JPH0635469B2 - Diethylenetriamine triacetic acid compound, production intermediate thereof and production method thereof - Google Patents

Description

TECHNICAL FIELD The present invention relates to a diethylenetriaminetriacetic acid compound,
More specifically, it relates to an ursodeoxycholyldiethylenetriaminetriacetic acid compound having a gallstone dissolving action, an intermediate for producing the same, and a method for producing the same.

BACKGROUND ART Ursodeoxycholic acid and chenodeoxycholic acid are known as drugs commonly used as therapeutic agents for gallstones.

Further, in JP-A-60-161996, a compound in which ursodeoxycholic acid or chenodeoxycholic acid and aspartic acid, glutamic acid, serine or carboxymethylglycine are amide-bonded dissolves outer shell calcified cholesterol gallstones. It has been reported to be effective.

However, ursodeoxycholic acid and chenodeoxycholic acid are effective only for pure cholesterol stones, and other cholesterol gallstones such as calcium-containing cholesterol mixed stones or cholesterol mixed stones, and Is suspected of its dissolving effect on bilirubin calcium stone, calcium carbonate stone, and the like.

On the other hand, the compound described in JP-A-60-161996 discloses N-ursodeoxycholyl-N-, which is said to have the highest dissolution effect on cholesterol gallstones with calcified outer shell.
Even with carboxymethylglycine (tentatively referred to as “compound A” hereinafter), it is only about 2 to 3 times that of glycochenodeoxycholic acid, which is a typical compound present in the living body.

As a result of earnest studies on bile acid derivatives, the present inventors have found that the ursodeoxycholyldiethylenetriaminetriacetate compound strongly dissolves calcium-containing gallstones, particularly calcium carbonate-containing gallstones in bile, and arrived at the present invention. .

Means for Solving the Problems According to the present invention, the following structural formula [I] And a physiologically acceptable salt thereof, a production intermediate thereof, and a production method thereof.

In the present invention, physiologically acceptable salts are mono,
A di- or tri-alkali salt, a mono- or di-mineral salt or a triammonium salt. Examples of the alkali salt include sodium salt and potassium salt, and examples of the mineral acid salt include hydrochloride, sulfate and nitrate.

The diethylenetriaminetriacetic acid compound of the above structural formula [I] has the following structural formula [II] And a halogenated acetic acid represented by the general formula [III] X-CH ₂ COOH [III] (wherein X represents chlorine, bromine or iodine) in the presence of a base. Can be manufactured.

The reaction ratio is such that the halogenated acetic acid [III] is 3 to 10 times the molar amount of the triamine compound [II]. Water is suitable as the reaction solvent. Examples of the halogenated acetic acid [III] include chloroacetic acid, bromoacetic acid and iodoacetic acid, and chloroacetic acid or bromoacetic acid is preferable. Examples of the base used include sodium hydrogen carbonate, sodium carbonate, potassium hydroxide and the like. The reaction temperature is 30-
The temperature is 95 ° C, preferably 40 to 60 ° C, and the reaction time is 1 to 48 hours, preferably 5 to 24 hours.

Diethylenetriamine triacetic acid compound [I] is prepared by using a basic solution at the end of the reaction with a mineral acid such as hydrochloric acid or sulfuric acid.
It can be separated in the form of the free acid by adjusting the pH to about 2.5. The obtained free acid is purified by subjecting it to column chromatography, if necessary.

The physiologically acceptable salt of diethylenetriaminetriacetate compound [I] is obtained by adding diethylenetriaminetriacetate compound [I] or an aqueous solution thereof to an excess amount of aqueous ammonia,
It can be produced by adding 1, 2 or 3 times equivalent of alkali or 1 or 2 times equivalent of mineral acid, and then drying this under reduced pressure to dryness. Examples of the alkali used include sodium hydrogen carbonate, potassium carbonate, sodium hydroxide or potassium hydroxide, and examples of the mineral acid include hydrochloric acid, sulfuric acid or nitric acid.

The above triamine compound [II] has the following general formula [IV] (In the formula, R ¹ represents a hydrogen atom, a benzyloxycarbonyl group or a t-butyloxycarbonyl group, and R 1
² represents a benzyloxycarbonyl group, a t-butyloxycarbonyl group or a trityl group. ) Is produced by catalytic reduction or hydrolysis with an acid (hereinafter referred to as "first method"), or by the following general formula [V] (In the formula, R ³ represents a linear or branched alkyl group having 1 to 4 carbon atoms.) By condensing the mixed acid anhydride, the mixed acid anhydride, and 10 to 50 times the molar amount of diethylenetriamine. It can be manufactured (hereinafter referred to as "second method").

For the catalytic reduction in the first method, a commonly used method can be adopted. Examples of the acid used for hydrolysis include hydrochloric acid, acetic acid, or a mixed solution thereof.

The reaction solvent in the second method is suitably tetrahydrofuran, dioxane, methanol, chloroform or water, or a mixed solution of two or more thereof. The reaction temperature is −30 to 20 ° C., preferably −10 to 10 ° C., and the reaction time is 10 minutes to 48 hours, preferably 1
Approximately 12 hours.

The triamine compound [II] can be produced by the above-mentioned first method or second method, but the first method is adopted in consideration of the purity of the obtained triamine compound [II] and the treatment in the next step. Is desirable.

The amide compound [IV] used as the raw material in the first method is the mixed acid anhydride represented by the general formula [V] and the following general formula [VI]. (In the formula, R ¹ and R ² have the same meanings as described above.) And can be produced by condensation.

The reaction ratio is such that the compound [VI] is 0.5 to 2 times the molar amount of the mixed acid anhydride [V]. Suitable reaction solvent is tetrahydrofuran, dioxane, methanol, chloroform or water, or a mixed solution of two or more of these. The reaction temperature is −30 to 20 ° C., preferably −10 to
Within the range of 10 ° C, the reaction time is 10 minutes to 48 hours,
It is preferably about 1 to 12 hours.

The compound [VI] uses diethylenetriamine and benzyloxycarbonyl chloride, di-t-butyl-dicarbonate or trityl chloride, and a general method for introducing an amino protecting group (the basics of peptide synthesis and experiments 17 to 39).
Page published on Mar. 20, 1985, issued by Maruzen Co., Ltd.).

The mixed acid anhydride [V] used as the starting material and the starting material for the amide compound [IV] in the second method can be produced by reacting ursodeoxycholic acid and alkyl chloroformate in the presence of an acid acceptor.

The reaction ratio is such that the alkyl chloroformate is approximately equimolar to ursodeoxycholic acid. Examples of the alkyl chloroformate used include ethyl chloroformate or isobutyl chloroformate. Acid acceptors include triethylamine, tributylamine or N-methylmorpholine. Dioxane or tetrahydrofuran is suitable as the reaction solvent. The reaction temperature is −20 to 10 ° C., and the reaction time is 1 minute to 3 hours. This reaction proceeds almost quantitatively and the obtained mixed acid anhydride [V] is unstable. Therefore, the reaction mixture is used as it is in the next step without isolation.

Action The dissolving action of the diethylenetriamine triacetic acid compound [I] on calcium carbonate-containing gallstones will be described in detail below. The lytic effect was evaluated by the ability to dissolve calcium carbonate (CaCO ₃ ) in bile.

The test was carried out by adding an excessive amount of calcium carbonate to 2 m of the artificial bile model, incubating at 37 ° C. for 18 hours in a tightly closed cap, and then centrifuging this at 3000 rpm to determine the amount of calcium carbonate dissolved in the obtained supernatant by atomic absorption. It was performed by measuring with a meter. The artificial bile model had a diethylenetriaminetriacetate compound [I] of 16 mM, lecithin of 8 mM and cholesterol of 4 mM.
It was prepared by dissolving in 1M phosphate buffer (pH 7.4 and 8.3). The diethylenetriamine triacetic acid compound [I] was treated in the same manner as described above except that glycochenodeoxycholic acid or compound A was changed, and the calcium carbonate-dissolving ability of these two compounds was tested for comparison. The results are shown in the table below.
Incidentally, in the table, the values in parentheses are those in JP-A-60-1619.
It is described in Japanese Patent Publication No. 96, and is also shown for reference.

As is clear from the above table, it is recognized that the diethylenetriaminetriacetic acid compound [I] of the present invention has far superior calcium carbonate-solubilizing ability as compared with glycochenodeoxycholic acid and compound A.

The present invention will be further described with reference to examples and examples.

Reference Example 1 (Compound [VI]) 215 m (1.99 mol) of diethylenetriamine was dissolved in 200 m of chloroform, and 111.7 g (0.401 mol) of trityl chloride was dissolved in 8 ml of chloroform.
The solution dissolved in 60 m was added dropwise under ice cooling, and the mixture was stirred overnight at room temperature. The reaction solution was washed with water, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The obtained residue was subjected to alumina column chromatography using a chloroform-methanol mixed solution (volume ratio 5: 1) as a developing solution to obtain 124.2 g of oily N-trityldiethylenetriamine. The yield based on trityl chloride is 89.
It was 7%.

Reference Example 2 (Compound [VI]) Diethylenetriamine 21.5 m (199.0 mmol) was dissolved in 40 m of chloroform, and 35% benzyloxycarbonyl chloride toluene solution 4.8 was dissolved therein.
A solution of 5 m (10.0 mmol) dissolved in 50 m of chloroform was added dropwise under ice cooling, and the mixture was stirred overnight at room temperature.
The reaction solution was washed with water and dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The obtained residue was subjected to alumina column chromatography using a chloroform-methanol mixed solution (volume ratio 20: 1) as a developing solution to obtain oily N, N '.
-0.56 g of dibenzyloxycarbonyldiethylenetriamine was obtained. The yield based on benzyloxycarbonyl chloride was 30.3%. Next, the developing solution was mixed with a chloroform-methanol mixture (volume ratio 5:
Alumina column layer was eluted by changing to 1) and oily N-
Benzyloxycarbonyldiethylenetriamine 0.2
7g was also obtained. The yield based on benzyloxycarbonyl chloride was 11.4%.

Reference Example 3 (Compound [VI]) Diethylenetriamine 21.5 m (199.0 mmol) was mixed with dioxane-water mixture (volume ratio 2: 1) 40 m
To the solution dissolved in was added an aqueous 1N sodium hydroxide solution (10 m) and di-t-butyl-dicarbonal (2.4 g, 11.0 mmol) under ice-cooling stirring, and the mixture was stirred at room temperature for 30 minutes. The reaction solution was extracted with chloroform, the chloroform layer was washed with water and dried, and then the solvent was distilled off under reduced pressure. The obtained residue was subjected to alumina column chromatography using a chloroform-methanol mixed solution (volume ratio 20: 1) as a developing solution, to obtain 0.46 g of oily N, N'-di-t-butyloxycarbonyldiethylenetriamine.
Got The yield based on di-t-butyl-disicarbonate was 27.6%.

Reference Example 4 (mixed acid anhydride [V]) In a mixed liquid of 11.8 g (30.1 mmol) of ursodeoxycholic acid, 50 m of tetrahydrofuran and 4.30 m (30.8 mmol) of triethylamine, -4
Isobutyl chloroformate 4.00 m (30.
(8 mmol) was added dropwise, and the mixture was stirred at -2 to 1 ° C for 2 hours, and isobutyl ursodeoxycholyl carbonate 14.8 was added.
A reaction mixture containing g (quantitative yield) was obtained.

Reference Example 5 (mixed acid anhydride [V]) 5.0 g (12.7 mmol) of ursodeoxycholic acid, 20 m of dioxane and 3.6 of tributylamine.
To a mixed solution of m (15.1 mmol), 1.4 m (14.7 mmol) of ethyl chloroformate was added dropwise at 5 to 10 ° C., and the mixture was stirred at the same temperature for 15 minutes, and ethyl ursodeoxycholyl carbonate 5.9 g ( A reaction mixture containing (quantitative yield) was obtained.

Example 1 (Amide Compound [IV]) Isobutyl ursodeoxycholyl carbonate 14.8 g (30.1 mmol) was added to a solution of 11.5 g (33.3 mmol) of N-trityldiethylenetriamine dissolved in 40 m of tetrahydrofuran by -6. The mixture was added at ˜0 ° C., and further stirred at −8 to −3 ° C. for 1 hour. The solvent of the obtained reaction solution was distilled off under reduced pressure, and the residue was subjected to alumina column chromatography using a chloroform-methanol mixed solution (volume ratio 50: 1) as a developing solution to obtain N-trityl-N ″.
-15.1 g (yield 69.8%) of white crystals of ursodeoxycholyldiethylenetriamine were obtained.

Melting point: 113 to 117 ° C. Infrared absorption spectrum (KBr, cm ⁻¹ ): 1645, 1550 Example 2 (amide compound [IV]) N, N′-dibenzyloxycarbonyl instead of 11.5 g of N-trityldiethylenetriamine Except that 12.4 g (33.3 mmol) of diethylenetriamine was used and treated in substantially the same manner as in Example 1, 11.1 g of N, N′-dibenzyloxycarbonyl-N ″ -ursodeoxycholyldiethylenetriamine glass-like substance was obtained. (Yield 4
9.5%).

Example 3 (amide compound [IV]) 11.5 g of N-trityldiethylenetriamine was added to N,
N'-Di-t-butyloxycarbonyldiethylenetriamine was treated in substantially the same manner as in Example 1 except that 10.1 g (33.3 mmol) was used, and N, N'-di-t-
Butyloxycarbonyl-N "-ursodeoxycholyldiethylenetriamine glassy material 6.3 g (yield 3
0.9%) was obtained.

Example 4 (triamine compound [II]) 3.08 g (4.28 mmol) of N-trityl-N "-ursodeoxycholyldiethylenetriamine was added to acetic acid 15
m, water (5 m) was added, and the mixture was stirred at 36 to 40 ° C for 1.2 hr. After cooling, the precipitate was filtered off, 140 m of water was added to the filtrate, the pH was adjusted to 11 with a 17% (W / V) sodium hydroxide aqueous solution, and then this was extracted with n-butanol. The n-butanol layer was washed with water, dried over anhydrous sodium sulfate, and then dried under reduced pressure to obtain N ″ -ursodeoxycholyldiethylenetriamine colorless glassy substance 2.
04 g (yield quantitative) was obtained.

Infrared absorption spectrum (KBr, cm ^-1 ); 1640, 1550 Nuclear magnetic resonance spectrum (DMSO-d ₆ ) δ; 0.61 (3H, s), 0.87 (3H, s), 0.8
8 (3H, d), 0.90 to 2.20 (26H, m),
2.42 to 2.60 (6H, m), 3.09 (2H,
q, J = 6Hz), 3.10 to 3.56 (5H, m),
3.90 (1H, br), 4.40 (1H, br),
7.86 (1 H, t, J = 6 Hz) Example 5 (triamine compound [II]) N, N′-dibenzyloxycarbonyl-N ″ -ursodeoxycholyldiethylenetriamine 3.2 g (4.2)
(8 mmol) 5m of methanol, 2m of acetic acid and 3m of water.
It was dissolved in the mixed solution of m. 30 mg of this dissolved palladium black
Was added and catalytically reduced at room temperature and atmospheric pressure for 5 hours. Then, the catalyst was filtered off, 140 m of water was added to the filtrate, and the same treatment as in Example 4 was performed below to obtain 1.92 g (yield 94.2%) of a colorless glassy substance of N ″ -ursodeoxycholyldiethylenetriamine. The infrared absorption spectrum and nuclear magnetic resonance spectrum of this material were in agreement with those described in Example 4.

Example 6 (triamine compound [II]) N, N'-di-t-butyloxycal-N "-ursodeoxycholyldiethylenetriamine 2.9 g (4.28)
Mmol) was dissolved in 20 m of a 4N hydrochloric acid-dioxane mixed solution (volume ratio 1: 1), and the mixture was stirred at room temperature overnight. To this reaction solution, 140 m of water was added and treated in substantially the same manner as in Example 4 to obtain 1.88 g of a colorless glassy substance of N ″ -ursodeoxycholyldiethylenetriamine (yield 92.0).
%) Was obtained. The infrared absorption spectrum and nuclear magnetic resonance spectrum of this material were in agreement with those described in Example 4.

Example 7 (triamine compound [II]) A small amount of a mixed solution of 20 m of dioxane and 5.9 g (12.7 mmol) of ethyl ursodeoxycholyl carbonate in 25.2 m (233.3 mmol) of diethylenetriamine at 5 to 10 ° C. Each of them was added dropwise and stirred at the same temperature for 3 hours. The reaction solution was poured into water and extracted with ethyl acetate. The ethyl acetate layer was washed with saturated saline and dried over anhydrous sodium sulfate, and then the solvent was distilled off to obtain N ″.
3.6 g of a viscous substance of ursodeoxycholyldiethylenetriamine were obtained. The purity of this material was 68%.

Example 8 (diethylenetriaminetriacetic acid compound [I]) Butylacetic acid 2.43 g (17.5 mmol) was added to water (15 m).
And was adjusted to pH 7.2 with 8% aqueous sodium carbonate solution. This adjustment liquid is N "at 50 ° C.
-Ursodeoxycholyldiethylenetriamine 1.60
g (3.35 mmol) in 15 m of water. Then, at 50 ° C with stirring, while adding 8% sodium carbonate aqueous solution dropwise to this mixed solution, the pH was first taken for 1.5 hours.
Was adjusted to 7.5-8.5 and the final pH was taken for another 12 hours.
Was adjusted to 8.0 to 8.5. Cool the reaction mixture to 1
It was adjusted to pH 2.5 with normal hydrochloric acid and extracted with n-butanol. The extract was washed with saturated saline and dried over anhydrous sodium sulfate, and then the solvent was distilled off. The residue was subjected to silica gel column chromatography using a mixture solution of ethanol-28% ammonia water (volume ratio 9: 1) as a developing solution,
The eluate was dried under reduced pressure. 20 m of water was added to the obtained residue, and the pH was adjusted to 2.5 with 1N hydrochloric acid. The resulting precipitate was collected by filtration and dried under reduced pressure to obtain N ″ -ursodeoxycholyldiethylenetriamine-N, N, N′-triacetic acid colorless powder 1.
05 g (yield 48.1%) was obtained.

Melting point; 137 to 140 ° C. Infrared absorption spectrum (KBr, cm ⁻¹ ); 2700 to 2300, 1730, 1640, 1545 Elemental analysis value (as C ₃₄ H ₅₇ N ₃ O ₉ ); Theoretical value (%) C, 62.65 H, 8.81 N, 6.45 Actual value (%) 62.59 8.78 6.40 Example 9 (diethylenetriaminetriaminetriacetic acid compound [I]) Using 3.6 g of the viscous substance of N ″ -ursodeoxycholyldiethylenetriamine obtained in Example 7. , Except that the conditions were slightly changed, the process was performed in substantially the same manner as in Example 8 and N ″ −
Ursodeoxycholyldiethylenetriamine-N, N,
0.86 g of N'-triacetic acid colorless powder (yield 17.5%)
Got The melting point and infrared absorption spectrum of this powder were in agreement with those described in Example 8.

Elemental analysis value (as C ₃₄ H ₅₇ N ₃ O ₉ ); theoretical value (%) C, 62.65H, 8.81N, 6.45 actual measurement value (%) 62.55 8.84 6.40 Example 10 (diethylenetriaminetriacetic acid compound [I]
Physiologically acceptable salt of) The eluate obtained by the silica gel column chromatography of Example 8 was concentrated, and an appropriate amount of toluene was added thereto,
The water in the concentrate was removed azeotropically and then dried. Then, the residue was dissolved in a small amount of methanol, and an appropriate amount of ethyl acetate was added to the residue for crystallization to crystallize N ″ -ursodeoxycholyldiethylenetriamine-N, N, N′-triacetic acid triammonium salt (1.30 g, yield). 55.3%).

Infrared absorption spectrum (KBr, cm ⁻¹ ); 1595,1400 Elemental analysis value (as C ₃₄ H ₆₆ N ₆ O ₉ ); Theoretical value (%) C, 58.10H, 9.46N, 11.96 Measured value (%) 58.38 9.47 11.89 Effect of the Invention The diethylenetriaminetriacetic acid compound [I] and its physiologically acceptable salt of the present invention can be used as a solubilizer for various gallstones containing calcium.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-60-161996 (JP, A) Pharmaceutical Research Vol. 38 No. 12 P. 409 ~ 421 (1967)

Claims (7)

[Claims] 1. A formula A diethylenetriaminetriacetic acid compound represented by and a physiologically acceptable salt thereof. 2. A formula Triamine compound (wherein, X is chlorine, bromine or iodine.) In formula X-CH ₂ COOH, shown in the halide acid represented by, characterized by reacting in the presence of a base formula And a physiologically acceptable salt thereof. 3. A formula The triamine compound represented by. 4. A general formula (In the formula, R ¹ represents a hydrogen atom, a benzyloxycarbonyl group or a t-butyloxycarbonyl group, and R 1
² represents a benzyloxycarbonyl group, a t-butyloxycarbonyl group or a trityl group. ) Catalytic reduction or hydrolysis with an acid of an amide compound represented by the formula The manufacturing method of the triamine compound shown by. 5. A general formula (In the formula, R ³ represents a linear or branched alkyl group having 1 to 4 carbon atoms) and 10 to 50
Formula characterized by condensing with a double molar amount of diethylenetriamine The manufacturing method of the triamine compound shown by. 6. A general formula (In the formula, R ¹ represents a hydrogen atom, a benzyloxycarbonyl group or a t-butyloxycarbonyl group, and R 1
² represents a benzyloxycarbonyl group, a t-butyloxycarbonyl group or a trityl group. The amide compound shown by these. 7. General formula (In the formula, R ³ represents a linear or branched alkyl group having 1 to 4 carbon atoms) and a general formula (In the formula, R ¹ represents a hydrogen atom, a benzyloxycarbonyl group or a t-butyloxycarbonyl group, and R 1
² represents a benzyloxycarbonyl group, a t-butyloxycarbonyl group or a trityl group. ) A general formula characterized by being condensed with a compound represented by (In the formula, R ¹ and R ² have the same meanings as described above.) A method for producing an amide compound.