SALT OF FUSED HETEROCYCLIC DERIVATIVE AND CRYSTAL THEREOF

Abstract

The present invention provides 3-[2-fluoro-5-(2,3-difluoro-6-methoxybenzyl-oxy)-4-methoxyphenyl]-2,4-dioxo-1,2,3,4-tetrahydrothieno[3,4-d]pyrimidine-5-carboxylic acid choline salt having excellent solubility and storage stability.

Claims

1. A method for preventing or treating a sex hormone-dependent disease in a subject, said method comprising administering to said subject a therapeutically effective amount of a compound represented by the formula: ##STR00003##

2. The method of claim 1, wherein said sex-hormone dependent disease is selected from the group consisting of benign prostatic hypertrophy, hysteromyoma, endometriosis, metrofibroma, precocious puberty, amenorrhea, premenstrual syndrome, dysmenorrhea, polycystic ovary syndrome, lupus erythematosis, hirsutism, short stature, sleep disorders, acne, baldness, Alzheimer's disease, infertility, and irritable bowel syndrome.

3. The method of claim 1, wherein said sex-hormone dependent disease is a cancer.

4. The method of claim 3, wherein said cancer is selected from the group consisting of prostate cancer, ovary cancer, uterine cancer, and breast cancer.

5. The method of claim 1, wherein said compound is administered orally.

6. The method of claim 1, wherein said subject is a human.

7. The method of claim 1, wherein said compound is crystalline.

8. The method of claim 7, wherein said compound exhibits characteristic peaks at diffraction angles (2()) of 7.1, 11.5, 19.4, 20.3, 21.5, 22.0, 22.6, 23.5, and 26.2 in a powder X-ray diffraction diagram.

9. The method of claim 7, wherein said compound is characterized by an X-ray powder diffraction spectrum substantially as depicted in FIG. 1.

10. The method of claim 7, wherein said compound exhibits characteristic peaks at chemical shift values ((ppm)) of 155.8, 149.8, 145.3, 118.0, 113.7, 111.6, 110.3, 98.1, 69.8, 58.7, 57.1, and 55.5 in a .sup.13C solid-state nuclear magnetic resonance (NMR) spectrum.

11. The method of claim 7, wherein said compound is characterized by a .sup.13C solid-state NMR spectrum substantially as depicted in FIG. 3.

12. The method of claim 7, wherein said compound exhibits characteristic peaks at chemical shift values ((ppm)) of 131.6, 145.2, and 151.8 in a .sup.19F solid-state NMR spectrum.

13. The method of claim 7, wherein said compound is characterized by a .sup.19F solid-state NMR spectrum substantially as depicted in FIG. 5.

14. The method of claim 7, wherein said compound exhibits an endotherm at about 213 C. as measured by differential thermal analysis.

15. The method of claim 7, wherein said compound is characterized by a differential thermal analysis curve substantially as depicted in FIG. 2.

16. The method of claim 7, wherein said compound is characterized by a thermogravimetric analysis curve substantially as depicted in FIG. 2.

17. The method of claim 1, said method comprising administering to said subject a pharmaceutical composition comprising said therapeutically effective amount of said compound.

18. The method of claim 17, wherein said pharmaceutical composition is a tablet or capsule.

Description

BEST MODE FOR CARRYING OUT THE INVENTION

[0020] Compound (A) of the present invention can be prepared, for example, using the following method. Specifically, for example, a free compound (B), which can be produced by using the method described in Patent reference 1 or by using methods according to this method, is mixed with an equal amount (1.0 equivalent) or a small excess amount of choline hydroxide in a suitable solvent. The mixture is then dissolved under heat, and the solvent is concentrated or added as appropriate, as required. Compound (A) precipitated upon cooling can then be isolated. Further, compound (A) may be purified by recrystallization using the same or similar solvent.

[0021] The solvent may be any solvent, provided that it does not interfere with salt formation. Examples of usable solvents include organic solvents, including alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol and 2-butanol, ethers such as tetrahydrofuran and diisopropyl ether, and water. A mixed solvent of these also may be used.

[0022] Compound (A) of the present invention is extremely useful as an agent for the prevention or treatment of sex hormone-dependent diseases such as benign prostatic hypertrophy, hysteromyoma, endometriosis, metrofibroma, precocious puberty, amenorrhea, premenstrual syndrome, dysmenorrhea, polycystic ovary syndrome, lupus erythematosis, hirsutism, short stature, sleep disorders, acne, baldness, Alzheimer's disease, infertility, irritable bowel syndrome, prostate cancer, uterine cancer, ovary cancer, breast cancer and pituitary tumor, a reproduction regulator, a contraceptive, an ovulation inducing agent, an agent for the prevention of post-operative recurrence of sex hormone-dependent cancers or the like.

[0023] Compound (A) according to the present invention may be appropriately mixed with a pharmaceutical carrier used conventionally to prepare a pharmaceutical composition. The pharmaceutical carrier may be appropriately in combination according to a dosage form as described below. As the pharmaceutical carrier, for example, excipients such as lactose or the like; lubricants such as magnesium stearate or the like; disintegrators such as carboxymethylcellulose or the like; binders such as hydroxypropylmethylcellulose or the like; surfactants such as Macrogol or the like; foamings such as sodium hydrogene carbonate or the like; dissolving aids such as cyclodextrin or the like; acidities such as citric acid or the like; stabilizers such as sodium edentate or the like; pH adjusters such as phosphoric acid salt or the like can be illustrated.

[0024] Examples of the dosage form of the pharmaceutical composition according to the present invention include orally-administered agents such as powders, granules, fine granules, dry syrups, tablets, capsules and the like; and parenterally-administered agents such as injections, poultices, suppositories and the like, and orally-administered agents are preferable.

[0025] Preferably, the above formulations are prepared in such a way that compound (A) according to the present invention is administered in 0.1 to 1,000 mg per day for adults in the case of orally-administered agents, and in 0.01 to 100 mg per day for adults for injections.

EXAMPLES

[0026] The present invention is further illustrated in more detail by way of the following Examples and Test Examples. However, the present invention is not limited thereto.

Example 1

Compound (A)

[0027] 3-[2-Fluoro-5-(2,3-difluoro-6-methoxybenzyloxy)-4-methoxyphenyl]-2,4-dioxo-1,2, 3,4-tetrahydrothieno[3,4-d]pyrimidine-5-carboxylic acid (3.07 g) and a 46% choline hydroxide aqueous solution (1.64 g) were suspended in a mixed solution of 1-propanol and water (about 1:1 volume ratio; 30 mL), and the mixture was heated and stirred for 15 min at 60 C. 1-Propanol (30 mL) was added to the mixture at 60 C., and the mixture was stirred at room temperature for 1 hour, and for another one hour under ice-cooled conditions. After the precipitate was collected by filtration, and washed twice with 1-propanol (1 mL). The resulting solid was dried under reduced pressure at 40 C. to obtain compound (A) (2.93 g). Further, this compound was heated and stirred at 60 C. in a mixed solvent of 1-propanol and water (about 1:1 volume ratio; 30 mL), and 1-propanol (30 mL) was added to the solution obtained after hot filtration. The mixture was cooled to room temperature, and stirred for 1 hour, and for another one hour under ice-cooled conditions. The precipitated crystals were collected by filtration, and washed twice with a mixed solvent of 1-propanol and water (about 3:1 volume ratio; 1 mL). The resulting crystals were air-dried for 4 days to obtain compound (A) (2.16 g).

[0028] .sup.1H-NMR (DMSO-d.sub.6)((ppm)): 3.10 (9H, s), 3.35-3.45 (2H, m), 3.70-3.90 (8H, m), 4.95 (2H, s), 5.47 (1H, brs), 6.44 (1H, s), 6.85-6.95 (1H, m), 7.05 (1H, d, J=11.5 Hz), 7.11 (1H, d, J=7.4 Hz), 7.48 (1H, dd, J=9.7 Hz, 19.5 Hz), 11.14 (1H, brs)

[0029] The obtained compound (A) was measured with regard to powder X-ray diffraction, thermal analysis, .sup.13C solid-state NMR spectrum and .sup.19F solid-state NMR spectrum under the conditions below to obtain data.

[0030] For the powder X-ray diffraction, the crystals were ground with a mortar, and measured with a powder X-ray diffraction apparatus X'Pert Pro MPD (Spectris plc, PANalytical Department) (reflection method; CuK rays, tube voltage 45 kV, tube current 40 mA).

[0031] The resulting diffraction diagram is shown in FIG. 1, and diffraction angles (2()) and relative intensities (%) of peaks that had relative intensities of about 20% or higher are shown in Table 1.

TABLE-US-00001 TABLE 1 Diffraction angle Relative intensity Diffraction angle Relative intensity (2 ()) (%) (2 ()) (%) 7.1 38 21.0 31 10.4 32 21.5 83 11.5 48 22.0 82 13.9 31 22.6 46 14.1 29 23.2 29 14.3 24 23.5 39 15.5 31 25.1 22 16.1 23 26.2 57 16.4 20 26.7 22 17.4 22 28.3 29 19.0 34 29.6 27 19.4 86 30.1 27 20.0 22 31.2 22 20.3 100

[0032] For reasons related to the nature of data in powder X-ray diffraction, the 20 values and the overall diffraction pattern are important for the recognition of crystal identity. It is common knowledge that the relative intensity in X-ray diffraction patterns fluctuates in a manner that depends on sample conditions and measurement conditions. It should be noted that the 2 values of diffraction patterns in powder X-ray diffraction may slightly fluctuate depending on sample conditions and measurement conditions.

[0033] For the thermal analysis, measurements were made using a thermogravimetric differential thermal analyzer TG-DTA TG8120 (Rigaku Corporation) (Heating rate: 10 C./min; reference material: aluminum oxide). The resulting chart is shown in FIG. 2.

[0034] Endothermic peak: around 213 C.

[0035] Note that the endothermic peak in thermal analysis may slightly fluctuate depending on sample conditions and measurement conditions.

[0036] For the .sup.13C solid-state NMR spectral measurement, a sample was charged into a 4-mm zirconia rotor, and measured with a Bruker Avance DRX500 (rotation speed 10 kHz) using the CP/MAS technique. The resulting spectrum chart is shown in FIG. 3.

[0037] Note that the chemical shift values in the .sup.13C solid-state NMR spectrum may slightly fluctuate depending on sample conditions and measurement conditions.

[0038] For the .sup.19F solid-state NMR spectrum measurement, a sample was charged into a 2.5-mm zirconia rotor, and measured with a Bruker Avance III 400 WideBore (rotation speed 30 kHz) using the MAS technique. The spectrum was observed with reference to the external standard sample polyvinylidene fluoride (PVDF) set to resonate at 91.2 ppm. The resulting spectrum chart is shown in FIG. 5.

[0039] Note that the chemical shift values in the .sup.19F solid-state NMR spectrum may slightly fluctuate depending on sample conditions and measurement conditions.

Example 2

Compound (A)

[0040] 3-[2-Fluoro-5-(2,3-difluoro-6-methoxybenzyloxy)-4-methoxyphenyl]-2,4-dioxo-1,2, 3,4-tetrahydrothieno[3,4-d]pyrimidine-5-carboxylic acid (4.07 g), a 46% choline hydroxide aqueous solution (2.18 g), 2-propanol (200 mL) and water (100 mL) were mixed, and heated for 15 min at 40 C. After removing the insoluble matter by filtration, the resulting solution was concentrated, and 2-propanol (80 mL) and diisopropyl ether (80 mL) were added. The mixture was then stirred at room temperature for about 1 hour, and then for about 4 hours under ice-cooled conditions. The precipitated solid was collected, and dried at 90 C. overnight under reduced pressure (yield 3.59 g). A mixed solution of ethanol and water (about 1:1 volume ratio; 25 mL) was then added to the resulting solid (3.56 g). The mixture was heated to 65 C., and subjected to hot filtration after adding a mixed solution of ethanol and water (1:1 volume ratio; 10 mL) at the same temperature. The resulting solution was stirred while being allowed to cool at room temperature for 2 hours, and further stirred at room temperature for about 1 hour after adding ethanol (20 mL). The mixture was further stirred at room temperature for about 1 hour after adding ethanol (20 mL), and further stirred overnight under ice-cooled conditions. The solid was collected from the mixture, washed with ethanol (5 mL), and dried by blowing nitrogen. The solid was further dried at 40 C. overnight under reduced pressure to obtain compound (A) (2.43 g). The obtained compound (A) was measured for powder X-ray diffraction in the same manner as in Example 1. The result confirmed that the compound (A) had the same crystal form observed in Example 1.

[0041] .sup.1H-NMR (DMSO-d.sub.6)((ppm)): 3.10 (9H, s), 3.35-3.45 (2H, m), 3.70-3.90 (8H, m), 4.96 (2H, s), 5.38 (1H, brs), 6.43 (1H, s), 6.85-6.95 (1H, m), 7.05 (1H, d, J=11.5 Hz), 7.10 (1H, d, J=7.4 Hz), 7.47 (1H, dd, J=9.7 Hz, 19.5 Hz), 11.11 (1H, brs)

Comparative Example 1

Crystals of Compound (B)

[0042] Ethyl acetate (0.1 mL) was added to an amorphous solid of 3-[2-fluoro-5-(2,3-difluoro-6-methoxybenzyloxy)-4-methoxyphenyl]-2,4-dioxo-1,2,3,4-tetrahydrothieno[3,4-d]pyrimidine-5-carboxylic acid (10 mg), and the suspension was heated to 50 C. The mixture was then dried by blowing nitrogen gas. The resulting solid was further heated to 70 C., and dried overnight under reduced pressure to obtain crystals of compound (B) (10 mg). The obtained crystals of compound (B) were measured for powder X-ray diffraction in the same manner as in Example 1. The resulting diffraction diagram is shown in FIG. 4.

Comparative Example 2

Crystals of Compound (B)

[0043] 3-[2-Fluoro-5-(2,3-difluoro-6-methoxybenzyloxy)-4-methoxyphenyl]-2,4-dioxo-1,2, 3,4-tetrahydrothieno[3,4-d]pyrimidine-5-carboxylic acid (0.69 g) was suspended in diisopropyl ether (10 mL), and the mixture was stirred at 60 C. for 3 hours under heat. The mixture was stirred at room temperature overnight, further for 1 hour under ice-cooled conditions. The solid was collected by filtration, and dried under reduced pressure to obtain crystals of compound (B) (0.65 g). The obtained crystals of compound (B) were measured for powder X-ray diffraction in the same manner as in Example 1. The result confirmed that the compound (B) had the same crystal form observed in Comparative Example 1.

Comparative Example 3

3-[2-Fluoro-5-(2,3-difluoro-6-methoxybenzyloxy)-4-methoxyphenyl]-2,4-dioxo-1,2,3,4-tetrahydrothieno[3,4-d]pyrimidine-5-carboxylic acid.1/2N,N-dibenzylethylenediamine salt (Hereinafter, Referred to as Compound (C))

[0044] Acetonitrile (10 mL), N,N-dibenzylethylenediamine (94.5 mg), and 3-[2-fluoro-5-(2,3-difluoro-6-methoxybenzyloxy)-4-methoxyphenyl]-2,4-dioxo-1,2,3,4-tetrahydrothieno[3,4-d]pyrimidine-5-carboxylic acid (200 mg) were mixed. The mixture was suspended at about 60 C., and stirred while being allowed to cool at room temperature. The solid was collected from the mixture by filtration, and dried at about 60 C. overnight under reduced pressure (247 mg).

[0045] .sup.1H-NMR (DMSO-d.sub.6)((ppm)): 2.83 (2H, s), 3.79 (3H, s), 3.80 (3H, s), 3.87 (2H, s), 4.95 (2H, s), 6.85-6.95 (1H, m), 6.98 (1H, s), 7.09 (1H, d, J=11.5 Hz), 7.19 (1H, d, J=7.5 Hz), 7.25-7.55 (6H, m)

Test Example 1

Saturation Solubility Test

[0046] The compound (A) obtained in Example 2, the compound (B) crystals obtained in Comparative Example 2, and the compound (C) obtained in Comparative Example 3 were suspended in water, or in 1st fluid for dissolution test (hereinafter, referred to as 1st fluid) or 2nd fluid (hereinafter, referred to as 2nd fluid) described in the Reagents-Test Solutions for General Tests, the Japanese Pharmacopoeia, 15th Edition. The suspensions were incubated at 37 C. After filtering a part of the suspensions, the concentrations of the resulting filtrates were measured by HPLC, and the saturation solubilities were calculated and compared. The HPLC measurement conditions are as follows.

Measurement Conditions

[0047] Detector: Ultraviolet and visible spectrophotometer/wavelength: 225 nm

Column: GL Science Inertsil ODS-3, 5 m, 4.6250 mm

[0048] Column temperature: a constant temperature of around 35 C.
Flow rate: 1.0 mL/min
Mobile phase A: 10 mM potassium dihydrogen phosphate aqueous solution adjusted to pH 5.5 with potassium hydroxide aqueous solution
Mobile phase B: Acetonitrile
Mobile phase ratio 0 to 30 min: mobile phase A/mobile phase B=70/30

[0049] The saturation solubility values of compound (A), compound (B) crystals, and compound (C) in water, 1st fluid, and 2nd fluid are shown in Table 2. In water, compound (A) had a saturation solubility about 600 times and about 60 times higher than those of compound (B) crystals and compound (C), respectively. The saturation solubility of compound (A) in 1st fluid was about 30 times and about 2 times higher than those of compound (B) crystals and compound (C), respectively. In 2nd fluid, the saturation solubility of compound (A) was about 10 times and about 70 times higher than those of compound (B) crystals and compound (C), respectively. These results thus confirmed significant improvements in the solubility of compound (A) over compound (B) crystals and compound (C).

TABLE-US-00002 TABLE 2 The crystals of the The compound (C) The compound (A) compound (B) obtained in obtained in obtained in Example 2 Comparative Example 2 Comparative Example 3 Water 4165 7 68 1st fluid 4 0.14 2 2nd fluid 3738 367 52 Unit: g/mL

Test Example 2

Assay for Oral Absorbability

1) Preparation of Sample for Drug Concentration Measurement by Administration Through Tail Vein

[0050] SD rats (Charles River, male, 7 weeks of age, 170 to 210 g) were used as experimental animals after fasting the rats overnight. N,N-dimethylacetamide (0.2 mL), saline (0.798 mL) and 2NNaOH (0.002 mL) were added in these amounts with respect to 1 mg of the compound (B) to prepare a 1.0 mg/mL solution. The solution was then administered in a 1 mL/kg dose (1 mg/kg) through the tail vein under no anesthesia (3 samples). The intravenous administration through tail was performed using a 26 G injection needle and a 1 mL syringe. Blood was collected through the subclavian veins after 2, 15, 60, 120, 240 and 360 minutes from the intravenous administration through tail. The blood was centrifuged, and the plasma was used as a sample for blood drug concentration measurement. 2) Preparation of Sample for Drug Concentration Measurement by Oral Administration

[0051] SD rats (Charles River, male, 7 weeks of age, 220 to 290 g) were used as experimental animals after fasting the rats overnight. A 0.5% methylcellulose aqueous solution (5 mL) was added with respect to 3 mg of compound (A) or compound (B) (in terms of a free compound) to prepare a 0.6 mg/mL drug solution. Each solution was orally administered to the rats in a 5 mL/kg dose (3 mg/kg) (3 samples each). The oral administration was performed using a rat sonde and a 2.5-mL syringe. Blood was collected through the subclavian veins under no anesthesia after 15, 30, 60, 120, 240, 360 and 480 minutes from the oral administration. The blood was centrifuged, and the blood plasma was used as a sample for blood drug concentration measurement.

3) Drug Concentration Measurement

[0052] A suitable internal standard substance solution (0.1 mL) was added to the blood plasma (0.025 mL) obtained in 1) and 2) using an ordinary method. Then, acetonitrile (0.875 mL) was added to remove the protein. After centrifugation, the supernatant (0.005 mL) was injected to LC-MS/MS. Blood plasma drug concentration was measured using the LC-MS/MS technique under the conditions below. Note that the standard curve was created by appropriately adding an internal standard substance and a target substance to a blank blood plasma (0.05 mL) using an ordinary method, followed by the foregoing procedures.

LC

Device: Agilent 1100

Column: Capcellpak MGIII 5 m 4.650 mm

[0053] Mobile phase A: 10 mM ammonium acetate aqueous solution
Mobile phase B: Acetonitrile

(Mobile Phase Ratios are Presented in Table 3)

[0054] Column temperature: 40 C.
Flow rate: 0.5 mL/min

MS/MS

Device: API-4000

[0055] Ionization method: ESI (Turbo Ion Spray)

TABLE-US-00003 TABLE 3 Time (minutes) A (%) B (%) 0.0 90 10 3.0 90 10 4.0 10 90 7.0 10 90 7.1 90 10 12.0 90 10

[0056] The bioavailability of the compound (A) was about 59%, and desirable oral absorbability was confirmed. Further, the maximum drug concentration time (Tmax) was 35 min in the compound (A), compared to 200 min in the compound (B), and because the compound (A) was quickly absorbed after the administration, a fast onset of action is expected.

[0057] Bioavailability (%) was calculated from the value of the area under the blood drug concentration-time curve determined by using a WinNonlin Professional (Pharsight Corporation) from the blood drug concentration at each time point obtained as above after the intravenous administration of the compound (B) through tail and the oral administration of the compound (A) or the compound (B).

Test Example 3

Stability Test

[0058] The compound (A) obtained in Example 2 was stored under 90 C. open conditions to examine stability. In stability measurement, the purity of a sample was measured by HPLC at the initial point and after 8 days, and the results were compared. The HPLC measurement conditions are as follows.

Measurement Conditions

[0059] Detector: Ultraviolet and visible spectrophotometer/wavelength: 225 nm

Column: GL Science Inertsil ODS-3, 5 m, 4.6250 mm

[0060] Column temperature: Constant temperature around 35 C.
Flow rate: 1.0 mL/min
Mobile phase A: 10 mM potassium dihydrogen phosphate aqueous solution adjusted to pH 5.5 with potassium hydroxide aqueous solution
Mobile phase B: Acetonitrile

(Mobile Phase Ratios are Shown in Table 4)

[0061] Area measurement range: 54 minutes from the start of the analysis. Areas of the peaks in the blank were excluded from the calculations.

[0062] The measurement results are shown in Table 5.

TABLE-US-00004 TABLE 4 Time (minutes) A (%) B (%) 0 70 30 20 70 30 40 30 70 60 30 70 60.1 70 30 80 70 30

TABLE-US-00005 TABLE 5 The compound (A) obtained in Example 2 Measurement point Initial point After 8 days Purity (%) 99.6 99.6

[0063] As described above, the results of Test Examples 1 to 3 show that the compound (A) of the present invention has excellent solubility, oral absorbability and storage stability, and thus represents an excellent compound capable of solving the problems relating to the physical properties of the free compound (B).

INDUSTRIAL APPLICABILITY

[0064] The compound (A) according to the present invention has excellent solubility and other desirable physical properties, and is useful as drug material, and suited for the industrial production of drugs.

BRIEF DESCRIPTION OF THE DRAWINGS

[0065] FIG. 1 is a powder X-ray diffraction diagram of compound (A) obtained in Example 1. The vertical axis shows X-ray diffraction intensity (Counts); and the horizontal axis shows diffraction angle (2()).

[0066] FIG. 2 is a diagram representing the TG-DTA measurement of compound (A) obtained in Example 1. The vertical axis (left) shows weight (%) in a thermogravimetric (TG) curve; the vertical axis (right) shows heat flux (v) in a differential thermal analysis (DTA) curve; and the horizontal axis shows temperature ( C.).

[0067] FIG. 3 is a .sup.13C solid-state NMR spectrum chart of compound (A) obtained in Example 1. The vertical axis shows intensity; and the horizontal axis shows chemical shift value ((ppm)).

[0068] FIG. 4 is a powder X-ray diffraction diagram of compound (B) obtained in Comparative Example 1. The vertical axis shows X-ray diffraction intensity (Counts); and the horizontal axis shows diffraction angle (2()).

[0069] FIG. 5 is a .sup.19F solid-state NMR spectrum chart of compound (A) obtained in Example 1. The vertical axis shows intensity; and the horizontal axis shows chemical shift value ((ppm)).