cl approximately Br > OCH3, whereas the 4-substituted 3-oxoestratropones exhibited activity according to OCH3 > Br approximately Cl. It is unclear if these substituent factors are purely electronic or steric effects or if the substituent operates indirectly by altering the conformation of the nonplanar troponoid ring. The estratropones represent a new class of tubulin binding agents with potential antiangiogenic utility.

Comparative effect of synthetic aminoestrogens with estradiol on platelet aggregation.

The in vitro effect upon platelet aggregation of estradiol and synthetic estrogens (prolame, buame, and proacame) is described. Prolame and buame produced a dose-dependent inhibition on platelet aggregation. Estradiol and proacame did not show anti-aggregating effects. The results suggest that the use of prolame and buame in estrogen therapy could reduce the risk of thrombo-embolic accidents.

C-nor-9,11-secoestranes as modified estrogens and fertility regulation.

The synthesis of C-nor-9,11-secoestradiol (4) has been achieved from 17 beta-acetoxy-11-chloro-3-methoxy-C-nor-9,11-secoestra-1,3,5(10)-tr ien-9-one (1) through a sequence of reactions without affecting the stereochemistry of estradiol-17 beta. Removal of the 9-keto function of 1 by hydrogenolysis and its subsequent treatment with Na/NH3 gives C-nor-9,11-secoestradiol 3-(methyl ether) (3), which has been demethylated under alkaline conditions to furnish C-nor-9,11-secoestradiol (4). Pyridinium chlorochromate oxidation of 3 gives the corresponding 17-ketone 6. Jones' oxidation of 4 to the ketone 5 and reaction of 5 and 6 with lithium acetylide gives corresponding 17 alpha-ethynyl derivatives 7 and 8. Relative binding affinity to estradiol-17 beta receptors and uterotropic, antiuterotrophic, and antiimplantation activities of compounds 3-8 have been studied. The effect of conformational flexibility on ligand-receptor interaction of these compounds is discussed.

The differential susceptibility of gonococcal opacity variants to sex hormones.

Neisseria gonorrhoeae exist in transparent (Tr) and opaque (Op) colony forms. Op forms are recovered from patients early in the menstrual cycle; Tr colonies predominate late in the cycle. The mechanism for this colonial variation was examined by determining the influence of gonodal hormones on growth inhibition of Op and Tr isogenic variants of gonococci. The estrogens, estrone and estradiol, enhanced growth whereas 19-nortestosterone, testosterone, and progesterone significantly inhibited gonococcal growth. Testosterone and progesterone inhibited growth of the Op variants to a greater degree than the Tr variants. Mixture of Tr and Op colonies grown in the presence of progesterone became predominantly Tr, as occurs in the luteal phase of the menstrual cycle. This study supports the hypothesis of hormonal influence on colonial variation but employed artificial in vitro conditions and high hormone levels.

[Selection of preparations with anticipated antiestrogen activity]. / Otbor preparatov s prepolagaemoi antiéstrogennoi aktivnost'iu.

Specificity of interaction between estrogen-receptor systems of guinea pig uterus and oviducts and six estrogens was studied. The obtained datas suggest existence of tissue specificity in estrogen-receptor interaction in uterus and oviducts. Modifications of the steroid molecule led to decrease in extent of estrogen affinity. Introduction of an ethyl group into 17 alpha-position resulted in the effect, by the rate of estrogen-receptor binding equal to reduction of 17 beta-hydroxyl. Low but distinct affinity of 17 alpha-estradiol, its structure and stability, enabled to select this substance as a preparation with anticipated anti-estrogenic activity.

[11-azasteroids. V. Hormonal properties of 11-azaestranes]. / 11-azasteroids. V. Hormonal properties of 11-azaéstranes.

Experiments were conducted on sexually-immature female rats, ovariectomized mice and castrated male rats. Hormonal properties of some 11-azaestran were studied. As shown, introduction of azafunction into the position of the 11 molecule of the steroid sharply reduced the hormonal activity, irrespective of other functions in the molecule determining the reference of the steroid to the estron, estradiol, or 19-nortestosterone series.

Chemical synthesis and bioassay of anordrin and dinordrin I and II.

The chemical synthesis of 2 alpha, 17 alpha-diethynyl-A-nor-5 alpha-androstane-2 beta, 17 beta-diol dipropionate (Anordrin) and the corresponding diacetate is reported. Similarly, the preparation of the 2 alpha, 17 alpha-diethynyl-A-nor-5 alpha-estrane-2 beta, 17 beta-diol, its diacetate and dipropionate (Dinordrin I), along with the corresponding 2 beta-epimer (Dinordrin II) from 17 beta-hydroxy-A-nor-5 alpha=estran-2-one is described. In rat uterotrophic activity bioassay, the slope of ethynylestradiol differed significantly from the slopes of the other three compounds, thus vitiating potency estimates with this reference compound. Dinordrin I was 20 times more potent than Anordrin and considerably more potent then Dinordrin II. The single-dose oral antifertility effect in rats generally paralleled uterotrophic activity. Immediate postovulatory contraceptive effectiveness was assessed in adult cycling female baboons given two doses daily for 4 days. Both Anordrin and Dinordrin I showed antifertility activity worthy of further study. Moreover, a definite luteolytic effect, with depression of both plasma estrogen and progesterone levels, was observed with these two steroids.

[Inhibition of rat liver delta 4-3-ketosteroid-5 alpha-reductase by steroids in vitro]. / Hemmung der delta4-3-Ketosteroid-5alpha-reduktase der Rattenleber durch Steroide in vitro.

The inhibition of the hydrogenation of testerone to 5alpha-dihydrotestosterone in microsomes of female rat liver has been studied by progesterone-, testosterone- and estradiol derivatives. In the pregnane series, progesterone, 17alpha-OH-progesterone and its acetate were the strongest inhibitors. In the estradiol series, 3-hydroxy-compounds with a CH2X-substituent at 17alpha-position were stronger inhibitors than the respective 3-methoxy derivatives. The most potent inhibitor till now is an estradiol derivative with a SeCN group in 16alpha-position.

Synthesis, antifertility activity, and protein binding affinity of 7(8 leads to 11 alpha)abeo steroids.

A series of 7(8 leads to 11 alpha)abeo steroids was synthesized by a modification of the previously described total synthesis of this class of compounds and evaluated for biological activity. In general, there was a marked reduction in the relative binding affinities of these compounds for the rabbit uterus estrogen and progestin receptor proteins. None of the compounds which were subjected to uterotropic, antiuterotropic, postcoital, progestational, antiprogestational, or antiandrogenic assays showed any significant activity.

PIP: Synthesis, antifertility activity, and protein binding afinity of 7(8 to 11alph) abeo-estranes and -pregnanes are described. There was a marked reduction in the relative binding affinities of these compounds for the rabbit uterus estrogen and progestin receptor proteins. None of the compounds subjected to uterotropic, antiuterotropic, postcoital, progestational, antiprogestational, or antiandrogenic assays revealed any marked activity.

Steroids and hematopoiesis. I. The effect of steroids on in vitro erythroid colony growth: structure/activity relationships.

When substituted steroids of several classes are added to cultures of rat bone marrow cells in the presence of erythropoietin a consistent enhancement of the number of colonies of hemoglobin synthesizing cells is obtained. Maximum steroid effectiveness was found to be between 10(-6) and 10(-7) M. Representative compounds of several classes of steroids were examined for their ability to enhance colony growth, including delta 4-estrenes, delta 4-androstenes, 5alpha-H androstanes and estranes, 5beta-H estranes, pregnanes and androstanes. While testosterone and its 5alpha-H derivatives had little or no activity, many synthetic derivatives of testosterone were highly active in increasing erythroid colony growth. All 5beta-H androstanes, estranes, and all but one 5beta-H pregnane were active. Cortisol consistently inhibited colony growth and estradiol and progesterone had no significant effect.