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    • 专利摘要:
    The invention relates to 6-hydroxy-3- (4- [2- (piperidin-1-yl) ethoxy] -phenoxy) -2- (4-methoxyphenyl) benzo [b] thiophene hydrochloride Novel crystal hydrates and uses thereof. Its use includes suppression of diseases associated with estrogen deficiency, including cardiovascular disease, hyperlipidemia and osteoporosis; And inhibiting other pathological conditions such as endometriosis, uterine fibrosis, estrogen-dependent cancers (breast cancer and uterine cancer), prostate cancer, benign prostatic hyperplasia, central nervous system disorders including Alzheimer's disease, prevention of breast cancer and increasing ChAT Up-regulating ChAT.
    公开号:KR20010049911A
    申请号:KR1020000043586
    申请日:2000-07-28
    公开日:2001-06-15
    发明作者:줄리 케이 부쉬；프레스톤 찰스 콘라드；메를린 제라드 플롬
    申请人:피터 지. 스트링거；일라이 릴리 앤드 캄파니；
    IPC主号:
    专利说明:

    Novel of 6-hydroxy-3- (4- [2- (piperidin-1-yl) ethoxy] -phenoxy) -2- (4-methoxyphenyl) benzo [iii] thiophene hydrochloride Crystal form {A Novel Crystalline Form of 6-Hydroxy-3- (4- [2- (Piperidin-1-Yl) Ethoxy] -Phenoxy) -2- (4-Methoxyphenyl) Benzo [b] Thiophene Hydrochloride}
    6-hydroxy-3- (4- [2- (piperidin-1-yl) ethoxy] -phenoxy) -2- (4-methoxyphenyl) benzo [b] thiophene hydrochloride (arzoxifene, Aroxifene) was first described generally in US Pat. No. 5,510,357, and specifically disclosed in US Pat. No. 5,723,474 ('474) and European Patent Application 0729956. Aroxysifen is a nonsteroidal mixed estrogen antagonist / agonist, especially the central nervous system including serum cholesterol lowering and hyperlipidemia, osteoporosis, estrogen dependent cancers including breast and uterine cancers, endometriosis, Alzheimer's disease It is useful for suppressing disorders, smooth muscle cell proliferation in the aorta, and restenosis.
    Specifically, aroxifen is used for the treatment of receptor-positive metastatic breast cancer, adjuvant therapy of receptor-positive patients receiving appropriate systemic or local treatment, reduction of invasive and non-invasive breast cancer recurrence, invasive breast cancer and its location It is useful and clinically evaluated to reduce the incidence of ductal carcinoma in situ (DCIS). Aroxifene is also useful in combination with radiotherapy, aromatase inhibitors, luteinizing hormone free hormone (LHRH) analogs and acetylcholine esterase (AChE) inhibitors.
    X-ray powder diffraction (XRD), thermogravimetric analyser (TGA), proton nuclear magnetic resonance ( 1 H NMR) and Karl Fischer of aroxifene bulk separated by the procedure described in '474. KF) analysis showed that aroxifene was hydrated and insufficient in crystallinity and contained varying amounts of volatile organic substances (ethyl acetate) in the lattice.
    Substances lacking crystallinity are typically less desirable than high crystalline materials in the formulation process. In addition, formulating a drug comprising a significant amount of organic solvent (eg, ethyl acetate) is generally undesirable because of potential solvent toxicity to the recipient of the drug and changes in the efficacy of the drug as a function of the solvent.
    Although aroxyphene prepared by the procedure described in '474 can be used as a medicament, a highly crystalline form of aroxysi, which does not contain organic solvents in the crystal lattice and is produced efficiently and reproducibly on a commercial scale It would be more desirable and advantageous to find a pen.
    1 shows differential scanning calorimetry (DSC) / TGA results of S-II.
    2 shows differential scanning calorimetry (DSC) / TGA results of F-I.
    3 shows the differential scanning calorimetry (DSC) / TGA results of F-III.
    4 shows the differential scanning calorimetry (DSC) / TGA results of F-I and F-III.
    5 shows the desolvation of S-II as a function of drying time and temperature.
    The present invention, when measured from a copper radiation source at 25 ± 2 ℃, 35 ± 10% relative humidity (RH), 4.6 ± 0.2, 7.8 ± 0.2, 9.3 ± 0.2, 14.0 ± 0.2, 17.6 ± 0.2, 20.8 ± 0.2, 6-hydroxy-3- (4- [2- (piperidin-1-yl) ethoxy] -phenoxy) with an X-ray diffraction pattern comprising a peak at 24.3 ± 0.2 ° (in 2θ) Novel chemically equivalent hydrated crystalline forms of -2- (4-methoxyphenyl) benzo [b] thiophene hydrochloride (F-III).
    The invention further comprises F-III, at least one pharmaceutical carrier, diluent or excipient, optionally estrogen, optionally progestin, optionally aromatase inhibitor, optionally LHRH analog and optionally acetylcholine esterase (AChE) inhibitor Relates to pharmaceutical formulations
    Further, the present invention provides for uterine fibrosis, endometriosis, smooth muscle cell proliferation of the aorta, restenosis, breast cancer, uterine cancer, prostate cancer, benign prostate hyperplasia, bone loss, osteoporosis, cardiovascular disease, hyperlipidemia, central nervous system disorders, Alzheimer's disease A method of using F-III to inhibit a pathological condition such as a disease and a method of using F-III in the manufacture of a medicament for inhibiting the condition.
    The present invention further relates to a method of using F-III to up-regulate choline acetyltransferase (ChAT) and a method of using F-III to prepare a medicament for increasing said choline acetyltransferase (ChAT). will be.
    Aroxifene bulk prepared by the procedure described in '474 (Example 41, crystallization from ethanol and ethyl acetate mixture, filtration and vacuum drying of filter cake at constant weight at room temperature) was tested by XRD. The results showed a lack of crystallinity. 1 H NMR confirmed that the bulk material contained 6% ethyl acetate.
    The crystallization procedure described in '474 was then modified to add ethanol to a suspension of crude aroxifene when refluxing ethyl acetate. After cooling and vacuum filtration, the solid obtained by this modified procedure is a mixed ethyl acetate / water solvate of highly crystalline aroxifene (hereinafter referred to as S-II) and S-II is FI It was later found to be the starting material for another, non-chemically equivalent hydrated crystalline form of zoxyfen.
    F-I can be obtained by removing ethyl acetate from the crystal lattice of S-II by annealing S-II in a vacuum drying / heating state. The time and temperature for annealing S-II to obtain F-I will vary from lot to lot, but typically about 5 days and about 100 ° C. A high temperature is required to convert S-II to FI through this procedure because slurrying S-II in water at ambient temperature or storing the sample for three weeks at 98% relative humidity does not convert to FI at all. Do. Furthermore, drying S-II at high temperatures in a convection oven did not desolvate S-II, suggesting that vacuum is also required to remove ethyl acetate from the lattice of S-II. F-I is preferably prepared separately from the tetrahydrofuran by crystallization of aroxifene (or any polymorph / sulfate thereof) at ambient temperature.
    According to the invention, aroxifene of the F-III form is particularly preferred. F-III is easily prepared and separated at ambient temperature. In the preparation of F-III, only mild drying conditions are necessary to remove low levels of crystallization solvent residues. These mild drying conditions consistently yield high purity and high crystalline solids (ie, no organic solvent residues), thus using F-III eliminates the toxicity problems associated with residues and organic solvents in the crystal lattice. Furthermore, manufacturing F-III is simple and efficient. That is, easy to mass production.
    F-III is readily prepared and separated from the mixture of isopropyl alcohol (IPA) and water at ambient temperature by crystallization of aroxifene (or any polymorph / solvate thereof). Typically, in order to affect the dissolution of the starting material of aroxyphene, the aroxifene can be suspended and heated in a mixture of IPA and water. Once dissolved, the solution is slowly cooled to room temperature and then cooled to 0-5 ° C. using an ice bath or cooler. After sufficient time has elapsed for crystallization to occur, the crystals are collected by vacuum filtration and vacuum dried to a constant weight to obtain F-III in a yield of at least 80%.
    Aroxyphene starting materials suitable for the crystallization process include, but are not limited to, aroxifene, or mixtures thereof, prepared according to the procedures described in S-II, F-I and '474. It is not important to start with what form of aroxifen since crystallization from IPA and water according to the procedure described herein yields F-III crystals. The volume ratio of water and IPA is generally about 1: 1 to 9: 1. More preferably, it is 2.5: 1 to 5.6: 1. Most preferably 3: 1 to 5.6: 1. After collecting the crystals by vacuum filtration, the wet F-III cake can be washed with cold demineralized water before vacuum drying. In addition, the drying temperature is preferably slightly high (12 to 24 hours at about 50 ° C). In order to synthesize F-III on a commercial scale, it is advantageous to use F-III as the crystallization seed.
    In a preferred process, 6-isopropoxy-3- (4- [2- (piperidin-1-yl) ethoxy] -phenoxy) -2- (4-methoxyphenyl) benzo [b] thiophene F-III is prepared, isolated and purified by chemical removal of 6-isopropyl hydroxy protecting group from hydrochloride (precursor A). When the deprotection reaction is monitored for complete removal of the isopropyl protecting group and it is determined that the removal is almost complete, the post-treatment of the reaction preferably includes crystallization under conditions that provide F-III as discussed above and below. Do. Methods of making precursor A and removing isopropyl groups are described in US Pat. No. 5,723,474 and incorporated herein by reference.
    In another preferred process, the reduction reaction of S-oxides with 6-benzyloxy-3- (4- [2- (piperidin-1-yl) ethoxy] -phenoxy) -2- (4-methoxyphenyl F-III is prepared, isolated and purified via chemical removal of benzyl protecting groups from 6-hydroxyl in benzo [b] thiophene- (S-oxide) (precursor B). The reduction and deprotection reactions are monitored to ensure that the sulfoxide is fully reduced to sulfide and to completely remove the benzyl hydroxy protecting group. When it is judged that the removal and reduction reactions are almost finished, it is preferred that the workup of the reaction comprises crystallization under the conditions providing F-III as discussed herein. Methods of preparing Precursor B, removing benzyl groups, and reducing 1-sulfoxide to the corresponding sulfides can be found in US Pat. No. 5,723,474, referenced above, by reference.
    Regardless of the chemical reaction used in the deprotection and reduction steps, the crystallization method of aroxifene from the IPA / water solution disclosed herein consistently produces F-III crystals with high purity.
    Characterization and differentiation of S-II, F-I and F-III
    DSC / TGA and XRD methods were used to characterize S-II, F-I and F-III. TGA is often very useful for distinguishing between different solid forms of a material because the temperature at which physical changes occur in a material is usually the nature of the polymorph or solvate. DSC is a technique often used to screen polymorphism and solvate formation of compounds. Finally, XRD is a technique for detecting a wide range of orders in crystalline materials.
    XRD measurements of the aroxifene prepared by the procedure described in '474 resulted in a low signal-to-noise ratio, high baseline XRD pattern, indicating a low crystallinity. Thus, a comparison of FI and F-III with respect to the material (S-II) prepared according to the modified aroxyphene crystallization procedure described above (adding ethanol to the suspension of aroxifene with reflux of ethyl acetate) Was done.
    Representative DSC / TGA traces of S-II, F-I and F-III are shown in FIGS. 1, 2 and 3, respectively. DSC results of S-II show a wide endothermic curve starting at about 62 ° C, indicating that ethyl acetate and water evaporate from the lattice. The endothermic curve starting at 152 ° C. shows melting. At the same time as the first transition, a TGA weight loss of about 2.5% by weight occurs and the remaining 0.5% weight loss occurs up to an onset of melting, suggesting that some solvent molecules are held more firmly in the lattice.
    DSC results of F-I show a broad endothermic curve starting at about 75 ° C., followed by a second endothermic curve corresponding to melting at about 155 ° C. The TGA results of F-I show a gradual weight loss up to 0.3% followed by a sudden mass loss of 1.5%, both showing dehydration of the lattice. The TGA weight loss corresponding to the onset of the primary DSC transition is offset in part by the difference in heating rate. Initial weight loss indicates water that was weakly trapped in the hydrate and secondary weight loss is consistent with about 0.5 moles of water present in the lattice under very low relative humidity (below 5%-see moisture sorption data). .
    DSC results of F-III show a wide endothermic curve at a low temperature of about 30 ° C., followed by a wide and relatively weak second endotherm curve starting at about 70 ° C. and the final transition corresponds to melting and starts at about 146 ° C. An abrupt 1.5% (~ 0.5 mole) weight loss in the TGA corresponding to the first endothermic curve corresponds to a decrease in weakly trapped water molecules, with an additional ~ 1.6% weight loss above 60 ° C being held tight. Loss of water molecules (ie those that exist even under very low relative humidity). The weight loss observed above 170 ° C. corresponds to the degradation of F-III.
    The XRD patterns of FI and F-III show sharp peaks and flat baselines, which are indicative of high crystalline material. The locations of angular peaks and corresponding I / I 0 data in 2θ for representative samples of FI, F-III and S-II are shown in Table 1. Although many strong reflections generally appear at similar diffraction angles, each form exhibits a different powder pattern and can clearly distinguish between S-II, FI and F-III.
    It is well known in the crystallography field that for any polymorph, the relative intensity of the diffraction peaks may change due to the preferred direction due to factors such as crystal morphology. Where the effect of the preferred direction is present, the peak intensity changes but the polymorphic characteristic peak position does not change. See, eg, The United States Pharmacopeia # 23, National Formulary # 18, pages 1843-1844, 1995. Therefore, based on the peak intensity as well as the peak intensity, when measured at 25 ± 2 ° C., 35 ± 10% relative humidity (RH) from the copper radiation source, F-III is 4.6 ± 0.2, 7.8 ± 0.2, 9.3 ± 0.2, It can be confirmed from the peak which exists in 14.0 ± 0.2, 17.6 ± 0.2, 20.8 ± 0.2, 24.3 ± 0.2 ° (indicated by 2θ).
    Table 1 S-IIF-IF-III 2θ (°)I / I 0 (%)2θ (°)I / I 0 (%)2θ (°)I / I 0 (%) 4.671.34.922.64.6320.8 5.0367.6934.67.82100 6.835.87.911009.2916.9 7.1716.19.892.510.1622.7 7.7310010.22210.355.4 9.031.310.747.413.7710.7 9.311.714.869.113.9715.2 9.662.415.452.315.066.9 10.271.615.9215.915.7122.3 10.472.216.671.715.877.4 10.916.316.983.116.3534.5 13.632.118.2817.816.7712.3 14.094.618.56717.2810 15.104.120.5813.117.6247.9 15.5210.520.858.818.0943.9 16.459.121.643.920.4342 16.677.622.194.820.8033.6 17.214.922.652.921.3142.7 17.532.423.283.421.7113 18.5328.223.9711.821.8514.5 18.6911.124.316.322.1312.8 19.373.525.523.922.2616.3 20.298.626.203.423.5113.2 20.6417.226.473.123.6915.9 21.0212.728.846.423.9125.6 21.685.130.133.524.3138.7 22.018.331.122.925.228 22.298 25.678.9 23.177.8 27.0518.9 23.399.1 27.8913.3 24.3013.6 28.248.6 25.763.4 28.7121.3 26.054 29.898.9 26.635.5 30.2418.7 27.013.1 30.885.8 27.492.8 31.447.6 28.101.8 33.064.5 28.7310.9 34.366 29.423.2 30.003.7 30.892.1 31.342.4 31.701.1 32.81One 32.910.8 33.482
    Additional Characterization of F-I and F-III
    Hygroscopicity studies were performed for F-I and F-III. Water absorption isotherms for F-I and F-III are shown in FIG. 4. As soon as the sample was first exposed to about 5% relative humidity, there was an immediate weight increase of 1.5% for F-I and 1.7% for F-III, which is equivalent to about 0.5 moles of water, respectively. Both forms (F-I and F-III) continue to absorb moisture through the entire humidity range, likely due to the incorporation of water molecules into the lattice.
    The difference in water absorption between the two forms seems to reflect the amount of water that can fit into the two grids (ie the amount of space available in the grid that can accept water molecules). The absence of hysteresis in the adsorption-desorption isotherms of F-I and F-III indicates that the crystal forms of F-I and F-III quickly equilibrate under any humidity.
    Water absorption curves for F-I and F-III reveal that these forms are essentially non-chemically equivalent hydrates. Under about 50% ambient relative humidity, F-I comprises about 1.7% water (corresponding to 0.5 moles of water) and F-III absorbs about 3.0% of water (corresponding to 0.85 moles of water). Since the bulk forms of F-I and F-III quickly reach equilibrium with the atmosphere, the water content observed by the analytical technique reflects the relative humidity at the time of data collection. The difference from lot to lot in the DSC data seems to be due to different degrees of hydration of the samples due to different storage conditions.
    XRD patterns were obtained for samples of F-I and F-III stored under different relative humidity (0, 22, 50 and 80%). As the relative humidity increases, there is a gradual shift of the F-III peak (expressed as about 13.8, 17.6, 18.0, 20.5, 24.0 °, 2θ) as well as a weak shift of the less intense peak. This change in the XRD pattern of the F-III indicates that the unit cell dimensions are changing, presumably in order to accept weakly held water molecules as the relative humidity increases. The continuous shift of peaks with humidity correlates well with moisture absorption data showing a gradual increase in weight over this humidity range and is evidence of the formation of various hydrates.
    Similar experiments were conducted on F-I to determine if changing the relative humidity had a similar effect on the lattice of F-I (0, 25, 52, 73 and 95% RH). As the relative humidity increased, very little shift of 0% RH peaks was observed at about 7.7, 18.3, 18.5, 20.5, 20.8 ° (in 2θ). The peaks at about 7.7, 20.8, and 24.1 at higher relative humidity also appear to be slightly wider and less resolved, indicating that water is absorbed into the amorphous component, especially at 73 and 95% RH (or its solids). Plasticize). The shift in peaks in the XRD pattern of F-I is less dramatic than the peak shift observed when F-III is exposed to different relative humidity. This suggests that the F-I grating does not have the same expansion and / or contraction as the F-III grating.
    F-I and F-III have been found to be stable over the entire relative humidity range, although F-III can absorb nearly twice as much water. These two forms were found to have comparable crystal size, morphology, water solubility and dissolution rate.
    A drying study was performed to monitor the desolvation of S-II as a function of drying time and temperature (see FIG. 5). XRD patterns were obtained several times during the desolvation experiment. Many diffraction peaks from the desolvation studies of S-II appear at similar angles to F-I, confirming that the lattice of S-II and F-I are nearly similar. After minimal drying, the disappearance of diffraction peaks at about 6.8, 7.2 and 14.0 ° (in 2θ) suggests that this reflection may be due to the crystallographic plane including the partial electron density of the ethyl acetate molecule. do.
    F-I is obtained through extended annealing under high temperature vacuum of the solvated material. F-I obtained in this manner showed high crystallinity as measured by XRD. Thus, the material obtained by crystallization from a solution of ethanol and ethyl acetate followed by vacuum drying for only a few hours as described in '474 shows very low crystallinity, which procedure results in partially desolvated S-II. Because it causes.
    F-I and F-III have several advantages over the prior art forms of aroxyphene described above. Compared to aroxifene produced by the procedure described in '474, F-I and F-III are more stable at ambient temperature and therefore easier to develop drugs (eg development of dosage formulations). In addition, F-I and F-III are much more crystalline than the form disclosed in '474. Crystalline materials are generally less hygroscopic and more stable (i.e., less prone to chemical degradation and maintain a consistent effect) than amorphous materials, and therefore are more desirable for formulation processes. Furthermore, unlike the form of aroxifene produced by the procedure described in '474, which contains water and ethyl acetate in its lattice, F-I and F-III contain only water.
    Characterization method
    DSC measurements were taken on a TA Instruments 2920 Modulated DSC attached to a Thermal Analyst 3100 and equipped with a cooling system. Samples (3-5 mg) were heated from 10 ° C. to 240 ° C. at a heating rate of 2 ° C./min in a crimped aluminum pan.
    TGA analysis was performed on TA Instruments' 2050 Thermogravimetric Analyser, attached to Thermal Analyst 3100. Samples (5-10 mg) were heated from 25 ° C. to 250 ° C. at a heating rate of 5 ° C./min in an open pan.
    The XRD pattern is equipped with a CuKα source (λ = 1.54056 μs) and a Kevex solid-state detector, operating at 50 kV and 40 mA, Siemens D5000 X-ray powder diffractometer (X-ray) powder diffractometer). Each sample was scanned between 4 ° and 35 ° when expressed in 2θ. Allow samples to equilibrate for at least 30 minutes at the desired temperature and / or relative humidity before taking data.
    Hygroscopicity measurements for F-I and F-III were made using the VTI method as follows. Each sample was vacuum dried at 60 ° C. until no further weight loss was detected, at which time the sample chamber had a relative humidity of 0%. Water absorption isotherms were obtained using a VTI vacuum moisture balance at 25 ° C. under the following conditions: sample size 10-15 mg, adsorption / desorption range 0-95% relative humidity, humidity interval 5%, sample interval 10 minutes. .
    The following examples further illustrate the process for preparing the hydrate of the present invention. These examples are not intended to limit the scope of these processes in any way and should not be so appreciated.
    Example
    Example 1
    F from 6-isopropoxy-3- (4- [2- (piperidin-1-yl) ethoxy] -phenoxy) -2- (4-methoxyphenyl) benzo [b] thiophene hydrochloride Preparation of -III
    6-isopropoxy-3- (4- [2- (piperidin-1-yl) ethoxy] -phenoxy) -2- (4-methoxyphenyl) at -10 to -20 ° C under nitrogen atmosphere BCl 3 (g) (4.23 g, 34 mmol) was added to 100 ml of a methylene chloride solution of benzo [b] thiophene hydrochloride (10 g, 18 mmol) at a rate such that the temperature of the reaction was kept below -10 ° C. . After the addition was complete, further stirred for 2 hours. To the reaction, isopropyl alcohol (IPA, 12.35 ml, 167 mmol) was slowly added below −10 ° C. and stirred for 30 minutes. A separate flask was filled with 100 ml of water and placed in an ice bath to cool to about 0 ° C. The solution of the product was transferred through the cannula to a flask of water above while continuing vigorous stirring. The resulting white slurry was stirred at 0 ° C. for 1 hour. The product was recovered by filtration and washing with 25 ml 40% CH 2 Cl 2 / water followed by 25 ml of cold water. The product was suspended in 60 ml IPA and 60 ml water and heated to 60 ° C. A solution was obtained at 48 ° C. An additional 120 ml of water was added. The solution was cooled to 35 ° C. and the slurry was slowly cooled further to 0-5 ° C. and stirred for several hours. The product was isolated by filtration and washed with cold demineralized water (125 ml). The wet F-III cake was dried in vacuo at about 50 ° C. for 12 to 24 hours to constant weight to give F-III.
    Example 2
    6-benzyloxy-3- (4- [2- (piperidin-1-yl) ethoxy] -phenoxy) -2- (4-methoxyphenyl) benzo [b] thiophene- (sulfur-oxide Preparation of F-III from
    At ambient temperature, demineralized water (5.25 ml), 1 M HCl (7.74 ml, 7.75 mmol), 10% Pd / C (type A32110, 1.37 g, 1.29 mmol Pd), 6-benzyloxy in 250 ml parr bottles -3- (4- [2- (piperidin-1-yl) ethoxy] -phenoxy) -2- (4-methoxyphenyl) benzo [b] thiophene- (sulfur-oxide) (3 g , 5.16 mmol) and isopropyl alcohol (32 ml) were added. The bottle was connected snugly to a wave shaker and then evacuated and filled with nitrogen twice, after which it was filled with hydrogen gas at a pressure of 30 psig. The shaker was run and the reaction mixture was heated to 60 ° C. After about 4 hours the reaction was confirmed to be complete by HPLC analysis. The reaction mixture was filtered through a pad of diatomaceous earth, which was washed twice with 10 ml of 0.1 M HCl. The solvent was removed in vacuo at about 50 ° C. The remaining residue was dissolved in 50% isopropyl alcohol / demineralized water (30 ml) and heated gently on a steam bath until one solution was obtained. Demineralized water (22 ml) was added to the solution and cooled to ambient temperature. The product slurry was further cooled to 0 ° C. The product was isolated by filtration and washed twice with 15 ml of cold demineralized water and dried to about constant weight at about 50 ° C. in vacuo to give F-III.
    Example 3
    6-isopropoxy-3- (4- [2- (piperidin-1-yl) ethoxy] -phenoxy) -2- (4-methoxyphenyl) benzo [b] thiophene- (sulfur- From F-III)
    Methylene chloride (105 l) and 6-isopropoxy-3- (4- [2- (piperidin-1-yl) ethoxy] -phenoxy) -2- (4-methoxyphenyl) benzo [b ] Thiophene- (sulfur-oxide) (10.5 kg) was combined and cooled to -15 to -20 ° C. BCl 3 (4.6 kg) was added while maintaining the reaction temperature between -10 and -20 ° C. Stirring is continued until the area ratio by HPLC of the starting material is less than 1%. HPLC system (4.6 mm ID × 25 cm Zorbax SB-Phenyl column 30 ° C., 70:30 methanol: 0.01 N sulfuric acid; flow rate 1.5 ml / min; detector 300 nm). Isopropanol (10.28 kg) was added while maintaining the reaction temperature between -10 and -20 ° C. The reaction mixture was stirred for 30-45 minutes. The crude product was separated by adding the reaction mixture to an additional 105 l of water cooled to 2-7 ° C while maintaining the reaction temperature at 2-7 ° C. Then washed with methylene chloride (120 l) cooled to 2-7 ° C. The crystalline slurry was stirred for 2-3 hours, filtered and then washed with methylene chloride (26 kg) and water (53 l) cooled to 2-7 ° C. The wet crude cake is combined with water (42 l) and isopropanol (40 l) and heated to 65-70 ° C. to affect dissolution. The hot solution was filtered off. The filtrate was then washed with a mixture of isopropanol (20 l) and water (21 l) heated to 65-70 ° C., followed by water (126 l) heated to 65-70 ° C. The solution was cooled to 40-45 [deg.] C. and seeded to grow crystals and stirred at this temperature for 2-3 hours. The slurry was cooled to 0-5 ° C, stirred for 3-4 hours and filtered. The filter cake was washed with water (122.6 l) cooled to 2-7 ° C. The product was vacuum dried at a maximum temperature of 50 ° C. until the weight change of the cake was less than 0.05 kg for 2-4 hours. Yield: 8.468 kg (87.3%). HPLC potency: 98.5%. Water by Karl Fischer method: 3.0%. Total related material by HPLC: 1.79%.
    Example 4
    6-benzyloxy-3- (4- [2- (piperidin-1-yl) ethoxy] -phenoxy) -2- (4-methoxyphenyl) benzo [b] thiophene- (sulfur-oxide Preparation of F-III from
    Tetrahydrofuran (261 ml), water (45 ml), concentrated sulfuric acid (6.14 g) and 6-benzyloxy-3- (4- [2- (piperidin-1-yl) ethoxy] -phenoxy)- 2- (4-methoxyphenyl) benzo [b] thiophene- (sulfur-oxide) (99% HPLC potency, 0.35% HPLC total relevant material level) was combined and stirred until homogeneous. 10% Pd / C (5.6 g slurried in 22 ml of water) was added with 5 ml of water rinse. Air was removed from the resulting slurry and overlaid with 60 psi of hydrogen. The reaction temperature was adjusted to 30 ° C. After 2 hours, 10% Pd / C (5.6 g) was added with water (30 ml). Thereafter, the hydrogenation reaction at 60 psi and 30 ° C. was continued for 22 hours. An additional 4.40 g of 10% Pd / C in 30 ml water was added and the hydrogenation reaction at 60 psi and 30 ° C. was continued for an additional 2.5 hours. The catalyst was removed by filtration and the pH of the filtrate was adjusted to 7.24 with 50% sodium hydroxide. Sodium chloride (8.66 g) dissolved in water (18 ml) was added and the biphasic solution was stirred for 30 minutes. The phases were separated and the aqueous phase was extracted with 50 ml of tetrahydrofuran. The organic phases were combined and concentrated by atmospheric distillation to a volume of 50 ml. 180 ml of methanol were added to the concentrate at 24 ° C. over 1 hour. The resulting crystalline slurry was stirred at 24 ° C. for 30 minutes, cooled to 0 ° C. and stirred for 1 hour. The solids were separated by filtration, washed successively with 39 ml of water, 39 ml of methanol and vacuum dried at 50 ° C. overnight. Yield 15.52 g (67.8%)
    Isopropanol (33 ml), water (66 ml) and 10 g of the solid separated above were collected. 1.8 M HCl (21 ml) was added to the stirred mixture at 25 ° C. The solids dissolve rapidly and then recrystallization of the hydrochloride salt occurs. After stirring for 30 minutes, the slurry is heated to 70 ° C. to affect the dissolution of all solids. After cooling the solution to 60 ° C., 33 ml of water were added. The resulting solution was cooled to 25 ° C. over 3 hours, during which time the hydrochloride salt precipitated. The slurry was stirred at 25 ° C. for about 3 hours, filtered, washed with 30 ml of water and then vacuum dried at 50 ° C. overnight. 8.9 g (82.7%) of F-III yield. HPLC potency: 96.5%. Water by the Karl Fischer method: 2.44%. HPLC related substances: 1.09%
    Glossary of Terms
    The term 'effective amount' as used herein refers to the amount of F-III that can inhibit the adverse effects of the conditions or conditions described herein. Even when F-III is administered with estrogens, progestins, aromatase inhibitors, LHRH analogs or AChE inhibitors, the term 'effective amount' refers to the amount of the agent that can produce its intended effect.
    The terms' inhibiting 'and' inhibiting 'refer to their generally accepted meanings, i.e., as described herein,' inhibiting, inhibiting, alleviating the severity or progression of a pathological condition or its sequelae, Improving, slowing, stopping or reversing.
    The terms 'preventing, preventing, precautionary, precautionary, and prevent' are used interchangeably and for a dose of F-III to cause or develop any pathological condition or sequelae described herein. It means reducing the possibility.
    The term 'sufficient estrogen, estrogen deficiency' means that women do not produce enough endogenous estrogen hormones to maintain estrogen-dependent functions such as menstruation, bone homeostasis, neuronal function, and cardiovascular status, It refers to a naturally occurring or clinically induced condition. This estrogen deficiency situation arises due to, but is not limited to, menopause, surgical or chemical ovarian resection and its functional equivalents (eg, administration of aromatase inhibitors, GnRH agonists or antagonists, ICI 182780, and the like). Disease conditions associated with estrogen deficiency include, but are not limited to, bone loss, osteoporosis, cardiovascular disorders, and hyperlipidemia.
    The term 'estrogen' as used herein refers to, for example, 17β-estradiol, estrone, conjugated estrogen (Premarin) ), Steroid compounds having estrogen activity such as equine estrogen 17β-ethynyl estradiol and similar substances. Preferred estrogen based compounds are Premarin And norethylnoderel.
    The term 'progestin' as used herein includes compounds having progesterone activity such as, for example, progesterone, norethylnodrel, nongestrel, megestrol acetate, noethine drone and the like. Noethynedrone is a preferred progestin based formulation.
    The term aromatase inhibitor, as used herein, is a commercially available inhibitor, for example, aminoglutamide (CYTANDREN). Anastrazole (ARIMIDEX) ), Letrozole (FEMARA) ), Formesstan (LENATRON) ), Exemestane (AROMASIN) ) And compounds capable of inhibiting aromatase, such as similar substances.
    As used herein, the term 'LHRH analogue' inhibits the production of estrogens in premenopausal women, eg, goserine (ZOLADEX). ), Leproprolide (LUPRON And analogues of luteinizing hormone free hormone, including analogues.
    The term 'AChE inhibitor' as used herein includes compounds that inhibit acetylcholine esterase and include, for example, pizostigmine salicylate, tacrine hydrochloride, donepezil hydrochloride and similar substances. .
    As used herein, the term 'up-regulate ChAT' refers to increasing the enzyme activity of ChAT, ie, promoting the conversion of choline to acetylcholine. Such promotion may include an increase in efficiency and / or an increase in the rate of reaction of ChAT and choline and / or an increase in the amount of ChAT present at the site of action. This increase in the amount of enzyme present may be due to other synthetic steps of genetic factor regulation or enzyme formation and / or reduced inactivation and metabolism of the enzyme.
    Selected inspection procedure
    General Rat Manufacturing Procedure: A 75 day old (unless otherwise noted) Sprague Dawley female rat (200-225 g in weight) was placed in Charles River Labora, Portage, MI, USA. Obtained from Charles River Laboratories. These rats were shipped a week after ovarian ablation (OVX) or Sham surgery at Charles River Laboratories. Upon arrival, these mice were divided into groups of 3 to 4 so that they could be placed in metal hanging cages for free access to food (about 0.5% calcium content) and water for a week. The relative humidity was 40% or more and the room temperature was maintained at 22.2 ° C ± 1.7 ° C. The photoperiod in the room was 12 hours day and 12 hours night.
    Dosing Regimen Tissue Collection: F-III was started daily after a week of acclimatization (thus 2 weeks after ovarian resection). 17α-ethynyl estradiol or F-III is administered orally, unless otherwise stated, in a suspension of 1% carboxymethylcellulose or dissolved in 20% cyclodextrin. Rats are administered daily for 4 days. After the dosing regimen, rats were weighed and anesthetized with a ketamine: Xylazine (2: 1 volume) mixture and a blood sample was taken through the heart. Rats were then suffocated with carbon dioxide and sacrificed. The uterus of the rats is removed through a midline incision and the wet uterus is weighed. 17α-ethynyl estradiol is obtained from Sigma Chemical Co., St. Louis, MO.
    Cardiovascular Disorders / Hyperlipidemia
    The blood samples obtained above are allowed to coagulate at room temperature for 2 hours, centrifuged at 3000 rpm for 10 minutes to obtain serum. Serum cholesterol is measured using a high performance cholesterol assay from Boehringer Mannheim Diagnostics. In brief, the cholesterol is oxidized to choles-4-en-3-one and hydrogen peroxide. Hydrogen peroxide is then reacted with phenol and 4-aminophenazone in the presence of peroxidase to produce a p-quinone imine dye which is read by spectrometer at 500 nm. The cholesterol concentration is then calculated against the standard curve. The overall analysis was obtained using a Biomek Automated Workstation.
    Uterine Eosinophil Peroxidase (EPO) Assay
    The uterus is stored at 4 ° C. until enzymatic analysis. The uterus are then homogenized in 50 volumes of 50 mM Tris buffer solution (pH-8.0) containing 0.005% Triton X-100. After addition of 0.01% hydrogen peroxide and 10 mM O-phenylenediamine (final concentration) to the Tris buffer solution, the increase in absorbance at 450 nm is monitored for 1 minute. The presence of eosinophils in the uterus indicates the estrogen activity of the compound. The maximum velocity at 15 second intervals was determined for the linear section of the initial response curve.
    Bone loss (osteoporosis) inhibition test procedure
    After the above general preparation procedure, rats (6 per treatment group) were treated daily for 35 days and sacrificed by choking with carbon dioxide on the 36th day. The 35 day period is sufficient for the maximum reduction in bone density to occur and is measured as described herein. At the time of sacrifice, the uterus is removed, incised to have no external tissues and the fluid contents drained before weighing the wet to check for estrogen deficiency associated with a complete incision of the ovary. The weight of the uterus is routinely reduced by about 75% because of ovarian resection. The uterus is then placed in 10% neutral buffered formalin for subsequent histological analysis.
    The right femurs were dissected, digitalized X-rays generated and analyzed by image analysis program (NIH image) in distal mataphysis. They also scan the proximal side of the tibia from these animals by quantitative computed tomography. According to the above procedure, F-III or ethynyl estradiol (EE 2 ) in 20% hydroxypropylβ-cyclodextrin is orally administered to experimental animals. F-III is also useful in combination with estrogen or progestin.
    MCF-7 Proliferation Assay
    MCF-7 adenocarcinoma cells (ATCC HTB 22) were treated with 10% fetal bovine serum (FBS) (v / v), L-glutamine (2 mM), sodium pyruvate (1 mM), HEPES {(N- [2-hydroxyethyl] piperazine-N '-[2-ethanesulfonic acid] 10 mM}, MEM supplemented with non-essential amino acids and bovine insulin (1 ug / mL) (phenol red -free) minimal essential medium (purchased from Sigma) (maintenance medium) 10 days prior to analysis, MCF-7 cells were depleted of 10% FBS instead of 10% FBS to deplete internally stored steroids. Transfer to maintenance medium supplemented with dextran coated charcoal stripped fetal bovine serum (DCC-FBS) (assay medium) MCF-7 cells are free of cell dissociation medium (Ca ++ / Mg ++ and 10 mM HEPES and 2 mM EDTA). Using HBSS (without phenol red) supplemented, remove from the holding flask, wash cells twice with assay medium and adjust to 80,000 cells per mL. . Was added to about 100 mL (8,000 cells) the micro culture well (Costar 3596) on a stable surface allows cell adhesion and equilibrium after being moved by incubation at 37 ℃, 5% CO 2, humidified incubator for 48 hours. Diluent Serial dilutions of DMSO or drug as a control are prepared in assay medium, 50 mL in three microcultures and 50 mL of assay medium to a final volume of 200 mL 37 ° C., 5% CO 2 , humidification. After 48 hours of further incubation in the incubator, the microcultures are pulsed with tritiated thymidine (1 uCi / well) for 4 hours.The culture is terminated by freezing at −70 ° C. for 24 hours and then ska Microcultures are thawed and harvested using a Skatron Semiautomatic Cell Harvester. Samples are counted by liquid scintillation using a Wallac Betaplace β counter.
    DMBA Induced Breast Tumor Suppression
    Estrogen-dependent breast tumors were induced in Sprague Doll female rats purchased from Harlan Industries, Indianapolis, Indiana, USA. At about 55 days of age, rats are fed orally with 20 mg of 7,12-dimethylbenz [a] anthracene (DMBA) once. About 6 weeks after the administration of DMBA, there is a palpation of tumors in the mammary glands at weekly intervals. If more than one tumor appears, the longest and shortest diameters of each tumor are measured and recorded with calipers and the animal is selected for testing. Attempts are made to uniformly distribute tumors of various sizes in the treated and control groups so that tumors of average size are distributed evenly among the test groups. Control groups and test groups for each experiment included 5 to 9 animals.
    Intraperitoneal injection or oral administration of 2% F-III to acacia. Orally administered compounds were dissolved or suspended in 0.2 mL of corn oil. Each treatment, including acacia and corn oil control treatments, is administered once a day to each test animal. After the initial tumor measurement and the selection of the experimental animals, the tumors are measured by the method each week. Treatment and measurement for the animals is continued for 3 to 5 weeks and then the final area of the tumor is measured. Changes in mean tumor area for each compound and control treatment are measured.
    Uterine fibrosis test procedure
    Test 1: F-III is administered to between 3 and 20 women with uterine fibrosis. The amount of compound administered is 0.1-1000 mg / day and the administration period is 3 months. The women observe the effect on uterine fibrosis until the administration period and up to 3 months after the end of the administration.
    Trial 2: Follow the same procedure as in Trial 1, but with a 6-month administration period.
    Trial 3: Follow the same procedure as in Trial 1, but with a dosing period of one year.
    Test 4: Prolonged estrogen stimulation causes leiomyomas in sexually mature female guinea pigs. Animals are injected estradiol 2 to 4 months or 3 to 5 times a week until tumors develop. Treatment consisting of F-III or vehicle is administered daily for 3 to 16 weeks, followed by the sacrifice of pigs and obtaining a uterus for analysis of tumor decay.
    Trial 5: Tissues from human leiomyomas are implanted into the peritoneal cavity and / or myometrium of female nude mice that are sexually mature and fertile. Exogenous estrogen is supplied to induce growth of the explanted tissue. In some cases, the collected tumor cells are cultured in vitro before implantation. Treatment consisting of F-III or vehicle is provided by gastric lavage daily for 3 to 16 weeks and the graft is removed to measure for growth or decay. At the time of sacrifice, the uterus is collected to assess the condition of the organs.
    Test 6: Tissues from human uterine leiomyoma tumors are collected and maintained in primary non-transformed cultures in vitro. To make a single cell suspension, surgical samples are passed through a sterile mesh or sieve, or alternatively, sliced thin from surrounding tissue. Cells are maintained in medium containing 10% serum and antibiotics. The growth rate with and without estrogen is measured. Cells are analyzed for their ability to produce the complement constituent protein C3 and their response to growth factors and growth hormone. Cultures are evaluated in vitro for the proliferative response of the cultures after treatment with progestin, GnRH, F-III and vehicle. Levels of steroid hormone receptors are measured weekly to ensure that important cellular properties are maintained in vitro. Tissues from 5 to 25 patients are used.
    Test 7: The ability of F-III to inhibit estrogen-stimulated proliferation of ELT cell lines induced in leiomyomas is described by Fuchs-Young, et al., "Inhibition of Estrogen-Stimulated Growth of Uterine Leiomyomas by Selective". Estrogen Receptor Modulators ", Mol. Car., 17 (3): 151-159 (1996), measured substantially the same and is hereby incorporated by reference.
    Endometriosis Test Procedures
    Test 1: 12 to 30 grown CD strain female rats were used as test animals. They were divided into three groups of equal numbers. The estrous cycle of all animals is monitored. Surgical operation is performed for each rat on the day of estrus. The left uterine horns of female rats of each group are removed, cut into small squares, and the squares loosely closed in several places adjacent to the mesenteric blood stream. In addition, the ovaries of two groups of female rats are removed. The day after surgery, group 1 and 2 rats were intraperitoneally injected with water for 14 days, while group 3 rats were intraperitoneally injected with 1.0 mg of F-III per kg of body weight for the same period. After 14 days of treatment, the mice are sacrificed and the endometrial explants, adrenal glands, remaining uterus and ovaries (if appropriate) are removed and prepared for histological examination. Weigh the ovaries and adrenal glands.
    Test 2: 12-30 grown CD strain female rats were used as test animals. These were divided into two groups of equal numbers. Monitor the estrous cycle of all mice. On the day of estrus, each female undergoes a surgical operation. The left uterine horns of the females of each group are removed, cut into small squares, and the squares loosely sutured in several places adjacent to the mesenteric blood stream. Approximately 50 days after surgery, Group 1 animals are injected intraperitoneally with water for 21 days, while Group 2 mice are injected intraperitoneally with 1.0 mg of F-III per kg of body weight for the same period. After 21 days of treatment, rats are sacrificed, explants and adrenal glands of the endometrium are removed and weighed. Explants are measured as evidence of growth. Monitor the heat cycle.
    Test 3: An autograph of endometrial tissue is used to cause endometriosis in rats and / or rabbits. Reproductive females undergo bilateral ovarian extraction and exogenously supply estrogen to provide certain constant hormone levels. Voluntary endometrial tissue is implanted into the peritoneum of 5 to 150 animals and fed with estrogen to induce growth of explanted tissue. Treatment consisting of the compounds of the invention is provided by gastric lavage daily for 3 to 16 weeks and the graft is removed to measure for growth or decay. At the time of sacrifice, the intact horns of the uterus are collected to assess the condition of the endometrium.
    Test 4: Tissues collected from human endometrial lesions are implanted into the peritoneal cavity of female nude mice that are sexually mature and fertile. Exogenous estrogen is supplied to induce growth of explanted tissue. In some cases, the collected endometrial cells are cultured in vitro before implantation. Treatment consisting of F-III is provided by gastric lavage daily for 3 to 16 weeks and the graft is removed to measure for growth or decay. At the time of sacrifice, the uterus is collected to assess the condition of the intact endometrium.
    Test 5: Tissues harvested from human endometrial lesions are maintained in a non-converted primary culture in vitro. To make a single cell suspension, surgical samples are passed through a sterile mesh or sieve or otherwise sliced thin from surrounding tissue. Cells are maintained in medium containing 10% serum and antibiotics. The growth rate with and without estrogen is measured. Cells are analyzed for their ability to produce the complement constituent protein C3 and their response to growth factors and growth hormone. Cultures are evaluated in vitro for the proliferative response of the cultures after treatment with progestin, GnRH, F-III and vehicle. Levels of steroid hormone receptors are measured weekly to ensure that important cellular properties are maintained in vitro. Tissues from 5 to 25 patients are used.
    Central nervous disorder including Alzheimer's disease
    Estrogens, such as 17β-estradiol, regulate gene transcription by binding to estrogen receptors (ER), which are present in the cytoplasm of certain cell populations. Ligand activation of the ER reaches its highest upon activation of the appropriate target gene when bound to a 13 base-pair palindromic DNA consensus sequence (estrogen response element (ERE)). Is a prerequisite for nuclear transport of the complex that initiates the assembly of the transfer device. Many genes regulated by estrogen have been identified. Among these are cytoskeleton proteins, neurotransmitters, biosynthetic and metabolic enzymes and receptors, as well as other hormones and neuropeptides. Estrogen response elements have been identified in many estrogen response genes, including vitelogenin, c-fos, prolactin and luteinizing hormone.
    Sequences similar to estrogen response elements, important in the central nervous system, have been identified at p75 ngr and trkA, both of which are neurotrophin (nerve growth factor (NGF), brain-induced nerve growth factor (BDNGF), and neurotrophin- It functions as a molecule that gives a signal to 3).
    BDNF as well as NGF have been shown to enhance the survival of cholinergic neurons in culture. If the interaction between neurotrophin and estrogen is important for the development and survival of basal forebrain neurons (which are degenerated in Alzheimer's disease), clinical conditions in which estrogen deficiency is present (such as postmenopausal) It is natural that it can contribute to the loss of these neurons.
    In order to determine the similarities and / or differences between F-III and estrogen in affecting gene expression in various brain regions, the following experiments were performed on ovarian excised rats (prepared as above). It was.
    Six week old rats were injected subcutaneously with estradiol benzoate (0.03 mg / kg), F-III or vehicle (control) daily. After 5 weeks of treatment, animals were sacrificed and their brains removed and hippocampus was collected by brown rice dissection surgery. The hippocampus is rapidly frozen in liquid nitrogen and stored at -70 ° C. Total RNA is prepared from pooled tissues obtained from appropriate treatments and controls and reverse transcribed using 3 ′ oligonucleotide primers selected as specific mRNA (poly-A +) populations. Polymerase chain reaction (PCR) was performed in a cocktail consisting of random 5 ′ oligonucleotides (length: 10 base pairs, total 150), reaction buffer solution, Taq polymerase and 32 PdTCP.
    After 40 amplifications, the reaction product was sized on 6% TBE-urea gel, dried and exposed to X-ray film. The resulting mRNA display pattern is compared between treatment groups.
    Combination of Estrogen with F-III
    During or after menopause, women often undergo hormone replacement therapy (HRT) to eliminate negative consequences associated with circulating endogenous estrogens, such as hot flashes. However, HRT is associated with an increased risk of developing certain cancers, including uterine and breast cancers. F-III can be used with HRT to suppress this risk.
    Combination of Aromatase Inhibitors with F-III
    By definition, ovaries do not function in postmenopausal women. The only source of estrogen in women is through the conversion of adrenal androgens to estrogens by the enzyme aromatase found in surrounding tissues (including fat, muscle and breast tumors). Thus, drugs that inhibit aromatase (aromatase inhibitors) deplete circulating estrogen in postmenopausal women. Because aromatase inhibitors cause estrogen deficiency, the use of aromatase inhibitors is an important treatment option for patients with metastatic breast cancer. During treatment with an aromatase inhibitor, lack of circulating estrogen can lead to negative unintended side effects (eg side effects on serum lipid levels). F-III can be used to suppress this negative effect.
    Combination of LHRH Analogues with F-III
    Continued exposure to LHRH (luteinizing hormone free hormone) makes the pituitary gland insensitive and no longer stimulates the ovaries to produce estrogens, thus inhibiting estrogen production in premenopausal women. The clinical effect is "medical ovarian extraction," which is reversible following discontinuation of the LHRH analogue. During treatment with LHRH analogues, the lack of circulating estrogen can lead to negative unintended side effects (eg side effects on serum lipid levels). F-III can be used to suppress this negative effect.
    Increased levels of acetylcholine
    In the hippocampus of patients with Alzheimer's disease, it is known that there are significantly lower levels of cholinergic neurons compared to the general population. The gradual loss of these cholinergic neurons seems to reflect a gradual loss of memory and cognitive function in these patients. One reason for the reduction of these neurons is thought to be the loss or degraded function of the neurotransmitter acetylcholine.
    The level of acetylcholine in neurons is basically determined by where there is an equilibrium between its biosynthesis and biodegradation. Choline acetyltransferase (ChAT) primarily results in the synthesis of acetylcholine and acetylcholinesterase results in degradation of acetylcholine.
    In order to determine the effect of F-III on the level of ChAT, the following experiments were performed: 40 rats daily after subcutaneous injection or oral gavaz 10% cyclodextrins after daily rat preparation procedure. Dissolved in a vehicle containing 3 mg / kg / day or administered in estradiol benzoate (0.03 or 0.3 mg / kg / day) or vehicle control. Animals are treated for 3 or 10 days. There are 20 animals per dosing regimen. At appropriate time intervals, animals are sacrificed and the brain dissected. Homogenize and analyze specific parts of the brain. Homogenates from the hippocampus and the frontal cortex are processed and the biosynthesis of acetylcholine is radiolabeled to determine the activity of ChAT. This procedure is described in Chef et al., Schöpp et al., J. Neural Transmiss., 78: 183-193, 1989, which is incorporated by reference.
    As expected, ChAT levels in bilateral ovary incisions (OVX) animals are reduced to> 50% (p <0.001) compared to the simulated surgical control group.
    In another embodiment of the present invention, F-III is used in combination with an AChE inhibitor. With the use of AChE inhibitors, the action of AChE is inhibited and the degradation of acetylcholine is prevented, increasing the level of acetylcholine.
    Benign prostatic hyperplasia (BPH)
    For a background on the association between estrogen action and treatment of BPH and prostate cancer, see PCT application WO 98/07274 (International Publication Date: October 15, 1998).
    The following experiments assess the ability of F-III to bind to estrogen receptors in various human prostate cancer cell lines.
    Lysates of LNCaP, DU-45 and PC-3 human prostate cancer cell lines, ie 50 nM tris-hydrochloric acid pH 7.4, 1.5 mM ethylenediamine tetraacetic acid (EDTA), 0.4 M potassium chloride, 10% glycerol, 0.5 mM 2-ME And protease inhibitor pepstatin (1 mg / mL), reupeptin (2 mg / mL), aprotinin (5 mg / mL) and phenylmethylsulfonyl fluoride (TEG medium containing 10 mM sodium molybdate) Prepare in medium (TEGP) containing PMSF, 0.1 mM).
    The cell lysate was centrifuged and the pellet resuspended in cold TEGP (1 mL TEGP / pellet 100 mg) and sonicated for 30 seconds on a Branson Model 450 Sonifier (70% utilization, Output 1.8). The lysate is pelleted by centrifugation at 10,000 x G for 15 minutes at 4 ° C and then the supernatant is removed and used immediately or stored at -70 ° C.
    Competitive binding assay: Binding buffer is TEG with 50 mM sodium chloride instead of 0.4 M potassium chloride, plus 1 mg / mL of egg white albumin (TEGO). F-III was diluted in TEGO to 20 nM and a triple series dilution solution was made from it. Analyze in round bottom polypropylene microplates in three microwells. Each well 35 mL of tritiated 17β-estradiol (0.5 nM, inactive 60.1 Ci / mmol, manufactured by Dupont-New England Nuclear, Boston, MA, USA), cold competition test compound (0.1 nM-5) mM) or TEGO, incubate with shaking at 4 ° C. for 5 minutes and add 70 mL of MCF-7 cell lysate.
    The plates are incubated at 4 ° C. for 24 hours and then 70 mL of dextran coated charcoal (DCC) is added to each well and shaken vigorously at 4 ° C. for 8 minutes. The plates are centrifuged at 1500 x G for 10 minutes at 4 ° C. Supernatants from each well are collected in flexible polystyrene microplates and counted by flash using a Wallac Microbeta Model 1450 counter. After correcting for counting efficiency (35-40%) and background, radioactivity is expressed as disintegrations per minute (DPM). An additional control is DCC total and total + DCC to define the lower limit of DCC extractable coefficients. The results of this competitive binding analysis are expressed as the average percent binding (% binding ± standard deviation) using the following equation.
    Prevention of breast cancer
    The invention also relates to administering F-III to a person at risk for de novo breast cancer. As used herein, the term "di novo" means firstly a lack of alteration or transformation of normal breast cells into cancer cells or malignant cells. Such a change may occur step by step in the same cell or daughter cell via an evolutionary process or in a single important event. This de novo process is in contrast to spreading from the primary tumor location of metastasis, metastasis or already changed or malignant cells.
    A person who has no risk of developing breast cancer is a person who can develop di novo breast cancer, has no evidence or suspicion of the possibility of breast cancer at a risk above average, and has never been diagnosed with breast cancer. Although there is no evidence that breast cancer exists and is recovering, the greatest risk factor contributing to the development of breast cancer is the personal history of breast cancer or the development of previous breast cancer. Another risk factor is a family history of breast cancer.
    Rats that induced breast tumors by administering the carcinogen N-nitroso-N-methylurea are well-accepted animal models for breast cancer research and have been known to be suitable for analyzing the effects of chemopreventive agents. .
    In two separate studies, 55-day-old Sprague Doll female rats received N-nitroso-N-methylurea intravenously (study 1) or intraperitoneally (study 2) at a dose of 50 mg / kg body weight. A week after administration, varying amounts of F-III, (Z) -2- [4- (1,2-diphenyl-1-butenyl) phenoxy] -N, N-dimethylethanamine base (tamoxifen base Or freely served special meals with mixed controls.
    In Study 1, the special diet dose 60 mg / kg, 20 mg / kg is interpreted as approximately 3 mg / kg, 1 mg / kg to the experimental animals.
    In study 2, the special diet doses of 20, 6, 2 and 0.6 mg / kg are interpreted to be approximately 1, 0.3, 0.1 and 0.15 mg / kg to experimental animals.
    Rats are examined for evidence of toxicity, weighed once a week and promoted to determine tumor formation. Rats are sacrificed and dissected after 13 weeks (study 1) or 18 weeks (study 2) to identify and weigh tumors.
    Formulation
    The term 'pharmaceutical' as used herein as an adjective means that the drug is not inherently harmful to the mammal to which it is administered. The term 'pharmaceutical formulation' means that the carrier, diluent, excipient and active ingredient must be compatible with the other ingredients of the formulation and not be harmful to its recipient.
    F-III is preferably formulated before administration. The choice of dosage form should be determined by the attending physician taking into account the same factors as those involved in determining the effective amount.
    Total active ingredients in such formulations comprise 0.1 to 99.9% by weight of the formulation. It is preferred that only two active ingredients be included in the formulation. That is, it is desirable to formulate a second active ingredient selected from F-III with estrogens, progestins, aromatase inhibitors, LHRH analogs and AChE inhibitors. Most preferred are formulations in which F-III is the only active ingredient.
    The pharmaceutical formulations of the present invention are prepared according to procedures known in the art using well known and readily available ingredients. For example, F-III alone or in combination with estrogens, progestins, aromatase inhibitors, LHRH analogs and AChE inhibitor compounds to formulate with conventional excipients, diluents or carriers to provide tablets, capsules, suspensions, solutions, injections, aerosols, Made from powder and similar forms.
    Pharmaceutical compositions of the invention for parenteral administration include sterile powders and sterile aqueous or sterile non-aqueous solutions, dispersions, suspensions or emulsions which are reconstituted immediately prior to use as sterile solutions or sterile suspensions. Examples of suitable sterile aqueous or sterile non-aqueous carriers, diluents, solvents or vehicles include water, saline, ethanol, glycerol, propylene glycol, polyethylene glycols and similar polyols, suitable mixtures thereof, vegetable oils (olive oils), ethyl oleate There are organic esters that are injectable as well. For example, using a coating material such as lecithin, maintaining the proper particle size when dispersed or suspended, or using a surfactant to maintain proper fluidity.
    Parenteral compositions may include adjuvant such as preservatives, wetting agents, emulsions, dispersants. Antimicrobial and antifungal agents such as parabens, chlorobutanol, phenol sorbic acid and similar substances are added to prevent the activity of microorganisms. It is also desirable to include isotonic agents, such as sugars, sodium chloride and the like. Prolonged absorption of the injectable formulation can be extended by including agents that delay absorption, such as aluminum monostearate and gelatin.
    In some cases, it is desirable to slow the absorption of the drug after subcutaneous or intramuscular injection in order to prolong the effect of the drug. This can be accomplished by using a liquid suspension of crystalline material with low solubility in water or by dissolving or suspending the drug in an oil vehicle. In the case of subcutaneous or intramuscular injection of a suspension comprising a form of a drug with low solubility in water, the rate of absorption of the drug varies with the rate of dissolution of the drug.
    'Depot' formulations of injectable F-III are biodegradable polymers such as poly (lactic acid), poly (glycolic acid), copolymers of lactic acid and glycolic acid, poly (orthoesters), poly (anhydrides) It is made by forming microencapsulated matrices of drugs in the stomach. The above materials are described in the art. The rate of drug release can be controlled depending on the ratio of drug to polymer and the nature of the particular polymer employed.
    Injectable formulations are sterilized at the time of preparation or shortly before administration (as in the example of a double chamber injection package), for example by filtration through bacterial catch filters, or by pre-sterilization of the individual components before making into a mixture.
    Solid forms for oral administration include capsules, tablets, pills, powders, and granules. In this solid form, F-III is admixed with one or more inert materials below. Excipients or extenders such as sodium citrate, dicalcium phosphate, and / or (a) sugars and starches, including lactose, glucose, mannitol and silicic acid, (b) carboxymethyl-cellulose and other cellulose derivatives as pharmaceutical carriers, Binders such as alginate, gelatin, poly (vinylpyrrolidine), sucrose and acacia, (c) wetting agents such as glycerol, (d) agar-agar, calcium carbonate, sodium bicarbonate, potato or tapioca starch, alginic acid, silicate, Disintegrants, such as sodium carbonate, (e) moisturizers, such as glycerol, (f) dissolution retardants, such as paraffin, (g) absorption accelerators, such as quaternary ammonium compounds, (h) cetyl alcohol and glycerin monostearate Wetting agents such as (i) absorbents such as kaolin and bentonite clay, (j) talc, calcium stearate, magnesium stearate, solid poly (ethylene glycol), sodium lauryl Lubricants such as sulfates and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may comprise a buffer.
    Solid compositions of a similar type may include fills in soft or hard gelatin capsules, using excipients such as lactose as well as high molecular weight poly (ethylene-glycol) and the like.
    Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared using coatings or shells, such as enteric coatings or other coatings well known in the pharmaceutical formulation art. The coatings may include opacifying agents or agents which release the active substance in certain parts of the digestive tract, for example acid soluble coatings for releasing the active substance in the stomach or base soluble coatings for releasing the active substance in the intestinal tract.
    The active substance may also be microencapsulated in a sustained release coating, the microcapsules making up part of the pill of the capsule formulation.
    Liquid dosage forms for oral administration of F-III include solutions, emulsions, suspensions, syrups and elixirs. In addition to the active ingredient, liquid formulations are inert diluents commonly used in the art, such as water or other pharmaceutical solvents, solubilizers and ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol , 1,3-butylene glycol, dimethyl formamide, oils (especially cotton yarn, peanuts, corn, germ, olives, castor and sesame oil), glycerol, tetrahydrofurfuryl alcohol, poly (ethylene glycol), sorbitol Emulsions such as fatty acid esters and mixtures thereof.
    In addition to inert diluents, liquid oral formulations may include wetting agents, emulsions, suspending agents, sweetening agents, fragrances, flavoring agents, and the like.
    Liquid suspensions, in addition to the active substances, include ethoxylated isostearyl alcohol, polyoxyethylene sorbitol, sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite clay, agar-agar, tragacanth and mixtures thereof. The same suspending agent may be included.
    Compositions for rectal or vaginal administration may be prepared by mixing F-III with suitable non-irritating excipients, such as cocoa butter, polyethylene glycol, or suppository waxes that are solid at room temperature or liquid at body temperature and will melt in the rectum or vagina to release the active ingredient. Manufacture. The compound is dissolved in the molten wax, made into the desired shape and cured into the final suppository formulation.
    F-III can also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomal formulations are formed by single or multiple lamella hydrated liquid crystals dispersed in an aqueous medium. Lipids that are nontoxic and capable of forming pharmaceutically metabolizable liposomes can be used. Compositions of the present invention in liposome form may comprise, in addition to F-III, stabilizers, excipients, preservatives and similar materials. Phospholipids and natural or synthetic phosphatidyl choline (lecithin) are the preferred lipids.
    Methods of forming liposomes are known in the art, for example, as described in Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, NY (1976), p. 33 et seq. It is.
    The following formulation examples are illustrative only and are not intended to limit the scope of the invention.
    Formulation 1: Gelatin Capsule
    Hard gelatine capsules are prepared using the following ingredients.Amount (mg / capsule) F-III0.1-1000 Starch, NF0-650 Starch fluid powder0-650 Silicon Fluid 350 Centistoke0-15
    The formulation can be varied according to reasonable modifications.
    Tablet formulations are prepared using the following ingredients.
    Formulation 2: Tablet
    ingredientAmount (mg / tablet) F-III2.5-1000 Cellulose, microcrystalline200-650 Silicon dioxide, fumigation10-650 Stearic acid5-15
    The ingredients are mixed and compressed to make tablets.
    Formulation 3: A tablet comprising about 10 and 50 mg of F-III, respectively, was prepared as follows.
    ingredientAmount (mg / tablet)Amount (mg / tablet) F-III11.356.5 Anhydrous lactose176.8128.2 Spray Dried Special Lactose44.232.0 Povidone11.013.0 Polysorbate 802.52.6 Crospovidone (inside)6.256.24 Crospovidone (outside)6.256.5 Magnesium stearate1.51.7 Microcrystalline cellulose (external)0.013.0
    The ingredients are mixed and compressed to make tablets.
    Alternatively, tablets each containing 2.5-1000 mg of F-III are prepared as follows.
    Formulation 4: Tablet
    ingredientAmount (mg / tablet) F-III25-1000 starch45 Cellulose, microcrystalline35 Polyvinylpyrrolidone (10% aqueous solution)4 Sodium carboxymethyl cellulose4.5 Magnesium stearate0.5 talcOne
    Pass F-III, starch and cellulose through a 45 mesh (U.S. Sieve) and mix well. The polyvinylpyrrolidone solution is mixed with the resulting powder and then passed through mesh 14 (U.S. Sieve). The granules so produced are dried at 50-60 ° C. and passed through No. 18 mesh (U.S. Sieve). Sodium carboxymethyl starch, magnesium stearate and talc previously passed through 60 mesh (U.S. Sieve) are compressed on a tablet machine, added to granules, and mixed before obtaining tablets.
    Suspensions containing 0.1-1000 mg of drug per 5 ml of each dose are prepared as follows.
    Formulation 5: Suspension
    ingredientVolume (mg / 5ml) F-III0.1-1000 mg Sodium carboxymethyl cellulose50 mg syrup1.25 mg Benzoic acid solution0.10 ml Flavorq.v. Colorq.v. Purified waterVolume to make with 5 ml
    The agent is passed through mesh 45 (U.S. Sieve) and mixed with sodium carboxymethyl cellulose and syrup to form a soft paste. The benzoic acid solution, flavor and pigment are diluted with some water and added to the paste with stirring. Then sufficient water is added to make the desired volume.
    An aerosol solution containing the following components is prepared.
    Formulation 6: aerosol
    ingredientAmount (wt%) F-III0.25 ethanol25.75 Propeller 22 (Chlorodifluoromethane)70.00
    F-III is mixed with ethanol and the mixture is added to a portion of propeller 22, cooled to 30 ° C. and transferred to the filling device. The required amount is sent to a stainless steel container and diluted with the remaining propeller. The valve unit is then fitted to the vessel.
    Suppositories are prepared as follows.
    Formulation 7: Suppositories
    ingredientAmount (mg / suppository) F-III250 Saturated Fatty Acid Glyceride2,000
    F-III is passed through mesh 60 (U.S. Sieve) and suspended in dissolved fatty acid glycerides with minimal required heat. The mixture is then poured into a nominal 2 g capacity suppository mold and cooled.
    Intravenous formulations are prepared as follows.
    Formulation 8: Intravenous Solution
    ingredientamount F-III25 mg Isotonic saline1,000 ml
    The solution of the above components is administered to the patient intravenously at a rate of about 1 ml / minute.
    Formulation 9: Combination Capsule I
    ingredientAmount (mg / capsule) F-III50 PremarinOne Avicel pH 10150 Starch 1500117.50 Silicone oil2 Tween 800.50 Cab-o-sil0.25
    Formulation 10: Combination Capsule II
    ingredientAmount (mg / capsule) F-III50 Norethylnoderel5 Avicel pH 10182.50 Starch 150090 Silicone oil2 Tween 800.50
    Formulation 11: Combination Tablet
    ingredientAmount (mg / capsule) F-III50 PremarinOne Corn Starch NF50 Povidone, K29-326 Avicel pH 10141.50 Avicel pH 102136.50 Crospovidone XL102.50 Magnesium stearate0.50 Cab-o-sil0.50
    Dosage
    The specific dosage of F-III administered according to the present invention is determined by the particular circumstances surrounding each situation. Such circumstances include the route of administration, the patient's previous history, the pathological condition or condition being treated, the severity of the condition and the condition being treated and the age and sex of the patient.
    In general, the minimum daily effective amount of F-III is about 1, 5, 10, 15, 20 mg. Typically, the maximum effective amount is about 800, 100, 60, 50, 40 mg. Most typically, the dosage is 15 to 60 mg. The exact dosage can be determined according to standard practice in the medical arts that dose titrating a patient; That is, initially a small dose is administered and the dose is gradually increased until the desired therapeutic effect is achieved.
    Although it may be necessary for the patient to titrate the dose with respect to the combination treatment described above, typical dosages of active ingredients other than F-III are as follows. Ethinyl Estrogen (0.01-0.03 mg / day), Mestranol (0.05-0.15 mg / day), Conjugated Estrogen Hormone (e.g. Premarin , Wyeth-Ayerst; 0.3-2.5 mg / day), methoxyprogesterone (2.5-10 mg / day), norethylnodrel (1.0-10.0 mg / day), nonethyn drone (0.5-2.0 mg / day), aminoglutamide ( 250-1250 mg / day, preferably 250 mg 4 times a day), anastazole (1-5 mg / day, preferably 1 mg once a day), letrozole (2.5-10 mg / day, Preferably 2.5 mg once a day), forestane (250-1250 mg / week, preferably 250 mg twice a week), exemestane (25-100 mg / day, preferably 25 mg 1 Once daily), goserine (3-15 mg / 3 months, preferably 3.6-7.2 mg once every 3 months), reprolide (3-15 mg / month, preferably 3.75-7.5 mg Once a month).
    Route of administration
    F-III can be administered by a number of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, nasal. Methods of administration of each of the estrogen and progestin based formulations are consistent with those known in the art. F-III is generally administered in a convenient formulation either alone or in combination with estrogens, progestins or AChE inhibitors.
    The pharmaceutical compositions of the invention can be used orally, rectally, intravaginally, parenterally, topically, buccally, sublingually to humans and other mammals (eg, dogs, cats, horses, pigs and the like). It can be administered as either nasal or nasal. Parenteral administration herein refers to a mode of administration that includes intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous or intraarticular injection or infusion.
    Dosage Method / Duration
    For most of the methods of the invention, F-III is administered continuously one to three times a day or as often as necessary to deliver an effective amount of F-III to the patient. Periodic therapy can be particularly effective in the treatment of endometritis and can be used acutely during painful seizures. In the case of restenosis, treatment should be limited to short intervals (1-6 months) after medical procedures such as angioplasty.
    6-hydroxy-3- (4- [2- (piperidin-1-yl) ethoxy] -phenoxy) -2- (4-methoxyphenyl) benzo [b] thiophene hydrochloride of the present invention The novel crystalline forms of are easier to formulate pharmaceutical formulations compared to substances lacking crystallinity and inhibit the diseases associated with estrogen deficiency, including cardiovascular disease, hyperlipidemia and osteoporosis; And inhibiting other pathological conditions such as endometriosis, uterine fibrosis, estrogen-dependent cancers (breast cancer and uterine cancer), prostate cancer, benign prostatic hyperplasia, central nervous system disorders including Alzheimer's disease, prevention of breast cancer and increasing ChAT Useful for
    权利要求:
    Claims (7)
    [1" claim-type="Currently amended] 4.6 ± 0.2, 7.8 ± 0.2, 9.3 ± 0.2, 14.0 ± 0.2, 17.6 ± 0.2, 20.8 ± 0.2, 24.3 ± 0.2 °, measured from a copper radiation source at 25 ± 2 ° C, 35 ± 10% relative humidity (RH) Crystalline 6-hydroxy-3- (4- [2- (piperidin-1-yl) ethoxy] -phenoxy)-having an X-ray diffraction pattern comprising a peak (when expressed in 2θ)- 2- (4-methoxyphenyl) benzo [b] thiophene hydrochloride hydrate (F-III).
    [2" claim-type="Currently amended] A crystalline compound of claim 1 and at least one pharmaceutical carrier, diluent or excipient; And optionally an estrogen, optionally a progestin, optionally an aromatase inhibitor, optionally an LHRH analog and optionally an acetylcholine esterase (AChE) inhibitor.
    [3" claim-type="Currently amended] 6-hydroxy-3- (4- [2- (piperidin-1-yl) ethoxy] -phenoxy) -2- (4-methoxyphenyl) benzo [b] thiophene from a mixture of isopropanol and water A process for preparing the compound of claim 1 comprising crystallizing hydrochloride.
    [4" claim-type="Currently amended] From uterine fibrosis, endometriosis, smooth muscle cell proliferation of the aorta, restenosis, breast cancer, uterine cancer, prostate cancer, benign prostatic hyperplasia, bone loss, osteoporosis, cardiovascular disease, hyperlipidemia, central nervous system disorders, Alzheimer's disease A compound of claim 1 for inhibiting a selected pathological condition.
    [5" claim-type="Currently amended] The compound of claim 4 for inhibiting breast cancer.
    [6" claim-type="Currently amended] The compound of claim 4 for inhibiting ovarian cancer.
    [7" claim-type="Currently amended] The compound of claim 4 for inhibiting endometrial cancer.
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    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    1999-07-29|Priority to US14618499P
    1999-07-29|Priority to US60/146,184
    1999-08-06|Priority to US14764299P
    1999-08-06|Priority to US60/147,642
    1999-08-19|Priority to US14982099P
    1999-08-19|Priority to US60/149,820
    2000-07-28|Application filed by 피터 지. 스트링거, 일라이 릴리 앤드 캄파니
    2001-06-15|Publication of KR20010049911A
    2007-06-27|Application granted
    2007-06-27|Publication of KR100733094B1
    优先权:
    申请号 | 申请日 | 专利标题
    US14618499P| true| 1999-07-29|1999-07-29|
    US60/146,184|1999-07-29|
    US14764299P| true| 1999-08-06|1999-08-06|
    US60/147,642|1999-08-06|
    US14982099P| true| 1999-08-19|1999-08-19|
    US60/149,820|1999-08-19|
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