KR20010013631A - Estrogen receptor ligands - Google Patents

Abstract

Crystals comprising at least a portion of an ERα ligand binding region that optionally binds to a ligand, ligands that bind to the ER receptor and methods of designing them, and homologue models of the ERβ receptor.

Description

Estrogen Receptor Ligand {ESTROGEN RECEPTOR LIGANDS}

The thyroid hormone receptor (TR) is known and its three-dimensional structure, and therefore its ligand binding region, is determined. Three-dimensional structural knowledge enables a better understanding of the ligand binding mode and the determination of the optimal structure of the ligand that binds to the receptor. This understanding will provide a pharmacophore model that can be used in the design of ligands such as drugs that bind to thyroid hormone receptors. It is common in the art that TR structures also provide guidance for the design of ER ligands.

Estrogen steroid hormones, thus estrogen receptors (ER), are members of the steroid hormone receptor class. The first natural ligand is estradiol (E2). However, raloxifene, centchroman, coumestrol, diethylstilbesterol, esculin, tamoxifen, zearalenone It is known that a number of structurally diverse nonsteroidal compounds such as) and zindoxifen also bind to estrogen receptors (FIG. 8). Most of these nonsteroidal estrogen receptor ligands include 2-4 carboxy, aromatic, and / or heterocyclic rings linked by 1-3 atomic chains. One or more rings may be fused with the central atom chain or with each other.

It is proposed that the receptor possesses a multifunctional modular structure with the potential to have separate regions for DNA binding, ligand binding, and transactivation. The ligand binding region (LBD) is designated as region E and is the largest region of the estrogen receptor. Ligand binding regions include ligand recognition sites and regions for receptor dimerization, interaction with heat shock proteins, nuclear localization and ligand dependent activation transfer.

An overview of the structure and function of estrogen receptors is provided in Katzenellenbogen, J. et al., Steroids, (1997) 62 (3): 268-303.

Compounds that bind to estrogen receptors are likely to be useful for treating a wide range of disease states. These include estrogen agonists for the treatment of diseases associated with estrogen deficiency (eg osteoporosis, myocardial and neurodegenerative diseases in postmenopausal women) and estrogen antagonists for the treatment of breast and uterine cancers. Included. In addition, certain types of ligands, such as tamoxifen, exhibit mixed efficacy / antagonism (ie, they are also estrogen agonists, estrogen antagonists, or partial estrogen antagonists when bound to estrogen receptors in other tissues), It can also prevent bone loss and reduce the risk of breast cancer. It is also known that benzothiophenes can be used as steroid hormone agonists or antagonists and that there is the possibility of changing their binding mechanisms, eg binding affinity, by changing substituents at various positions on the molecule. Thus, it would be desirable to be able to design ligands recognizable by estrogen receptors and capable of binding to estrogen receptors. It would also be desirable to know the three-dimensional structure of the estrogen receptor. This knowledge will be useful in the design of compounds to bind to estrogen receptors. We were able to produce estrogen receptor crystals and determine the three-dimensional structure of the estrogen receptor. Unexpectedly, the ER structure thus determined revealed that the TR structure did not provide a good model for ligand binding to ER.

The present invention relates to estrogen receptor ligands. More specifically, the present invention relates to ligands that bind to estrogen receptors, determination of such receptors, including determination of receptors and ligands, synthetic ligands, methods of using such synthetic ligands, and methods of designing ligands that bind to estrogen receptors. do.

1A shows a depiction portion of the 2.6 Å resolution composite crystal mean map of the RAL-ER-LBD complex;

1B is a 3.1 Å resolution 6 × average map of the E2-ER-LBD complex. In Figures 1A and 1B the map is drawn in lines in 1F and doubled in the final refined model;

FIG. 2A is a schematic representation of ER-LBDa showing the location of various secondary structural elements, where the helix is grey, the extended portion is very light gray and the coil portion is dark gray. E2 paints in very dark gray and is emphasized in space-filled form;

2B is a topology diagram of the ER-LBD. Helix is shown as a rectangle and β strands are shown as an arrow. The central core layers H5, H6, H9 and H10-hatched are sandwiched between the outer flanking areas H1-4 H7, H8 and H11. The structural elements on the side of the layer motifs S1 / S2 and H12 are S1, S2, H12 and are indicated by crossing lines. N and C termini are also indicated. All secondary structural elements are numbered following the nomenclature established for known nuclear receptor LBDs;

3A is a stereoscopic view of a ligand binding cavity. The cavity is shown in an orientation similar to that given in FIG. 1A. Side chains for the residues that outline the cavity are illustrated. Hydrophobic residues are shown in gray, basic residues in dots, and acidic residues in parallel. E2 is black (core) and dark gray (terminal hydroxyl group);

3B is a schematic diagram of a ligand binding cavity. Residues that directly hydrogen bond in the hydroxyl radius range are shown in ball and bar representations along hydrophobic residues that have nonpolar interaction with E2 (shown in gray as radial wheels). The atomic name and ring nomenclature of E2 are also given;

3C shows the molecular volume (dark gray dotted surface) and accessible binding cavity volume (light gray dotted surface) of E2;

4A is a schematic representation of an ER-αLBD amorphous dimer shown perpendicular to the dimer axis. N and C termini are indicated;

4B shows the dimer of FIG. 4A along the dimer axis. E2 is highlighted in the middle gray in the form of space filling. The helix included in the dimer interface is displayed;

4C shows the H11 helix forming the center of the dimer interface. Interacting residues are shown to be distinguished according to their polarity (gray-hydrophobic residues; parallel-polar residues; crossed parallel-basic residues);

5A is a schematic diagram of the interaction by the binding cavity and E2. The picture was created using the LIGPLOT software;

5B is a comparison of E2 and RAL binding mode (E2-dark grey; RAL-light grey);

FIG. 6 is a schematic of ER-LBD showing different positions of helix 12 in E2 (cross-parallel) and RAL (parallel) complexes. The rest of the ER-LBD is shown in gray. The dashed lines represent non-model parts of the structure. Helix interacts with H12 in both complexes is shown;

7 is a space filling diagram of a) E2 complex and b) RAL complex. H12 (black) is located above the hormone binding cavity in the E2 complex. Raloxifene induces a structural change such that H12 occupies a hydrophobic groove between H3 and H5. The hydrophobic side chains of all residues between residues 354 (H3) and 380 (H5) are shown in dark gray. Other highlighted residues are Lys362 (parallel), Glu380 and Tyr537 (crossed parallel), Asp351 (dot), and ligand RAL (grey). The remaining atoms of the LBD monomer are white. Note that differences in other parts of the ER-LBD complex may be due to sites lost from the current model;

8 shows the structure of several representative estrogen receptor ligands;

8A, 8B and 8C show the alterations made in the steroid nucleus of a ligand that binds to an estrogen receptor;

8D and 8E show how ligand affinity can be enhanced by the introduction of substituents;

9A-9F show enhanced selectivity by using different substituents on estrogen receptor ligands;

10-19 show structural reactions of the chemical reactions included in Examples 1-51 below, which are non-limiting and are given by way of example only.

20 shows the crystal coordinates of the estrogen receptor alpha (ERα) binding region in complex with raloxifene.

21 shows the crystal coordinates of the estrogen receptor alpha (ERα) binding region in complex with 17-beta-estradiol.

22 shows a homology model of estrogen receptor alpha (ERα) complexed with raloxifene.

FIG. 23 shows a homology model of estrogen receptor beta (ERβ) complexed with estradiol.

Accordingly, the present invention provides an estrogen receptor ligand binding region determination in a first aspect.

In a second aspect the invention provides ligands of the estrogen receptor, in particular synthetic ligands, by the use of crystals.

In a third aspect of the invention, a method of designing a ligand to bind to an estrogen receptor is provided. This method uses a three-dimensional model based on the determination of estrogen receptors. In general, this method involves determining a compound that is likely to bind to the receptor based on the estrogen receptor and in particular the three-dimensional shape compared to that of the ligand binding region of the estrogen receptor. Preferably, these compounds have a structure complementary to that of the estrogen receptor. Such methods include selecting a compound or modifying a compound present to determine which amino acid or amino acids in the ligand binding region of the estrogen receptor interact with the binding ligand and to improve the interaction. Preferably, the improvement in interaction results from an improvement in binding affinity but may also include enhancing receptor selectivity and / or modulating potency.

Preferably, the ligand binds to ER with a high binding affinity, for example in the range of 20-2000 pmol.

Ligands may bind tightly to ER but do not upregulate gene expression and do not inhibit the action of estradiol and estradiol mimetics. Accordingly, the present invention provides a method of inhibiting the activity of estradiol or estradiol pseudomechanism by providing a ligand that binds ER with high affinity to block the activity of estrogen. Alternatively, binding of the ligand to ER may result in a structural change to ER, further inhibiting further binding. The present invention also provides a method of inhibiting estradiol activity in an animal, the method comprising administering to the animal a ligand that binds at least the LBD of the ER with a high affinity and blocks further ligand binding to the LBD of the ER. It involves doing. Such ligands are useful, for example, in the treatment of diseases in which the estrogen receptor is mediated in women.

Design based on the structure of the ER ligand

This 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 with improved properties of known ER ligands. Such enhanced properties include one or more of the following: (1) higher affinity, (2) improved selectivity to either the α- or β-isoform of ER, and / or (3) Effect of designed degree (efficacy versus partial efficacy versus antagonist). Without knowledge of the ER structure, modifications to produce ligands with enhanced properties and reasonable chances of success would not be possible for those skilled in the art. ER receptor structures also have utility in the discovery of new, structurally novel types of ER ligands. Electronic screening of a large library of structurally diverse compounds, such as the Available Chemical Directory (ACD), will identify a structurally new class of ER ligands that will bind to the three-dimensional structure of the estrogen receptor. In addition, the ER structure allows for "reverse-engineering" or "new design" of compounds that bind to ER.

(1) enhanced affinity

This work revealed the presence of receptors defined by β- and α-face cavities with centers above and below the B- and C-rings of estradiol, respectively.

The present invention provides new ligands that exploit this discovery by filling the α- and β-face cavities.

Preferably, the ligand fills at least one of the α- and β-face cavities to exclude water from the cavity or cavities.

The ligands produced according to the invention bind more effectively to ER than estradiol. This ligand can bind at twice the affinity of estradiol, preferably at three times the affinity, and most preferably at least ten times the affinity.

Alteration of the steroid nucleus is at the positions indicated by R in FIGS. 8A and 8B (α-substitution at 7-, 9-, 12-, 14-, 16-, and 17-positions; β-substitution at 8-, 11 -, 15-, and 18-positions). Preferably, these substituents are hydrophobic substituents, for example methyl, ethyl, iso-propyl, chlorine, bromine, or iodine.

Modifications to the 2-aryl benzothiophene can occur to fill the α- and β-face cavities of the ER at the 2'-, 3'-, and 6'-positions (FIG. 8C). Preferably, the substituent is present at at least two positions in the following three positions such that the vertical structure between the B- and C- rings of the 2-aryl benzothiophene nucleus is forced: 3, 2 ', or 6'. This vertical structure facilitates the localization of 2'-, 3'-, and 6'-substitutions in the α- and β-face cavities of the ER.

In a manner similar to the benzothiophene family, the affinity of the other family of nonsteroidal ER ligands can be enhanced by substitution of small hydrophobic substituents at the positions indicated by R 2 ′, R 3 ′ and / or R 6 ′ in FIG. 8C.

Preferably, the ligands produced according to the invention fill at least one of the α- and β-cavities of the ER without disturbing the rest of the ER structure.

Another aspect of the invention reveals an unfilled hydrophobic cavity in the raloxifene / ER complex. Filling these cavities with hydrophobic substituents to rule out water will enhance binding affinity. This cavity can be filled by placing a hydrophobic substituent with a piperidinyl nitrogen atom on the ethoxyphenyl side chain α of raloxifene. This hydrophobic substituent is a linear alkyl or perfluoroalkyl group (-CH ₃ to -C ₁₀ H ₂₁ , -CF ₃ to -C ₁₀ F ₂₁ ), benzyl (-CH ₂ Ph), or methylenecyclohexyl (-CH ₂ C ₆ H ₁₁ ).

In a third aspect of the invention, examination of the ER structure reveals that the hydroxyl group at position 3 of estradiol or position 6 of raloxifene forms a hydrogen bond interaction with Glu-353 and Arg-394 ( 5a and 5b). It is known that substitution of hydroxyl groups at the 3 position of estradiol or the 6 position of raloxifene results in a decrease in affinity for ER. The present invention reveals the reason for this reduction of affinity: the second hydrogen atom has an adverse electrostatic interaction with Arg-394 while one of the hydrogen atoms of the amino group forms a favorable hydrogen bond interaction with Glu-353. Form. The invention also discloses a method for enhancing the affinity of the 3-amino analogue of estradiol and the 6-amino analogue of raloxifene: one of the two hydrogen atoms of the amino group to produce a secondary amino group Substituting with an alkyl group. Alternatively, the amino group may be substituted with a guanidino group (FIG. 8E), which obtains two additional hydrogen bond interactions, the first being a salt bridge with Glu-353 and the second Leu-387 residue. The central structure at is a hydrogen bond interaction with a carbonyl group. Similar enhancement of affinity for ER can be achieved by substitution of the guanidino group with fused 2-aminopyrrole (FIG. 8E).

In a closely related aspect of the present invention, an understanding of the reduction in affinity for ER seen in ether derivatives at either the 3-position of estradiol or the 6-position of raloxifene is provided: Ligand and Glu at ER Electrostatic repulsion between ether oxygen atoms of -353. The present invention discloses a method for increasing the affinity of estradiol 3-position or raloxifene 6-position ether derivatives: substitution of ether oxygen atoms with amino (NH) groups.

In a fourth aspect of the invention, the substitution of the 4-hydroxyl group of raloxifene is affinity by obtaining a second hydrogen bond interaction between the amino group and the central structural carbonyl group in Gly-521 of the ER. Will be enhanced (FIG. 8D).

(2) improved selectivity

Estrogen receptors are known to have two separate forms known as ERα and ERβ. In addition, the ratio of α- to β-forms of ER is quite different in other cell and tissue forms. Thus, by designing a ligand that selectively binds to one or another isoform of the estrogen receptor, it may be possible to separate the desired therapeutic effect from the unwanted side effects of the estrogen receptor ligand.

The α- and β-forms of the estrogen receptors differ significantly in their primary sequence and in their tertiary structure. As a result of these receptor differences, the ligand can bind to the two isoforms with different affinity.

We were able to isolate, fractionate and produce crystals for ERα. From these determinations, we determined the three-dimensional structure at high resolution. In addition, we generated a partial homology model of ERβ based on experimentally derived ERα coordinates. This partial ERβ homologue model captures the essential difference in binding properties between ERα and ERβ. The difference between ERα and ERβ is determined based on the comparison of the experimental ERα coordinates with the partial homologue model of ERβ, and using this difference the ability of the ligand to bind to either or both of the ERα and ERβ receptors is expected. Can be. Thus, if it is known that one tissue has only one type of estrogen receptor, it is possible to impart a degree of tissue specificity for the ligand by designing the ligand to bind to the predominant form of the receptor. Advantageously, the ligand can be designed to specifically bind ERα or ERβ.

In addition, the detailed understanding of other receptors makes it possible to understand the different behaviors of compounds in different tissues, for example the dependence of raloxifene (RAL) on tissues (which are active) of estrogen or estrogen antagonistic behavior. There is this.

In another aspect, the present invention provides an estrogen receptor ligand binding region determination for ERα and a partial homologue model for ERβ. Specificity of the ligands is also provided up to a specific ratio of ERα and ERβ or ERα to ERβ. Its advantage is that tissue specificity is imparted to the ligand. Accordingly, the present invention also provides methods for their design with ligands, in particular synthetic ligands of ERα and ERβ.

The present invention provides novel ligands that exploit this difference by closely positioning the ligand substituents to one or more amino acid residues between the α- and β-isoforms of the ER.

The ligands produced according to the invention bind more efficiently to one of the α- or β-isoforms of the ER. The selectivity of the bond between the α- or β-isoforms may be 10 times, more preferably 100 times, and most preferably 1000 times greater.

For example, the amino acid residue at position-384 in the β-face cavity of ERα is Leu (side chain volume = 76.6 Å ³ ), and the amino acid residue at the corresponding position of ERβ is Met (side chain volume = 79.3 Å ³ ). Thus the β-face cavity of ERβ is smaller. In conclusion, ERα selectivity can be enhanced by placing larger substituents than methyl groups in the β-face cavity close to residue-384. The interaction between the ligand and residue-384 can be enhanced by introducing a substituent at the β 8-, 15-, or 18-position on the steroid nucleus.

In the α-face cavity of ERα, the amino acid residue at position-421 is Met (side chain volume = 79.3 Å ³ ), and for ERβ is Ile (side chain volume = 77.3 Å ³ ). Thus, the α-face cavity of ERα is smaller. This difference can be used to produce β-selective compounds through substitutions larger than the methyl group at the α 14-, 16-, or 17-position of the steroid nucleus.

Similarly, from either the 2'- or 3'-position of the 2-arylbenzothiophene nucleus for interaction with residue-384 in the β-face cavity, or with residue-421 in the α-face cavity Substitution can be done from the 6'-position for this purpose (FIGS. 9A and 9B). However, free rotation around the C2-C1 'bond will efficiently exchange substitutions at the 2'- and 6'-positions, thus reducing selectivity. Moving the hydroxyl group from position-4 ′ (FIG. 9A) to position-5 ′ (FIG. 9B) deflects the bond orientation such that the R ₂ substituent is in the β-face pocket and the R ₆ substituent is in the α-face pocket I will. This bias results from the fact that only one of the two possible rotamers around the C2'C1 'bond will allow hydrogen bond formation between the 5'-hydroxyl group and the receptor residue His-524.

The invention also provides a means for enhancing the selectivity of other classes of nonsteroidal ER ligands. In a method analogous to the benzothiophene family of ER ligands, substituents larger than methyl can be introduced at either the R2 'or R3' position to produce ERα selective compounds or ERβ selective compounds at R ₆ '(FIG. 8C).

To exploit the difference between ERα and ERβ at position-326 (Ile at ERα and Val at ERβ) and position-445 (Phe at ERα and Tyr at ERβ), the position-3 of the steroid nucleus or the position of the benzothiophene nucleus Substitutions can be made from -6.

The present invention also provides a means for specifically producing an ERα selective ligand. A six-membered linker between the hydroxyl group at position-3 or position-6 raloxifene of the A-ring of estradiol and the aromatic or heteroaromatic ring on the side chain will place the side chain ring very close to residue-445 ( 9c). The edges of the ERα Phe-445 and the side of the side chain rings can form advantageous "π-teeing" interactions. This advantageous interaction is not possible with ERβ Tyr-445, so the analog of this form is ERα selective (FIG. 9D).

Another aspect of the invention provides a greater means of enhancing ERα selectivity. Introduction of the carboxylate or amino group on the meta or para position of the aforementioned aromatic or heteroaromatic ring will form a hydrogen bond interaction between the conserved Glu-323 or Lys-449 (FIG. 9E). Alternatively, the heteroaromatic ring may be a pyridone ring, which will simultaneously form advantageous hydrogen bond interactions with both Glu-323 or Lys-449 (FIG. 9F). Either amino, carboxylate, or pyridone ring substitutions will enhance the advantageous "π-teeing" interaction between the aromatic or heteroaromatic ring of the ligand and Phe-445 at ERα.

(3) control of effect

The present invention provides an understanding of the difference between estrogen and antiestrogen binding and thus provides a means of designing an ER ligand with the desired degree of potency. Investigations of the differences between the ER / estradiol and ER / raroxifene complexes reveal large shifts in Helix-12 (H12, FIG. 6). H12 employs an "effective" structure defined by the structure of the ER / estradiol complex and an "antagonistic" structure defined by the structure of the ER / raroxifene complex. These two structures are in thermodynamic equilibrium. When the ER complexes with a full agonist such as estradiol, the equilibrium is located far in the direction of the "effective" structure. In contrast, when forming complexes with antagonists, the equilibrium is pushed in the direction of the “antagonistic” structure. In the case of raloxifene, the large side chain at position-3 collides structurally with H12 in its potent structure, thus strongly pushing the equilibrium in the antagonistic direction. By introducing progressively smaller side chains at position-3 of raloxifene, the equilibrium will be returned towards the braille potency structure. Accordingly, the present invention provides a means for developing ligands (agonists, partial agonists, or antagonists) with the desired degree of potency.

In particular, the importance of H12 is determined as playing a central role in determining the potency (efficacy versus antagonism) of the ligand. Thus, ligands that can bind and / or alter the structure of H12 are particularly important when designing ligands for estrogen receptors or when evaluating binding of ligands.

The inventors also found a reason why raloxifene has a different binding structure than that of estradiol, the difference being in its active structure but unpredictable by antagonistic action. It has been shown by the inventors that antagonism is caused by protruding part on the raloxifene structure, which results in a large substitution of H12 for its structure in the ER / estradiol complex.

It has also been found that at least most of these receptor proteins are in dimeric form. This dimerization leads to breakable pathways. This form of destruction can be used to predict antagonism or to produce antagonists. Destruction can take the form of ligand binding, which alters the structure of the helix, including directly binding to the dimerization interface or dimerization interface to inhibit dimerization.

In addition, the orientation of the ligand can be a hot chain to the receptor in dimeric or monomeric form. Moreover, using the crystals of the present invention, the effect of ligand binding to LBD on the receptor structure can be seen to affect the behavior of the receptor, which is a co-activator, co-inhibitor ( co-repressor) or heat-shock protein. Previously, this was not possible.

Production of Estrogen Receptor Crystals and Their Applications

Preferably, the crystal is produced from a sequence comprising at least 150 amino acids of the selected estrogen receptor. More preferably, the sequence comprises at least 200 amino acids. Most preferably, the sequence comprises at least 250 amino acids. Preferably, the sequence comprises at least a portion of the ligand binding region of the estrogen receptor. More preferably, the sequence comprises the entire ligand binding region of the estrogen receptor.

Typically ER LBD is homogeneously purified for crystallization. Purity of ER LBD is determined by SDS-PAGE, mass spectroscopy, and hydrophobic HPLC. Purified ER for crystallization should have at least 95.5% purity, preferably at least 99,0% purity, most preferably at least 99.5% purity.

Preferably, the crystal used should be able to withstand exposure to the X-ray beam used to produce the diffraction pattern data needed to interpret the X-ray crystallographic structure. For example, crystals grown using estrogen receptor sequences bound to various ER ligands have been found to degrade during exposure to X-ray beams at room temperature, while estrogen receptor sequences bound to various ER ligands have been found to degrade. The grown crystals are freezing and can withstand exposure to the X-ray beam.

Advantageously, the crystal has a resolution determined by X-ray crystallography of less than 3.5 Hz and most preferably less than 2.8 Hz. Preferably, crystals grown using naturally occurring estradiol have an effective resolution of less than 3.1 Hz and crystals grown using raloxifene have an effective resolution of less than 2.6 Hz.

The production of such crystals enables the three-dimensional structure of the ligand binding region of the estrogen receptor to be mapped. Using these crystals in conjunction with the map will give a detailed understanding of how estradiol and other estrogens bind to estrogen receptors. This technique may also allow the design of binding sites to enable the design of estrogen antagonists.

For example, in the prior art it has been proposed to prepare raloxifene analogs using many substituents on 2-aryl groups (Grease et al., J. Med. Chem. (1997), 40: 146-147). One has been shown to be effective in preventing bone loss in exchange for a loss of binding affinity using 2-naphthyl, eg 4'-OH substituents (causing a weaker affinity loss compared to naphthyl). By mapping the estrogen receptor and examining the following structural formula X, it is intuitively obvious to align this compound with the estrogen binding site, i.e., the fusion of the D-ring of the estradiol with the sub position-2 aryl substituent, using the map Found that even 6'-OH, or even 5'-OH, is more favorable for affinity.

For example, the use of this method has allowed the inventors to determine different binding modalities of different steroid hormones to the estrogen receptor, e.g. the binding of testosterone to the estrogen receptor, which is an incomplete binding, is equivalent to that of estradiol. And how it is different. In particular, these models include (1) electrostatic repulsion between the C-3 carbonyl oxygen atom of testosterone and the carboxylate of Glu-353 and (2) between the side chain of the C-18 methyl group of testosterone and the side chain of Leu-387. Shows that there is a stereostructural repulsion, which explains the much lower affinity of testosterone compared to estradiol for the estrogen receptor. It has been found that steric structural disturbances and other stereochemical modalities of molecules affect the flexibility of ligand regions, the ability to alter tertiary structure, thereby affecting the disturbance of ligand binding regions. Thus, using the crystals of the present invention, it is clearly shown how estradiol binds to the estrogen receptor and thus it becomes possible to understand as well as to predict the structural reasons why the compound behaves like an estrogen. This allows us to understand the irregularities of the estrogen receptors-the ability to bind to many structurally diverse ligands. This understanding can be applied to many steroid hormone receptors, especially glucocorticoid receptors, to a greater or lesser extent.

Determination of estrogen receptor binding regions can be used as a model in methods for the design of synthetic compounds to bind to receptors. This model shows why very small differences in the chemical structure of the ligands have potentially widely varying binding affinities. Thus, the three-dimensional structure of the ligand binding region can be used in pharmaceutical models for compounds that bind to estrogen receptors.

DETAILED DESCRIPTION Hereinafter, embodiments of the present invention will be described in more detail with reference to the accompanying drawings, which are given by way of example only.

Example 1

material

Protein Purification and Crystallization of Estrogen Receptor α

Human EP-LBD-α was expressed in Escherichia coli (Hegy G.B. et al., Steroids, 1961, 61, June, 367-373). Fermented in batch and incubated at 30 ° C. in purified glycerol / salt medium. The temperature was raised to 39 ° C to induce the production of recombinant protein. After 2 hours, the cells were harvested and frozen by centrifugation, and the thawed cells were 100 mM Tris-HCl (pH 7.8), 100 mM KCl, 10% glycerol, 4 mM EDTA, 4 mM DTT, 5 μg / ml at 0 ° C. In antipans, it was crushed with a Bead Beater homogenizer (Biospec, Bartlesville, OK, USA) (6 × 22 seconds, 3 minutes rest between breaks). 250 ml of buffer per 1200 ml of fermentation volume were used with 210 ml of acid washed with glass beads (212-300 microns). After centrifugation, the supernatant was introduced into a 25 ml column of estradiol-sepharose fast flow (Green G. et al., Proc. Natl. Acad. Sci. USA (1980) 77, 5115-5119). . The column was washed with 130 ml of 10 mM Tris-HCl, pH 7.8, 700 mM KCl, 1 mM EDTA, followed by 130 ml of 10 mM Tris-HCl, pH 7.8, 250 mM NaSCN, 10% dimethylformamide, Washing with 1 mM EDTA followed by 110 ml of 10 mM Tris-HCl, pH 8.0. The reactive Cys residues were modified by washing the column with 120 ml 30 mM Tris-base, 15 mM iodoacetic acid, pH 8.1. The remaining reagent was washed with 50 ml of Tris-base, 15 mM iodoacetic acid, pH 8.1. The remaining reagent was washed with 50 ml of Tris-HCl, pH 8.0 followed by 20 ml of 10 mM Tris-HCl, pH 7.8, 250 mM NaSCN, 10% dimethylformamide, 1 mM EDTA. ET-LBD-α was eluted by including 100 μM of the desired ligand in the final buffer. Fractions containing ET-LBD-α were pooled (65 mL) and concentrated to 4 mL (Centriprep 30, Amicon). Final purification was performed using the Bio-Rad 491 preliminary PAGE instrument according to the user manual. One dilution of the Ormstein / Davies buffer system is used. The stack (0.7 cm) and dissolution (70 cm) gels were 5.6% (acrylamide / bis). Elution buffer was 10 mM Tris-HCl, pH 8.0 and electrophoresis was performed at 12 W. Fractions containing ER-LBD-α were pooled and concentrated to the desired protein concentration (Centriprep 30).

Data collection, organization and purification

ER-LBD-α-RAL Complex:

Natural datasets were collected from single frozen crystals on beamline X11 in DESY / Hamburg (l = 0.905 μs). Diffraction data were recorded on a 30 cm Mar Research image plate at 120 K with a crystal-detector distance of 245 mm or 390 mm. Heavy atom derivatives were collected in-house from flash frozen crystals. Data were integrated and converted using DENZO and SCALPACK programs. MIR analysis was performed using the CCP4 program (Table 2). Diffraction data for alternating C2 (York) and C2221 (DESY, Hamburg) crystal forms were collected at resolutions of 3.0 Hz and 3.1 Hz, respectively. Initial phase was calculated at 3 μs using MLPHARE, followed by 2-fold averaged amorphous matrix purification and phase expansion using DM. Initial polyalanine traces were used to generate a dimer investigation model using purified amorphous crystal symmetry and used in Molecular Replacement (AmoRe) Collaborative Computation Project No. 4 (The CCP4 Suit: Program for Protein Crystallography, Acta Cryst D50, 760-763 (1994)) was used to place the alternating C2 and C2221 crystal forms. 20 cycles of cross crystals averaging all three crystal forms were obtained using DMMULTI (Supra and Cowtan, K, dm: An automated procedure for phase improvement by density modification, In Joint CCP4 and ESF-EACBM Newsletter on Proten Crystallo-). graphy 31 pp 34-38 (1994)). The resulting electron density map showed no bias towards the input model and allowed for clear tracking of the rest of the molecule and assignment of most amino acid sequences. Purification was performed by REFMAC using bulk solvent and anisotropic scaling (Murshudou et al., Acta. Cryst. D53, 240-255 (1997)). Strict non-crystalline restraints were maintained during the initial cycle but loosened in the final purification step. Individual atomic temperature components were purified isotropically. Residues Asp332, Phe337, Lys416, Lys467, Ser468, Leu469, Glu470 and Glu471 were poorly resolved in the electron density map and were not modeled on their C $ atoms.

ER-LBD-α-E2 Complex:

Diffraction data were collected at room temperature from a single ER-LBD-E2 crystal using a 128 cm Mar Research image plate located at 280 mm crystal-detector distance on the beamline BW7AB in DESY / Hamburg (l = 916 Hz). Initial phase predictions were obtained with AMoRe using a purified ER-LBD RAL dimer (cut following Met528) as a survey model. All data between 15 and 4 ms was used for the recursion and translation functions, and in the cross-circulation function model, Patterson self-vectors were selected within a radius of 30 ms. The correct interpretation corresponding to three ER-LBD dimers had correlation coefficient 69.8 and R-factor 40.6 after AMoRe rigid-body purification. A six-fold averaging was performed using DM and the structure was purified by REFMAC using strict amorphous crystallites, volume solvents and anisotropic scaling and averaged phases from DM. A single overall B value was applied to the initial stages of purification until Rfree converged. In the following cycle, a strict, constrained, total isotropic B value tablet was employed. Full model assembly was performed using a graphics package QUANTA (Molecular Simulations, Inc. San Diego). Side chains Leu306, Leu466, Leu469, Lys492, Lys531 and Leu346 were poorly resolved in the electron density map and were not modeled on their C $ atoms.

TABLE 1

ER-Raroxifene ER-Estradiol

Space group C2 P21

Unit cell dimensions

a (Å) 104.53 61.48

B (Å) 53.68 115.16

C (Å) 102.71 137.38

β (o) 116.79 103.01

Molecular No./AU 2 6

Resolution 25-2.6 20-3.1

Unique reflection No. 15,497 34,025

% Complete 94.6 99.1

Multiples 4.5 2

Rsym (I) 8 10

Reflections used in tablets 13,868 30,583

Rcryst 23.98 22

Rfree 30.4 25.3

Non-H atom 3,741 11,508

Water 66 126

% A, B, L (a, b, l, p) 92.4 (7.6) 93.0 (7.0)

Rmsd Coupling Length (Å) 0.01 0.01

Rmsd Coupling Angle 0.04 0.03

Mean B Protein (Å2) 48.3 37.8

Rmsd NCS Protein 0.57 0.08

Rmsd NCS B (Å2) 8.2 1.1

Rsym (I) = 100x G _h G 1G _{i hi} - <I> 1 / _h G G I _{i h, i} where I is the observed intensity, <I> is the average intensity of a plurality of observation of symmetry-related reflections.

Rcryst = 100 x E11F ₀ 1-1F _c 11 / E1f ₀ 1 ..

Rfree is the same as Rcryst but is calculated using a reflection test set (10% of the total data set) derived from the purification process.

R.m.s. The deviation is the combined length and each length from Engh and Huber anomaly values.

R.m.s. The distance is between all non-crystalline symmetry (NCS) related atomic positions.

R.m.s. The difference is between all non-crystalline symmetry (NCS) related atomic temperature factors.

TABLE 2

Data set PCMBS-1 PCMBS-2 KAuCN

Resolution (Å) 20-3 20-3 20-3.6

Unique reflection No. 10,335 9,316 5,835

% Complete 97.6 89 94.2

Multiple 4 3.1 2.5

Rsym (I) 8.1 9.2 7

Reagent concentration (mM) 4 4 4

Immersion time (days) 5 14 2

Resolution (Å) 20-3 20-3 20-3.6

Riso 16.9 20.7 13.7

Position No. 2 2 1

Cullis R 0.75 / 0.68 0.76 / 0.66 0.90 / 0.85

(centric / acentric)

Phasing power 1.22 / 1.88 1.23 / 2.02 0.71 / 0.94

(centric / acentric)

F.O.M (20-3Å) 0.67 / 0.48 / 0.49

(centric / acentric / overall)

Cullis R = E1E1 / G11F _PH 1-1FP _P 11 centric (c) and acentric (a) reflections.

FOM = <EP (α) e ^1α / EP (α) where α is phase and P (α) is the phase probability distribution. Phasing power.

Example 2

ER-E2 crystallization

Prior to crystallization the protein was buffer exchanged to 20 mM using Tris / HCl buffer pH 7.8 and concentrated to 12-13 mg / ml. Crystals were grown by vapor diffusion using a hanging and sitting drop technique. The best crystals were obtained by buffering with 0.1 M Tris / HCl buffer using 2.4 M ammonium formate or 80-90 mM magnesium formate as precipitant. Unbuffered stock solution of 4 M ammonium formate or 200 mM magnesium formate was used. The magnesium formate stock solution was stored at 4 ° C. and filtered before use. The optimum pH ranged from 7.9 to 8.3 and the best crystals grew at pH 8.1. Although a suitable X-ray crystal was obtained at 13 mg / ml, the protein concentration in the drop was 8 mg / ml. However, the crystals obtained under these conditions were often paired and adding DMSO up to 8% significantly improved the quality. The size of the crystals correlated with the size of the seating / hanging drop. The optimal size of the drop was obtained by mixing 2.5 ml of protein with 2.5 ml of stock solution. All crystallizations were performed at 18 ° C. The best crystals with a size of 0.5 × 0.05 × 0.05 mm ³ were mounted in the X-ray quartz capillary.

ER-α-raroxifene (ER-R) crystallization

After purification as above, the protein buffer was replaced with 20 mM Tris / HCl pH 7.8-7.9 and the protein was usually concentrated to 10-12 mg / ml. Along with the hanging drop technique, steam dispersion was used for the crystallization. The best conditions for crystallization are using the following solvents: 0.1 M Tris / HCl buffer pH 8.3, 12% (w / v) PEG 4000, 0.1 M maltose, 50 mM lysine, 0.2 M MgCl ₂ , 5% dioxane . The concentration of the protein solution used for crystallization was diluted to 20 mM Tris / HCl buffer pH 8.3 to 7.3-7.5 mg / ml. Crystallization was performed at different drop sizes and protein-storage buffer ratios. The best crystals were grown from the drop obtained by mixing 2 ml of protein with 2 ml or 3 ml of well buffer. The best temperature for crystal growth was 18 ° C. Under these conditions, the main C2 crystal form (a = 104.53Å b = 53.68Å c = 102.71Å b = 116.79Å) was obtained in the form of monoclinic plates (0.1 × 0.1 × 0.02 mm ³ ), which was then It was used for heavy element derivative investigation and structural purification.

Other crystal forms were also produced by fine tuning of the conditions. Lowering the concentration of PEG 4000 to 10-11% w / v led to another C2 crystal form: a = 89.9 ′ b = 75.09 ′ c = 87.50 ′ b = 103.01 Hz. These crystals grew in a single pyramid at 18 ° C. and, despite severe twin formation, were able to mechanically separate into small, unpaired pieces of crystals suitable for X-ray data collection.

Alteration of other conditions, for example increasing the dioxane concentration from 5 to 7.5 and 10%, replacing 50 mM Lys with 50 mM Arg, and replacing 0.1 M maltose with 0.1 M sucrose or glucose, was performed in C2221 square. Orthorhombic crystal forms were produced: a = 65.47 Å b = 95.99 Å c = 164.14 Å. The crystals reached a size of 0.2 × 0.03 × 0.03 mm ³ and they were preferably established at 18 ° C.

It is also possible to obtain crystals of SeMet derivatives or ER-R complexes. They can grow from typical conditions for the main C2 crystal form, but the concentration of dioxane usually rises to 7.5%. These crystals became very fragile and the quality of the X-ray data was poor, which was only used as additional information to locate Met residues.

All ER-RAL complex crystal forms were suitable for flash cooling using N ₂ flows at 100 K and 120 K. In all cases, cryopro-tectant consisted of mother liquor (well buffer composition) and 25% v / v MPD.

Due to the sensitivity of the ER-R crystals all heavy element soaks were done in the correct mother liquor (from well buffer) and the heavy element compounds were always dissolved as solid material in these solutions. PCMBS-1 and KAuCN soaking were performed for 3 days and PCMBS-2 for 3 weeks. All dipping was done at 18 ° C. The cryo-solution contained heavy element compounds in immersion concentration.

Pure ER-LBD was particularly resistant to crystallization so that suitable crystals were obtained after carboxymethylation of three thiol groups.

Example 3

Determination of the Structure of the Estrogen Receptor α Ligand Binding Region

Crystals of ER-LBD complexed with estradiol or raloxifene will be diffracted at medium resolution, monoclinic and asymmetric with dimers for raloxifene or 3 dimers for estrogen Included as a unit (see Table 1). Isomorphous replacement was used to determine the crystal structure of the ER-LBD-RAL complex. The initial isomorphic substitution / density altered electron density map shows an amorphous double axis of rotation and the initial polyalanine traces are allowed to build on the resulting doubled mean map. Subsequent averaging between three different crystal forms of the RAL complex allowed modifications to the initial trace. The remaining proteins that are not yet explicitly traced and most amino acid sequences are assigned. The resulting model had an R value of 45%. A final model with R values and geometrical parameters that were acceptable with the maximum possible purification and manual reassembly was obtained. Initial phase estimation was obtained for estradiol (E2) complexes by molecular substitution using ER-LBD RAL as a survey model. The recursion in the translation function gave the correct interpretation. Six-fold averaging between the three dimers in the crystal line asymmetric unit allowed the lossy portion of the structure to be traced and the position of E2 in the binding cavity to be determined. The structure was refined using strict non-crystalline restraints and mean phase information to obtain a final model with an Rcryst of 22.0 and an R3 of 25.3 in all data between 20 and 3.1 μs (Table 1).

result

The crystals produced in Examples 1 and 2 were subjected to X-ray crystallographic studies, whereby it was found that the LBD overlapped with characteristic “wedge” spherical units. It has three layers of antiparallel α-helix sandwich motifs and is constructed of eight main helixes. This motif comprises a central core layer of three helixes (H5 / 6, H9 and H10) sandwiched between two additional layers of helix (H2-3 and H7, H8, H11). The arrangement of structural elements in this fashion forms a "molecular scaffold" that retains a significant amount of ligand binding cavity at the "toe end" of the wedge region. The remaining two structural elements, the two small strands of unbalanced β-sheets (S1 and S2) and helix H12, are located at the "ligand bond ends" of the molecule and stand on the side of the main three-layer motif (see Figure 2). From the N-terminus, the chain follows one turn of the twisted helix (H1) and rotates 90 ° into the short helix (H2), which lies parallel to the longest axis of the LBD. Following helix H2, the chain continues in an irregularly expanding structure in the same direction until it is covered under the bottom of the molecule. At this stage, the chain returns itself through a long, curved helix (H3). The N-terminal portion of this helix forms part of the ligand binding cavity. The sequence has a proline at 365, which is constant and passes through 310 helix (H4) before the main chain turns sharp (90 °) to form the first three central helixes (H5 / 6). Helix H5 / 6 can be described geometrically as a single unit, although its C-terminal is twisted by 40 ° at the alanine residue at position 382 so that it is correctly positioned to form part of the E2 binding cavity. This helix is twisted and prominent and maintains a series of hydrophobic interactions between leucine at 378 and 379 (H5), phenol at 367 and leucine at 453, all of which are highly conserved and 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 exits to the other side of the molecule via 3 ₁₀ helix H7. The helix H8 runs three quarters up to the long axis of the LBD, passing through the second central helix (H9) and turning back through a chaotic loop to form the final helix H10. At the end of H10 the polypeptide central structure changes direction and runs the full length ligand binding region in the form of helix 11 up to H8 in the non-parallel direction. After a small rotation, the chains appear as S1 / S2β hairpins on the helix H12, the central positive helix of the AF-2 site on the opposite side.

Dimerization

Crystallographic studies also reveal that the receptor is dimerized. ER is sequestered with an inactive complex with heat shock protein 90 (hsp90) and other ancillary factors in the absence of E2. Ligand binding initiates the degradation of this complex and receptor dimerization occurs through region E. Ligand binding forms of ER exist as strict homodimers in solution and ER-LBD is arranged as an amorphous dimer in both the E2 and RAL complex crystals. This quaternary configuration almost certainly reflects the physiological state of ER-LBD in vivo as all crystal forms of the ligand ER-LBD containing amorphous dimer. The dimer axis coincides with the longest axis of the LBD and each molecule is inclined approximately 15 ° away from the two position overlaps. The symmetrical arrangement produces molecules having dimensions of approximately 55 mm 3 width 50 mm thickness 35-60 mm 3. The quaternary arrangement observed places the N and C termini of each monomer on the opposite "face" of the dimer. The C terminus of each monomer protrudes toward the dimer axis while the N terminus is away from the interface. Dimerization surfaces are very broad and encompass about 15% (1,703, ² ) of the surface area accessible to each monomer. LBD is positioned so that the H18 / H11 side of each monomer forms an additional intermolecular helix layer. The contact between the two molecules is primarily entangled through the H11 helix to form a tight central structure, but also includes H8 from one monomer and H9 and H10 from neighboring monomers. The H11 helix is arranged as a branched coil with side chains of Leu 504, Ala 505, Leu 508, Leu 509 and Leu 511 residues that form a partial "leucine zipper" motif at terminal N. Hydrophobic patches are formed on both sides by a network of hydrogen bonding residues. Arg 545 and Asn 519 make direct hydrogen bonds with Ser 512 and His 516, respectively. This overall monomer-monomer arrangement appears to be maintained within the receptor superfamily without being affected by the nature of the ligand. The observed ER-LBD dimer makes the dimer interface with that of the same and crystallographic unligated RXR-α homodimer in terms of overall monomer orientation (58% hydrophobicity / 42% hydrophilicity).

Thus, the invariant nature of the LBD quaternary structure suggests providing a stable entity that facilitates the separation of two DNA binding regions in a manner that allows optimal binding to ERE.

The elucidation of the three-dimensional structure of such estrogen receptor ligand binding regions provides a useful mechanism for designing ligands that bind to estrogen receptors. This detailed knowledge of receptor structure allows for predicting whether ligand-induced structural changes can be anticipated, so that ligand binding to the receptor will act as an antagonist, partial antagonist, agonist or partial agonist.

Example 4

Partial homology model of ERβ

Coordinates obtained in Example 1 (estradiol or complex of raloxifene with ERα) were used to generate two partial homology models of ERβ (complex of estradiol and raloxifene, respectively). This was done by importing ERα coordinates into Sybyl (Tripos Associates, St. Louis, MO, U.S.A.) of version 6.4. The "change" command was used in the Sybyl biopolymer module to vary amino acids between ERα and ERβ and near the ligand binding pocket. Four such residues were mutated: 1326V (val at Ile-326), L384M (Met at Leu-384), M421I (Ile at Met-421), F445Y (Tyr at Phe-445). These partial ERβ homology models that matched the experimental ERα coordinates were used to design the isoselective ligands described in Example 5-51.

Ligand Design

Examples of ligands designed to match the receptor were produced as follows:

Example 5

2- (2,6-dimethylphenyl) -6-hydroxybenzo [β] thiophene (1)

(a) to a solution (6 g, 36.5 mmol) of 6-methoxybenzo [β] thiophene (Graham et al., J. med. Chem., 1989, 32, 2548) in 50 mL of anhydrous tetrahydrofuran- At 60 ° C. n-butyllithium (20.5 mL, 41 mmol, 2.0 M solution in cyclohexane) was added dropwise through a dropping funnel. After stirring for 30 minutes, trimethyltin chloride (41 mL, 41 mmol, 1.0 M solution in hexane) was added dropwise through a dropping funnel. The resulting mixture was warmed to 0 ° C. and stirred for 1 h and then wetted with 100 ml of 1 M hydrochloric acid. The aqueous phase was extracted with ethyl acetate. The organic phases were combined, dried over sodium sulfate and concentrated in vacuo. This resulted in 9.24 g (28 mmol, 77%) of 2-trimethylstannyl-6-methoxybenzo [β] thiophene as white crystals. ¹ H NMR (CDCl ₃ ) 7.66 (d, J = 8.6 Hz, 1H), 7.34 (d, J = 2.3 Hz, 1H), 7.29 (s, 1H), 6.95 (dd, J = 8.6 Hz, 2.2 Hz, 1H), 3.86 (s, 3H), 0.39 (s, 9H).

(b) 370 mg (2 mmol) of 2-bromo-m-xylene in 8 ml of N, N-dimethylformamide, 115 mg (0.1 mmol) of tetrakistriphenylphosphinepalladium (0) and 160 mg of cupric oxide (2 mmol) was stirred at 100 ° C. in nitrogen. After 5 minutes 981 mg (3 mmol) of 2-trimethylstannyl-6-methoxybenzo [β] thiophene (Example 1a) in 2 ml of N, N-dimethylformamide were added all at once in the reaction mixture. The reaction was heated for 2 hours and then brought to room temperature. The resulting mixture was concentrated, dissolved in ethyl acetate, filtered through a pad of silica and concentrated. The crude product was purified by chromatography (silica, 99: 1, petroleum ether / ethyl acetate) to give 328 mg (1.22 mmol, 61% 2- (2,6-dimethylphenyl) -6-methoxybenzo [β] thiophene. ) Was obtained as yellow crystals. ¹ H NMR (CDCl ₃ ) 7.89 (d, J = 8.7 Hz, 1H), 7.32-7.59 (m, 4H), 7.23 (dd, J = 8.7 Hz, 2.2 Hz, 1H), 7.18 (s, 1H), 4.12 (s, 3 H), 2.46 (s, 6 H).

(c) 145 mg (1.54 mmol) of 2- (2,6-dimethylphenyl) -6-methoxybenzo [β] thiophene (Example 1b) was dissolved in 15 ml of dichloromethane and stirred in a solution of boron trifluoro. Ride dimethylsulfide complex (1.5 mL) was added. The solution was stirred for 15 h at room temperature in nitrogen in the dark. The reaction mixture was wetted with 10 ml of water, extracted with dichloromethane, dried over magnesium sulfate and concentrated. The crude product was purified by chromatography (silica, 80:20, petroleum ether / ethyl acetate) to give 94.1 mg (0.37 mmol, 69%) of 2- (2,6-dimethylphenyl) -6-hydroxybenzo [β] thiophene. ) Was obtained as white crystals. MP 95-96 ° C. ¹ H NMR (CDCl ₃ ) 7.63 (d, J = 8.6, 1H), 7.08-7.31 (m, 4H), 6.92 (s, 1H), 6.91 (dd, J = 8.6, 2.2 Hz, 1H), 4.91 ( s, 1 H), 2.20 (s, 6 H).

Example 6

2- (2-ethyl-6-methylphenyl) -6-hydroxybenzo [β] thiophene (2)

(a) Cross-coupling of 492 mg (2 mmol) of 6-ethyl-6-methyliodobenzene and 981 mg (3 mmol) of the product from 1 (a) was carried out by the method of Example 1 (b) It was. The crude product was purified on chromatography (silica, 99: 1, petroleum ether / ethyl acetate) to give 438 mg (1.55 mmol, 78) of 2- (2-ethyl-6-methylphenyl) -6-methoxybenzo [β] thiophene. %) Was obtained as a colorless oil. ¹ H NMR (CDCl ₃ ) 7.67 (d, J = 8.9 Hz, 1H), 7.08-7.36 (m, 4H), 7.01 (dd, J = 8.9 Hz, 2.2 Hz, 1H), 6.96 (s, 1H), 3.89 (s, 3H), 2.54 (q, J = 7.6 Hz, 2H), 2.19 (s, 3H), 1.12 (t, J = 7.6 Hz, 3H),

(b) Deprotection of 100 mg (0.35 mmol) of 2- (2-ethyl-6-methylphenyl) -6-methoxybenzo [β] thiophene (Example 2 (a)) in Example 1 (c) It was carried out by the method of. The crude product was purified by chromatography (silica, 90:10, petroleum ether / ethyl acetate) to give 69 mg (0.26 mmol, 73 2- (2-ethyl-6-methylphenyl) -6-hydroxybenzo [β] thiophene. %) Was obtained as white semicrystals. ¹ H NMR (CD ₃ OD) 7.59 (d, J = 8.7, 1H), 7.06-7.25 (m, 4H), 6.90 (s, 1H), 6.88 (dd, J = 8.7, 2.2 Hz, 1H), 2.51 (q, J = 7.6 Hz, 2H), 2.15 (s, 3H), 1.09 (t, J = 7.6 Hz, 3H).

Example 7

2- (2,6-dimethyl-4-hydroxyphenyl) -6-hydroxybenzo [β] thiophene (3)

(a) Cross-coupling of 402 mg (2 mmol) of 4-bromo-3,5-dimethylphenol and 981 mg (3 mmol) of the product from 1 (a) by the method of Example 1 (b) Was performed. The crude product was purified by chromatography (silica, 90:10, petroleum ether / ethyl acetate) to give 210 mg of 2- (2,6-dimethyl-4-hydroxyphenyl) -6-methoxybenzo [β] thiophene. 0.74 mmol, 37%) was obtained as yellow crystals. ¹ H NMR (CDCl ₃ ) 7.88 (d, J = 8.7 Hz, 1H), 7.55 (d, J = 2.5 Hz, 1H), 7.22 (dd, J = 8.7 Hz, 2.5 Hz, 1H), 7.14 (s, 1H), 6.83 (s, 2H), 4.94 (s, 1H), 4.11 (s, 3H), 2.38 (s, 6H),

(b) deprotection of 100 mg (0.35 mmol) of 2- (2,6-dimethyl-4-hydroxyphenyl) -6-methoxybenzo [β] thiophene (Example 3 (a)) It was carried out by the method in (c). The crude product was purified by chromatography (silica, 80:20, petroleum ether / ethyl acetate) to give 52 mg of 2- (2,6-dimethyl-4-hydroxyphenyl) -6-hydroxybenzo [β] thiophene. 0.19 mmol, 54%) was obtained as white crystals. MP 202-204 ° C, ¹ H NMR (CD ₃ OD) 7.56 (d, J = 8.7, 1H), 7.19 (d, J = 2.2 Hz, 1H), 6.86 (dd, J = 8.7, 2.2 Hz, 1H) , 6.84 (s, 1 H), 6.54 (s, 2 H), 2.10 (s, 6 H).

Example 8

2- (2-methylphenyl) -6-hydroxybenzo [β] thiophene (4)

(a) Cross-coupling of 340 mg (2 mmol) of 4-bromotoluene and 981 mg (3 mmol) of the product from 1 (a) was carried out by the method of Example 1 (b). The crude product was purified by chromatography (silica, 99: 1, petroleum ether / ethyl acetate) to give 500 mg (1.97 mmol, 98%) of 2- (2-methylphenyl) -6-methoxybenzo [β] thiophene. Obtained as a crystal. ¹ H NMR (CDCl ₃ ) 7.66 (d, J = 8.7 Hz, 1H), 7.19-7.49 (m, 5H), 7.15 (s, 1H), 6.99 (dd, J = 8.7, 2.3 Hz, 1H), 3.88 (s, 3H), 2.48 (s, 3H),

(b) Deprotection of 125 mg (0.49 mmol) of 2- (2-methylphenyl) -6-methoxybenzo [β] thiophene (Example 4 (a)) by the method in Example 1 (c) Was performed. The crude product was purified by chromatography (silica, 90:10, petroleum ether / ethyl acetate) to give 60 mg (0.23 mmol, 47%) of 2- (2-methylphenyl) -6-hydroxybenzo [β] thiophene as white. Obtained as a crystal. MP 97-98 ° C., ¹ H NMR (CDCl ₃ ) 7.63 (d, J = 8.4 Hz, 1H), 7.18-7.48 (m, 5H), 7.14 (s, 1H), 6.91 (dd, J = 8.4, 2.3 Hz, 1H), 4.86 (s, 1H), 1.56 (s, 3H).

Example 9

2- (2-chloro-6-methylphenyl) -6-hydroxybenzo [β] thiophene (5)

(a) Cross-coupling of 505 mg (2 mmol) of 3-chloro-2-iodotoluene and 981 mg (3 mmol) of product from 1 (a) was carried out by the method of Example 1 (b). . The crude product was purified by chromatography (silica, 99: 1, petroleum ether / ethyl acetate) to give 439 mg (1.52 mmol, 76) of 2- (2-chloro-6-methylphenyl) -6-methoxybenzo [β] thiophene. %) Was obtained as a yellow oil. ¹ H NMR (CDCl ₃ ) 7.68 (d, J = 8.7 Hz, 1H), 7.15-7.36 (m, 4H), 7.03 (s, 1H), 7.01 (dd, J = 8.7, 2.2 Hz, 1H), 3.88 (s, 3H), 2.25 (s, 3H),

(b) Deprotection of 100 mg (0.35 mmol) of 2- (2-chloro-6-methylphenyl) -6-methoxybenzo [β] thiophene (Example 5 (a)) in Example 1 (c) It was carried out by the method of. The crude product was purified by chromatography (silica, 90:10, petroleum ether / ethyl acetate) to give 44 (2- (2-chloro-6-methylphenyl) -6-hydroxybenzo [β] thiophene (0.16 mmol, 46). %) Was obtained as a yellow oil. ¹ H NMR (CD ₃ OD) 7.62 (d, J = 8.7 Hz, 1H), 7.18-7.35 (m, 4H), 6.99 (s, 1H), 6.89 (dd, J = 8.7, 2.2 Hz, 1H), 2.23 (s, 3 H).

Example 10

2- (2-methylnaphth-1-yl) -6-hydroxybenzo [β] thiophene (6)

(a) Cross-coupling of 221 mg (1 mmol) of 1-bromo-2-methylnaphthalene and 491 mg (1.5 mmol) of the product from 1 (a) was carried out by the method of Example 1 (b). . The crude product was purified by chromatography (silica, 99: 1, petroleum ether / ethyl acetate) to give 159 mg (0.52 mmol) of 2- (2-methylnaph-1-yl) -6-methoxybenzo [β] thiophene. , 52%) was obtained as white crystals. ¹ H NMR (CDCl ₃ ) 7.66-7.88 (m, 4H), 7.30-7.48 (m, 4H), 7.11 (s, 1H), 7.04 (dd, J = 8.7, 2.2 Hz, 1H), 3.91 (s, 3H), 2.40 (s, 3H),

(b) deprotection of 110 mg (0.36 mmol) of 2- (2-methylnaph-1-yl) -6-methoxybenzo [β] thiophene (Example 6 (a)) By the method). The crude product was purified by chromatography (silica, 90:10, petroleum ether / ethyl acetate) to give 52 mg (0.18 mmol of 2- (2-methylnaph-1-yl) -6-hydroxybenzo [β] thiophene. , 50%) was obtained as white semi-crystals. ¹ H NMR (CD ₃ COCD ₃ ) 8.60 (s, 1H), 7.87-8.05 (m, 2H), 7.74 (d, J = 8.7 Hz, 1H), 7.65-7.71 (m, 1H), 7.38-7.54 ( m, 4H), 7.18 (s, 1H), 7.02 (dd, J = 8.7, 2.2 Hz, 1H), 2.39 (s, 3H).

Example 11

2- (2,5-dimethyl-4-hydroxyphenyl) -6-hydroxybenzo [β] thiophene (7)

(a) Cross-coupling 248 mg (1 mmol) of 2,5-dimethyl-4-iodophenol and 491 mg (1.5 mmol) of the product from 1 (a) was carried out by the method of Example 1 (b) Was performed. The crude product was purified by chromatography (silica, 9: 1, petroleum ether / ethyl acetate) to give 130 mg of 2- (2,5-dimethyl-4-hydroxyphenyl) -6-methoxybenzo [β] thiophene. 0.46 mmol, 46%) was obtained as white crystals. ¹ H NMR (CDCl ₃ ) 8.34 (s, 1H), 7.69 (d, J = 8.8 Hz, 1H), 7.45 (d, J = 2.3 Hz, 1H), 7.19 (s, 1H), 7.17 (s, 1H ), 6.98 (dd, J = 8.8, 2.3 Hz, 1H), 6.78 (s, 1H), 3.87 (s, 3H), 2.34 (s, 3H), 2.20 (s, 3H),

(b) Example 1 Deprotection of 35 mg (0.12 mmol) of 2- (2,5-dimethyl-4-hydroxyphenyl) -6-methoxybenzo [β] thiophene (Example 6 (a)) It was carried out by the method in (c). The crude product was purified by chromatography (silica, 70:30, petroleum ether / ethyl acetate) to give 26 mg of 2- (2,5-dimethyl-4-hydroxyphenyl) -6-hydroxybenzo [β] thiophene. 0.096 mmol, 80%) was obtained as white crystals. MP 134-136 ° C, ¹ H NMR (CD ₃ COCD ₃ ) 8.41 (s broad, 2H), 7.63 (d, J = 8.7, 1H), 7.30 (d, J = 2.2 Hz, 1H), 7.18 (s, 1H), 7.13 (s, 1H), 6.93 (dd, J = 8.7, 2.2 Hz, 1H), 6.77 (s, 1H), 2.34 (s, 3H), 2.19 (s, 3H).

Example 12

2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thiophene (8)

Prepared according to the method of Hauser et al., WO 96/30361.

Example 13

2- (2-benzylphenyl) -6-hydroxybenzo [β] thiophene (9)

Cross-coupling of 124 mg (0.5 mmol) of 4-bromodiphenylmethane and 246 mg (0.75 mmol) of the product from 1 (a) was carried out by the method of Example 1 (b). The crude product was deprotected by the method of Example 1 (c). This was purified by chromatography (silica, 92: 8, petroleum ether / ethyl acetate) to give 108 (2.2- mmol, 68%) of 2- (2-benzylphenyl) -6-hydroxybenzo [β] thiophene as pink crystals. Got as. ¹ H NMR (CD ₃ COCD ₃ ) 8.55 (s, 1 H), 7.62 (d, J = 8.4 Hz, 1 H), 7.04-7.51 (m, 11 H), 6.93 (dd, J = 8.5, 2.5 Hz, 1 H) , 4.19 (s, 2 H).

Example 14

2- (4-hydroxynaphth-1-yl) -6-hydroxy [β] thiophene (10)

Cross-coupling of 119 mg (0.5 mmol) of 1-bromo-4-methoxynaphthalene and 246 mg (0.75 mmol) of the product from 1 (a) was carried out by the method of Example 1 (b). The crude product was deprotected by the method of Example 1 (c). This was purified by chromatography (silica, 8: 2, petroleum ether / ethyl acetate) to obtain 9 mg (0.03) of 2- (4-hydroxynaphth-1-yl) -6-hydroxy [β] thiophene (10). mmol, 6.2%) as dark brown crystals. ¹ H NMR (CD ₃ COCD ₃ ) 9.34 (s, 1H), 8.55 (s, 1H), 8.31-8.38 (m, 1H), 8.20-8.27 (m, 1H), 7.72 (d, J = 8.4 Hz, 1H), 7.48-7.58 (m, 2H), 7.48 (d, J = 7.7, 1H), 7.37 (d, J = 2.2, 1H), 7.34 (s, 1H), 7.00 (d, J = 7.7, 1H ), 6.98 (dd, J = 8.4, 2.2 Hz, 1H).

Example 15

2- (2-methyl-3-chlorophenyl) -6-hydroxybenzo [β] thiophene (11)

Cross-coupling 126 mg (0.5 mmol) of 2-chloro-6-iodotoluene and 246 mg (0.75 mmol) of the product from 1 (a) was carried out by the method of Example 1 (b). The crude product was deprotected by the method of Example 1 (c). This was purified by chromatography (silica, 92: 8, petroleum ether / ethyl acetate) to give 88 mg (0.32 mmol) of 2- (2-methyl-3-chlorophenyl) -6-hydroxybenzo [β] thiophene (11). , 64.1%). ¹ H NMR (CDCl ₃ ) 7.72 (d, J = 8.7 Hz, 1H), 7.29-7.41 (m, 2H), 7.27 (d, J = 2.5, 1H), 7.17 (d, J = 7.91, 1H), 7.10 (s, 1 H), 6.92 (dd, J = 8.7, 2.5 Hz, 1 H), 2.46 (s, 3 H).

Example 16

2- (2-methyl-5-chlorophenyl) -6-hydroxybenzo [β] thiophene (12)

Cross-coupling of 126 mg (0.5 mmol) of 4-chloro-2-iodotoluene and 246 mg (0.75 mmol) of the product from 1 (a) was carried out by the method of Example 1 (b). The crude product was deprotected by the method of Example 1 (c). This was purified by chromatography (silica, 92: 8, petroleum ether / ethyl acetate) to give 88 mg (0.32 mmol) of 2- (2-methyl-5-chlorophenyl) -6-hydroxybenzo [β] thiophene (12). , 64.1%). ¹ H NMR (CDCl ₃ ) 7.63 (d, J = 8.7 Hz, 1H), 7.43 (d, J = 2.0 Hz, 1H), 7.27 (d, J = 2.2, 1H), 7.19-7.23 (m, 2H) , 7.14 (s, 1 H), 6.92 (dd, J = 8.5, 2.2 Hz, 1 H), 2.41 (s, 3H).

Example 17

2- (2-methyl-4-chlorophenyl) -6-hydroxybenzo [β] thiophene (13)

Cross-coupling of 103 mg (0.5 mmol) of 2-bromo-5-chlorotoluene and 246 mg (0.75 mmol) of the product from 1 (a) was carried out by the method of Example 1 (b). The crude product was deprotected by the method of Example 1 (c). This was purified by chromatography (silica, 92: 8, petroleum ether / ethyl acetate) to give 118 mg (0.43 mmol) of 2- (2-methyl-4-chlorophenyl) -6-hydroxybenzo [β] thiophene (13). , 85.9%) was obtained as pink crystals. ¹ H NMR (CD ₃ COCD ₃ ) 8.63 (s, 1H), 7.69 (d, J = 8.7 Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H), 7.26-7.41 (m, 4H), 6.92 (dd, J = 8.4, 2.2 Hz, 1H), 2.47 (s, 3H).

Example 18

2- (2,5-hydroxy-4-bromophenyl) -6-hydroxybenzo [β] thiophene (14)

The cross-coupling of 222 mg (0.75 mmol) of 1,4-dibromo-2,5-dimethoxybenzene and 367 mg (1.125 mmol) of the product from 1 (a) was carried out in the method of Example 1 (b) Was performed. This was then purified by chromatography (silica, 95: 5, petroleum ether / ethyl acetate) to give 2- (2,5-methoxy-4-bromophenyl) -6-methoxybenzobenzo [β] thiophene 65.5 Mg (0.17 mmol, 23%). 25 mg (0.066 mmol) was deprotected to give 14.1 mg (0.042 mmol, 63.4%) of 2- (2,5-hydroxy-4-bromophenyl) -6-hydroxybenzo [β] thiophene. ¹ H NMR (CD ₃ COCD ₃ ) 8.84 (s, 1H), 8.54 (s, 1H), 8.33 (d, J = 8.74 Hz, 1H), 7.81 (s, 1H), 7.69 (d, J = 8.4 Hz , 1H), 7.34 (d, J = 2.2 Hz, 1H), 7.28 (s, 1H), 7.14 (s, 1H), 6.92 (dd, J = 8.4, 2.2 Hz, 1H).

Example 19

2- (2-methyl-4-nitrophenyl) -6-hydroxybenzo [β] thiophene (15)

(a) Cross-coupling of 432 mg (2.0 mmol) of 2-bromo-5-nitrotoluene and 982 mg (3.0 mmol) of product from 1 (a) was carried out by the method of Example 1 (b). . The crude product was purified by chromatography (silica, 8: 2, petroleum ether / ethyl acetate) to give 681 mg of 2- (2-methyl-4-nitrophenyl) -6-methoxybenzo [β] thiophene (about 75% Purity).

(b) Deprotection of 100 mg (0.33 mmol) of 2- (2-methyl-4-nitrophenyl) -6-methoxybenzo [β] thiophene (Example (15a)) was carried out in Example 1 (c). It was carried out according to the method. The crude product was purified by chromatography (silica, 70:30, petroleum ether / ethyl acetate) to give 73 mg (0.26 mmol, 2- (2-methyl-4-nitrophenyl) -6-hydroxybenzo [β] thiophene, 78%). ¹ H NMR (CDCl ₃ ) 8.16 (d broad, J = 2.1 Hz, 1H), 8.08 (dd, J = 8.5, 2.1 Hz, 1H), 7.68 (d, J = 8.5 Hz, 1H), 7.60 (d, J = 8.5, 1H), 7.29 (d, J = 2.4 Hz, 1H), 7.27 (s, 1H), 6.94 (dd, J = 8.5, 2.4 Hz, 1H), 5.15 (s, 1H), 2.59 (s , 3H).

Example 20

2- (2-methyl-4-aminophenyl) -6-hydroxybenzo [β] thiophene (16)

50 mg (0.18 mmol) of 2- (2-methyl-4-nitrophenyl) -6-hydroxybenzo [β] thiophene (Example 15 (b)) was dissolved in 5 ml of ethanol and tin dichloride dihydrate 198. Mg (0.88 mmol) was added. The mixture was heated at 70 ° C. for 3 hours in a nitrogen atmosphere. Hydrochloric acid (1 M) was added and the aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with bromine water, dried over magnesium sulfate and concentrated in vacuo. The crude product was purified by chromatography (silica, 6: 4, petroleum ether / ethyl acetate) to give 22 mg (0.086 mmol, 2- (2-methyl-4-aminophenyl) -6-hydroxybenzo [β] thiophene. 48%). ¹ H NMR (CD ₃ OD) 7.54 (d, J = 8.6, 1H), 7.18 (d, J = 2.4 Hz, 1H), 7.17 (d, J = 8.6, 1H), 7.00 (s, 1H), 6.84 (dd, J = 8.6, 2.4 Hz, 1H), 6.64 (d, J = 2.4 Hz, 1H), 6.58 (dd, J = 8.6, 2.4 Hz), 2.37 (s, 3H).

Example 21

2- (2-methyl-3-nitrophenyl) -6-hydroxybenzo [β] thiophene (17)

(a) Cross-coupling of 432 mg (2.0 mmol) of 2-bromo-6-nitrotoluene and 982 mg (3.0 mmol) of product from 1 (a) was carried out by the method of Example 1 (b). . The crude product was purified by chromatography (silica, 95: 5, petroleum ether / ethyl acetate) to give 114 (2- (2-methyl-3-nitrophenyl) -6-methoxybenzo [β] thiophene (0.38 mmol, 13%).

(b) deprotection of 200 mg (0.67 mmol) of 2- (2-methyl-3-nitrophenyl) -6-methoxybenzo [β] thiophene (Example (17a)) of Example 1 (c) It was carried out according to the method. The crude product was purified by chromatography (silica, 70:30, petroleum ether / ethyl acetate) to give 101 mg (0.35 mmol, 2- (2-methyl-3-nitrophenyl) -6-hydroxybenzo [β] thiophene, 53%). ¹ H NMR (CDCOCD ₃ ) 8.67 (s, 1H), 7.86 (dd, J = 8.0, 1.2 Hz, 1H), 7.76 (dd, J = 7.7, 1.2 Hz, 1H), 7.73 (d, J = 8.7 Hz , 1H), 7.54 (m, 1H), 7.38 (d, J = 2.1 Hz, 1H), 7.36 (s, 1H), 7.00 (dd, J = 8.7, 2.1 Hz, 1H), 5.15 (s, 1H) , 2.52 (s, 3H).

Example 22

2- (2-methyl-3-aminophenyl) -6-hydroxybenzo [β] thiophene (18)

350 mg (1.23 mmol) of 2- (2-methyl-3-nitrophenyl) -6-hydroxybenzo [β] thiophene (Example 17 (b)) was dissolved in 10 ml of ethanol and tin dichloride dihydrate 1384. Mg (6.1 mmol) was added. The mixture was heated at 70 ° C. for 3 hours in a nitrogen atmosphere. Hydrochloric acid (1 M) was added and the aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with bromine water, dried over magnesium sulfate and concentrated in vacuo. The crude product was purified by HPLC (reverse phase, C18, concentration gradient acetonitrile / water + 0.05 trifluoroacetic acid) to give 2- (2-methyl-3-aminophenyl) -6-hydroxybenzo [β] thiophene 27 Mg (0.10 mmol, 8.1%) was obtained. ¹ H NMR (CD ₃ OD) 8.93 (s, 1 H broad), 7.65 (d, J = 8.8, 1 H), 7.33 (d, J = 2.5 Hz, 1 H), 7.12 (s, 1 H), 6.92-7.00 ( m, 2H), 6.71-6.78 (m, 2H), 4.69 (s, 2H broad), 2.20 (s, 3H).

Example 23

2- (2-methyl-3-bromo-5-hydroxyphenyl) -6-hydroxybenzo [β] thiophene (19)

(a) The method of Example 1 (b) was cross-coupled with 369 mg (1.3 mmol) of 2,6-dibromo-4-methoxytoluene and 636 mg (1.95 mmol) of product from 1 (a). Was performed by. The crude product was purified by chromatography (silica, 98: 2, petroleum ether / ethyl acetate) to give 2- (2-methyl-3-bromo-5-methoxyphenyl) -6-methoxybenzo [β] thiophene 220 mg (0.61 mmol, 46.6%) were obtained.

(b) Deprotection of 70 mg (0.19 mmol) of 2- (2-methyl-3-bromo-5-methoxyphenyl) -6-methoxybenzo [β] thiophene (Example (19a)) It was carried out according to the method of Example 1 (c). The crude product was purified by chromatography (silica, 8: 2, petroleum ether / ethyl acetate) to give 2- (2-methyl-3-bromo-5-hydroxyphenyl) -6-hydroxybenzo [β] thiophene 55 mg (0.16 mmol, 86%) were obtained. ¹ H NMR (CDCOCD ₃ ) 8.66 (s, 1H), 7.69 (d, J = 8.4, 1H), 7.34 (d, J = 2.2 Hz, 1H), 7.23 (s, 1H), 7.15 (d, J = 2.5 Hz, 1H), 6.99 (d, J = 2.2 Hz, 1H), 7.00 (dd, J = 8.4, 2.2 Hz, 1H), 6.94 (d, J = 2.5 Hz, 1H), 2.38 (s, 3H) .

Example 24

2- (2-methyl-5-hydroxyphenyl) -6-hydroxybenzo [β] thiophene (20)

(a) 30 mg (0.08 mmol) of 2- (2-methyl-3-bromo-5-methoxyphenyl) -6-methoxybenzo [β] thiophene (Example (19 a)) was added to tetrahydrofuran. It was dissolved in 2 ml. The mixture was cooled to -70 ° C and butyllithium (0.12 mmol) was added to the reaction mixture. The reaction mixture was stirred at -70 ° C for 2.5 hours and then at room temperature overnight. The reaction mixture was wetted with aqueous ammonium chloride solution, extracted with ethyl acetate, and dried over magnesium sulfate. This gave 30 mg of crude 2- (2-methyl-5-methoxyphenyl) -6-methoxybenzo [β] thiophene.

(b) Example 1 (c) deprotection of 30 mg (0.10 mmol) of 2- (2-methyl-5-methoxyphenyl) -6-methoxybenzo [β] thiophene (Example (20a)) It was carried out according to the method. The crude product was purified by HPLC (reverse phase, C18, concentration gradient, acetonitrile / water + 0.05 trifluoroacetic acid) to give 2- (2-methyl-5-hydroxyphenyl) -6-hydroxybenzo [β] thiophene. 6.7 mg (0.026 mmol, 24%) was obtained. ¹ H NMR (CD ₃ COCD ₃ ) 8.55 (s, 1H), 8.29 (s, 1H), 7.67 (dd, J = 8.5, 2.2 Hz, 1H), 7.33 (t, 1H), 7.24 (d, J = 2.5 Hz, 1H), 7.13 (dd, J = 8.2, 2.0 Hz, 1H), 6.92-6.99 (m, 2H), 6.76 (dt, 1H), 2.35 (d, J = 2.2 Hz, 3H).

Example 25

2-phenyl-6-hydroxybenzo [β] thiophene (21)

Cross-coupling of 157 mg (1.0 mmol) of bromobenzene and 491 mg (1.5 mmol) of the product from 1 (a) was carried out by the method of Example 1 (b). The crude product was deprotected according to the method of Example 1 (c). This was purified by chromatography (silica, 9: 1, petroleum ether / ethyl acetate) to give 81 mg (0.36 mmol, 36%) of 2-phenyl-6-hydroxybenzo [β] thiophene. ¹ H NMR (CD ₃ COCD ₃ ) 8.65 (s, 1 H), 7.60-7.75 (m, 4 H), 7.28-7.48 (m, 4H), 6.95 (dd, J = 8.5, 2.5 Hz, 1 H).

Example 26

2- (4-hydroxyphenyl) -6-benzo [β] thiophene (22)

(a) Stanylation of 3 g (22.4 mmol) of benzo [β] thiophene was carried out according to the method of Example 1 (a). This gave 6.3 g (21.3 mmol) of 2-trimethylstannylbenzo [β] thiophene.

(b) Cross-coupling of 468 mg (2.0 mmol) of 4-iodophenol and 889 mg (3.0 mmol) of the product from 22 (a) was carried out by the method of Example 1 (b). The crude product was deprotected according to the method of Example 1 (c), then purified by chromatography (silica, 9: 1, petroleum ether / ethyl acetate) and recrystallized (petroleum ether / ethyl acetate) 2- (4- 20 mg (0.09 mmol, 4%) of hydroxyphenyl) -benzo [β] thiophene was obtained. ¹ H NMR (CD ₃ COCD ₃ ) 8.70 (s, 1H), 7.88 (m, 1H), 7.79 (m, 1H), 7.56-7.69 (m, 3H), 7.25-7.38 (m, 2H), 6.90- 7.00 (m, 2 H).

Example 27

2- (2-trifluoromethyl-6-fluorophenyl) -6-hydroxybenzo [β] thiophene (23)

Prepared by the parallel solution phase method. 61 mg (0.25 mmol) of 2-bromo-3-fluorobenzotrifluoride in 1 ml of N, N-dimethylformamide, 15 mg (0.013 mmol) of tetrakistriphenylphosphinepalladium (0) and cupric oxide 20 mg (0.25 mmol) of the mixture was stirred at 100 ° C. in nitrogen. After 5 minutes, 123 mg (0.38 mmol) of 2-trimethylstannyl-6-methoxybenzo [β] thiophene (Example 1 (a)) in 2 mL of N, N-dimethylformamide were added all at once with the reaction mixture. Added. The solution was heated to 100 ° C. for 3 hours, concentrated on a speed-vac, dissolved in dichloromethane, filtered through a pad of silica and then concentrated again. The product was dissolved in 1.5 mL of dichloromethane and 1 mL of borontrifluoride dimethylsulfide complex was added. The reaction mixture was heated in the dark overnight, wet with water and extracted with dichloromethane. The organic phase was dried through a sodium sulfate drying tube and then concentrated to a speed-bag. The crude product was purified by HPLC (silica, n-heptane + 0.5% acetic acid to ethyl acetate + 0.5% acetic acid concentration eluent) to give 2- (2-trifluorophenyl) -6-hydroxybenzo [β] thiophene 1.5 Mg (0.005 mmol, 2%) was obtained. ¹ H NMR (CD ₃ COCD ₃ ) 8.72 (s, 1H broad), 7.71-7.78 (m, 3H), 7.55-7.65 (m, 1H), 7.37 (d, J = 2.2 Hz, 1H), 7.29 (s , 1H), 6.99 (dd, J = 8.7, 2.2 Hz, 1H).

Example 28

6- (6-hydroxy-2-benzo [β] thienyl) -4,5-dimethylbenzo-2,1,3-thiadiazole (24)

Cross-coupling and subsequent deprotection of 61 mg (0.25 mmol) of 6-bromo-4,5-dimethylbenzo-2,1,3-thiadiazole and 123 mg (0.38 mmol) of product from 1 (a) Was carried out according to the method of Example 23. The crude product was purified by HPLC (silica, n-heptane + 0.5% acetic acid to ethyl acetate + 0.5% acetic acid concentration eluent) to give 6- (6-hydroxybenzo [β] thien-2-yl) -4,5- 3.6 mg (0.012 mmol, 4.6%) of dimethylbenzo-2,1,3-thiadiazole were obtained. ¹ H NMR (CD ₃ COCD ₃ ) 7.92 (s, 1 H), 7.74 (d, J = 8.5, 1 H), 7.38 (d, J = 2.2 Hz, 1 H), 7.37 (s, 1 H), 7.01 (dd, J = 8.5, 2.2 Hz, 1H), 2.76 (s, 3H), 2.50 (s, 3H).

Example 29

2- (4-methyl-3-thienyl) -6-hydroxybenzo [β] thiophene (25)

Cross-coupling of 44 mg (0.25 mmol) of 3-bromo-4-methylthiophene and 123 mg (0.38 mmol) of the product from 1 (a) was followed according to the method of Example 23. The crude product was purified by HPLC (silica, n-heptane + 0.5% acetic acid to ethyl acetate + 0.5% acetic acid concentration eluent) to give 2- (4-methyl-3-thienyl) -6-hydroxybenzo [β] ti. Offen 22 mg (0.09 mmol, 36%) was obtained. ¹ H NMR (CD ₃ COCD ₃ ) 8.57 (s, 1 H), 7.66 (d, J = 8.4, 1 H), 7.53 (d, J = 3.2 Hz, 1 H), 7.35 (s, 1 H), 7.32 (m, 1H), 7.23 (m, 1H), 6.94 (dd, J = 8.4, 2.2 Hz), 2.43 (d, J = 0.74 Hz, 3H).

Example 30

2- (3,4,5-trimethyl-2-thienyl) -6-hydroxybenzo [β] thiophene (26)

Cross-coupling of 252 mg (1.0 mmol) of 2-iodo-3,4,5-trimethylthiophene and 491 mg (1.5 mmol) of the product from 1 (a) was carried out according to the method of Example 1 (b) Was performed. Crude 2- (3,4,5-trimethyl-2-thienyl) -6-methoxybenzo [β] thiophene was deprotected according to the method of Example 1 (c), followed by chromatotron (silica, 9 1, petroleum ether / ethyl acetate) to give 180 mg (0.625 mmol, 63%) of 2- (3,4,5-trimethyl-2-thienyl) -6-hydroxybenzo [β] thiophene. . ¹ H NMR (CD ₃ COCD ₃ ) 8.55 (s, 1 H), 7.67 (d, J = 8.7 Hz, 1 H), 7.32 (d, J = 2.2 Hz, 1 H), 7.06 (d, J = 0.7 Hz, 1 H ), 6.95 (dd, J = 8.7, 2.2 Hz, 1 Hz), 2.34 (s, 3H), 2.30 (s, 3H), 1.96 (s, 3H).

Example 31

2- (5- (1,3-dimethylurasilyl) -6-hydroxybenzo [β] thiophene (27)

(a) Cross-coupling 266 mg (1.0 mmol) of 5-iodo-1,3-dimethyluracil and 491 mg (1.5 mmol) of the product from 1 (a) by the method of Example 1 (b) Was performed. The crude product was purified by chromatography (silica, 40: 1, dichloromethane / ethyl acetate) to give 211 mg (0.625) of 2- (5- (1,3-dimethylurasilyl) -6-methoxybenzo [β] thiophene. mmol, 63%).

(b) deprotection of 30 mg (0.10 mmol) of 2- (5- (1,3-dimethylurasilyl) -6-methoxybenzo [β] thiophene (Example (27a)) in Example 1 (c) The crude product was purified by chromatography (silica, 9: 1, petroleum ether / ethyl acetate) to give 2- (5- (1,3-dimethylurasilyl) -6-hydroxybenzo [ β] thiophene 1.2 mg (0.004 mmol, 4.2%) ¹ H NMR (CDCl ₃ ) 7.74 (s, 1H), 7.61 (d, J = 8.7 Hz, 1H), 7.54 (s, 1H), 7.10 -7.30 (m, 1H), 7.13 (dd, J = 8.7, 2.3 Hz, 1H), 3.87 (s, 3H), 3.51 (s, 3H).

Example 32

[2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [phenyl] methanone (28)

(a) 200 mg of 6-methoxy-2- (4-methoxyphenyl) benzo [β] thiophene (Hauser et al., WO 96/30361) in dichloromethane (5 mL) and 110 mg (0.78) of benzoyl chloride mmol) was added 740 mg (5.6 mmol) of aluminum chloride. The reaction mixture was stirred at rt for 5 h. Ethyl acetate and 1 M hydrochloric acid were added to stop the reaction. The organic layer was separated and the aqueous phase was extracted with ethyl acetate. The combined organic phases were dried over magnesium sulfate, filtered and concentrated. The crude product was purified by chromatography (silica, 95: 5, petroleum ether / ethyl acetate) to give [2- (4-methoxyphenyl) -6-methoxybenzo [β] thien-3-yl] phenylmethanone 131 Mg (0.35 mmol, 47%) was obtained as yellow crystals. ¹ H NMR (CDCl ₃ ) 7.72-7.80 (m, 2H), 7.59 (d, J = 8.9 Hz, 1H), 7.36-7.43 (m, 1H), 7.21-7.34 (m, 5H), 6.97 (dd, J = 8.9, 2.5 Hz, 2H), 6.72 (m, 1H), 3.88 (s, 3H), 3.72 (s, 3H).

(b) 70 mg (0.19 mmol) of [2- (4-methoxyphenyl) -6-methoxybenzo [β] thien-3-yl] phenylmethanone (Example 28a) was diluted with dichloromethane (5 mL). Dissolved and placed under nitrogen atmosphere and cooled to -5 ° C. 0.56 mL (0.56 mmol) of 1 M BBr ₃ was added dropwise to the stirred solution. The reaction mixture was stirred at 5 ° C. for 1 hour and then poured into ice water and extracted with ethyl acetate. The organic phase was dried over magnesium sulfate, filtered and concentrated. The crude product was purified by chromatography (silica, petroleum ether / ethyl acetate in a concentration gradient of 75:25 to 50:50) to [2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thiene-3- 46] (0.13 mmol, 71%) of il] phenylmethanone were obtained as yellow crystals. MP 214-217 ° C., ¹ H NMR (CD ₃ COCD ₃ ) 8.74 (s, 1H), 8.64 (s, 1H), 7.70-7.77 (m, 2H), 7.20-7.54 (m, 7H), 6.96 (dd) , J = 8.7, 2.4 Hz, 1H), 6.68-6.76 (m, 2H).

Example 33

[2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [2-naphthyl] methanone (29)

(a) 150 mg (0.55 mmol) of 6-methoxy-2- (4-methoxyphenyl) benzo [β] thiophene (Hauser et al., WO 96/30361) and 111 mg (0.58 mmol) of 2-naphthoylchloride Acylation of mmol) was carried out according to the method of Example 28 (a). The crude product was purified by chromatography (silica, 95: 5, petroleum ether / ethyl acetate) to give [2- (4-methoxyphenyl) -6-methoxybenzo [β] thien-3-yl] [2-nap 105 mg (0.25 mmol, 45%) of methyl] methanone were obtained as yellow crystals. ¹ H NMR (CDCl ₃ ) 8.21 (s, 1H), 7.95 (dd, J = 8.7, 1.7 Hz, 1H), 7.71 (m, 3H), 7.31-7.61 (m, 6H), 6.96 (dd, J = 8.9, 2.5 Hz, 1H), 6.65 (m, 2H), 3.88 (s, 3H), 3.63 (s, 3H).

(b) 70 mg (0.17 mmol) of [2- (4-methoxyphenyl) -6-methoxybenzo [β] thien-3-yl] [2-naphthyl] methanone (Example 29 (a)) Deprotection of was carried out according to the method of Example 28 (b). The crude product was purified by chromatography (silica, petroleum ether / ethyl acetate in a concentration gradient of 75:25 to 50:50) to [2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thiene-3- 47 mg (0.12 mmol, 72%) of [1] [2-naphthyl] methanone were obtained as yellow crystals. MP 229-232 ° C., ¹ H NMR (CD ₃ COCD ₃ ) 8.73 (s, 1H), 8.52 (s, 1H), 8.24 (s, 1H), 7.83-7.98 (m, 4H), 7.41-7.63 (m , 4H), 7.22-7.33 (m, 2H), 6.96 (dd, J = 8.8, 2.2 Hz, 1H), 6.65 (m, 2H).

Example 34

[2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [4-tert-butylphenyl] methanone (30)

(a) 150 mg (0.55 mmol) of 6-methoxy-2- (4-methoxyphenyl) benzo [β] thiophene (Hauser et al., WO 96/30361) and 115 mg of 4-tert-butylbenzoyl chloride (0.58 mmol) acylation was carried out according to the method of Example 28 (a). The crude product was purified by chromatography (silica, 95: 5, petroleum ether / ethyl acetate) to give [2- (4-methoxyphenyl) -6-methoxybenzo [β] thien-3-yl] [4-tert 125 mg (0.29 mmol, 52%) of -butylphenyl] methanone was obtained as yellow crystals. ¹ H NMR (CDCl ₃ ) 7.64-7.73 (m, 2H), 7.55 (d, J = 8.9 Hz, 1H), 7.22-7.34 (m, 5H), 6.95 (dd, J = 8.9, 2.5 Hz, 1H) 6.72 (m, 2H), 3.87 (s, 3H), 3.71 (s, 3H), 1.22 (s, 9H).

(b) 70 mg (0.17) of [2- (4-methoxyphenyl) -6-methoxybenzo [β] thien-3-yl] [4-tert-butylphenyl] methanone (Example 30 (a)) deprotection) was carried out according to the method of Example 28 (b). The crude product was purified by chromatography (silica, petroleum ether / ethyl acetate in a concentration gradient of 75:25 to 50:50) to [2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thiene-3- Il] [2-tert-butylphenyl] methanone 30 mg (0.07 mmol, 46%) was obtained as yellow crystals. MP 197-200 ° C., ¹ H NMR (CD ₃ COCD ₃ ) 8.69 (s, 1H), 8.60 (s, 1H), 7.63-7.70 (m, 2H), 7.35-7.46 (m, 4H), 7.21-7.28 (m, 2H), 6.94 (d, J = 8.8, 2.2 Hz, 1H), 6.72 (m, 2H), 1.28 (s, 9H).

Example 35

[2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [4-methoxyphenyl] methanone (31)

(a) 150 mg (0.55 mmol) of 6-methoxy-2- (4-methoxyphenyl) benzo [β] thiophene (Hauser et al., WO 96/30361) and 99 mg of 4-methoxybenzoyl chloride ( 0.58 mmol) acylation was carried out according to the method of Example 28 (a). The crude product was purified by chromatography (silica, 95: 5, petroleum ether / ethyl acetate) to give [2- (4-methoxyphenyl) -6-methoxybenzo [β] thien-3-yl] [4-methoxy 112 mg (0.27 mmol, 50%) of oxyphenyl] methanone was obtained as yellow crystals.

(b) 70 mg (0.17 mmol) of [2- (4-methoxyphenyl) -6-methoxybenzo [β] thien-3-yl] [4-methoxyphenyl] methanone (Example 31 (a)) ) Deprotection was carried out according to the method of Example 28 (b). The crude product was purified by chromatography (silica, 50:50, petroleum ether / ethyl acetate) to give [2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [2-meth 40 mg (0.07 mmol, 63%) of oxyphenyl] methanone was obtained as yellow crystals. ¹ H NMR (CD ₃ COCD ₃ ) 8.71 (s, 2H broad), 7.73 (m, 2H), 7.38-7.42 (m, 2H), 7.28 (m, 2H), 6.94 (d, J = 8.4, 2.4 Hz , 1H), 6.86 (m, 2H), 6.75 (m, 2H), 3.79 (s, 3H).

Example 36

[2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [4-carboxyphenyl] methanone (32)

(a) 506 mg (1.87 mmol) of 6-methoxy-2- (4-methoxyphenyl) benzo [β] thiophene (Hauser et al., WO 96/30361) and 390 mg (1.97 terephthalic acid monomethyl ester chloride) Acylation of mmol) was carried out according to the method of Example 28 (a). The crude product was purified by chromatography (silica, 8: 2, petroleum ether / ethyl acetate) to give [2- (4-methoxyphenyl) -6-methoxybenzo [β] thien-3-yl] [4-methoxy 442 mg (1.02 mmol, 55%) of oxycarbonylphenyl] methanone were obtained.

(b) [2- (4-methoxyphenyl) -6-methoxybenzo [[beta] thien-3-yl] [4-methoxycarbonylphenyl] methanone (Example (32a)) 406 mg (0.94) deprotection) was carried out according to the method of Example 1 (c). The crude product was purified by recrystallization (acetic acid / dichloromethane / methanol) to give [2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [4-carboxyphenyl] methanone 270 Mg (0.69 mmol, 73%) was obtained. ¹ H NMR (CD ₃ COCD ₃ ) 8.76 (s, 1 H broad), 8.63 (s, 1 H broad), 7.92-7.98 (m, 2H), 7.76-7.83 (m, 2H), 7.63 (d, J = 8.8 Hz, 1H), 7.41 (d, J = 2.5 Hz, 1H), 7.21 (m, 2H), 6.99 (dd, J = 8.8, 2.5 Hz, 1H), 6.69 (m, 2H).

Example 37

[2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [4-methoxycarbonylphenyl] methanone (33)

100 mg (0.25 mmol) of [2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [4-carboxyphenyl] methanone (Example 32 (b)) was added to methanol. Dissolve and add 5 drops of thionylchloride. The reaction mixture was stirred at room temperature for 24 hours, wetted with water, extracted with ethyl acetate and dried over magnesium sulfate. The crude product was purified by chromatography (silica, 9: 1, petroleum ether / ethyl acetate) to give [2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [4-meth 42 mg (0.1 mmol, 42%) of oxycarbonylphenyl] methanone were obtained. ¹ H NMR (CD ₃ OD) 8.76 (s, 1 H broad), 7.82-7.88 (m, 2H), 7.66-7.72 (m, 2H), 7.61 (d, J = 8.9 Hz, 1H), 7.28 (d, J = 2.4 Hz, 1H), 7.08-7.14 (m, 2H), 6.90 (dd, J = 8.9, 2.4 Hz, 1H), 6.53-6.60 (m, 2H), 3.85 (s, 3H).

Example 38

[2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [4-ethoxycarbonylphenyl] methanone (34)

50 mg (0.12 mmol) of [2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [4-carboxyphenyl] methanone (Example 32 (b)) was added to ethanol. Dissolve and add 5 drops of thionylchloride. The reaction mixture was stirred at room temperature for 24 hours, wetted with water, extracted with ethyl acetate and dried over magnesium sulfate. The crude product was purified by chromatography (silica, 9: 1, petroleum ether / ethyl acetate) to give [2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [4- 39 mg (0.1 mmol, 74%) of oxycarbonylphenyl] methanone were obtained. ¹ H NMR (CD ₃ OD) 8.72 (s, 2H broad), 7.87-7.94 (m, 2H), 7.76-7.82 (m, 2H), 7.62 (d, J = 8.8 Hz, 1H), 7.42 (d, J = 2.2 Hz, 1H), 7.17-7.24 (m, 2H), 6.99 (dd, J = 8.8, 2.2 Hz, 1H), 6.66-6.72 (m, 2H), 4.31 (q, 2H), 1.32 (t , 3H).

Example 39

[2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [4-cyanophenyl] methanone (35)

(a) 300 mg (1.11 mmol) of 6-methoxy-2- (4-methoxyphenyl) benzo [β] thiophene (Hauser et al., WO 96/30361) and 193 mg of 4-cyanobenzoyl chloride ( Acylation of 1.17 mmol) was carried out according to the method of Example 28 (a). The crude product was purified by chromatography (silica, 8: 2, petroleum ether / ethyl acetate) to give [2- (4-methoxyphenyl) -6-methoxybenzo [β] thien-3-yl] [4-sia Nophenyl] methanone 206 mg (0.52 mmol, 46%) was obtained.

(b) 30 mg (0.075 mmol) of [2- (4-methoxyphenyl) -6-methoxybenzo [β] thien-3-yl] [4-cyanophenyl] methanone (Example (35a)) Deprotection of was carried out according to the method of Example 1 (c). The crude product was purified by chromatography (silica, 5: 5, petroleum ether / ethyl acetate) to give [2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [4-sia 24 mg (0.06 mmol, 86%) of nophenyl] methanone was obtained. ¹ H NMR (CD ₃ COCD ₃ ) 8.77 (s, 1 H broad), 8.73 (s, 1 H broad), 7.78-7.85 (m, 2H), 7.65-7.73 (m, 3H), 7.43 (d, J = 2.2 Hz, 1H), 7.16 (m, 2H), 7.02 (dd, J = 8.8, 2.2 Hz, 1H), 6.69 (m, 2H).

Example 40

[2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [4- (1H-tetrazol-5-yl) phenyl] methanone (36)

30 mg (0.08 mmol) of [2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [4-cyanophenyl] methanone (Example 35 (b)) It was dissolved in 1 ml of N-dimethylformamide and kept under nitrogen. 49 mg (0.08 mmol) of sodium azide and 40 mg (0.08 mmol) of ammonium chloride were added to the reaction mixture, and the mixture was heated to reflux for 2 hours. N, N-dimethylformamide was removed in a speed-vac. The compound was deprotected according to the method of Example 1 (c). The crude product was purified by HPLC (reverse phase, concentration gradient of C18, acetonitrile / water + 0.05 trifluoroacetic acid) to [2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl ] 24 mg (0.06 mmol, 72%) of [4- (1H-tetrazol-5-yl) phenyl] methanone. ¹ H NMR (CD ₃ COCD ₃ ) 8.80 (s, 1H), 8.65 (s, 1H), 8.00-8.12 (m, 2H), 7.86-7.92 (m, 2H), 7.62 (d, J = 8.8 Hz, 1H), 7.42 (d, J = 2.2 Hz, 1H), 7.18-7.28 (m, 2H), 7.00 (dd, J = 8.8, 2.2 Hz, 1H), 6.65-6.75 (m, 2H).

Example 41

5-oxo-5- [2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] pentanoic acid methyl ester (37)

(a) 200 mg (0.74 mmol) of 6-methoxy-2- (4-methoxyphenyl) benzo [β] thiophene (Hauser et al., WO 96/30361) and 139 mg (0.78 mmol) of methyladipoyl chloride Acylation) was performed according to the method of Example 28 (a). The crude product was purified by chromatography (silica, 9: 1, petroleum ether / ethyl acetate) to give 5-oxo-5- [2- (4-methoxyphenyl) -6-methoxybenzo [β] thiene-3- 91 mg (0.22 mmol, 30%) of il] pentanoic acid methyl ester was obtained.

(b) 80 mg (0.19 mmol) of 5-oxo-5- [2- (4-methoxyphenyl) -6-methoxybenzo [β] thien-3-yl] phenanoic acid methyl ester (Example (37a)) Deprotection) was carried out according to the method of Example 1 (c). The crude product was purified by chromatography (silica, 98: 2, chloroform / methanol + acetic acid) to give 5-oxo- 5- [2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thiene-3 38 mg (0.10 mmol, 52%) of -yl] pentanoic acid methyl ester was obtained. ¹ H NMR (CD ₃ COCD ₃ ) 8.86 (s, 1H broad), 8.67 (s, 1H broad), 7.82 (d, J = 8.8 Hz, 1H), 7.28-7.40 (m, 3H), 6.95-7.05 ( m, 3H), 3.56 (s, 3H), 2.37-2.46 (m, 2H), 2.11-2.20 (m, 2H), 1.36-1.60 (m, 4H).

Example 42

5-oxo-5- [2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] pentanoic acid (38)

25 mg (0.06 mmol) of 5-oxo-5- [2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] pentanoic acid methyl ester (Example 37 (b)) It was dissolved in 5 ml of methanol and 0.5 ml of 1 M sodium hydroxide. The reaction mixture was stirred for 1 hour to neutralize, extracted with ethyl acetate and dried over magnesium sulfate. This gave 11 mg (0.03 mmol, 49%) of 5-oxo-5- [2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] pentanoic acid. ¹ H NMR (CD ₃ COCD ₃ ) 7.82 (d, J = 8.8 Hz, 1H), 7.28-7.40 (m, 3H), 6.90-7.05 (m, 3H).

Example 43

[2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [4-propylphenyl] methanone (39)

(a) 200 mg (0.74 mmol) of 6-methoxy-2- (4-methoxyphenyl) benzo [β] thiophene (Hauser et al., WO 96/30361) and 142 mg of 4-propylbenzoyl chloride (1.17) Acylation of mmol) was carried out according to the method of Example 28 (a). The crude product was purified by chromatography (silica, 9: 1, petroleum ether / ethyl acetate) to give [2- (4-methoxyphenyl) -6-methoxybenzo [β] thien-3-yl] [4-iso 128 mg (0.52 mmol, 42%) of propylphenyl] methanone were obtained.

(b) 100 mg (0.24 mmol) of [2- (4-methoxyphenyl) -6-methoxybenzo [β] thien-3-yl] [4-isopropylphenyl] methanone (Example (39a)) Deprotection of was carried out according to the method of Example 1 (c). The crude product was purified by chromatography (silica, 5: 5, petroleum ether / ethyl acetate) to give [2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [4-propyl Phenyl] methanone 28 mg (0.07 mmol, 30%) was obtained. ¹ H NMR (CD ₃ COCD ₃ ) 8.71 (s, 1H), 8.62 (s, 1H), 7.62-7.70 (m, 2H), 7.45 (d, J = 8.8 Hz, 1H), 7.39 (d, J = 2.2 Hz, 1H), 7.21-7.28 (m, 2H), 7.14-7.20 (m, 2H), 6.94 (dd, J = 8.8, 2.2 Hz, 1H), 6.68-6.75 (m, 2H), 2.25 (t , 2H), 1.58 (m, 2H), 0.88 (t, 3H).

Example 44

[2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [4-iodophenyl] methanone (40)

(a) 200 mg (0.74 mmol) of 6-methoxy-2- (4-methoxyphenyl) benzo [β] thiophene (Hauser et al., WO 96/30361) and 207 mg of 4-iodobenzoyl chloride ( 0.77 mmol) acylation was carried out according to the method of Example 28 (a). The crude product was purified by chromatography (silica, 9: 1, petroleum ether / ethyl acetate) to give [2- (4-methoxyphenyl) -6-methoxybenzo [β] thien-3-yl] [4-io Dophenyl] methanone 258 mg (0.52 mmol, 70%) was obtained.

(b) 100 mg (0.20 mmol) of [2- (4-methoxyphenyl) -6-methoxybenzo [β] thien-3-yl] [4-iodophenyl] methanone (Example (40)) Deprotection of was carried out according to the method of Example 1 (c). The crude product was purified by chromatography (silica, 5: 5, petroleum ether / ethyl acetate) to give [2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [4-io 43 mg (0.09 mmol, 45%) of dophenyl] methanone were obtained. ¹ H NMR (CD ₃ COCD ₃ ) 8.71 (s, 2H, broad), 8.62 (s, 1H), 7.69-7.78 (m, 2H), 7.54 (d, J = 8.8 Hz, 1H), 7.45-7.50 ( m, 2H), 7.40 (d, J = 2.2 Hz, 1H), 7.17-7.24 (m, 2H), 6.96 (dd, J = 8.8, 2.2 Hz, 1H), 6.68-6.75 (m, 2H).

Example 45

[2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [4-acetylphenyl] methanone (41)

(a) 246 mg (0.75 mmol) in hexamethylditin, [2- (4-methoxyphenyl) -6-methoxybenzo [β] thien-3-yl] [4-iodophenyl] methanone (Example (39a)) A mixture of 250 mg (0.50 mmol), 6 mg (0.005 mmol) of tetrakis triphenylphosphine palladium (O) and 20 ml of toluene was heated under reflux for 20 hours in a nitrogen atmosphere. The reaction mixture was concentrated and dissolved in diethyl ether, washed twice with water, dried over magnesium sulfate, filtered and concentrated. This gave 241 mg (0.45 mmol, 90%) of the desired [2- (4-methoxyphenyl) -6-methoxybenzo [β] thien-3-yl] [4-trimethylstannylphenyl] methanone.

(b) 100 mg (0.19 mmol) of [2- (4-methoxyphenyl) -6-methoxybenzo [β] thien-3-yl] [4-trimethylstannylphenyl] methanone (Example 41a); 15 mg (0.19 mmol) of acetylchloride was dissolved in 5 ml of toluene. 4.6 mg (0.0044 mmol) of tris (dibenzylideneacetone) palladium (O) chloroform was added to the reaction mixture. The reaction mixture was then heated to 70 ° C. under nitrogen atmosphere for 20 hours, filtered and extracted with ethyl acetate, washed with saturated sodium bicarbonate and dried over magnesium sulfate. Deprotection of the crude [2- (4-methoxyphenyl) -6-methoxybenzo [β] thien-3-yl] [4-acetylphenyl] methanone was carried out according to the method of Example 1 (c). . The product was purified by chromatography (silica, 5: 5, petroleum ether / ethyl acetate) to give [2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [4-acetylphenyl ] 61 mg (0.16 mmol, 82%) were obtained. ¹ H NMR (CD ₃ COCD ₃ ) 7.85-7.92 (m, 2H), 7.76-7.83 (m, 2H), 7.59 (d, J = 8.8 Hz, 1H), 7.41 (d, J = 2.2 Hz, 1H) , 7.17-7.24 (m, 2H), 6.98 (dd, J = 8.8, 2.2 Hz, 1H), 6.66-6.73 (m, 2H), 2.54 (s, 3H).

Example 46

[2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [4-propionylphenyl] methanone (42)

200 mg (0.38 mmol) of [2- (4-methoxyphenyl) -6-methoxybenzo [β] thien-3-yl] [4-trimethylstannylphenyl] methanone (Example 41 (a)) 34 mg (0.38 mmol) of propionyl chloride was dissolved in 10 ml of toluene. 9.2 mg (0.0088 mmol) of tris (dibenzylideneacetone) palladium (O) chloroform was added to the reaction mixture. The reaction mixture was then heated to 70 ° C. under nitrogen atmosphere for 20 hours, filtered and extracted with ethyl acetate, washed with saturated sodium bicarbonate and dried over magnesium sulfate. Deprotection of the crude [2- (4-methoxyphenyl) -6-methoxybenzo [β] thien-3-yl] [4-propionylphenyl] methanone was carried out according to the method of Example 1 (c) It was. The crude product was purified by HPLC (reverse phase, C18, concentration gradient acetonitrile / water + 0.05 trifluoroacetic acid) to [2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl ] [4-propionylphenyl] methanone 9 mg (0.02 mmol, 6%) was obtained. ¹ H NMR (CD ₃ COCD ₃ ) 8.72 (s, 1H), 8.61 (s, 1H), 7.85-7.92 (m, 2H), 7.76-7.83 (m, 2H), 7.59 (d, J = 8.8 Hz, 1H), 7.41 (d, J = 2.2 Hz, 1H), 7.17-7.24 (m, 2H), 6.98 (dd, J = 8.8, 2.2 Hz, 1H), 6.66-6.73 (m, 2H), 2.99 (q , 2H), 1.08 (t, 3H).

Example 47

[2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [4-buturylphenyl] methanone (43)

100 mg (0.19 mmol) of [2- (4-methoxyphenyl) -6-methoxybenzo [β] thien-3-yl] [4-trimethylstannylphenyl] methanone (Example 41 (a)); 22 mg (0.20 mmol) of butilyl chloride was dissolved in 5 ml of toluene. 4.6 mg (0.0044 mmol) of tris (dibenzylideneacetone) palladium (O) chloroform was added to the reaction mixture. The reaction mixture was then heated to 70 ° C. under nitrogen atmosphere for 20 hours, filtered and extracted with ethyl acetate, washed with saturated sodium bicarbonate and dried over magnesium sulfate. The crude product was purified by chromatography (silica, 9: 1, petroleum ether / ethyl acetate). Deprotection of the crude [2- (4-methoxyphenyl) -6-methoxybenzo [β] thien-3-yl] [4-buturylphenyl] methanone was carried out according to the method of Example 1 (c) It was. The product was purified by chromatography (silica, 9: 1, petroleum ether / ethyl acetate) to give [2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [4-buturyl Phenyl] methanone 17 mg (0.04 mmol, 22%) was obtained. ¹ H NMR (CD ₃ COCD ₃ ) 8.72 (s, 1H), 8.61 (s, 1H), 7.85-7.92 (m, 2H), 7.76-7.83 (m, 2H), 7.60 (d, J = 8.8 Hz, 1H), 7.42 (d, J = 2.2 Hz, 1H), 7.17-7.24 (m, 2H), 6.99 (dd, J = 8.8, 2.2 Hz, 1H), 6.66-6.73 (m, 2H), 2.96 (q , 2H), 1.67 (m, 2H), 1.08 (t, 3H).

Example 48

[2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [4-ethylthiocarbonylphenyl] methanone (44)

50 mg (0.12 mmol) of [2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [4-carboxyphenyl] methanone (Example 32 (b)) was added to ethanol. Dissolve and add 5 drops of thionylchloride. The reaction mixture was stirred at room temperature under nitrogen atmosphere for 24 hours, wetted with water, extracted with ethyl acetate and dried over magnesium sulfate. The crude product was purified by HPLC (reverse phase, C18, concentration gradient acetonitrile / water + 0.05 trifluoroacetic acid) to [2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl ] 26 mg (0.06 mmol, 54%) of [4-ethylthiocarbonylphenyl] methanone were obtained. ¹ H NMR (CD ₃ OD) 8.72 (s, 1 H broad), 8.60 (s, 1 H), 7.89-7.98 (m, 2H), 7.75-7.84 (m, 2H), 7.61 (d, J = 8.8 Hz, 1H), 7.42 (d, J = 2.5 Hz, 1H), 7.17-7.24 (m, 2H), 6.99 (dd, J = 8.8, 2.5 Hz, 1H), 6.66-6.72 (m, 2H), 2.89 (q , 2H), 1.32 (t, 3H).

Example 49

[2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [4-hydroxyphenyl] methanone (45)

Of [2- (4-methoxyphenyl) -6-methoxybenzo [β] thien-3-yl] [4-methoxyphenyl] methanone (Example (31a)) described in Example 31 (b) After deprotection, [2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [4-hydroxyphenyl] methanone was purified as a by-product. ¹ H NMR (CD ₃ COCD ₃ ) 8.60-9.00 (s, 3H broad), 7.62-7.72 (m, 2H), 7.36-7.40 (m, 2H), 7.22-7.28 (m, 2H), 6.92 (dd, J = 8.6, 2.4 Hz, 1H), 6.68-6.80 (m, 4H).

Example 50

[2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [4-methylaminocarbonylphenyl] methanone (46)

[2- (4-methoxyphenyl) -6-methoxybenzo [β] thien-3-yl] [4-methoxycarboxyphenyl] methanone (Example 32 (b)) was dissolved in ethanol and 1 M Stir with sodium hydroxide for 4 hours to deprotect with [2- (4-methoxyphenyl) -6-methoxybenzo [β] thien-3-yl] [4-carboxyphenyl] methanone. Ethanol was evaporated and the aqueous phase was extracted with ethyl acetate, dried over magnesium sulfate and evaporated. The reaction was then run in parallel solution phase way. 20 mg (0.048 mmol) of [2- (4-methoxyphenyl) -6-methoxybenzo [β] thien-3-yl] [4-carboxyphenyl] methanone was converted to benzotriazol-1-yl-oxy- Tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP) 30 mg (0.058 mmol), N-hydroxybenzotriazole H ₂ O (HOBt) 3.6 mg (0.024 mmol), N, N-dimethylformamide 2.5 Ml, N, N-diisopropylethylamine 12.4 mg (0.096 mmol) and 2.23 mg (0.096 mmol) methylamine hydrochloric acid were mixed in this order at room temperature in a nitrogen atmosphere for 3 days. The reaction was diluted with ethyl acetate. The organic phase was washed with 10% citric acid, dried over 3 ml Xtube (extube, Varian Chem. Elut) and concentrated to speed-bag. Deprotection of [2- (4-methoxyphenyl) -6-methoxybenzo [β] thien-3-yl] [4-methylaminocarbonylphenyl] methanone according to the method of Example 1 (c) And purified by HPLC (reverse phase, C18, concentration gradient acetonitrile / water + 0.05 trifluoroacetic acid) to [2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl ] 6 mg (0.015 mmol, 31%) was obtained. ¹ H NMR (CD ₃ COCD ₃ ) 7.70-7.85 (m, 4H), 7.55 (d, J = 8.8 Hz, 1H), 7.41 (d, J = 2.2, 1H), 7.19-7.25 (m, 2H), 6.99 (dd, J = 8.8, 2.2 Hz, 1H), 6.66-6.74 (m, 2H).

Example 51

[2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [4-isobutylaminocarbonylphenyl] methanone (47)

The reaction was carried out in a parallel solution phase way by the method of Example 46 using isobutylamine hydrochloric acid (5.24 mg (0.096 mmol)) instead of methylamine. The crude product was purified by HPLC (reverse phase, C18, concentration gradient acetonitrile / water + 0.05 trifluoroacetic acid) to [2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl ] 1.2 mg (0.0027 mmol, 5.6%) of [4-isobutylaminophenyl] methanone were obtained. ¹ H NMR (CD ₃ COCD ₃ ) 7.72-7.92 (m, 4H), 7.51 (d, J = 8.8 Hz, 1H), 7.42 (d, J = 2.2, 1H), 7.18-7.26 (m, 2H), 6.97 (dd, J = 8.8, 2.2 Hz, 1H), 6.66-6.76 (m, 2H).

Example 52

[2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [4-benzylaminocarbonylphenyl] methanone (48)

The reaction proceeded in a parallel solution phase way by the method of Example 46 using benzylamine hydrochloric acid (7.68 mg (0.096 mmol)) instead of methylamine. The crude product was purified by HPLC (reverse phase, C18, concentration gradient acetonitrile / water + 0.05 trifluoroacetic acid) to [2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl 1.7 mg (0.0035 mmol, 7.4%) of] [4-benzylaminophenyl] methanone were obtained. ¹ H NMR (CD ₃ COCD ₃ ) 8.33 (s, broad), 7.85-7.91 (m, 2H), 7.75-7.82 (m, 2H), 7.53 (d, J = 8.8 Hz, 1H), 7.42 (d, J = 2.2 Hz, 1H), 7.18-7.36 (m, 7H), 6.98 (dd, J = 8.8, 2.2 Hz, 1H), 6.68-6.74 (m, 2H).

Example 53

15 mg (0.040 mmol) of [2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [4-carboxyphenyl] methanone (Example 32 (b)) Zol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP) 40 mg (0.077 mmol), N-hydroxybenzotriazole H ₂ O (HOBt) 4.9 mg (0.032 mmol), 3.0 ml of N, N-dimethylformamide, 16.5 mg (0.128 mmol) of N, N-diisopropylethylamine, and 0.015 g (0.096 mmol) of L-serine methyl ester hydrochloric acid were sequentially mixed at room temperature in a nitrogen atmosphere for 3 days. It was. The reaction was diluted with ethyl acetate. The organic phase was washed with 1 M hydrochloric acid and bromine water. Then dried over magnesium sulfate and evaporated to dryness. Purification by HPLC (reverse phase, C18, concentration gradient acetonitrile / water + 0.05 trifluoroacetic acid) gave 7.4 mg (0.015 mmol, 38%) of compound 49. ¹ H NMR (CD ₃ COCD ₃ ) 7.78-7.92 (m, 4H), 7.55 (d, J = 8.8 Hz, 1H), 7.41 (d, J = 2.2, 1H), 7.19-7.28 (m, 2H), 6.98 (dd, J = 8.8, 2.2 Hz, 1H), 6.66-6.74 (m, 2H), 4.69 (m, 1H), 3.95 (m, 2H), 3.69 (s, 3H).

Example 54

15 mg (0.040 mmol) of [2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [4-carboxyphenyl] methanone (Example 32 (b)) Zol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP) 40 mg (0.077 mmol), N-hydroxybenzotriazole H ₂ O (HOBt) 4.9 mg (0.032 mmol), 3.0 ml of N, N-dimethylformamide, 16.5 mg (0.128 mmol) of N, N-diisopropylethylamine, and 0.013 g (0.096 mmol) of L-alanine methylester hydrochloric acid were mixed in this order at room temperature in a nitrogen atmosphere for 3 days. It was. The reaction was diluted with ethyl acetate. The organic phase was washed with 1 M hydrochloric acid and bromine water. Then dried over magnesium sulfate and evaporated to dryness. Purification by HPLC (reverse phase, C18, concentration gradient acetonitrile / water + 0.05 trifluoroacetic acid) yielded 17.4 mg (0.037 mmol, 91%). ¹ H NMR (CD ₃ COCD ₃ ) 8.12 (s, 1H broad), 8.10 (s, 1H broad), 7.75-7.89 (m, 4H), 7.53 (d, J = 8.8 Hz, 1H), 7.41 (d, J = 2.2, 1H), 7.17-7.23 (m, 2H), 6.98 (dd, J = 8.8, 2.2 Hz, 1H), 6.69-6.74 (m, 2H), 4.59 (m, 1H), 3.66 (s, 3H), 1.45 (d, 3H).

Example 55

15 mg (0.040 mmol) of [2- (4-hydroxyphenyl) -6-hydroxybenzo [β] thien-3-yl] [4-carboxyphenyl] methanone (Example 32 (b)) Zol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP) 40 mg (0.077 mmol), N-hydroxybenzotriazole H ₂ O (HOBt) 4.9 mg (0.032 mmol), 3.0 ml of N, N-dimethylformamide, 16.5 mg (0.128 mmol) of N, N-diisopropylethylamine and 0.021 g (0.096 mmol) of L-phenylalanine methylester hydrochloric acid were mixed in this order at room temperature in a nitrogen atmosphere for 3 days. It was. The reaction was diluted with ethyl acetate. The organic phase was washed with 1 M hydrochloric acid and bromine water. Then dried over magnesium sulfate and evaporated to dryness. Purification by HPLC (reverse phase, C18, concentration gradient acetonitrile / water + 0.05 trifluoroacetic acid) gave 10.9 mg (0.020 mmol, 51%) of compound 51. ¹ H NMR (CD ₃ COCD ₃ ) 8.01 (s, 1H broad), 7.98 (s, 1H broad), 7.75 (s, 4H), 7.53 (d, J = 8.8 Hz, 1H), 7.41 (d, J = 2.2, 1H), 7.15-7.31 (m, 7H), 6.98 (dd, J = 8.8, 2.2 Hz, 1H), 6.69-6.74 (m, 2H), 4.82 (m, 1H), 3.65 (s, 3H) .

The biological properties of the compounds prepared according to Examples 1 to 26 and 28 to 40 and estradiol for comparative purposes were determined by radioligand substitution assay. The affinity of ERα and ERβ was determined by IC ₅₀ , the concentration of ligand required to replace 50% of tritated 17-β-estradiol from hERα (human estrogen receptor α) or hERβ (human estrogen receptor β). In this analysis, the IC ₅₀ of the compound was found to vary from 2.0 nM to 20 μM in ERα and from 2.0 nM to 12 μM in ERβ. The ERα / ERβ selectivity ratio varied from 0.2 to 23.

Experimental technique of ER binding assay

The affinity of ER (by substitution of ³ [H] -estradiol) was measured using a scintistrip assay ¹ . Human estrogen receptor (hER) alpha and beta were extracted from the nuclei of SF9-cells infected with recombinant baculovirus delivery vectors containing cloned hER genes ² . The concentration of hER in the extract was determined by E2-analysis coupled with specific ³ [H] -G25 ³ .

1) Haggblad, J., Carlsson, B., Kivela, P., Siitari, H., (1995) Biotechniques 18, 146-151.

2) Barkhem, T., Carlsson, B., Simons, J., Moller, B., Berkenstam, A., Gustafsson J.A.G., Nilsson, S. (1991) J. Steroid Biochem. Molec. Biol. 38, 667-75.

3) Salononsson, m., Carlsson, B., Haggblad, J., (1994) J. Steroid Biochem. Molec. Biol. 50, 313-318.

Claims (63)

A crystal comprising at least a portion of an ERα ligand binding region. The crystal of claim 1, comprising at least 200 amino acids of ERα. The crystal of claim 1 or 2 comprising at least 250 amino acids of ERα. The crystal according to any one of claims 1 to 3, comprising the entire ERα. The crystal according to any one of claims 1 to 4, which is produced using a sequence comprising the helix H ₁₂ of ERα. The crystal according to any one of claims 1 to 5, which can be used in X-ray crystallography techniques. The crystal of any one of claims 1 to 6 comprising a ligand bound to ERα or a portion thereof. 8. The crystal of claim 7, wherein the ligand is estradiol, raloxifene, or other ligand that binds with high affinity (<10 μM) to ERα. The crystal of ERαLBD according to any one of claims 1 to 8, belonging to spatial group P21 and having a unit cell dimension of a = 61.48 ms, b = 115.16 ms, c = 137.38 ms. The determination of ERαLBD according to any one of claims 1 to 9, belonging to spatial group P2 and having unit cell dimensions of a = 104.53 ms, b = 53.68 ms, c = 102.71 ms and β = 116.79 degrees. 10. The determination of ERαLBD according to claim 1, which belongs to spatial group C2 and has unit cell dimensions of a = 89.91 μs, b = 75.09 μs, c = 87.50 μs and β = 103.01 °. 11. The determination of ERαLBD according to any one of claims 1 to 9, belonging to spatial group C2221 and having unit cell dimensions of a = 65.47 ms, b = 95.99 ms, c = 164.14 ms. Design of a ligand to bind to the estrogen receptor, comprising determining the amino acid or amino acids of the ligand binding region of the estrogen receptor that interacts with the binding ligand and selecting a ligand that is likely to bind to the receptor depending on the structure of the potential ligand. Way. The method of claim 13, wherein the interaction with ERα and ERβ is determined separately by which ER-type selective ligand can be selected. The method according to claim 13 or 14, wherein for the ERα selective ligand, the crystal according to any one of claims 1 to 12 is used in the design of the potential ligand. A ligand for an estrogen receptor designed using the method according to any one of claims 13-15. Ligand designed according to the method according to claim 14, specific for ERα or ERβ. Ligand that binds at least LBD of ER with affinity between 20 pmol and 200 nM. Ligand that reversibly binds to at least LBD of ER. A method of inhibiting estradiol activity in an animal comprising administering to the animal the ligand of claim 19 or 20. 21. The method of claim 20, comprising administering to the animal the ligand of claim 18 or 19. 20. A pharmaceutical compound comprising the ligand of any one of claims 16-19. An estrogen agonist, estrogen antagonist, partial estrogen agonist, or partial estrogen antagonist designed using the method according to any of claims 13-15. ERα selective ligands having a structural group larger than methyl that can fit into the β cavity of ERα. An ERα selective ligand having the general formula Z and having a hydrophobic substituent at one or more of the 8β, 15β or 18 positions. An ERβ selective ligand having the general formula Z of claim 25 and having a hydrophobic substituent in at least one of the 9α or 12α positions. The ERα selective ligand according to claim 25 or the ERβ selective ligand according to claim 26, wherein the hydrophobic substituent is selected from methyl group, ethyl group, isopropyl group, chlorine, bromine or iodine. ERα or ERβ selective ligand wherein the ligand is 2'-, 3'-, 5'- and / or 6'- substituted 2-aryl benzothiophene. The ERα or ERβ selective ligand of claim 28 substituted at one or more of the 2 ′, 3 ′, 5 ′ and 6 ′ positions. The ERα selective ligand of claim 28, wherein the substituted 2-aryl benzothiophene fills the α- and β-side cavities of the ER. ERα selective ligand, which is 2-aryl benzothiophene having a small hydrophobic substituent at one or more of the 2 ', 3', 5 'and 6' positions. An ER ligand capable of filling the hydrophobic cavity of ER-α. 33. The ligand of claim 32 having a hydrophobic substituent on the ethoxyphenyl side chain of the piperidinyl nitrogen atom of raloxifene. 33. The method of claim 31 or 32, wherein the ligand is a straight chain alkyl group, perfluoroalkyl group (-CH ₃ to -CH ₁₀ H ₂₁ , -CF ₃ to -C ₁₀ F ₂₁ ), benzyl- (CH ₂ Ph) And a ligand with a hydrophobic substituent selected from benzyl- (methylene cyclohexyl group). An ER ligand having a structure capable of interacting with Glu-353 of ERα or Glu-262 of ERβ. An ER ligand having a structure capable of interacting with Arg-394 of ERα or Arg-303 of ERβ. An ER ligand having a structure capable of interacting with residues His-524 of ERα or His-432 of ERβ. An ER ligand having a structure capable of interacting with Met-421 or Leu-384 of ERα or Ile-330 or Met-293 of ERβ. ERα selective ligands having a structure capable of interacting with Met-421 and / or Leu-384 of ERα. An ERβ selective ligand having a structure capable of interacting with Ile-330 and / or Met-293 of ERβ. 41. The ERβ selective ligand of claim 40, wherein a substitution greater than the methyl group is provided at the α 14, 16 or 17 position of the steroid nucleus. An ER ligand having a structure capable of interacting with Leu-384 of ERα or Met-293 of ERβ. ERα selective ligand that can interact with Leu-384 of ERα. ERβ selective ligand that can interact with Met-293 of ERβ. 41. The ERβ selective ligand of claim 40, further comprising a substituent at the 2 'or 3' position of the 2-aryl benzothiophene nucleus. ERβ selective ligand having a substituent larger than the methyl group at the R ₂ 'position of 6,3'-dihydroxybenzothiophene. ERα selective ligand having a substituent larger than the methyl group at the R ₂ 'and / or R ₃ ' position of 6,5'-dihydroxybenzothiophene. The ligand is selective for ERα or ERβ comprising a position-6 substituent from the benzothiophene nucleus or a position-3 substituent from the estradiol nucleus, arranged to selectively bind amino acids Ile-326 of ERα or Asn-236 of ERβ Ligand. The ligand is selective for ERα or ERβ comprising a position-6 substituent from the benzothiophene nucleus or a position-3 substituent from the estradiol nucleus, arranged to selectively bind amino acids Phe-445 of ERα or Tyr-354 of ERβ Ligand. An ERα selective ligand having a structure capable of preferentially interacting with Glu-323 and Phe-445 of ERα over Glu-262 and Tyr-354 of ERβ. An ER ligand having a structure arranged to promote binding of the ER structure to helix H12. The crystal according to any one of claims 1 to 12, wherein the resolution determined by X-ray crystallography is less than 3.5 Hz. Data encrypted with mechanically decodable data that can represent a three-dimensional graphical representation of a crystal according to any one of claims 1 to 12 or a homologue of the crystal when using a programmed machine with instructions for using the data. A mechanically decodable data storage medium comprising a storage material. a) employing computational means for performing the correct operation between the binding site of the receptor and the chemical entity; And b) analyzing the result of the correct action to predict the association between the binding site and the chemical entity. Human ER-a ligand binding region amino acid residues MET343, LEU346, THR347, LEU349, ALA350, ASP351, GLU353, LEU354, TRP383, LEU384, LEU387, MET388, LEU391, ARG394, PHE404, MET421, ILE424, PHE425, LEU428, GLY521, GLY521, GLY521 , A crystallized molecule or molecular complex comprising a binding pocket defined by the structural coordinates of LEU525, or said molecule or molecule whose root mean square deviation from the central structural atom of said amino acid is not greater than 1.5 ms Homologues of the complexes. Human ER-β ligand binding region amino acid residues MET343, LEU346, THR347, LEU349, ALA350, ASP351, GLU353, LEU354, TRP383, MET384, LEU387, MET388, LEU388, LEU391, ARG394, PHE404, ILE421, ILE424, PHE425, L428 A homolog model comprising a binding pocket defined by the structural coordinates of HIS524, LEU525. Rat ER-α ligand binding region amino acid residues MET252, LEU255, THR256, LEU258, ALA259, ASP260, GLU262, LEU263, TRP292, LEU293, LEU296, MET297, LEU300, ARG303, PHE313, ILE330, ILE333, PHE334, LEU337, A crystallized molecule or molecular complex comprising a binding pocket defined by the structural coordinates of GLY429, HIS423, LEU433. Rat ER-β ligand binding region amino acid residues MET252, LEU255, THR256, LEU258, ALA259, ASP260, GLU262, LEU263, TRP292, MET293, LEU296, MET297, LEU300, ARG303, PHE313, ILE330, ILE333, PHE334, LEU337, Homolog model including binding pockets defined by the structural coordinates of GLY429, HIS432, LEU433. A method of antagonizing or antagonizing the action of ERα or ERβ comprising administering to a mammal a compound that is spatially fit to the binding pocket of ERβ. 60. The method of claim 59, wherein the compound has at least one of the following: a) a group capable of functioning as a hydrogen bond donor to HIS432; b) groups acting as hydrogen bond acceptors and donors to Arg-394 and Glu-353 of ERα or Arg-303 and Glu-262 of ERβ; c) Met-252, Leu-255, Leu-258, Ala-259, Leu-263, Trp-292, Met-293, Leu-296, Met-297, Leu-300, Phe-313, Ile- of ERβ. 330, Ile-333, Phe-334, Leu-337, Leu-433, or Met-343, Leu-346, Leu-349, Ala-350, Leu-354, Trp-383, Leu-384, Leu of ERα -A group capable of forming hydrophobic contact with at least one of -387, Met-388, Leu-391, Phe-404, Met-421, Ile-424, Phe-425, Leu-428, Leu-525. 61. The method of claim 59 or 60, wherein the compound has a group capable of forming a hydrogen bond or salt bridge with ASP260. 61. The method of claim 59 or 60, wherein the compound has a group capable of forming hydrogen bonds or salt bridges with Asp-351. ER ligand according to any one of examples 5 to 55.