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  • C07D333/52—Benzo[b]thiophenes; Hydrogenated benzo[b]thiophenes
  • C07D333/54—Benzo[b]thiophenes; Hydrogenated benzo[b]thiophenes with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to carbon atoms of the hetero ring
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  • C07D333/52—Benzo[b]thiophenes; Hydrogenated benzo[b]thiophenes
  • C07D333/54—Benzo[b]thiophenes; Hydrogenated benzo[b]thiophenes with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to carbon atoms of the hetero ring
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  • C07D333/52—Benzo[b]thiophenes; Hydrogenated benzo[b]thiophenes
  • C07D333/54—Benzo[b]thiophenes; Hydrogenated benzo[b]thiophenes with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to carbon atoms of the hetero ring
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  • C07K14/721—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor

Definitions

• This invention relates to estrogen receptor ligands. More particularly, the present invention relates to ligands which will bind to estrogen receptors, crystals of such receptors, including crystals of receptor and ligand, synthetic ligands, methods of using such synthetic ligands and methods for designing ligands which will bind to the estrogen receptor.
• the thyroid hormone receptor (TR) is known and its three-dimensional structure, and hence its ligand binding domain, has been determined. Knowledge of the three-dimensional structure has enabled a better understanding of the modes of ligand binding and the determination of the optimum conformation of ligand to bind to the receptor. This understanding will provide a pharmacophore model usable in the design of ligands, such as drugs, to bind to the thyroid receptor. It is generally believed in the art that the TR structure also provides a guide to the design of ER ligands.
• Estrogen steroid hormone and thus the estrogen receptor (ER) is a member of the steroid hormone receptor family. Its primary natural ligand is estradiol (E2). However, it is known that a large number of structurally diverse non-steroidal compounds such as raloxifene, centchroman, coumestrol, diethylstilbesterol, esculin, tamoxifen, zearalenone, and zindoxifen also bind to the estrogen receptor (Fig. 8). The majority of these non-steroidal estrogen receptor ligands contain 2-4 carboxyclic, aromatic, and/or heterocyclic rings connected by a 1-3 atom chain. One or more of the rings may be fused with the central atom chain or with each other.
• the receptor possesses a multi-functional modular structure potentially having discrete domains for DNA binding, ligand binding, and transactivation.
• the ligand binding domain (LDB) has been designated domain E and is the largest domain of the estrogen receptor.
• the ligand binding domain includes a ligand recognition site and regions for receptor dimerzation, interaction with heat shock proteins, nuclear localization and ligand dependent transactivation.
• estrogen agonists for treatment of disease linked to estrogen deficiency e.g., osteoporosis, cardiovascular and neurodegenerative diseases in post menopausal women
• estrogen antagonists for treatment of breast and uterine cancer e.g., certain ligands such as tamoxifen display mixed agonist/antagonist action (that is they are either estrogen agonists, estrogen antagonists, or a partial estrogen antagonists when binding to the estrogen receptors of different tissues) and such compounds may simultaneously prevent bone loss and reduce the risk of breast cancer.
• benzothiophenes are usable as agonists or antagonists to steroid hormones, and that it is possible to modify their binding mechanics, for example the binding affinity, by changing the substituent groups at various positions on the molecule. Therefore, it would be desirable to be able to design ligands which are recognizable by and able to bind to the estrogen receptor. Additionally, it would be desirable to know the three dimensional structure of the estrogen receptor. Such knowledge would be useful for the design of compounds intended to bind to the estrogen receptor. The present inventors have been able to produce an estrogen receptor crystal and to determine from that the three dimensional structure of the estrogen receptor. Unexpectedly, the thus determined ER structure reveals that the TR structure does not provide a good model for binding of ligands to ER.
• the present invention provides an estrogen receptor ligand binding domain crystal.
• the present invention provides ligands, particularly synthetic ligands, of estrogen receptors by use of the crystals.
• methods for designing ligands which will bind to the estrogen receptor are provided. Such methods use three dimensional models based on the crystals of the estrogen receptor. Generally, such methods comprise, determining compounds which are likely to bind to the receptor based on their three dimensional shape compared to that of the estrogen receptor and in particular the ligand binding domain of the estrogen receptor. Preferably, those compounds have a structure which is complementary to that of the estrogen receptor.
• Such methods comprise the steps of determining which amino acid or amino acids of the ligand binding domain of the estrogen receptor interacts with the binding ligand, and selecting compounds or modifying existing compounds, to improve the interaction.
• improvements in the interaction are manifested as increases in the binding affinity but may also include increases receptor selectivity and/or modulation of efficacy.
• the ligands bind to the ER with a high binding affinity, for example within the range of 20-2000 pmol.
• the ligands may bind tightly bind to the ER yet not up-regulate gene expression thereby inhibiting the action of estradiol and estradiol mimetics.
• the invention also provides a method of inhibiting the activity of estradiol or estradiol mimetics by providing ligands which bind to ER with a high affinity, blocking the activity of estrogens.
• binding of the ligand to the ER may cause conformational changes to the ER inhibiting further binding thereto.
• the invention further provides a method of inhibit estradiol activity in an animal, the method comprising administering to the animal a ligand which binds to at least the LBD, of the ER with high affinity and blocks binding of further ligands to at least the LDB of the ER.
• ligands are useful in, for example, the treatment of estrogen receptor mediated diseases in females. Structure Based Design of ER Ligands
• the present work has elucidated the structure of the ligand binding cavity of the estrogen receptor.
• Knowledge of the structure of this cavity has utility in the design of structurally novel ER ligands and in the design of non-obvious analogs of known ER ligands with improved properties.
• These enhanced properties include one or more of the following: (1) higher affinity, (2) improved selectivity for either the ⁇ - or ⁇ -isoform of the ER, and/or (3) a designed degree of efficacy (agonism vs. partial agonism vs. antagonism).
• the ER receptor structure also has utility in the discovery of new, structurally novel classes of ER ligands.
• Electronic screening of large, structurally diverse compound libraries such as the Available Chemical Directory (ACD) will identify new structural classes of ER ligands which will bind to the 3-dimensional structure of the estrogen receptor.
• ACD Available Chemical Directory
• the ER structure allows for "reverse-engineering” or “de novo design” of compounds to bind to the ER.
• the present work has revealed the presence of receptor defined ⁇ - and ⁇ -face cavities centered respectively above and below the B- and C-rings of estradiol.
• the present invention provides new ligands which exploit this discovery by filling the ⁇ - and ⁇ -face cavities.
• the ligand fills at least one of the ⁇ - and ⁇ -face cavities so as to exclude water from the cavity or cavities.
• the ligands produced in accordance with the invention bind more effectively to the ER than estradiol.
• the ligand may bind with twice the binding affinity of estradiol, preferably three times the affinity, and most preferably ten or more times the affinity.
• Modifications to the steroid nucleus may be made at the positions marked in R in Fig. 8a and 8b ( ⁇ -substitution at the 7-, 9-, 12-, 14-, 16-, and 17-positions; ⁇ -substitution at the 8-. 1 1-, 15-, and 18-positions).
• those substituents are hydrophobic substituents, e.g., methyl, ethyl, iso-propyl, chlorine, bromine, or iodine.
• Modifications to 2-aryl benzothiophenes may be made at the 2'-, 3'-, and 6' -positions (Fig. 8c) in order to fill the ⁇ - and ⁇ -face cavities of ER.
• substituents should be present in at least two of the following three positions: 3, 2', or 6' so that a perpendicular conformation between the B- and C-rings of the 2-aryl benzothiophene nucleus is enforced. This perpendicular conformation facilitates the positioning of the 2'-, 3'-, and 6' -substituents in the ⁇ - and ⁇ -face cavities of ER.
• the affinity of other classes of non-steroidal ER ligands may be enhanced by substitution of small hydrophobic substituents at positions marked R2', R3', and/or R6' in Fig. 8C.
• the ligand produce in accordance with the invention fills at least one of the ⁇ - and ⁇ -cavities of the ER without perturbing the remainder of the ER structure.
• Another aspect of this invention reveals an unfilled hydrophobic cavity in the raloxifene/ER complex. Filling this cavity with hydrophobic substituents so as to exclude water will enhance binding affinity.
• This cavity may be filled by positioning a hydrophobic substituent on the ethoxyphenyl sidechain to the piperidinyl nitrogen atom of raloxifene.
• This hydrophobic substituent may be a linear alkyl or perfluoroalkyl group (-CH 3 to -C 10 H 21 , -CF 3 to -C 10 F 21 ), benzyl (-CH : Ph, or methylene cyclohexyl (-CH 2 C 6 H ⁇ ).
• examination of the ER structure reveals that the hydroxyl group at position-3 of estradiol or position-6 of raloxifene form hydrogen bonding interactions with Glu-353 and Arg-394 (Fig. 5a and 5b). It is known that replacement of the hydroxyl group at position-3 of estradiol or position-6 of raloxifene results in a decrease in affinity for the ER.
• the invention reveals the reason for this reduction in affinity: while one of the hydrogen atoms of the amino group forms a favorable hydrogen bonding interaction with Glu-353, the second hydrogen atom forms an unfavorable electrostatic interaction with Arg-394.
• this invention reveals a method for enhancing the affinity of 3 -amino analogs of estradiol and 6-amino analogs of raloxifene: replacement of one of the two hydrogen atoms of the amino group with an alkyl group to produce a secondary amino group.
• the amino group may be replaced with a guanidino group (Fig. 8e) which will pick up two additional hydrogen bonding interactions, the first is a salt bridge to Glu-353 and the second is a hydrogen bonding interaction with a backbone carbonyl group in residue Leu-387.
• Similar enhancement of affinity for the ER may be achieved by replacement of the guanidino group with a fused 2-aminopyrrole (Fig. 8e).
• the estrogen receptor has been found to have two discrete forms, known as ER ⁇ and ER ⁇ . Furthermore the ratio of the ⁇ - to the ⁇ -forms of the ER may vary considerably in different cell and tissue types. Therefore it may be possible to dissociate desirable therapeutic effects from undesirable side effects of estrogen receptor ligands by designing ligands that selectively bind to one or the other isoforms of the estrogen receptor.
• the ⁇ - and ⁇ -forms of the estrogen receptor differ significantly in their primary sequence and slightly in their tertiary structure. As a consequence of these receptor differences, ligands may bind with different affinity to the two isoforms.
• the present inventors have been able to isolate, differentiate and produce crystals for the ER ⁇ . From these crystals, the present inventors have determined the three dimensional structure to high resolution. Further, the inventors have created a partial homology model of ER ⁇ based on the experimentally derived ER ⁇ coordinates. This partial ER ⁇ homology model captures the essential differences in binding properties between ER ⁇ and ER ⁇ . Based on a comparison of the experimental ER ⁇ coordinates and the partial homology model of the ER ⁇ , the differences between the ER ⁇ and ER ⁇ have been determined and using these differences, the ability of a ligand to bind to either the ER ⁇ and ER ⁇ receptors or to both receptors can be predicted.
• the ligands may be designed to specifically bind ER ⁇ ir ER ⁇ .
• RAL raloxifen
• the invention provides estrogen receptor ligand binding domain crystals for ER ⁇ and a partial homology model for ER ⁇ . Specificity of ligands for either the ER ⁇ and ER ⁇ or even to a specific ratio of ER ⁇ to ER ⁇ is also provided. The advantage of this is that tissue specificity is conferred to the ligand.
• the invention also provides ligands, particularly synthetic ligands of ER ⁇ and ER ⁇ together with methods for their design.
• the present invention provides new ligands which exploit these differences by positioning ligand substituents in close proximity to one or more amino acid residue that differ between the ⁇ - and ⁇ -isoforms of the ER.
• the ligands produced in accordance with the invention bind more effectively to either the ⁇ - or ⁇ -isoforms of the ER.
• the selectivity of the binding between the ⁇ - or ⁇ -isoforms may be ten-fold, more preferably one hundred-fold, and most preferably greater than one thousand-fold.
• substitutions may be made from either the 2'- or 3 '-positions of the 2-arylbenzothiophene nucleus to interact with residue-384 in the ⁇ -face cavity or from the 6' -position to interact with residue-421 in the ⁇ -face cavity (Fig. 9a and 9b).
• free rotation about the C2-C1' bond will effectively interchange the substituents at the 2'- and o p positions thereby reducing selectivity.
• Moving the hydroxyl group from position-4' (Fig. 9a) to position-5' (Fig. 9b) will bias the binding orientation such that the R 2 substituent will be positioned in the ⁇ -face pocket and the R 6 substituent in the ⁇ -face pocket. This bias results from the fact that only one of the two possible rotamers about the C2'C1' bond will allow hydrogen bond formation between the 5 '-hydroxyl group and the receptor residue His-524.
• This invention also provides a means of enhancing the selectivity of other classes of non-steroidal ER ligands.
• substituents larger than methyl may be introduced at either the R2' or R3' positions to produced ER- ⁇ selective compounds or at R 6 ' to produce ER- ⁇ selective compounds (Fig. 8c).
• Substitutions may be made from position-3 of the steroid nucleus or position-6 of the benzothiophene nucleus to exploit the differences between ER- ⁇ and ER- ⁇ at position-326 (He in ER- ⁇ and Val in ER- ⁇ ) and at position-445 (Phe in ER- ⁇ and Tyr in ER- ⁇ ).
• This invention also provides a means for producing specifically ER- ⁇ selective ligands.
• a six atom linker between the hydroxyl group at position-3 of the A-ring of estradiol or at position-6 raloxifene and an aromatic or heteroaromatic ring on the sidechain will position the sidechain ring in close proximity to residue-445 (Fig. 9c).
• the edge of ER- ⁇ Phe-445 and the face of the sidechain ring can form a favorable " ⁇ -teeing" interaction. This favorable interaction is not possible with the ER- ⁇ Tyr-445, therefore analogs of this type with be ER- ⁇ selective (Fig. 9d).
• Another aspect of this invention provides a means of further enhancing ER- ⁇ selectivity.
• Introduction of a carboxylate or amino group on the meta or para position of the above mentioned aromatic or heteraromatic ring will form a hydrogen bonding interaction between the conserved Glu-323 or Lys-449 (Fig. 9e).
• the heteroaromatic ring may be a pyridone ring which will simultaneously form favorable hydrogen bonding interactions with both Glu-323 or Lys-449 (Fig. 9f).
• Either of the amino, carboxylate, or pyridone ring substitutions will reinforce the favorable " ⁇ -teeing" interaction between the aromatic or heteroatomic ring of the ligand and Phe-445 in ER- ⁇ .
• This invention provides an understanding of the differences between estrogen and antiestrogen binding and therefore a means to design ER ligands with the desired degree of efficacy.
• An examination of the differences between the ER/estradiol and ER/raloxifene complexes reveals a large movement in Helix-12 (H12, Fig. 6).
• H12 adopts an "agonistic" conformation defined by the structure of the ER/estradiol complex and an "antagonistic" conformation defined by the structure of the ER/raloxifene complex. These two conformation are in thermodynamic equilibrium. When the ER is complexed with a full agonist, such as estradiol, the equilibrium lies far in the direction of the "agonistic" conformation.
• this invention provides a means of developing ligands with the desired degree of efficacy (agonist, partial agonist, or antagonist).
• H12 has been determined as playing a central role in determining the efficacy (agonism vs. antagonism) of a ligand.
• ligands which are able to bind to and/or alter the conformation of H12 are of particular importance when designing a ligand or assessing the binding of a ligand, for the estrogen receptor.
• raloxifene has a different binding conformation to that of estradiol, the distinction lying in its active conformation but being unpredictable by virtue of it antagonistic action.
• the antagonism has been shown, by the present inventors, to be caused by a protruding portion on the raloxifene molecule which causes a large displacement of H12 relative to its conformation in the ER/estradiol complex.
• Disruptions of this type can be used to predict antagonism or to produce antagonists. Disruptions may take the form of ligand binding which alters the conformation of the helices that comprise the dimerization interface or direct binding to the dimerization interface which then inhibits dimerization.
• the orientation of the ligand may be keyed to the receptor, in the dimeric or monomeric form.
• the influence of ligand binding to the LDB on the receptor conformation can now be shown to have influences on the behavior of the receptor since it may disrupt the binding of co-activator, co-repressor, or heat-shock proteins. Previously, such predictions could not me made.
• the crystal is produced from a sequence comprising at least one hundered and fifty amino acids of the selected estrogen receptor. More preferably, the sequence comprises at least two hundred amino acids. Most preferably, the sequence comprises at least two hundred and fifty amino acids. Preferably, the sequence comprises at least a portion of the ligand binding domain of the estrogen receptor. More preferably, the sequence comprises the whole ligand binding domain of the estrogen receptor.
• ER LBDs are purified to homogeneity for crystallization. Purity of ER LBDs is measured with SDS-PAGE, mass spectrometry, and hydrophobic HPLC. The purified ER for crystallization should be at least 97.5% pure, preferably at least 99.0% pure, and most preferably at least 99.5% pure.
• the crystals used can withstand exposure to X-ray beams used to produce the diffraction pattern data necessary to solve the X-ray crystallographic structure.
• crystals grown using estrogen receptor sequence bound to a various of ER ligands have been found to decompose during exposure to X-ray beams at room temperature, whereas crystals grown using estrogen receptor sequence bound to various ER ligands are freezable and are able to withstand exposure to X-ray beams.
• the crystals have a resolution determined by X-ray crystallography of less than 3.5 A and most preferably less than 2.8 A.
• crystals grown using naturally occurring estradiol have an effective resolution of lower than 3.1 A and crystals grown using raloxifene have an effective resolution of lower than 2.6 A.
• estradiol binds to the estrogen receptor and hence the structural reasons why a compound behaves as an estrogen can not only be understood but also predicted. This enables an understanding of the promiscuity of the estrogen receptor - its ability to bind a variety of structurally diverse ligands. This understanding can be applied to a greater or lesser extent to all steroid hormone receptors, especially the glucocorticoid receptor.
• Crystals of the estrogen receptor binding domain can be used as models in methods for the design of synthetic compounds intended to bind to the receptor. Such models show why very slight difference in chemical moieties of a ligand potentially have widely varying binding affinities. Hence, the three dimensional structure of the ligand binding domain can be used a pharmaceutical model for compounds which bind to estrogen receptors.
• Figure la shows representative portions of a 2.6 A resolution multicrystal averaged map for a RAL-ER-LBD complex
• Figure lb is a 3.1 A resolution six-fold averaged map for a E2-ER-LBD complex.
• the map is contoured at IF and superimposed on the final, refined models;
• Figure 2a is a schematic representation of the ER- LBDa indicating the locations of the various secondary structural elements " and 3 10 helices are coloured grey, extended regions are very light grey and coil regions are coloured in dark grey. E2 is coloured very dark grey and is highlighted in space-filling form;
• Figure 2b is a topology diagram for ER-LBD. Helices are represented as rectangles and ⁇ strands as arrows. The central core layer (H5,H6,H9 and H10 - striped) is sandwiched between the outer flanking layers (HI- 4) (H7, H8 ,H1 1). The structural elements which flank the layered motif (S1/S2 and H12) are S I, S2, H12 and are cross hatched. The N and C termini are also labelled. All secondary structural elements have been numbered in keeping with the nomenclature that has been established for other known nuclear receptor LBDs;
• Figure 3a is a stereoview of the ligand binding cavity.
• the cavity is viewed in a similar orientation to that given in Fig. la. Sidechains for residues that line the cavity are illustrated. Hydrophobic residues are shown in grey, basic residues are shown as spotted and acidic residues are shown in hatched.
• E2 is coloured black (core) and dark grey (terminal hydroxyl groups);
• Figure 3b is a schematic representation of the ligand binding cavity. Residues that make direct hydrogen bonds to the hydroxyl radius are shown in ball-and-stick representation along with hydrophobic residues that make non-polar interactions with E2 (shown as grey with radial spokes). The atom names and ring nomenclature of E2 are also given;
• Figure 3c is a representation of the molecular volume of E2 (dark grey dotted surface) and the accessible binding cavity volume (light grey dotted surface);
• Figure 4a is a schematic representations of the ER- ⁇ LBD non-crystallographic dimer viewed perpendicular to the dimer axis. The N and C termini are labelled;
• Figure 4b is a view of the dimer of Fig. 4a along the dimer axis. E2 is highlighted in mid grey in space-filling form. Helices that are involved in the dimer interface are labelled;
• Figure 4c is a view showing the Hl l helices that form the backbone of the dimer interface. Interacting residues are show coloured according to polarity (grey -hydrophobic residues; hatched - polar residues; cross-hatched - basic residues);
• Figure 5a is a schematic representation of the binding cavity and interactions made by E2. The figure was produced using LIGPLOT software;
• Figure 5b is a comparison of the E2 and RAL binding modes (E2 - dark grey; RAL - light grey);
• Figure 6 is a schematic representation of the ER-LBD showing the different positioning of helix 12 in the E2 (cross-hatched) and RAL (hatched) complexes. The remainder of the ER-LBD is shown in grey. Dashed lines indicate unmodelled regions of the structure. The helices which interact with H12 in the two complexes are marked: and
• Fig. 7 is a space filling representation of a) an E2 complex and b, an RAL complex.
• H12 black
• Raloxifene induces a conformational change so that H12 occupies a hydrophobic groove between H3 and H5.
• the hydrophobic sidechains of all residues that lie between residues 354 (H3) and 380 (H5) are drawn in dark grey.
• Other highlighted residues are Lys362 (hatched), Glu380 and Tyr537 (cross-hatched), Asp351 (spots) and the ligand RAL (grey).
• the remaining atoms of the LBD monomer are white. Note that differences in other parts of the ER-LBD complexes may be due regions missing from the current models;
• Fig. 8 shows the structure of several representative estrogen receptor ligands
• Figs. 8a, 8b and 8c show modifications made to the steroid nucleus of ligands which bind to the estrogen receptor;
• Figs. 8d and 8e show how affinity of the ligand can be enhanced by adding substituents
• Figs. 9a-9f show selectivity enhancement by using different substituents on the estrogen receptor ligand.
• Figs. 10 to 19 show by way of structural formulae the chemical reactions involved in the following Examples 1 to 51 , which are non-limiting and given by way of illustration only.
• Figure 20 shows crystal coordinates for estrogen receptor alpha (ER ⁇ ) binding domain in complex with raloxifene.
• Figure 21 shows crystal coordinates for estrogen receptor alpha (ER ⁇ ) binding domain in complex with 17-beta-estradiol.
• Figure 22 shows a homology model of estrogen receptor alpha (ER ⁇ ) beta complexed with raloxifene.
• Figure 23 shows a homology model of estrogen receptor-beta (ER ⁇ ) complexed with estradiol.
• the human EP-LBD- ⁇ was over expressed in Escherichia coli. (Hegy G.B. et al Steroids (1961 61 June 367-373). Fermentation was carried out as batch and fed batch cultivation in a defined glycerol/salt medium at 30°C. Production of recombinant protein was induced by raising the temperature to 39°C. After 2 h, cells were harvested by centrifugation, and frozen, thawed cells were disrupted by a Bead Beater homogenizer (6 x 22 sec, with a 3 min resting time between bursts) (Biospec.
• the column was first washed with 130 ml 10 mM Tris-HCl. (pH 7.8), 700 mM KCl, 1 mM EDTA, followed first by 130 ml 10 mM Tris-HCl (pH 7.8), 250 mM NaSCN, 10% dimethyl-formamide, 1 mM EDTA and then by 1 10 ml 10 mM Tris-HCl pH 8.0.
• Reactive Cys residues were modified by washing the column with 120 ml 30 mM Tris-base, 15mM iodoacetic acid, pH 8.1. Excess reagent was washed out by 50 ml Tris-base, 15 mM iodoacetic acid, pH 8.1.
• the stacking (0.7 cm) and resolving (70 cm) gels was 5.6% (acrylamide/bis).
• the elution buffer was lOmM Tris-HCl pH 8.0 and the electrophoresis was carried out at 12 W. Fractions containing ER-LBD- ⁇ was pooled and concentrated (Centriprep 30) to the desired protein concentration.
• Rfree is the same as Rcryst but was calculated using a test set of reflections (10% of the whole dataset) that was excluded from the refinement process.
• NCS non-crystallographic symmetry
• NCS non-crystallographic symmetry
• Phasing power 1.22 / 1.88 1.23 / 2.02 0.71 / 0.94
• Protein concentration in the drop was 8 mg/ml although X-ray suitable crystals were also obtained at 13 mg/ml. However, crystals obtained from such conditions were very often twinned and the addition of DMSO at up to 8% significantly improved their quality.
• the size of the crystals was correlated with the size of the sitting/hanging drop. The optimum size of the drop was achieved by mixing 2.5 ml of protein with 2.5ml of the reserved solution. All crystallisations were performed at 18°C. The best crystals, with a size of 0.5x0.05x0.05 mm 3 , were mounted in the X-ray quartz capillaries.
• the protein buffer was replaced with 20 mMTris/HCl pH 7.8-7.9 and the protein was concentrated usually to 10-12 mg/ml.
• the vapour diffusion method with the hanging drop technique was used for crystallations.
• the best conditions for crystallisation used the following medium: 0.1 M Tris/HCl buffer pH 8.3, 12% (w/v) of PEG 4000, 0.1 M Maltose, 50 mM Lysine, 0.2 M MgC12, 5% dioxane.
• the concentration of the protein solution used for crystallisation was brought up to 7.3-7.5 mg/ml by dilution with 20 mM Tris/HCl buffer pH 8.3. Cyrstallisations were performed with different drop sizes and protein-to-reservoir buffer ratios.
• cryoprotectant consisted of mother liquor (well buffer composition) and 25% v/v MPD.
• Pure ER-LBD is particularly refractive to crystallisation and suitable crystals were obtained after carboxymethylation of the three thiol groups.
• Crystals of the ER "-LBD complexed with either estradiol or raloxifen will diffract to medium resolution, are monoclinic and contain either a single dimer in the case of raloxifen or three dimers in the case of estrogen in the asymmetric unit (see Table 1).
• Multiple isomorphous replacement was used to determine the crystal structure of the ER-LBD-RAL complex.
• An initial multiple isomorphous replacement/density modified electron density map showed the position of the non-crystallographic two-fold rotation axis and allowed an initial polyalanine trace to be built on the resultant two-fold averaged map. Subsequent averaging between three different crystal forms of the RAL complex enabled corrections to be made to the initial trace.
• Example I and II The crystals produced in Example I and II were subjected to X-ray crystallographic studies which revealed that the LBD is folded into a characteristic "wedge-shaped" globular unit. It has a three-layered, anti-parallel ⁇ -helical sandwich motif and is constructed from 8 major helices The motif comprises a central core layer of 3 helices (H5/6, H9 and H10) sandwiched between two additional layers of helices (H2-3 and H7, H8, Hl l). The arrangement of structural elements in this fashion creates a "molecular scaffold” maintaining a sizable ligand binding cavity at the "toe end" of the wedge-shaped domain.
• the remaining secondary structural elements, a small two stranded anti-parallel ⁇ -sheet (SI and S2) and helix HI 2. are located at the "ligand binding end" of the molecule and flank the main three-layered motif (see Figure 2). From the N-terminus, the chain follows one turn of the distorted "-helix (HI), turns 90° and enters a short helix (H2) that lies parallel to the longest axis of the LBD. After helix H2, the chain continues in the same direction in an irregular extended conformation before tucking under the bottom of the molecule. At this stage, the chain turns back on itself through the long, bent, helix (H3). The N-terminal portion of this helix forms part of the ligand binding cavity.
• the sequence has a proline at position 365 which is invariable and it is at this residue that the main chain takes a sharp (90°) change in direction, passes through a 310 helix (H4) before forming the first of three central helices (H5/6).
• Helix H5/6 can be geometrically described as a single unit, although it is kinked by 40° at the alanine residue at position 382 in a manner such that its C-terminal end is correctly positioned to form part of the E2 binding cavity.
• This helix is kinked and is distinguishing and is maintained by a series of hydrophobic interactions between leucines at 378 and 379 (H5) with a phenol at 367 and leucine at 453 all of which are highly conserved and are part of the nuclear receptor LBD signature motif (Wurst). From this position the sequence passes through a small $ ⁇ hairpin (S1/S2) covering one side of the binding cavity, and emerges on the other side of the molecule via the 3 10 helix H7. Helix H8 runs three quarters of the way up the long axis of the LBD, passes through a second central helix (H9) before turning back via a disordered loop to form a final helix H10.
• the polypeptide backbone changes direction and runs the full length of the ligand binding domain, in an anti-parallel direction to H8 in the form of helix 1 1.
• the chain emerges on the opposite side to the S1/S2 ⁇ hairpin at helix HI 2, the core amphipathic helix of the AF-2 region.
• the dimer axis coincides with the longest axis of the LBD with each molecule tilted approximately 15° away from the two position fold. This symmetric arrangement generates a molecule with dimensions of approximately 55A high by 50A wide by 35-60A breadth.
• the observed quaternary arrangement locates the N and C termini of each monomer on the opposite "faces" of the dimer. The C terminus of each monomer projects towards the dimer axis while the N termini are far removed from the interface.
• the dimerisation surface is fairly extensive and encompasses about 15% ( 1,703 A 2 ) of each monomer's accessible surface area.
• the LBD's are positioned so that the H8/H1 1 face of each monomer lines up to form an additional, intermolecular helical layer.
• Hl l helices are arranged as a bifurcated coiled coil with the side chains of the residues Leu 504, Ala 505, Leu 508, Leu 509 and Leu 51 1 which are interdigitated to form a partial "leucine zipper" motif at the coils end terminal N.
• This hydrophobic patch is flanked on either side by a network of hydrogen bonding residues. Arg 545 and Asn 519 make direct hydrogen bonds with Ser 512 and His 516 respectively.
• LBD's quaternary structure therefore suggests that it provides a stable entity that facilitates separation of the two DNA binding domains in such a way as to allow optimal binding to EREs.
• Such an elucidation of the 3-dimensional structure of the estrogen receptor ligand binding domain provides a useful tool for designing ligands for binding to the estrogen receptor.
• Such a detailed knowledge of the structure of the receptor enables prediction with accuracy whether a ligand binding to the receptor will act as an antagonist, a partial antagonist, an agonist or a partial agonist since the ligand-induced conformational changes can be anticipated.
• Example I The coordinates obtained in Example I (ER ⁇ complexed with either estadiol or with raloxifine) were used to create two partial homology models of ER ⁇ (complexed with estradiol and raloxifene respectively). This was accomplished by importing the ER ⁇ coordinates into version 6.4 of Sybyl (available from Tripos Associates, St. Louis, MO, U.S.A.). The "change" command in the Sybyl biopolymer module was used to mutate amino acids which differ between ER ⁇ and ER ⁇ and which are in the vicinity of the ligand binding pocket.
• the crude product was purified on a chromatotron (silica, 6:4, petroleum ether/ ethyl acetate) produc i ng 22 mg (0.086 mmol , 48 % ) of 2 -( 2 -methyl -4- aminophenyl)-6-hydroxybenzo[ ⁇ ]thiophene.
• Example 24 2-(2-methyl-5-hydroxyphenyl)-6-hydroxybenzo[ ⁇ ]thiophene (20). a) 30 mg (0.08 mmol) of 2-(2-methyl-3-bromo-5-methoxyphenyl) -6-methoxybenzo[ ⁇ ]th ⁇ ophene (example (19a) was dissolved in 2 ml of tetrahydrofuran. The mixture was cooled to -70°C and butylhthium (0 12 mmol) was added to the reaction mixture The reaction mixture was stirred for 2.5 hours at -70°C and then at room temperature overnight. The reaction mixture was quenched with aqueous ammonium chloride, extracted with ethyl acetate and dried over magnesium sulphate. This produced 30 mg of crude 2-(2-methyl-5-methoxyphenyl) -6-methoxybenzo[ ⁇ ]th ⁇ ophene.
• the solution was heated to 100°C for 3 hours, concentrated on a speed-vac, dissolved in dichloromethane, filtered through a silica pad and then concentrated again.
• the product was dissolved in 1.5 ml of dichloromethane and 1 ml of boron trifluoride dimethylsulfide complex was added.
• the reaction mixture was stirred overnight in darkness, quenched with water and extracted with dichloromethane.
• the organic phase was dried by passing it through sodium sulphate dryingtubes and then it was concentrated in a speed-vac.
• the crude product was purified on a chromatotron (silica, 95:5, petroleum ether/ethyl a c e t a t e ) p r o d u c i n g 1 3 1 m g ( 0 . 3 5 m m o l , 4 7 % ) o f [2-(4-methoxyphenyl)-6-methoxybenzo[ ⁇ ] thien-3-yl]phenylmethanone as yellow crystals.
• example (32a) was accomplished by the procedure set forth in example 1 (c).
• the crude product was purified by recrystalhsation (acetic acid/ dichloromethane/ methanol) producing 270 mg (0.69 mmol, 73%) of [2-(4-hydroxyphenyl)-6- hydroxybenzot ⁇ ] th ⁇ en-3-yl][4-carboxyphenyl]methanone.
• the crude product was purified on a chromatothron (silica, 9:1 petroleum ether/ethyl acetate) producing 42 mg (0.1 mmol, 42%) of 2-(4-hydroxyphenyl)-6-hydroxybenzo[ ⁇ ] thien-3-yl][4- methoxycarbonylphenyljmethanone.
• example (39a) was accomplished by the procedure set forth in example 1 (c).
• the crude product was purified on a chromatotron (silica, 5:5 petroleum ether/ethyl acetate) producing 28 mg (0.07 mmol, 30%) of [2-(4-hydroxyphenyl)-6-hydroxybenzo [ ⁇ ]thien-3-yl][4-propyl ⁇ henyl]methanone.
• example (40) was accomplished by the procedure set forth in example 1 (c).
• the crude product was purified on a chromatotron (silica, 5:5 petroleum ether/ethyl acetate) producing 43 mg (0.09 mmol, 45%) of [2-(4-hydroxyphenyl)-6- hydroxybenzo[ ⁇ ]thien-3-yl][4-iodophenyl]methanone.
• the crude product was purified on a chromatotron (silica, 9: 1 petroleum ether/ethyl acetate).
• the deprotection of the crude [2-(4-methoxyphenyl)- -6-methoxybenzo[ ⁇ ] thien-3-yl][4-buturylphenyl]methanone was accomplished by the procedure set forth in example 1 (c).
• the product was purified on a chromatotron (silica, 9:1 petroleum ether/ethyl acetate) producing 17 mg (0.04 mmol, 22%) of [2-(4-hydroxyphenyl)-6-hydroxybenzo[ ⁇ ]thien-3-yl][4-buturylphenyl]methanone.
• the biological character of the compounds prepared in accordance with Examples 1 to 26 and 28 to 40 inclusive and also, for comparison purposes estradiol was measured in a radioligand displacement assay.
• the affinity for ER ⁇ and ER ⁇ was measured as an IC 50 , the concentration of ligand necessary to displace 50% of tritated 17- ⁇ - estradiol from either hER ⁇ (human estrogen receptor ⁇ ) or hER ⁇ (human estrogen receptor ⁇ ) respectively.
• IC 50 's of compounds varied from 2.0 nM to 20 ⁇ M for ER ⁇ and from 2.0 nM to 12 ⁇ M for ER ⁇ .
• the ER ⁇ /ER ⁇ selectivity ratio varied from 0.2 to 23.
• Affinity for the ER (by displacement of [H]-estradiol) ws measured using the scintistrip assay 1 .
• Human estrogen receptors (hER) alpha and beta were extracted from the nuclei from SF9-cells infected with a recombinant baculovirus transfer vector containing the cloned hER genes. 2 The concentration of hER's in the extract was measured as specific 3 [H]-E2 binding with the G25-assay. 3

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