JP2978590B2 - Ethylenediamine diacetate compound and production method thereof - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an ethylenediamine diacetate compound, and more particularly to a ursodeoxycholylethylenediamine diacetate compound having a gallstone dissolving action and a method for producing the same.

[0002]

2. Description of the Related Art Ursodeoxycholic acid is known as a drug widely used as a therapeutic agent for gallstones.

Further, JP-A-60-161996 discloses that a compound obtained by amide bonding ursodeoxycholic acid or chenodeoxycholic acid with aspartic acid, glutamic acid, serine or carboxymethylglycine is a cholesterol-based gallstone in which the outer shell is calcified. Is reported to have the effect of dissolving

[0004]

However, ursodeoxycholic acid and chenodeoxycholic acid are effective only on pure cholesterol stone, and other cholesterol gallstones, for example, cholesterol mixed stone containing calcium or cholesterol mixed stone. Further, for bilirubin calcium stone or calcium carbonate stone, etc.,
Its dissolution effect has been questioned.

On the other hand, the compound disclosed in Japanese Patent Application Laid-Open No. 60-161996 can be used to produce N-ursodeoxycorylol N-carboxymethylglycine, which is considered to have the highest dissolving effect on outer shell calcified cholesterol gallstones. Is only about 2 to 3 times that of glycochenodeoxycholic acid, which is a typical compound existing in the above.

The present inventors have conducted intensive studies on bile acid derivatives. As a result, they have found that ursodeoxycholylethylenediamine diacetate strongly dissolves calcium-containing gallstones, particularly calcium carbonate-containing gallstones, in bile. Reached.

[0007]

According to the present invention, the following structural formula

[0008]

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There is provided a ursodeoxycholylethylenediamine diacetate compound (hereinafter referred to as "compound I"), a physiologically acceptable salt thereof, and a process for producing the same.

In the present invention, the physiologically acceptable salt means a mono-, di- or tri-alkali salt, a mono- or di-mineral acid salt or a triammonium salt. Examples of the alkali salts include sodium salts and potassium salts, and examples of the mineral salts include hydrochlorides, sulfates, and nitrates.

Compound [I] can be produced through the following reaction steps.

First, the following general formula

[0013]

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(Wherein R ¹ represents a linear or branched alkyl group having 1 to 4 carbon atoms) (hereinafter referred to as “compound [II]”) and the following general formula:

[0015]

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(Wherein R ² represents a hydrogen atom, a benzyloxycarbonyl group, a t-butyloxycarbonyl group or a trityl group) (hereinafter referred to as “compound [III]”). General formula

[0017]

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(Wherein R ² has the same meaning as described above) (hereinafter referred to as “compound [IV]”) by catalytic reduction or hydrolysis with an acid (hereinafter referred to as “first method”). ) Or a method of directly condensing compound [II] with ethylenediamine (hereinafter referred to as “method 2”).

[0019]

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(Hereinafter referred to as "compound [V]").

The reaction ratio of the compound [II] to the compound [III] in the first method is 0.5 to 2 times the molar amount of the compound [III] to the compound [II]. As the reaction solvent, tetrahydrofuran, dioxane, methanol, chloroform, water, or a mixed solution containing two or more of these is appropriate. The reaction temperature is in the range of −30 to 20 ° C., preferably −10 to 10 ° C., and the reaction time is 10 to 10 ° C.
Minutes to 48 hours, preferably about 1 to 12 hours.

The compound [II] as a raw material can be produced by reacting ursodeoxycholic acid with an 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 to be used include ethyl chloroformate and isobutyl chloroformate. Examples of the acid acceptor include triethylamine, tributylamine and N-methylmorpholine. As a reaction solvent, dioxane or tetrahydrofuran is appropriate. The reaction temperature is-
The temperature is 20 to 10 ° C., and the reaction time is 1 minute to 3 hours.
This reaction proceeds almost quantitatively, and the obtained compound [II] is unstable. Therefore, the reaction mixture is used in the next step without isolation, without isolation.

Similarly, the compound [II
I] is a general method for introducing an amino-protecting group using ethylenediamine and benzyloxycarbonyl chloride, di-t-butyl-dicarbonate or trityl chloride (Basics of peptide synthesis and experiments, pages 17 to 39, Showa 60)
(January 20, Maruzen Co., Ltd.).

For the catalytic reduction in the first method, a commonly used method can be employed. Examples of the acid used in the hydrolysis include hydrochloric acid, acetic acid and a mixture thereof.

The reaction solvent in the second method is suitably tetrahydrofuran, dioxane, methanol, chloroform or water, or a mixed solution comprising two or more of these. The reaction temperature is from -30 to 20C, preferably from 110 to
The reaction temperature is in the range of 10 ° C., and the reaction time is 10 minutes to 48 hours, preferably about 1 to 12 hours.

The compound [V] can be produced by the first method or the second method described above. However, in consideration of the purity of the obtained triamine compound [V] and the treatment in the next step, the first method can be used. It is preferable to employ it.

Next, compound [V] is represented by the following general formula

[0029]

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(Wherein, X represents chlorine, bromine or iodine). The compound [I] is reacted with a halogenated acetic acid (hereinafter referred to as "compound [VI]") in the presence of a base. can get.

The reaction ratio is 3 to 10 times the molar amount of the compound [VI] with respect to the compound [V]. The reaction solvent is suitably water. Examples of the base used include sodium hydrogen carbonate, sodium carbonate, potassium hydroxide and the like. The reaction temperature is 30 to 95 ° C, preferably 40 to 60 ° C.
C., and the reaction time is 1 to 48 hours, preferably 5 to 24 hours.

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

The physiologically acceptable salt of the compound [I] is obtained by adding an excess amount of aqueous ammonia, 1, 2 or 3 equivalents of an alkali or 1 or 2 equivalents of a mineral acid to the compound [I] or an aqueous solution thereof. , Followed by drying under reduced pressure. Examples of the alkali used include sodium hydrogencarbonate, potassium carbonate, sodium hydroxide and potassium hydroxide, and examples of the mineral acid include hydrochloric acid, sulfuric acid and nitric acid.

[0034]

The dissolving effect of the ethylenediamine diacetate compound on calcium carbonate-containing gallstones will be described in detail below. The dissolution effect was evaluated by the ability to dissolve calcium carbonate (CaCO ₃ ) in bile.

In the test, an excess amount of calcium carbonate was added to 2 ml of the artificial bile model, and the mixture was incubated at 37 ° C. for 18 hours in a sealed stopper. The mixture was centrifuged at 3000 rpm, and the amount of calcium carbonate dissolved in the resulting supernatant was measured. Was measured by an atomic absorption spectrometer. It was prepared by dissolving in a 0.01 M phosphate buffer (pH 7.4 and 8.3) so that the diethylenetriamine triacetic acid compound became 16 mM, lecithin 8 mM, and cholesterol 4 mM.

The ethylenediamine diacetate compound is converted to glycochenodeoxycholic acid or N-ursodeoxycholyl-N
-The same treatment was carried out as described above except that carboxymethylglycine was changed, and the calcium carbonate dissolving ability of these two compounds was tested for comparison.

The results are shown in the following table. The values in parentheses in the table are those described in JP-A-60-161996 and are also shown for reference.

[0038]

[Table 1]

As is clear from the above table, the ethylenediamine diacetate compound of the present invention has much higher calcium carbonate dissolving ability than glycochenodeoxycholic acid and N-ursodeoxycholyl-N-carboxymethylglycine. Is admitted.

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

[0041]

Reference Example 1 (Compound [II]) Ursodeoxycholic acid 11.8 g (30.1 mmol), tetrahydrofuran 50 ml and triethylamine 4.30 ml (30.
4.0 mmol (30.8 mmol) of isobutyl chloroformate was added dropwise to the mixture at -4 to -1 ° C.
The mixture was stirred at −2 to 1 ° C. for 2 hours to obtain a reaction mixture containing 14.8 g of isobutyl ursodeoxycholyl carbonate (the yield was quantitative).

[0042]

Reference Example 2 (Compound [III]) Ethylenediamine 1
33 ml (1.99 mol) was dissolved in 200 ml of chloroform, and 111.7 g of trityl chloride (0.
(401 mol) dissolved in 860 ml of chloroform 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 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: 5: 1) as a developing solution to obtain 108.9 g of oily N-tritylethylenediamine. The yield based on trityl chloride was 89.9%.

[0043]

Example 1 (Compound [IV]) tetrahydrofuran 4
0 ml of N-tritylethylenediamine 10.1 g (3
(3.3 mmol) was dissolved in 14.8 g (30.1 mmol) of isobutyl ursodeoxycholyl carbonate at −6 to 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 a chloroform-methanol mixed solution (volume ratio: 50:50).
The mixture was subjected to alumina column chromatography using 1) as a developing solution to give 14.1 g of a glassy substance of N-trityl-N'-ursodeoxycholylethylenediamine (yield 69.
5%).

(Compound [V]) 2.89 g of N-trityl-N'-ursodeoxycholylethylenediamine (4.
28 mmol) was dissolved in 15 ml of acetic acid, and
and stirred at 36 to 40 ° C. for 1.2 hours. After cooling, the precipitate was filtered, 140 ml of water was added to the filtrate, and 17% (W
/ V) The pH was adjusted to 11 with an aqueous sodium hydroxide solution, and then 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 1.83 g (yield 98.5%) of a colorless glassy substance of N′-ursodeoxycholylethylenediamine.

(Compound [I]) 2.43 g of bromoacetic acid
(17.5 mmol) was dissolved in 15 ml of water, and the solution was adjusted to pH 7.2 using an 8% aqueous sodium carbonate solution. At 50 ° C., 1.45 g (3.35 mmol) of N′-ursodeoxycholylethylenediamine was prepared.
Was added to 15 ml of water containing Then 50 ℃ under stirring
Then, while dropwise adding an 8% aqueous sodium carbonate solution to this mixture, the pH was first adjusted to 7.5 to 8.5 over 1.5 hours, and the final pH was further increased to 8.0 to 8.0 over 12 hours. Adjusted to 5. The reaction solution was cooled and adjusted to pH 2.
5, and extracted with n-butanol. The extract was washed with brine, dried over anhydrous sodium sulfate, and the solvent was distilled off. The residue was subjected to siligal column chromatography using a mixed solution of ethanol-28% aqueous ammonia (volume ratio 9: 1) as a developing solution, and the eluate was dried under reduced pressure. 20 ml of water was added to the obtained residue, and the pH was adjusted to 2.5 with 1N hydrochloric acid. The resulting precipitate is collected by filtration and dried under reduced pressure to give N'-ursodeoxycholylethylenediamine-
0.87 g of a colorless powder of N, N-diacetic acid (yield 49.5)
%).

[0046]

[0047]

Example 2 (Compound [V]) dioxane 20 ml and isobutyl ursodeoxycholyl carbonate 6.2
g (12.7 mmol), a mixed solution was added dropwise to 17.2 ml (233.3 mmol) of ethylenediamine at 5 to 10 ° C., and the mixture was stirred at the same temperature for 3 hours. The reaction was poured into water and extracted with ethyl acetate. The ethyl acetate layer was washed with saturated saline, dried over anhydrous sodium sulfate, and then the solvent was distilled off to obtain 3.3 g of a viscous substance of N'-ursodeoxycholylethylenediamine. The purity of this material was 68%.

(Compound [I]) The same treatment as in Example 1 was carried out except that 3.3 g of the obtained viscous substance of N'-ursodeoxycholylethylenediamine was used and the conditions were slightly changed. -Colorless powder of ursodeoxycholylethylenediamine-N, N-diacetic acid 0.68 (yield 17.2)
%). Melting point, IR spectrum and ¹ H
-The NMR spectrum was consistent with that described in Example 1.

[0049]

Example 3 (Physiologically acceptable salt of compound [I]) The eluate obtained by silica gel column chromatography in Example 1 was concentrated, and an appropriate amount of toluene was added thereto. Was azeotropically removed and then dried. Next, the residue was dissolved in a small amount of methanol, and an appropriate amount of ethyl acetate was added thereto for crystallization, and N'-ursodeoxycholylethylenediamine-N, N-diacetate diammonium salt (1.05 g, yield: 56.4). %).

[0050]

[0051]

The ethylenediaminediacetic acid compound and the physiologically acceptable salt thereof of the present invention can be used as a dissolving agent for various calcium-containing gallstones.

Claims (2)

(57) [Claims] (1) Formula (1) And a physiologically acceptable salt thereof. 2. A compound of the general formula (Wherein, R ¹ represents a linear or branched alkyl group having 1 to 4 carbon atoms) and a mixed acid anhydride represented by the general formula: (Wherein R ² represents a hydrogen atom, a benzyloxycarbonyl group, a t-butyloxycarbonyl group or a trityl group.) A compound represented by the following general formula: (Wherein R ² has the same meaning as described above) by a method of catalytic reduction or hydrolysis with an acid of the amide compound represented by the following formula, or a method of directly condensing the above mixed anhydride with ethylenediamine. To produce an ethylenediamine compound represented by the general formula: (Wherein, X represents chlorine, bromine or iodine.) The ethylenediaminediacetic acid compound according to claim 1, which is reacted with a halogenated acetic acid represented by the following formula: Method for producing salt.