Discovery of desketoraloxifene analogues as inhibitors of mammalian, Pseudomonas aeruginosa, and NAPE phospholipase D enzymes.

Phospholipase D (PLD) hydrolyses cellular lipids to produce the important lipid second messenger phosphatidic acid. A PLD enzyme expressed by Pseudomonas aeruginosa (PldA) has been shown to be important in bacterial infection, and NAPE-PLD has emerged as being key in the synthesis of endocannabinoids. In order to better understand the biology and therapeutic potential of these less explored PLD enzymes, small molecule tools are required. Selective estrogen receptor modulators (SERMs) have been previously shown to inhibit mammalian PLD (PLD1 and PLD2). By targeted screening of a library of SERM analogues, additional parallel synthesis, and evaluation in multiple PLD assays, we discovered a novel desketoraloxifene-based scaffold that inhibited not only the two mammalian PLDs but also structurally divergent PldA and NAPE-PLD. This finding represents an important first step toward the development of small molecules possessing universal inhibition of divergent PLD enzymes to advance the field.

Selenium analogues of raloxifene as promising antiproliferative agents in treatment of breast cancer.

Synthetic protocols for the preparation of selenium analogues of raloxifene were elaborated. General aim of the current research is to improve the positive impact of selenium atom introduction in drug design. Antiproliferative activity on CCL-8 (mouse sarcoma), MDA-MB-435s (human melanoma), MES-SA (human uterus sarcoma), MCF-7 (human breast adenocarcinoma), HT-1080 (human fibrosarcoma), MG-22A (mouse hepatoma) tumor cell lines, and normal cell line NIH 3T3 (mouse fibroblasts) was studied. Influence of aminoethoxy "tail" and benzoyl group position on SAR was discussed. Results of in vivo studies on BALB/c female mice with 4T1 cell induced breast cancer model showed that selenium analogue of raloxifene is able to suppress estrogen-depending tumor growth.

Design, synthesis, and biological evaluation of cyclopropyl analogues of stilbene with raloxifene side chain as subtype-selective ligands for estrogen receptor.

We have designed the cyclopropane analog of stilbene as subtype-selective ligands for estrogen receptor based on the bioisosterism that cyclopropane could act as alkene bioisoster. Three cyclopropane analogs were prepared efficiently starting from 4-benzyloxybenzaldehyde, and evaluated for their binding to estrogen receptors ERα and ERß. These cyclopropane analogs were also found to be full agonists in estrogen receptor-mediated gene transcription assay. Compared to the stilbene analogs such as tamoxifen and raloxifene, the three cyclopropane analogs showed lower binding affinity for estrogen receptor, but higher subtype selectivity for ERα. The structure-activity relationship revealed from this study might provide clues for improving subtype selectivity for ERα.

Discovery of a new class of liver receptor homolog-1 (LRH-1) antagonists: virtual screening, synthesis and biological evaluation.

Targeting LRH-1: Virtual screening and molecular modeling were used to identify novel antagonists of liver receptor homolog-1 (LRH-1), an emerging therapeutic target for breast cancer. Hit compounds were synthesized and biologically assayed, and the preliminary results suggest that raloxifene-based analogues, substituted at the position C-7 of the benzothiophene ring, might generate an inactive protein conformation through binding and thus antagonize this nuclear receptor.

Application of 4D-QSAR studies to a series of raloxifene analogs and design of potential selective estrogen receptor modulators.

Four-dimensional quantitative structure-activity relationship (4D-QSAR) analysis was applied on a series of 54 2-arylbenzothiophene derivatives, synthesized by Grese and coworkers, based on raloxifene (an estrogen receptor-alpha antagonist), and evaluated as ERa ligands and as inhibitors of estrogen-stimulated proliferation of MCF-7 breast cancer cells. The conformations of each analogue, sampled from a molecular dynamics simulation, were placed in a grid cell lattice according to three trial alignments, considering two grid cell sizes (1.0 and 2.0 Å). The QSAR equations, generated by a combined scheme of genetic algorithms (GA) and partial least squares (PLS) regression, were evaluated by "leave-one-out" cross-validation, using a training set of 41 compounds. External validation was performed using a test set of 13 compounds. The obtained 4D-QSAR models are in agreement with the proposed mechanism of action for raloxifene. This study allowed a quantitative prediction of compounds' potency and supported the design of new raloxifene analogs.

Recent advances in the synthesis of raloxifene: a selective estrogen receptor modulator.

Estrogens are a group of steroids that exert important effects on reproductive and many non-reproductive tissues. Selective estrogen receptor modulators (SERM) are a class of therapeutic agents widely prescribed for the treatment and prevention of breast cancer, osteoporosis, and postmenopausal symptoms. Raloxifene, an example of oral SERM is prescribed primarily for the treatment and prevention of postmenopausal disorders in woman. The current review provides an outline of practical methodologies used to access benzothiophenyl scaffolds of raloxifene and relevant structural analogs. The contents are discussed in five sections: (a) synthesis of raloxifene, (b) organometallic analogs, (c) radiolabelled analogs, (d) constrained raloxifene analogs, and (e) other oxygen, sulfur, and nitrogen based raloxifene analogs. In addition to the synthesis, biological activity of a few synthetic analogs has been discussed.

Effect of selective estrogen receptor modulator/raloxifene analogue on proliferation and collagen metabolism of tendon fibroblast.

The selective estrogen receptor modulator raloxifene is therapeutically beneficial for postmenopausal connective tissue degradation, such as osteoporosis, vascular sclerosis, and dermal degradation; however, the effects of raloxifene on postmenopausal tendon metabolism have not been clarified. In this study, we investigated the effects of raloxifene analogue (LY117018) on cell proliferation and collagen metabolism using cultured rat Achilles tendon fibroblasts. 17beta-Estradiol (E(2); 10(-11)-10(-9) M) and LY117018 (10(-9)-10(-7) M) had no significant effects on tendon fibroblast proliferation, based on a BrdU (5-bromo-2'-deoxyuridine) incorporation assay (24 hr) and a WST-8 colorimetric assay (2 or 6 days). Neither E(2) nor LY117018 significantly altered the expression of type I collagen, which is a main component of the tendon extracellular matrix (ECM), whereas both E(2) and LY117018 significantly increased the expression of matrix metalloproteinase (MMP)-13, which is responsible for tendon collagen degradation in rat. Also, both E(2) and LY117018 increased the expression of type III collagen and elastin, which are minor components of tendon ECM, but are considered to govern the elastic properties of tendons. These changes in collagen and MMP induced by either E(2) or LY117018 were attenuated by the estrogen receptor alpha blocker ICI 182,780. The results of this study suggest that postmenopausal estrogen deficiency might downregulate tendon collagen turnover and decrease tendon elasticity. Further, raloxifene treatment might restore these changes to premenopausal levels.

In vitro evaluation, biodistribution in rats of radiolabeled raloxifene.

Raloxifene is a selective estrogen receptor modulator that produces both estrogen agonistic effects on bone and lipid metabolism and estrogen-antagonistic effects on uterine endometrium and breast tissue. In the present study, raloxifene was labeled with I-131 by iodogen method and investigated for its radiopharmaceutical potential. Radiolabeling yield is 91+/-0.7%, as determined by radio thin layer chromatography (RTLC). Results of in vitro study indicated (131)I-raloxifene has high stability (4h) in serum. Biodistribution study was carried out with Albino wistar female rats. The result has shown that the radioiodinated raloxifene has higher uptake in uterus than breast and ovarian.

In vivo activation of gene transcription via oestrogen response elements by a raloxifene analogue.

Raloxifene is a selective oestrogen receptor modulator with tissue-specific effects. The mechanisms behind the effects of raloxifene are partly unclear, and the aim of the present study was to investigate whether raloxifene can activate the classical oestrogen-signalling pathway in vivo in three known oestrogen-responsive organs, uterus (reproductive organ), bone (non-reproductive organ) and thymus (immune organ). For this purpose, we have used reporter mice with a luciferase gene under control of oestrogen-responsive elements (EREs), enabling detection of in vivo activation of gene transcription via the classical oestrogen pathway. Three-month-old ovariectomized ERE-luciferase mice were treated with the raloxifene analogue (LY117018), oestradiol (OE(2)) or vehicle for 3 weeks. Luciferase activation was measured in bone, uterus and thymus, and compared to bone parameters, and uterus and thymus weights. The raloxifene analogue affected bone mineral density (BMD) to the same extent as OE(2), and both treatments resulted in increased luciferase activity in bone. As expected, OE(2) treatment resulted in increased uterus weight and increased uterine luciferase activity, while the effect of LY117018 on uterus weight and luciferase activity was modest and significantly lower than the effect of OE(2). LY117018 and OE(2) treatment resulted in similar luciferase activation in thymus. However, only OE(2) treatment resulted in thymic atrophy, while no effect on thymus weight was seen after LY117018 treatment. In summary, the raloxifene analogue LY117018 can activate the classical oestrogen pathway in bone, uterus and thymus in vivo, and this activation is associated with BMD and uterus weight, but not thymus weight.

Raloxifene analogue LY117018 suppresses oxidative stress-induced endothelial cell apoptosis through activation of ERK1/2 signaling pathway.

A selective estrogen receptor modulator, raloxifene, has been shown to reduce cardiovascular events in relatively high-risk postmenopausal women with osteoporosis. However, the mechanisms by which raloxifene exerts a pharmacological effect on cardiovascular organs have not been fully elucidated. The present study was designed to examine whether the raloxifene analogue, 6-hydroxy-2-(p-hydroxyphenyl)-benzo(b) thien-3-yl-p-(2-(pyrrolidinyl)ethoxy phenyl ketone (LY117018), could inhibit apoptosis and to clarify the signaling pathway in vascular endothelial cells. LY117018 significantly inhibited hydrogen peroxide-induced apoptosis in bovine carotid artery endothelial cells. The anti-apoptotic effect of LY117018 was abolished by an estrogen receptor antagonist, 7alpha,7beta-(9[(4,4,5,5,5-Pentafluoropentyl)sulfinyl]nonyl) estra-1,3,5(10)-triene-3,17-diol (ICI 182,780). Mitogen-activated protein kinases (MAPK), including p38, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated protein kinase1/2 (ERK1/2), and Akt, have been shown to act as apoptotic or anti-apoptotic signals. Phosphorylation of p38, JNK, ERK1/2 and Akt was examined. LY117018 increased ERK1/2 phosphorylation but did not enhance the phosphorylation of p38, JNK, or Akt. The anti-apoptotic effect of LY117018 was prevented by treatment with 2-[2'-amino-3'-methoxyphenyl]-oxanaphthalen-4-one (PD98059), an upstream inhibitor of ERK1/2. LY117018 stimulated an increase in ERK1/2 phosphorylation, which was diminished by ICI 182,780. The activation of ERK/1/2 by LY117018 was not inhibited by the transcription inhibitor, actinomycin D. These results suggest that estrogen receptors and the ERK1/2 signaling pathway are involved in the anti-apoptotic action of LY117018 in vascular endothelial cells.

Increased antitumor potential of the raloxifene prodrug, raloxifene diphosphate.

Raloxifene (RAL) significantly reduced the incidence of breast cancer in women at high risk of developing the disease. Unlike tamoxifen (TAM), an increased incidence of endometrial cancer was not observed in women treated with RAL. However, RAL, having two hydroxyl moieties, can be conjugated rapidly through phase II metabolism and excreted, making it difficult to achieve adequate bioavailability by oral administration in humans. As a result, higher doses must be administered to obtain an efficacy equivalent to that achieved with TAM. To improve oral bioavailability and antitumor potential, RAL diphosphate was prepared as a prodrug. RAL diphosphate showed several orders of magnitude lower binding potential to both ER alpha and ER beta and weak antiproliferative potency on cultured human MCF-7 and ZR-75-1 breast cancer cells, as compared to RAL. However, RAL diphosphate has a much higher bioavailability than RAL, endowing it with higher antitumor potential than RAL against both 7,12-dimethylbenz(a)anthracene-induced mammary carcinoma in rats and human MCF-7 breast cancer implanted in athymic nude mice. The RAL prodrug may provide greater clinical benefit for breast cancer therapy and prevention.

Role of raloxifene as a potent inhibitor of experimental postmenopausal polyarthritis and osteoporosis.

OBJECTIVE: In postmenopausal rheumatoid arthritis (RA), both estrogen deficiency and the inflammatory disease contribute to the development of generalized osteoporosis. Hormone replacement therapy (HRT) with estradiol preserves bone mineral density (BMD) and ameliorates arthritis, but long-term therapy is no longer an option due to significant side effects. We therefore used a mouse model of human RA to test the hypothesis that a selective estrogen receptor modulator (SERM), the raloxifene analog LY117018, could be beneficial in the treatment of both arthritis and osteoporosis. METHODS: Female DBA/1 mice were ovariectomized and arthritis was induced with collagen immunization. Mice received an injection of raloxifene, estradiol, or vehicle control, administered prophylactically or therapeutically, and thereafter the clinical arthritis score was evaluated continuously. At termination, BMD was analyzed with peripheral quantitative computed tomography. Paws were collected for histology, and sera were analyzed for cytokines and markers of bone and cartilage turnover. Levels of cytokine messenger RNA (mRNA) were investigated with real-time polymerase chain reaction. RESULTS: Treatment with raloxifene dramatically decreased the frequency and severity of arthritis. Effective preservation of bone and cartilage was seen in raloxifene-exposed mice, as demonstrated by increased BMD and decreased serum levels of cartilage oligomeric matrix protein in the raloxifene-treated mice compared with controls. Decreased levels of mRNA for both tumor necrosis factor alpha and RANKL in spleen cells from raloxifene-treated arthritic mice indicated an immunosuppressive action of this SERM. CONCLUSION: In a well-established model of postmenopausal RA, the raloxifene analog LY117018 potently inhibits the progression of arthritis and the associated development of osteoporosis, both in a prophylactic and in a therapeutic regimen. Since long-term HRT has been associated with significant side effects, raloxifene may be a useful adjuvant treatment for postmenopausal RA.

Biological activities of a novel selective oestrogen receptor modulator derived from raloxifene (Y134).

BACKGROUND AND PURPOSE: Selective oestrogen receptor (ER) modulators (SERMs) are of great value in the treatment of breast cancer and osteoporosis. The aim of this study was to characterize pharmacologically a new class of SERMs synthesized based on the core structure of raloxifene. EXPERIMENTAL APPROACH: Competitive receptor binding and luciferase-based reporter methods were used to study the bioactivities of raloxifene analogues, followed by efficacy determination in breast cancer cell proliferation assay. ER antagonist effects were investigated in female rats by measuring uterine and mammary gland growth, using wet weight, BrdU incorporation and terminal end bud (TEB) as indicators. KEY RESULTS: Five analogues, belonging to two different structural series and display higher binding affinities for ERalpha than ERbeta were functionally evaluated. One such analogue, Y134, exhibited potent antagonist activity at ERs in CV-1 cells cotransfected with plasmids containing ERalpha or ERbeta and oestrogen-response element-driven luciferase. The estimated IC(50) value was 0.52 nM for ERalpha and 2.94 nM for ERbeta, comparable to that of raloxifene. Little cytotoxicity was observed at Y134 concentrations below 10 microM. Y134 suppressed oestrogen-stimulated proliferation of ER-positive human breast cancer MCF-7 and T47D cells. At an identical dose, administered to ovariectomized rats, Y134 was more effective than raloxifene at arresting oestrogen-induced outgrowth of TEB and mammary gland DNA synthesis, but their inhibitory effects on the uterus were comparable. CONCLUSIONS AND IMPLICATIONS: Y134 is a potent ER antagonist with better mammary gland selectivity than raloxifene and shows potential for development as a new SERM for therapeutic use.

Activity of estradiol and selective estrogen receptor modulators in the mouse N20.1 oligodendrocyte/astrocytes cell line.

OBJECTIVE: The mechanism through which estrogen exerts its neuroprotective and anti-neurodegenerative effects in the central nervous system is poorly understood. Human glial cells are implicated in the pathogenesis of Alzheimer's disease and have both alpha and beta estrogen receptors (ER). We developed a glial cell model for ER function using the N20.1 mouse oligodendroglial cell line to evaluate the response of ERalpha and ERbeta to estradiol (E2), a raloxifene analog LY117018 (LY) and 4-hydroxytamoxifen (4OHT). DESIGN: We tested the ability of exogenous ER to activate transcription in response to ligands (100 nM) using the glial cell line N20.1 in a transient cotransfection assay with an ERalpha or ERbeta expression vector, an ERE-driven reporter and a Renilla luciferase transfection control. RESULTS: Endogenous ER was not detected in the N20.1 cells by Western immunoblotting. E2 stimulated both ERalpha and ERbeta on both ERE- and AP-1 driven promoters. The transcription stimulation by E2 in the ERalpha and ERbeta through the AP-1driven promoter, though significant, was not of the same magnitude as the stimulation of the ERalpha through the ERE-driven promoter. 4OHT and LY did not show significant transcriptional activation of either the ERalpha or ERbeta, through either the ERE or AP-1 driven promoters. LY, at a 10-fold higher concentration than E2, showed a difference in its antagonist activity on the ERbeta through the AP-1 pathway when compared with the ERE- driven promoter, demonstrating not only promoter specificity, but also receptor specificity. CONCLUSIONS: This is the first description of the activity of 4OHT and LY on estrogen receptors in glia.

Benzothiophenes containing a piperazine side chain as selective ligands for the estrogen receptor alpha and their bioactivities in vivo.

The synthesis of benzothiophenes containing a piperazine side chain and their binding affinities for estrogen receptors are described. These compounds bearing piperazine side chains were identified to be high-affinity ligands with high selectivity for ER alpha subtype. They were also potent agonists in bone tissue.

Oxidation of raloxifene to quinoids: potential toxic pathways via a diquinone methide and o-quinones.

Raloxifene was approved in 1997 by the FDA for the treatment of osteoporosis in postmenopausal women, and it is currently in clinical trials for the chemoprevention of breast cancer. Before widespread use as a chemopreventive agent in healthy women, the potential cytotoxic mechanisms of raloxifene should be investigated. In the current study, raloxifene was incubated with GSH and either rat or human liver microsomes, and the metabolites and GSH conjugates were characterized using liquid chromatography-tandem mass spectrometry. Raloxifene was converted to raloxifene diquinone methide GSH conjugates, raloxifene o-quinone GSH conjugates, and raloxifene catechols. For comparison, three raloxifene catechols were synthesized and characterized. In particular, 7-hydroxyraloxifene was found to oxidize to the 6,7-o-quinone. As compared with raloxifene diquinone methide, which has a half-life of less than 1 s in phosphate buffer, the half-life of raloxifene 6,7-o-quinone was much longer at t(1/2) = 69 +/- 2.5 min. The stability offered by raloxifene 6,7-o-quinone implies that it may be more toxic than raloxifene diquinone methide. Cytotoxicity studies in the human breast cancer cell lines S30 and MDA-MB-231 showed that 7-hydroxyraloxifene was more toxic than raloxifene in both cell lines. These results suggest that raloxifene could be metabolized to electrophilic and redox active quinoids, which have the potential to cause toxicity in vivo.

Potent inhibition of human liver aldehyde oxidase by raloxifene.

The selective estrogen receptor modulator, raloxifene, has been demonstrated as a potent uncompetitive inhibitor of human liver aldehyde oxidase-catalyzed oxidation of phthalazine, vanillin, and nicotine-Delta1'(5')-iminium ion, with K(i) values of 0.87 to 1.4 nM. Inhibition was not time-dependent. Raloxifene has also been shown to be a noncompetitive inhibitor of an aldehyde oxidase-catalyzed reduction reaction of a hydroxamic acid-containing compound, with a K(i) of 51 nM. However, raloxifene had only small effects on xanthine oxidase, an enzyme related to aldehyde oxidase. In addition, several other compounds of the same therapeutic class as raloxifene were examined for their potential to inhibit aldehyde oxidase. However, none were as potent as raloxifene, since IC(50) values were orders of magnitude higher and ranged from 0.29 to 57 micro M. In an examination of analogs of raloxifene, it was shown that the bisphenol structure with a hydrophobic group on the 3-position of the benzthiophene ring system was the most important element that imparts inhibitory potency. The relevance of these data to the mechanistic understanding of aldehyde oxidase catalysis, as well as to the potential for raloxifene to cause drug interactions with agents for which aldehyde oxidase-mediated metabolism is important, such as zaleplon or famciclovir, is discussed.

Synthesis and binding affinities of fluoroalkylated raloxifenes.

Three fluoroalkylated derivatives (1-3) of the selective estrogen receptor modulator (SERM), raloxifene, have been synthesized. The key step in the synthesis is the C-C bond formation of benzo[b]thiophene and a substituted phenyl group (ring C) using a Stille reaction. The in vitro binding affinities of the substituted raloxifenes 1-3 are 45, 60, 89%, respectively, relative to the affinity of estradiol, which is higher than the affinity of raloxifene itself (25%). When labeled with the positron-emitting radionuclide, these compounds might be useful as PET imaging agents for estrogen receptor-positive breast tumors.

Raloxifene analog LY117018 enhances the regeneration of sciatic nerve in ovariectomized female mice.

The aim of this study was to examine the effects of LY117018, a selective estrogen receptor modulator, on peripheral nerve regeneration, using a model of sciatic nerve crush injury in mice. Sciatic functional index, an index of functional recovery, was significantly higher in LY117018 treated mice throughout regeneration. Analysis of semi-thin sections revealed a significant increase in both the total number of regenerating nerve fibers at day 7, and the mean axonal area of myelinated fibers at 7, 14, and 21 days after injury, in LY117018 treated mice. Analysis of axonal transport through retrograde labeling of motor neurons showed that LY117018 increased transport, and ICI 182,780 blocked the effects of LY117018, delineating estrogen receptors as its target. Our study suggests that LY117018 may markedly accelerate peripheral nerve regeneration and functional recovery through activation of estrogen receptors.

Synthesis of constrained raloxifene analogues by complementary use of Friedel-Crafts and directed remote metalation reactions.

New constrained heterocyclic analogues, 2a,b and 3, of Raloxifene (1) have been prepared by complementary Directed remote Metalation (DreM)/Friedel-Crafts cyclization approaches. Utilization of a benzylidene-thiolactone rearrangement was successfully implemented to construct benzothiophenes 13a-c in good yields. Selective deprotection of 13a and 13b induced by complexation followed by triflation gave 18 and 23, thereby allowing efficient Suzuki-Miyaura cross coupling with borolane 16 to give biaryls 19 and 24. Treatment of 19 with BCl(3) induced an intramolecular para Fridel-Crafts cyclization and concomitant double deprotection to furnish analogue 2a, a new 5,6,6,6-(C(4)S-C(6)-C(6)-C(6)) sulfur-containing heterocycle. Exposure of 25 with excess LDA induced a DreM cyclization delivering the ortho-substituted 5,6,6,6-(C(4)S-C(6)-C(6)-C(6)) heterocylic analogue 26 in 70% yield. Similar treatment of 13c and 27 afforded 30, representing the novel 5,5,6,6-(C(4)S-C(5)-C(6)-C(6)) ring system, which was subjected to Suzuki-Miyaura cross coupling with 16 to give the biaryl 31 in 93% yield; deprotection furnished the final constrained analogue 3.