JPH07119233B2 - Method for synthesizing the bioactive substance probestin - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial field of application) The present invention relates to a synthetic method for producing a physiologically active substance, Probestin, which has an inhibitory activity on aminopeptidase M as disclosed in JP-A-64-85995. .

(Prior art and problems to be solved by the present invention) The physiologically active substance probestin (Probestin) is a tetrapeptide containing an unnatural amino acid and is represented by the following formula: With {(2S, 3R) -3-amino-2-hydroxy-
It corresponds to 4-phenylbutanoyl} leucylprolylproline, and among the constituent amino acids of this compound, leucine and proline are natural types (L type). Probestin has the activity of inhibiting aminopeptidase M. An enzyme inhibitor that inhibits aminopeptidase M has a function of suppressing the metabolism of physiologically active substances such as enkephalin (see, for example, "Peptides" Vol. 8, pp. 523-532 (1986)). The aminopeptidase M inhibitory action of probestine is specific, and it has a stronger inhibitory activity than actinonin, which is a known aminopeptidase M inhibitor. On the other hand, Japanese Patent Laid-Open No. 64-85995 describes a method for producing probenestin by a microorganism, but it is difficult to obtain a large amount of highly pure probestin at one time by this method, and industrially available probenstine. Is desired.

(Means for Solving the Problems) In order to solve the above-mentioned problems, the present inventors have studied a method for producing probevestin by organic synthesis. As a result, bestatin is used as a raw material to obtain L in bestatin.
-Provestin was successfully produced in good yield by a short step in which two molecules of proline were sequentially condensed.

Bestatin is the following formula Having (2S, 3R) -3-amino-2-hydroxy-4
-A known substance corresponding to phenylbutanoyl leucine, and a method for producing bestatin by a culture method (JP-A-52-116435) and a synthetic method (JP-A-52-136118) has been established.

That is, the present invention has the following general formula [Wherein R ¹ is an amino-protecting group] is an amino-protected form of bestatin represented by the following formula or an activated derivative at the carboxyl group thereof. [Wherein R ² is a carboxyl protecting group] is condensed with a carboxyl protected form of proline to give the following general formula: [Wherein R ¹ and R ² have the above-mentioned meanings] to produce a compound, and the carboxyl protecting group (R ² ) of the compound of the formula (IV) is eliminated by a conventional method to give the following formula [Wherein R ¹ and R ² have the above-mentioned meanings] to produce a compound of formula (IV ′) or an activated derivative at the carboxyl group of the proline of formula (III) The following general formula can be obtained by condensing a carboxyl-protected compound. [Wherein R ¹ and R ² have the above-mentioned meanings], and an amino protecting group (R ¹ ) and a carboxyl protecting group (R ² ) are prepared from the compound of formula (V) by a conventional method. The following formula, which is characterized in that The gist of the present invention is a method for producing probevestin having

The amino-protected form (II) of bestatin used as a starting compound in the method of the present invention is prepared by a conventional method by reacting the raw material bestatin with a known amino-protecting group-introducing reagent commonly used in peptide synthesis. . For example, the amino protection method described in JP-A No. 52-136118 can be used. The amino protecting group (R ¹ ) in the amino protected form (II) of bestatin can be one commonly used in peptide synthesis and is an amino protecting group which can be eliminated by catalytic reduction but not by hydrolysis. It is preferable that it is an aralkoxycarbonyl group, for example, a benzyloxycarbonyl group, or a benzyl group. The carboxyl-protecting group (R ² ) in the carboxyl-protected proline (III) to be condensed can be generally one commonly used in peptide synthesis, and particularly, an ester-forming group which is easily removed by hydrolysis, such as an alkyl group. It may be a group or a substituted alkyl group, preferably a diphenylmethyl group or a benzyloxymethyl group, or an aryl group.

The condensation of the bestatin N-protected form of formula (II) with the proline C-terminal-protected form of formula (III) in the method of the present invention is carried out by using an active esterifying agent and a condensing agent commonly used for peptide synthesis in an organic solvent. Conveniently, it is carried out at 0 ° C to 30 ° C for 0.5 hours to several hours. Acetonitrile, TH as organic solvent
An aprotic solvent such as F or DMF can be used. The active esterifying agent is N-hydroxysuccinide, hydroxybenzotriazole or the like, and the condensing agent is 1-ethyl-
A general peptide synthesis reagent such as 3- (3-dimethylaminopropyl) carbodiimide hydrochloride (abbreviated as WSCD) or DCC is used. Similarly, the condensation of the compound of formula (IV ′) with the proline compound of formula (III) at 0 ° C to 30 ° C in the presence of an active esterifying agent and a condensing agent in an organic solvent.
It takes 0.5 to several hours. As the organic solvent, aprotic solvents such as acetonitrile, THF and DMF can be used. Again, active esterifying agents include N-hydroxysuccinide, hydroxybenzotriazole (HOB
As the condensing agent such as t), general peptide synthesis reagents such as WSCD and DCC are used.

In order to eliminate the carboxyl protecting group (R ² ) from the compound of formula (IV) produced by the first-stage condensation reaction in the method of the present invention, the compound of formula (IV) is hydrolyzed in the presence of an acid. Submit for reaction. Acids such as trifluoroacetic acid, hydrochloric acid, and acetic acid can be used as this acid.

In order to deprotect the compound of formula (V) produced in the second-stage condensation reaction in the method of the present invention, trifluoroacetic acid is first removed in order to remove the carboxyl protecting group (R ² ) by hydrolysis. After treatment with an acid such as hydrochloric acid or acetic acid, when the amino protecting group (R ¹ ) is an aralkoxycarbonyl group, methanol, ethanol, ethyl acetate, dioxane, etc. in the presence of a catalyst such as palladium or palladium carbon. Or in the presence of a catalyst such as palladium or palladium carbon in the presence of a catalyst such as methanol, ethanol, ethyl acetate, dioxane, etc. to reduce the amino protecting group.
A method of eliminating (R ¹ ) can be used. Formula (IV)
The deprotection from the compound of ¹ ) first removes the amino protecting group (R ¹ ) in the reverse order to the above, and then the carboxyl protecting group (R ² )
Can also be detached.

The invention will now be described in more detail by reference synthetic examples showing the preparation of starting compounds, as well as examples showing the preparation of compounds of the formula [I].

Reference Example 1 2042 mg (6.62 mmol) of bestatin was dissolved in 120 ml of water and 20 ml of dioxane, and 723 mg of sodium hydrogencarbonate was added. 20 ml of an ether solution containing 0.75 g of benzyloxycarbonyl chloride was added, and the mixture was stirred for 1 hour. Further, 20 ml of an ether solution of 723 mg of sodium hydrogen carbonate and 0.75 g of benzyloxycarbonyl chloride was added, and the mixture was stirred overnight.
The aqueous layer was separated, acidified with 6N HCl and then extracted with ether. The solvent was dried over magnesium sulfate, concentrated to dryness,
Recrystallization from ethyl acetate gave 2560 mg of N-benzyloxycarbonylbestatin. Yield 88%. mp212 ° C. TLC: Rf = 0.3 (chloroform-methanol = 10: 1) NMR (DMSO-d ₆ ); δ 0.72 to 0.97 (6H, m), 1.37 to 1.87
(3H, m), 2.59 to 2.92 (2H, t, J = 8Hz), 3.82 to 4.47 (3
H, m), 4.92 (2H, d, J = 5Hz), 6.73 (1H, d, J = 8Hz),
7.19 (5H, s), 7.27 (5H, s), 7.68 (1H, d, J = 8Hz) Example 1 (a) N-benzyloxycarbonylbestatin 1024 mg
(2.32 mmol) was dissolved in 230 ml of acetonitrile, and HOBt (345 mg), WSCD (490 mg) and proline diphenylmethyl ester (652 mg) were added under ice-cooling stirring, and the mixture was reacted for 4 hours.
The reaction solution was concentrated, ethyl acetate was added to the residue, the organic layer was washed with water, dried over magnesium sulfate and purified by silica gel column chromatography to give N-benzyloxycarbonylbestatinylproline diphenyl as an oil. 1280 mg of methyl ester was obtained. Yield 78
%.

This compound corresponds to the compound where R ¹ is a benzyloxycarbonyl group and R ² is a diphenylmethyl group in the above formula (IV).

TLC: Rf = 0.3 (chloroform-ethyl acetate = 2: 1) NMR (CDCl ₃ ): δ 0.75 to 0.95 (6H, m), 1.35 to 1.70 (3H,
m), 1.75 to 2.25 (4H, m), 2.78 to 3.00 (2H, m), 3.40
~ 3.95 (2H, m), 4.00-4.28 (2H, m), 4.97 (2H, s),
6.79 (1H, s), 7.13 (5H, s), 7.21 (15H, s) MS (FD): M ⁺ + 1 = 706 (b) N-benzyloxycarbonylbestatinylproline obtained in the above (a)・ Diphenyl methyl ester
1180 mg (1.674 mmol) of trifluoroacetic acid (11 ml) and anisole (1 ml) were added under ice cooling and stirred for 10 minutes. A solution of isopropyl ether-hexane (2: 1) (50 ml) was added and the precipitate was collected by filtration to give N-benzyl. 840 mg of oxycarbonylbestatinylproline was obtained. Yield 98%.

This compound corresponds to the case where R ¹ is a benzyloxycarbonyl group in the above formula (IV ′).

mp96 to 99 ° C. TLC: Rf = 0.2 (chloroform-methanol = 3: 1) NMR (CDCl ₃ ): δ 0.71 to 1.20 (6H, m), 1.36 to 1.76 (3H,
m), 1.81 to 2.36 (4H, m), 2.61 to 3.06 (2H, m), 3.26
~ 3.96 (2H, m), 4.00 ~ 4.81 (4H, m), 4.92 (2H, s),
7.16 (5H, s), 7.31 (5H, s) MS (FD): M ⁺ + 1 = 540 (c) 740 mg of N-benzyloxycarbonylbestatinylproline obtained in the above (b) was used as in Example 1. Similarly, it was condensed with proline diphenylmethyl ester to obtain 830 mg of N-benzyloxycarbonylbestatinylprolylproline diphenylmethyl ester as an oily substance. Yield 76%.

This compound corresponds to the compound when R ¹ is a benzyloxycarbonyl group and R ² is a diphenylmethyl group in the above formula (V).

TLC: Rf = 0.3 (ethyl acetate) NMR (CDCl ₃ ): δ 0.75 to 1.08 (6H, m), 1.40 to 1.65 (3H,
m), 1.70 to 2.20 (8H, m), 2.82 to 3.30 (2H, m), 3.43
~ 3.85 (4H, m), 4.05-4.80 (3H, m), 4.97 (2H, s),
6.79 (1H, s), 7.15 (5H, s), 7.23 (15H, s) MS (FD): M ⁺ + 1 = 803 (d) N-benzyloxycarbonylprolylproline obtained in the preceding item (c). Diphenyl methyl ester730 m
When g (0.911 mmol) was subjected to a treatment for eliminating a diphenylmethyl group in the same manner as in the above item (b), N-benzyloxycarbonylbestatinylprolylproline was obtained. Yield 573 mg (yield 99%) mp 105 to 106 ° C. TLC: Rf = 0.5 (chloroform-methanol = 3: 1) NMR (CDCl ₃ ): δ 0.69 to 1.01 (6H, m), 1.34 to 1.67 (3H,
m), 1.74 to 2.34 (8H, m), 2.59 to 3.04 (2H, m), 3.24
~ 3.87 (4H, m), 3.96 ~ 4.84 (5H, m), 4.91 (2H, s),
7.13 (5H, s), 7.19 (5H, s) MS (FD): M ⁺ = 636 (e) 475 mg (0.747 mmol) of N-benzyloxycarbonylbestatinylprolylproline obtained in (d) above was added. , 10% hydrous methanol dissolved in 50 ml, 10% Pd-C2
50 mg was added and catalytically reduced for 1 hour under hydrogen at atmospheric pressure. The N-benzyloxycarbonyl group was eliminated. After the catalyst was filtered off and the solvent was distilled off, the residue was purified by HP-20 resin chromatography to obtain 278 mg of probestin.

mp121 to 123 ° C. TLC: Rf = 0.3 (acetonitrile-water = 4: 1) NMR (D ₂ O): δ 0.80 to 1.05 (6H, m), 1.45 to 1.75 (3H,
m), 1.80 to 2.40 (8H, m), 2.85 to 3.15 (2H, m), 3.55
~ 3.85 (5H, m), 4.25 (2H, s), 4.50-4.70 (2H, m),
7.35 (5H, s) MS (FD): M ⁺ + 1 = 503 Example 2 70 mg (0.0873 mmol) of N-benzyloxycarbonylbestatinylprolylproline diphenylmethyl ester obtained in Example 1 (c) was used. It was dissolved in 20 ml of methanol, 70 mg of 10% Pd-C was added, and catalytic reduction was performed under hydrogen at atmospheric pressure for 1.5 hours. After the catalyst was filtered off and the solvent was distilled off, the residue was
Purification by LH-20 resin chromatography (chloroform-methanol = 1: 1) gave 35 mg of probestin.

Test Example 1 The synthetic probestin and the natural probestin obtained in Example 1 (e) or Example 2 were re-purified by the same purification method (that is, 0.1% trifluoroacetic acid-acetonitrile (7: 3) using a YMC-Gel column). ) As an elution solvent
Purified by HPLC once and purified by Dowex 50W column and second HPLC using 0.5N ammonia water as an elution solvent to obtain pure products. Regarding the synthetic probestin purified as described above and the purified probestin as a natural product, the aminopeptidase M (AP-M) inhibitory activity value (IC ₅₀ ) and optical rotation ▲ [α] ²⁵ _D ▼ (c0.2, (Methanol) was measured. The results are shown in the table below.

Since the physicochemical properties such as the results of the above table and NMR and MASS analysis were in agreement, the probestin synthesized by the method of the present invention was the same as the natural probestin derived from the microorganism shown in JP-A-64-85995. Turned out to be a substance.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Seiji Shibahara 760 Shimooka-cho, Kohoku-ku, Yokohama-shi, Kanagawa Meiji Seika Co., Ltd., Pharmaceutical Research Laboratory (72) Inventor Shigeharu Inoue 760 Meiji, Kohoku-ku, Yokohama-shi, Kanagawa Confectionery Co., Ltd., Pharmaceutical Research Laboratory (72) Inventor Shigumi Yoshida, 760, Shimooka-cho, Kohoku-ku, Yokohama, Kanagawa Prefecture ―1 Ichigao Annex 801 (72) Inventor Takaaki Aoyagi 3-3-6 Motokugenuma, Fujisawa City, Kanagawa Prefecture (72) Inventor Tomio Takeuchi 5-1-11 Higashigotanda, Shinagawa-ku, Tokyo 701 New Mansion 701

Claims (3)

[Claims] 1. The following general formula [Wherein R ¹ is an amino-protecting group] is an amino-protected form of bestatin represented by the following formula or an activated derivative at the carboxyl group thereof. [Wherein R ² is a carboxyl protecting group] is condensed with a carboxyl protected form of proline to give the following general formula: [Wherein R ¹ and R ² have the above-mentioned meanings] to produce a compound, and the carboxyl protecting group (R ² ) of the compound of the formula (IV) is eliminated by a conventional method to give the following formula [Wherein R ¹ and R ² have the above-mentioned meanings] to produce a compound of formula (IV ′) or an activated derivative at the carboxyl group of the proline of formula (III) The following general formula can be obtained by condensing a carboxyl-protected compound. [Wherein R ¹ and R ² have the above-mentioned meanings], and an amino protecting group (R ¹ ) and a carboxyl protecting group (R ² ) are prepared from the compound of formula (V) by a conventional method. The following formula, which is characterized in that And a method for producing probencin having. 2. A condensation reaction between an amino-protected form of bestatin represented by the formula (II) and a carboxyl-protected form of proline represented by the formula (III) is an active ester commonly used for peptide synthesis by coupling an amino acid. In the presence of an agent and a condensing agent to form an active ester of the amino protected form of bestatin of formula (II) as an intermediate, which is then reacted with the proline of formula (III). The condensation reaction between the compound of formula (IV ′) and the carboxyl protected form of proline represented by formula (III) is also commonly used for peptide synthesis by coupling of amino acids. The method according to claim 1, which is carried out in the same manner as the above-mentioned condensation step in the presence of an active esterifying agent and a condensing agent. 3. The following equation N-protected-vestatinylproline represented by the formula: wherein R ¹ is an amino-protecting group.