JPS5842863B2 - S,S'-biscyclotetrapeptide and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel biscyclotetrapeptide useful as an ion inclusion agent and a method for producing the same.

Antamanid, which is made up of 10 amino acids, is already known as a peptide having properties as an ion inclusion agent that forms a complex with a metal ion, but these are monocyclic compounds.

In these monocyclic peptides, the metal ion** complex-forming ability depends on the effective ion-dipole interaction between the peptide carbonyl group and the ion. must be a peptide with 10 or more constituent amino acids.

As a result of intensive research to develop a compound that has less than 10 constituent amino acids and has an ionic inclusion power greater than that of conventional peptides, the present inventor found that a specific biscyclotetrapeptide has an excellent ionic inclusion power. Based on this finding, the present invention has been completed.

That is, the present invention provides an s-s'-biscyclotetrapeptide represented by the general formula (R in the formula is a methyl group).

*7 All of the compounds represented by this general formula (I) are novel compounds that have not been published in any literature. It can be produced by cyclizing a tetrapeptide represented by the following formula, removing the sulfhydryl protecting group R//, and then trimerizing the tetrapeptide.

In addition, the tetrapeptide of general formula (I[) used as a raw material compound in this case can be prepared by, for example, first reacting sarcosine with an amino group protected with L-proline alkyl ester to form a dipeptide, and then removing the amino protecting group. After that, this is reacted with L-cysteine with protected amino groups and sulfhydryl groups to form a tripeptide, and after further removal of the amino protecting group, this is reacted with glycine with protected amine groups to form a tetrapeptide. The alkyl ester portion of this tetrapeptide is then hydrolyzed, converted into an active ester, and the amino protecting group is removed.

In the general formula (IF), the active ester group R' and shite are p-nitrophenyl ester group, hydroxysuccinimide ester group, and the sulfhydryl protecting group is a group that can be removed by treatment with anhydrous hydrofluoric acid. Examples include benzyl group and p-methoxybenzyl group.

As the amine protecting group when producing the tetrapeptide of general formula (n), -[tert-butyloxycarbonyl group, benzyloxycarbonyl group, p-methoxybenzyloxycarbonyl group, which are used in ordinary peptide synthesis, An o-nitrophenylsulfenyl group can also be used as an alkyl ester group (Higashi methyl ester, ethyl ester, etc.) for protection of a carboxyl group.

The peptide bond formation reaction when forming the tetrapeptide of general formula (n) can be carried out using conventionally known methods such as the DCC method (dicyclohexylcarbodiimide method), the active ester method, and the mixed acid anhydride method. Can be used in combination.

Synthesis of L-cystyl-sarcosyl-proline alkyl ester with protected amino group and sulfhydryl group of raw material synthesis intermediate and L-cystyl-sarcosyl-L-proline alkyl with protected glycyl-sulfhydryl group of raw material compound with amine group protected In the synthesis of esters, the DCC method is most suitable, and these peptides can be obtained in high yield and purity.

Peptides that are synthetic intermediates for the s-s'-biscyclotetrapeptide of the present invention can be easily isolated and purified by washing with acid and alkali and recrystallizing.

The tetrapeptide active ester with a protected sulfhydryl group, which is a raw material compound in the method of the present invention, is preferably used in the form of an acid salt.

Examples of this acid salt include hydrochloride, hydrobromide, and hydrofluoride.

The cyclization reaction in the method of the present invention is carried out in a large amount of an inert solvent, such as pyridine, dimethylformamide, etc.
It can be carried out at a temperature of 30-90°C.

The sulfhydryl protecting group can be removed from the cyclic tetrapeptide thus obtained by treatment with anhydrous hydrofluoric acid in the presence of anisole.

The trimerization reaction resulting from the formation of S-8 bonds involves air oxidation of a cyclic tetrapeptide having a free sulfhydryl group in an alcohol such as methanol or ethanol in the presence of an appropriate catalyst such as 1,2-short ethane or iodine. This is done by

The reaction is completed in 1 to 2 days, and s-s'-biscyclotetrapeptide is precipitated.

The compound of the present invention (''J,, 16 in the infrared absorption spectrum
60crrL-1 shows a characteristic absorption derived from the amide-bonded carbonyl group, and both the ninhydrin reaction and the nitroprusid reaction are negative, and the product can be identified from these.

Although the compound of the present invention has eight constituent amino acids, it has properties as an ion inclusion agent that forms a complex with alkali metal ions and alkaline earth metal ions, and can be analyzed as an ion sensor. , can be widely used in the fields of medicine and agriculture.

Next, the present invention will be explained in more detail with reference to Examples.

Reference Example (A) Synthesis of sarcosyl-L-proline methyl ester hydrochloride t-7'' Tyloxycarbonyl sarcosine 6, OOP was dissolved in 40 ml of chloroform, and 3.49 ml of N-methylmorpholine and imptylic ooformate 4 were dissolved therein.
．． 157d was added at a temperature of -18°C and stirred for 2 minutes.

Next, add proline methyl ester 5.21' to this solution.
Add 1 oml of a chloroform solution containing 3.49 ml of N-methylmorpholine and stir at -18°C for 1 hour.
The mixture was further stirred at room temperature overnight.

The reaction mixture was thoroughly filtered, and Solution F was washed successively with water, a 4% aqueous sodium bicarbonate solution, and saturated brine, and dried over anhydrous sodium sulfate.

Then, the solvent was distilled off under reduced pressure, leaving 9.5% of the remaining oil.
Dissolve 2P (yield 100%) in 20 ml of ethyl acetate,
280 ml of a solution prepared by dissolving dry hydrogen chloride gas in ethyl acetate and adjusting the concentration to 4N was added, and the mixture was reacted at room temperature for 1 hour.

After the reaction is complete, the solvent is distilled off and the residue is crystallized with a mixed solvent of ethyl acetate and ethyl ether.
The target compound 6.12f (yield 82%) was obtained at 168°C.

The physical properties of this material are as follows.

Rf value of thin layer chromatography Rf1 = 0.17 (Developing solvent chloroform: methanol = 5:1) Rf2 = 0.03 (developing solvent chloroform: methanol: acetic acid = 95:5:1) Elemental analysis value (C9H1□03N2C1 ) HN Calculated value (%) 45.64 7.24 11.83 Actual value (%) 45.64 7.23 11.81 (B)
Synthesis of 5-p-methoxybenzyl-L-cystyl-sarcosyl-L-florin methyl ester hydrochloride Tert-butyloxycarbonyl-8-p-methoxybenzyl-L-cystine dicyclohexylamine salt 4.9
4f and sarcosine-L-proline methyl ester hydrochloride 2.24P in 60ml of a solution dissolved in roform,
10 ml of a solution of 1.95 P of dicyclohexylcarbodiimide dissolved in chloroform was added at -10°C.

Next, the reaction mixture was stirred at 10° C. for 5 hours and then at room temperature overnight.

The reaction mixture was filtered, the p solution was concentrated under reduced pressure, the residue was dissolved in 50 ml of ethyl acetate, filtered again, and the filter solution was washed sequentially with 10% citric acid aqueous solution, 5% sodium bicarbonate aqueous solution, and water. It was dried with anhydrous sodium sulfate.

Then, the solvent was distilled off under reduced pressure, and the residue was solidified with n-hexane to obtain tert-butyloxycarbonyl-8-
p-methoxybenzyl-L-cystyl-sarcosyl-L
-70 phosphorus methyl ester 4.24P (yield 100%)
was dissolved in 70 ml of a solution prepared by dissolving dry hydrogen chloride gas in ethyl acetate to have a concentration of 4N, and reacted at room temperature for 1 hour.

After the reaction is complete, the solvent is distilled off and the residue is crystallized with ethyl ether to yield the target compound with a melting point of 83-84°C (3.85°C).
P (yield 86%) was obtained.

Rf1=0.54, Rf2=0.44 Elemental analysis value (as C20H3005N3SC1) CK
N Calculated value (%) 52.22 6.57 9.13 Actual value (%) 52.18 6.64 9.11 (q Tertiary-butyloxycarbonylglycyl-8-p-methoxybenzyl-L-cystyl- Synthesis of sarcosyl-L-7'loline-p-nitrophenyl ester Tert-butyloxycarbonylglycine dicyclohexylamine salt 1.43S' and 5-p-methoxybenzyl-
L-cystyl-sarcosyl-L-proline methyl ester 1.85 P dissolved in chloroform 50ml
To the solution, 3 ml of a solution of 0.83 P of dicyclohexylcarbodiimide dissolved in chloroform was added at -10°C.

tert-butyloxycarbonyl in "C1B)"
Tert-butyloxycarbonylglycyl-5-p-methoxybenzyl-L-cystylsarcosyl-L-florin was prepared in the same manner as the method for producing 8-p-methoxybenzyl-L-cystyl-sarcosyl-L-proline methyl ester. The methyl ester was obtained as an amorphous product (yield 77%).

This compound 1.65P was dissolved in 10 rILl of methanol, 5.7 ml of 1N-NaOH was added, and the mixture was reacted at room temperature for 1 hour.

After the reaction was completed, 101 rLl of water was added to the reaction mixture, methanol was removed under reduced pressure, the remaining aqueous solution was washed with ether, and the pH was adjusted to 3-4 with 10% citric acid aqueous solution to obtain an oil.

When this is solidified with petroleum ether, tert-butyloxycarbonylglycyl-8-p-methoxybenzyl-L-cystyl-sarcosyl-L-proline 1 has a melting point of 82-84°C.
．． 30f (yield 81%) was obtained.

Next, this compound 1.19f and p-nitrophenol 0
．． Dissolve 29F in chloroform 15- and add 0.43% of dicyclohexylcarbodiimide to this. 3 ml of a solution in chloroform was added at 0°C, and the mixture was stirred overnight at 0°C to react.

After the reaction is complete, the reaction mixture is filtered, the lachrymal fluid is concentrated under reduced pressure,
The residue was dissolved in acetone containing 2 drops of acetic acid and filtered again.

When the solvent of the p-liquid is distilled off, an oily substance is obtained, and when this is crystallized with a mixed solvent of ether and n-hexane, the melting point is 59~
The target product 1.341 (yield 93%) was obtained at 60°C.

Rf, = 0.76, Rf2-0.07 Elemental analysis value (as C32H410t OH5S) HN Calculated value (%) 55.88 6.01 10.18 Actual value (%) 56.18 5.98 9.92 Implemented Example (A) Synthesis of cyclo-CJ') tert-butyloxycarbonyl-8-p-methoxybenzyl-L-cystyl-sarcosyl-L-cystyl-sarcosyl-L -Prolyl-p-nitrophenyl ester 1.20S' was treated with a solution prepared by dissolving dry hydrogen chloride gas in ethyl acetate and adjusting the concentration to 4N under water cooling for 1 hour, and then the solvent was distilled off to leave a residue. When solidified with ether, glycyl-8-p-methoxybenzyl-L-cystyl-sarcosylproline-p-nitrophenyl ester hydrochloride 1.10S' (yield 100
%)was gotten.

This was dissolved in 8 ml of dimethylformamide, and the resulting solution was dropped into 600 ml of pyridine at 60° C. for 3 hours, followed by stirring overnight to react.

After the reaction, the solvent was distilled off, the residue was dissolved in a mixed solvent of water and methanol, and the H-type and OH-
The mixture was passed through a type ion exchange resin column to remove unreacted substances.

Then, the solvent was distilled off, the residue was dissolved in methanol,
After purification by gel filtration through a column of Sephadex LH-20 (registered trademark, Pharmacia Fine Chemicals), recrystallization from methanol yielded cyclotetrapeptide 480■ with a melting point of 142-143°C (yield 61%). was gotten.

This compound has a negative ninhydrin reaction and a negative nitropurgide reaction, and the infrared absorption spectrum shows simaX-1660cfrL 1520CrI.
There was absorption at L11255crIL', and the mass spectrum was M+-448.

Rf, = 0.47, Rf2-0 Elemental analysis value (as C21H2805N4S・1/2H20) HN Calculated value (%) 55.13 6.39 12.25 Actual value (%) 55.17 6.12 12.128 )
Synthesis of S-8'-biscyclo(glycyl-L-hemicistyl-sarcosyl-L-7"lolyl) cyclo(crysyl-8-p-methoxybenzyl-L-cystyl-sarcosyl-L-7"lolyl) 100rn9 Aniso-)'
It was dissolved in a mixture of 0.3 ml of Ly and 10 ml of anhydrous hydrofluoric acid, and reacted at 0° C. for 1 hour.

Next, the anhydrous hydrofluoric acid was distilled off at 0° C., and the resulting residue showed a positive nitropurgide reaction.

Dissolve this in methanol, 1,2-short ethane 6
5 m9 was added and dry air was introduced until the nitropurgide reaction became negative.

When the reaction was completed, the target product was precipitated. When this was washed with alcohol, 30 cm of the desired product (yield: 41%) was obtained as white crystals with a melting point (decomposition) of 280-285°C.

This compound has a negative ninhydrin reaction and a negative nitropurgide reaction, and the infrared absorption spectrum shows a max-1
There was absorption in 640CrIL ``1540CrfL''.

Rf1=0.11, Rf2=0 Elemental analysis value (as C26H3808N8 S2/3H20) HN

Claims (1)

[Scope of Claims] 1 An s-s'-biscyclotetrapeptite represented by the general formula (R in the formula is a methyl group). **2 General formula (R in the formula is a methyl group, RQIi active ester group, R1
' is a sulfhydryl protecting group) is cyclized and condensed, treated with anhydrous hydrofluoric acid to remove the sulfhydryl protecting group, and then trimerized. A method for producing s-s'-biscyclotetrapeptide represented by the general formula (R in the formula has the same meaning as above).