Synthesis and Pharmacological Evaluation of SNC80 Analogues with a Bridged Piperazine Ring

To discover novel δ‐opioid receptor ligands derived from SNC80 ( 1 ), a series of 6,8‐diazabicyclo[3.2.2]nonane derivatives bearing two aromatic moieties was prepared, and the affinity toward δ, μ, and κ receptors, as well as σ receptors, was investigated. After removal of the 4‐methoxybenzyl and 2,4‐dimethoxybenzyl protecting groups, the pharmacophoric N,N‐diethylcarbamoylbenzyl residue was attached to the 6,8‐diazabicyclo[3.2.2]nonane framework to yield the designed δ receptor ligands. In a first series of compounds the benzhydryl moiety of SNC80 was dissected, and one phenyl ring was attached to the bicyclic framework. In a second series of δ ligands the complete benzhydryl moiety was introduced into the bicyclic scaffold. The determined δ receptor affinities show that compounds based on an (R)‐glutamate‐derived bicyclic scaffold possess higher δ receptor affinity than their (S)‐glutamate‐derived counterparts. Furthermore, an intact benzhydryl moiety leads to δ receptor ligands that are more potent than compounds with two separated aromatic moieties. Compound 24 , with the same spatial arrangement of substituents around the benzhydryl stereocenter as SNC80, shows the highest δ receptor affinity of this series: K_i=24 nM . Whereas the highly potent δ ligands reveal good selectivity against μ and κ receptors, the σ₁ and/or σ₂ affinities of some compounds are almost in the same range as their δ receptor affinities, such as compound 25 (σ₂: K_i=83 nM ; δ: K_i=75 nM ). In [³⁵S]GTPγS assays the most potent δ ligands 24 and 25 showed almost the same intrinsic activity as the full agonist SNC80, proving the agonistic activity of 24 and 25 . The enantiomeric 4‐benzylidene derivatives 15 and ent‐ 15 showed selective cytotoxicity toward the 5637 (bladder) and A‐427 (small‐cell lung) human tumor cell lines.     

Challenging Approach to the Development of Novel Estrogen Receptor Modulators Based on the Chemical Properties of Guaiazulene

Tamoxifen, a therapeutic agent for breast cancer, has been associated with genetic polymorphisms in the metabolism of N,N-dialkylaminoethyl substituent, which plays an important role in the expression of selective estrogen receptor modulator (SERM) activity. To solve this problem, we developed a novel estrogen receptor (ER) modulator, Az-01, on the basis of the aromaticity, dipole moment, and isopropyl group of guaiazulene. Az-01 showed four-fold lower binding affinity for ER than E2 but had similar ER-binding affinity to that of 4-hydroxytamoxifen (4-HOtam). Unlike tamoxifen, Az-01 acted as a partial agonist with very weak estrogenic activity at high concentrations when used alone, and it showed potent anti-estrogenic activity in the presence of E2. The cell proliferation and inhibition activities of Az-01 were specific to ER-expressing MCF-7 cells, and no effect of Az-01 on other cell proliferation signals was observed. These findings are important for the development of new types of SERMs without the N,N-dialkylaminoethyl substituent as a privileged functional group for SERMs.     

Steroid conjugates of dichloro(6-aminomethylnicotinate)platinum(II): effects on DNA, sex hormone binding globulin, the estrogen receptor, and various breast cancer cell lines

Esters of 6-aminomethylnicotinic acid with various steroidal alcohols were treated with K(2)PtCl(4) to give the N,N-chelated dichloroplatinum(II) complex conjugates 4. Their interaction with plasmid DNA was monitored by electrophoretic mobility measurements. Their affinities towards sex hormone binding globulin (SHBG) and towards the nuclear estrogen receptor ER(alpha) were assessed by competitive displacement radioassays. The inhibitory effect of 4 on breast tumour cells MCF-7 ER(+)/ER(-) and MDA-MB-231 was investigated in vitro. Conjugates with 3-O-linked estrogens 4 a,b or 17-O-linked androgens 4 g bound strongly to SHBG. The conjugate complex 4 b, featuring a 3-O-linked estradiol, also bound strongly and agonistically to the estrogen receptor. It also elicited distinct growth retardation of MCF-7 (ER(+)) cells, presumably by a mechanism different from that of cisplatin.     

取代基物化参数用于2-苯基吲哚衍生物构效关系的研究

用取代基电性、立体性和疏水性物化参数对2-苯基吲哚衍生物进行了结构表征,并对化合物与雌激素受体相对亲和力进行定量结构活性相关(QSAR)研究。经逐步回归筛选变量后,所建多元线性回归方程的复相关系数R2及留一法交互检验相关系数R2cv分别为0.900和0.662。用预测集样本进行了外部预测,所得外部预测样本集复相关系数Re2xt和外部预测集交互检验Q2ext分别为0.896 6和0.897 4。模型结果显示:吲哚环1号N原子上立体效应强的取代基有利于亲和力的提高;而吲哚环1号N原子和3号C原子上电性强的取代基、2-苯基环上4′号位置有羟基取代等,均对化合物亲和力的提高不利。     

Structure-activity relationships for a family of benzothiophene selective estrogen receptor modulators including raloxifene and arzoxifene

The search for the "ideal" selective estrogen receptor modulator (SERM) as a substitute for hormone replacement therapy (HRT) or use in cancer chemoprevention has focused on optimization of estrogen receptor (ER) ligand binding. Based on the clinical and preclinical benzothiophene SERMs, raloxifene and arzoxifene, a family of SERMs has been developed to modulate activity and oxidative lability. Antiestrogenic potency measured in human endometrial and breast cancer cells, and ER ligand binding data were correlated and seen to provide a guide to SERM design only when viewed in toto. The in vitro studies were extended to the juvenile rat model, in which the desired antiestrogenic profile and putative cardiovascular benefits of SERMs were observed.     

N-(Anilinoethyl)amide Melatonergic Ligands with Improved Water Solubility and Metabolic Stability

The MT₂-selective melatonin receptor ligand UCM765 (N-(2-((3-methoxyphenyl)(phenyl)amino)ethyl)acetamide), showed interesting sleep inducing, analgesic and anxiolytic properties in rodents, but suffers from low water solubility and modest metabolic stability. To overcome these limitations, different strategies were investigated, including modification of metabolically liable sites, introduction of hydrophilic substituents and design of more basic derivatives. Thermodynamic solubility, microsomal stability and lipophilicity of new compounds were experimentally evaluated, together with their MT₁ and MT₂ binding affinities. Introduction of a m-hydroxymethyl substituent on the phenyl ring of UCM765 and replacement of the replacement of the N,N-diphenyl-amino scaffold with a N-methyl-N-phenyl-amino one led to highly soluble compounds with good microsomal stability and receptor binding affinity. Docking studies into the receptor crystal structure provided a rationale for their binding affinity. Pharmacokinetic characterization in rats highlighted higher plasma concentrations for the N-methyl-N-phenyl-amino derivative, consistent with its improved microsomal stability and makes this compound worthy of consideration for further pharmacological investigation.     

Putative biomarkers and targets of estrogen receptor negative human breast cancer

Breast cancer is a progressive and potentially fatal disease that affects women of all ages. Like all progressive diseases, early and reliable diagnosis is the key for successful treatment and annihilation. Biomarkers serve as indicators of pathological, physiological, or pharmacological processes. Her2/neu, CA15.3, estrogen receptor (ER), progesterone receptor (PR), and cytokeratins are biomarkers that have been approved by the Food and Drug Administration for disease diagnosis, prognosis, and therapy selection. The structural and functional complexity of protein biomarkers and the heterogeneity of the breast cancer pathology present challenges to the scientific community. Here we review estrogen receptor-related putative breast cancer biomarkers, including those of putative breast cancer stem cells, a minor population of estrogen receptor negative tumor cells that retain the stem cell property of self-renewal. We also review a few promising cytoskeleton targets for ER alpha negative breast cancer.     

Bis‐arylidene Oxindoles as Anti‐Breast‐Cancer Agents Acting via the Estrogen Receptor

We report a new family of bis‐arylidene oxindole derivatives that show highly selective estrogen receptor (ER)‐mediated anticancer activity at low‐nanomolar concentrations in ER‐positive (ER+) breast cancer cells. In terms of cell growth inhibition, IC₅₀ values for these compounds in ER+ breast cancer cells are two to three orders of magnitude lower than in ER‐negative (ER?) breast cancer cells and non‐cancer cells. In comparison with known bis‐arylidene drugs, these compounds are at least three orders of magnitude more toxic than tamoxifen and 1.5–4‐fold more toxic than 4‐hydroxytamoxifen in ER+ MCF‐7 cancer cells. These oxindoles inhibit ER transactivation, and their anticancer activities are inhibited in ER‐depleted MCF‐7 cells. Some of these nonsteroidal molecules also exhibit essential properties of selective ER down‐regulation. From the development of two series of bis‐arylidene oxindole‐based compounds, we report a new series of anticancer agents for estrogen‐responsive breast cancer.     

The predicted 3D structures of the human M1 muscarinic acetylcholine receptor with agonist or antagonist bound

The muscarinic acetylcholine G-protein-coupled receptors are implicated in diseases ranging from cognitive dysfunctions to smooth-muscle disorders. To provide a structural basis for drug design, we used the MembStruk computational method to predict the 3D structure of the human M1 muscarinic receptor. We validated this structure by using the HierDock method to predict the binding sites for three agonists and four antagonists. The intermolecular ligand-receptor contacts at the predicted binding sites agree well with deductions from available mutagenesis experiments, and the calculated relative binding energies correlate with measured binding affinities. The predicted binding site of all four antagonists is located between transmembrane (TM) helices 3, 4, 5, 6, and 7, whereas the three agonists prefer a site involving residues from TM3, TM6, and TM7. We find that Trp 157(4) contributes directly to antagonist binding, whereas Pro 159(4) provides an indirect conformational switch to position Trp 157(4) in the binding site (the number in parentheses indicates the TM helix). This explains the large decrease in ligand binding affinity and signaling efficacy by mutations of Trp 157(4) and Pro 159(4) not previously explained by homology models. We also found that Asp 105(3) and aromatic residues Tyr 381(6), Tyr 404(7), and Tyr 408(7) are critical for binding the quaternary ammonium head group of the ligand through cation-pi interactions. For ligands with a charged tertiary amine head group, we suggest that proton transfer from the ligand to Asp 105(3) occurs upon binding. Furthermore, we found that an extensive aromatic network involving Tyr 106(3), Trp 157(4), Phe 197(5), Trp 378(6), and Tyr 381(6) is important in stabilizing antagonist binding. For antagonists with two terminal phenyl rings, this aromatic network extends to Trp 164(4), Tyr 179(extracellular loop 2), and Phe 390(6) located at the extracellular end of the TMs. We find that Asn 382(6) forms hydrogen bonds with selected antagonists. Tyr381(6) and Ser 109(3) form hydrogen bonds with the ester moiety of acetylcholine, which binds in the gauche conformation.     

Conformational analysis of bivalent estrogen receptor ligands: from intramolecular to intermolecular binding

The estrogen receptor binding affinities of bivalent raloxifene ligands tethered by flexible spacers of different lengths have been evaluated in vitro. Two bivalent binding modes, intra- and intermolecular, were hypothesized to explain their different binding properties. The binding affinities of these bivalent ligands in an aqueous environment are influenced by their conformations, which can be determined by 2D NMR and UV spectral methods. Moreover, computer modeling and simulations were performed to explain the binding modes of these bivalent ligands and to estimate the conformational entropy difference between their unbound and bound states. It was found that bivalent ligands tethered by long spacers had weaker binding affinities because of the shielding of the binding moieties that results from their folded conformations; those tethered by short spacers had stronger affinities because they exposed their ligands to the receptor.     

Jostling for Position: Optimizing Linker Location in the Design of Estrogen Receptor‐Targeting PROTACs

Estrogen receptor‐α (ER) antagonists have been widely used for breast cancer therapy. Despite initial responsiveness, hormone‐sensitive ER‐positive cancer cells eventually develop resistance to ER antagonists. It has been shown that in most of these resistant tumor cells, the ER is expressed and continues to regulate tumor growth. Recent studies indicate that tamoxifen initially acts as an antagonist, but later functions as an ER agonist, promoting tumor growth. This suggests that targeted ER degradation may provide an effective therapeutic approach for breast cancers, even those that are resistant to conventional therapies. With this in mind, we previously demonstrated that proteolysis targeting chimeras (PROTACs) effectively induce degradation of the ER as a proof‐of‐concept experiment. Herein we further refined the PROTAC approach to target the ER for degradation. The ER‐targeting PROTACs are composed of an estradiol on one end and a hypoxia‐inducing factor 1α (HIF‐1α)‐derived synthetic pentapeptide on the other. The pentapeptide is recognized by an E3 ubiquitin ligase called the von Hippel Lindau tumor suppressor protein (pVHL), thereby recruiting the ER to this E3 ligase for ubiquitination and degradation. Specifically, the pentapeptide is attached at three different locations on estradiol to generate three different PROTAC types. With the pentapeptide linked through the C7α position of estradiol, the resulting PROTAC shows the most effective ER degradation and highest affinity for the estrogen receptor. This result provides an opportunity to develop a novel type of ER antagonist that may overcome the resistance of breast tumors to conventional drugs such as tamoxifen and fulvestrant (Faslodex).     

Analysis of high-affinity binding determinants in the receptor binding epitope of basic fibroblast growth factor

Basic fibroblast growth factor (bFGF) is implicated in the pathogenesis of several vascular and connective diseases. A key step in the discovery of bFGF receptor antagonists to mitigate these actions is to define the functional epitope required for receptor binding of the growth factor. In previous studies, we identified Glu96 as an essential residue in this epitope using site-directed mutagenesis. Here we examined the role of solvent accessible neighboring residues of Glu96 of bFGF on receptor binding affinity. Wild-type bFGF and its muteins were cloned and expressed in Escherichia coli and evaluated for FGF receptor binding affinity. Replacement of Asn104 of bFGF by alanine reduced receptor binding affinity over 400-fold compared with wild-type bFGF. We next explored the effect of neighboring residues of Asn104 on receptor binding affinity-Muteins in which Arg97, Leu98, Glu99, Asn101, Asn102, Thr105 and Pro141 were individually replaced by alanine exhibited receptor binding similar to wild-type bFGF. By contrast, substitution of Tyr103 or Leu140 by alanine reduced receptor binding affinity about 400- and 150-fold, respectively, in accord with a previous report. We conclude that at least six solvent-accessible residues in bFGF are crucial for high-affinity receptor binding, as evidenced by at least a 10-fold diminution in the affinity of the corresponding alanine muteins. The polar residues Glu96 and Asn104 appear to form an area important for facilitating the initial contact between ligand and receptor, whereas Tyr24, Tyr103, Leu140 and Met142 form a hydrophobic patch that may stabilize the complex. The detailed structure of this functional epitope can be employed in the discovery and design of bFGF antagonists using computational methods.      

Synthesis and investigations on the oxidative degradation of C3/C5-alkyl-1,2,4-triarylpyrroles as ligands for the estrogen receptor

In this study, we synthesized 1,2,4‐triarylpyrroles as ligands for the estrogen receptor (ER). Two pyrrole series were prepared with either C3‐alkyl or C3/C5‐dialkyl residues. Compounds from both series were susceptible to oxidative degradation—dialkylated compounds (t_1/2=33–66 h) to a higher extent than their monoalkylated congeners (t_1/2=140–211 h). Nevertheless, stability was sufficient for determination of in vitro ER binding affinity. The most active agonist in hormone‐dependent, ERα‐positive MCF‐7/2a and U2‐OS/α cells was 1,2,4‐tris(4‐hydroxyphenyl)‐3‐propyl‐1H‐pyrrole ( 6 d ) (MCF‐7/2a: EC₅₀=70 nM ; U2‐OS/α: EC₅₀=1.6 nM ). A corresponding inactivity in U2‐OS/β cells demonstrated the high ERα selectivity. This trend was confirmed in a competition experiment using estradiol (E2) and purified hERα and hERβ proteins (relative binding affinity (RBA) calculated for 6 d : RBA(ERα)=1.85 %; RBA(ERβ) <0.01 %). Generally, C3/C5‐dialkyl substitution led to reduction of activity, possibly due to lower stability.     

Discovering small-molecule estrogen receptor α/coactivator binding inhibitors: high-throughput screening, ligand development, and models for enhanced potency

Small molecules, namely coactivator binding inhibitors (CBIs), that block estrogen signaling by directly inhibiting the interaction of the estrogen receptor (ER) with coactivator proteins act in a fundamentally different way to traditional antagonists, which displace the endogenous ligand estradiol. To complement our prior efforts at CBI discovery by de?novo design, we used high-throughput screening (HTS) to identify CBIs of novel structure and subsequently investigated two HTS hits by analogue synthesis, finding many compounds with low micromolar potencies in cell-based reporter gene assays. We examined structure-activity trends in both series, using induced-fit computational docking to propose binding poses for these molecules in the coactivator binding groove. Analysis of the structure of the ER-steroid receptor coactivator (SRC) complex suggests that all four hydrophobic residues within the SRC nuclear receptor box sequence are important binding elements. Thus, insufficient water displacement upon binding of the smaller CBIs in the expansive complexation site may be limiting the potency of the compounds in these series, which suggests that higher potency CBIs might be found by screening compound libraries enriched with larger molecules.     

Synthesis,receptor binding,molecular modeling,and proliferative assays of a series of 17alpha-arylestradiols

A series of new derivatives of estradiol substituted at position 17alpha by various aryls has been synthesized. This was made possible by efficient activation methods for the addition of aryllithiums to the carbonyl group at position 17 of estrone by using tetramethylethylenediamine (TMEDA) or BF3 x OEt2. Their relative binding affinity (RBA) for the alpha and the beta forms of the estrogen receptor (ER) have been measured. All except one of the compounds synthesized had an RBA value of around 10 % which indicates a level of tolerance towards the bulky substituent at position 17. The lipophilicity values measured for these compounds are higher than that found for estradiol (E2). A study of their proliferative/antiproliferative effects was carried out on hormone-dependent (MCF7) and hormone-independent (MDA-MB231) breast cancer cell lines. It is interesting to note that all the compounds are estrogenic. The possibility of easily attaching an iodine at the end of a phenyl spacer opens up a route to new radiopharmaceuticals for use in radioimaging.     

Cyclopenta[d]pyrimidines and dihydropyrrolo[2,3-d]pyrimidines as potent and selective corticotropin-releasing factor 1 receptor antagonists

Two new classes of potent and selective CRF(1) receptor antagonists are presented. Exploration of general templates 3 and 4 through modifications of the top amine and bottom phenyl substituents led to optimization of the in vitro affinity and pharmacokinetic profiles. The typical alkyl chains present in the top region of CRF(1) antagonists were replaced by substituted heteroaryl moieties, leading to a dramatic improvement of the metabolic stability. This improvement was apparent when the compounds were dosed in vivo: several compounds exhibited low plasma clearance, good oral bioavailability, and high brain penetration. As a consequence of their outstanding pharmacokinetic profiles, these CRF(1) antagonists, as exemplified by compound 4 fi (4-(4-bromo-3-methyl-1H-pyrazol-1-yl)-7-(2,4-dichlorophenyl)-2-methyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidine), produced a dose-dependent "anxiolytic-like" effect when administered orally, decreasing the vocalization of rat pups.     

Structure–Activity Relationships of 1-Benzoylazulenes at the OX1 and OX2 Orexin Receptors

We previously demonstrated the potential of di- or trisubstituted azulenes as ligands (potentiators, weak agonists, and antagonists) of the orexin receptors. In this study we investigated 27 1-benzoylazulene derivatives, uncovering seven potentiators of the orexin response on OX₁ and two weak dual orexin receptor agonists. For potentiators, replacement of the azulene scaffold by indole retained the activity of four out of six compounds. The structure–activity relationships for agonism and potentiation can be summarized into a bicyclic aromatic ring system substituted with two hydrogen-bond acceptors (1-position, benzoyl; 6-position, carboxyl/ester) within 7–8 Å of each other; a third acceptor at the 3-position is also well tolerated. The same pharmacophoric signature is found in the preferred conformations of the orexin receptor agonist Nag26 from molecular dynamics simulations. Subtle changes switch the activity between weak agonism and potentiation, suggesting overlapping binding sites.     

Development of novel 1,2,3,4-tetrahydroisoquinoline derivatives and closely related compounds as potent and selective dopamine D3 receptor ligands

Based on N-alkylated 1,2,3,4-tetrahydroisoquinoline derivatives, which are structurally related to the partial agonist BP 897, a series of novel, selective dopamine D3 receptor antagonists has been synthesised. Derivatisation included changes in the arylamide moiety and the tetrahydroisoquinoline substructure leading to compounds with markedly improved selectivities and affinities in the low nanomolar concentration range. From the 55 structures presented here, (E)-3-(4-iodophenyl)-N-(4-(1,2,3,4-tetrahydroisoquinolin-2-yl)butyl)acrylamide (51) has high affinity (Ki(hD3)=12 nM) and a 123-fold preference for the D3 receptor relative to the D2 receptor subtype. Its pharmacological profile offers the prospect of a novel radioligand as a tool for various dopamine D3-receptor-related in vitro and in vivo investigations.     

A Scaffold-Hopping Strategy toward the Identification of Inhibitors of Cyclin G Associated Kinase

We recently reported the discovery of isothiazolo[4,3-b]pyridine-based inhibitors of cyclin G associated kinase (GAK) displaying low nanomolar binding affinity for GAK and demonstrating broad-spectrum antiviral activity. To come up with novel core structures that act as GAK inhibitors, a scaffold-hopping approach was applied starting from two different isothiazolo[4,3-b]pyridines. In total, 13 novel 5,6- and 6,6-fused bicyclic heteroaromatic scaffolds were synthesized. Four of them displayed GAK affinity with K_d values in the low micromolar range that can serve as chemical starting points for the discovery of GAK inhibitors based on a different scaffold.