JPS5931495B2 - Novel peptide production method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION X-G1u-His-Phe=Arg-Trp-G1y
=Ly: (wherein X is a benzyloxycarbonyl group or D
-Ser-Met, Y is n lysine residues 3
(where n is an integer of 2) *X
1-Glu(0But)-His-Phe-Arg-T
rp- (in the formula, BUt is a tertiary butyl group, Boc is a tertiary butyloxycarbonyl group, and X1 is a benzyloxycarbonyl group or Boc-D-Ser-Met). -Gly-Lys-Ly
s-Y-NH2I■', relating to peptides.

More specifically, the present invention provides a compound with the general formula ly-Lys(Boc)-Pro-Val-Gly-O
A peptide with the general formula H-Lys (
Boc)-Lys(Boc)-Y(Boc)n-NH2
(1) A peptide having the formula (BOc and Y have the meanings given above) is condensed by methods generally known in peptide chemistry, whereupon the protecting group is removed with a strong acid, such as trifluoroacetic acid. The present invention relates to a method for producing peptide 5 having the general formula as described above.

Even though only the first 18 amino acids counting from the N-terminus show its effect, the shorter N-terminal ACTH
- It is a known fact that peptides have an adrenocorticostimulating effect.

The peptides with general formula 1 produced according to the present invention show biological activity, some of them have high biological activity, i.e. 280E in murine subcutaneous test.
/ Has biological activity. In contrast, natural pig A
CTH and many synthetic ACTH-like peptides are commonly
It exhibits an activity on the order of ClOOlE/~. Due to its biological properties, the peptides produced according to the present invention are administered in small doses for various disease conditions.

This represents an important advance in terms of reduced side effects. Furthermore, the present invention requires a fairly complex ACT.
There is an advantage that H synthesis is simplified by producing peptides with reduced size in the peptide chain.

The peptide produced by this development has storage effect (Dep
It has physical and chemical properties that make it suitable as a pharmacological preparation.

According to the invention, it is clear that peptides with a group X acting as a biological protective group are not easily degraded by the body like other peptides. This partly explains the storage effect obtained with the peptides of the invention. The invention will now be described with reference to the following examples,
These are not limited.

Example 1 N-Cbz-Glu-His-Phe-Arg-Trp
Gly-Lys-PrO-Val-Gly-LysLy
Synthesis of s-Lys-Lys-NH2 acetate (Aq.) [
Cb, z, = carbobenzoxy (also benzyloxycarbonyl (・u), NCb7.-Clu...
... is Cb7. is bonded to N of glutamine] (a) 24.0 mmol of hydrochloric acid gas dissolved in 10 ml of ethyl acetate was dissolved in 13 ml of dimethylformamide.
-10 to 3,28y (6.00 mmol) dissolved in
0C. and then add isoamyl nitrite 0.9
Add 5 ml (7.20 mmol).

After 15 minutes, add 3.54 ml (25.
Neutralize with 3 mm monomer (c).

Add H-Arg-Trp-Gly-Lys (B
Oc)-PrO-Val-Gly~0H4.507(5
．． 01 mmol) and triethylamine 0.70 wL1
(5.00 mmol) of dimethylformamide 11 ml
A solution of is added at -1'C. After overnight storage in the refrigerator, the resulting triethylammonium chloride precipitate is separated and the remainder of the solution is evaporated in vacuo. By treating the evaporation residue with ethyl acetate, an amorphous powder is obtained, which after drying weighs 7.117 kg.

Recrystallization of this product from 64 ml of n-butanol containing 2.5 ml of methanol yields 5,627 blocked decapeptides (75% of theory). Melting point: 168°C (gas generation), (α): ~500,
Chromatographically single with c-0.55 (in methanol), RfA: 0.46 and RfB: 0.67.
Lys(BOc)-Lys(BOc)-NH2O. 37
27 (0.400 mmol), N-hydroxylsuccinimide 0.777 (0.67 mmol) and N-mericyclohexylcarbodiimide 0.1007 (0.4
9 mmol) are added to 0.5007 (0.333 mmol) of (1) dissolved in 3 ml of dimethylformamide.

After 48 hours at room temperature, the resulting N-mericyclohexylurea precipitate is separated and the resulting solution is evaporated in vacuo. By treating the evaporation residue with ethyl acetate, a large precipitate is formed, which after filtration, washing and drying weighs 0.8707 g. Dissolve the product in 17 ml of water-saturated butanol and add 5 x 2.5 ml of butanol-saturated water.
Extract using. The butanol phase was evaporated in vacuo, the evaporation residue was treated with ether, filtered, washed and dried (110,6207 (theoretical amount of 76
%) is obtained. (α): 134.

, C-0.56 (in methanol), RfA: 0.61 and RfB: 0.80. (c) Cbz-Glu-His-Phe-Arg-
TrpGly-Lys-PrO-a1-Gly-Lys
-Lys-Lys-Lys-NH2) acetate (Aq.)
0.5037 (0.205 mmol) of (I[) is dissolved in a mixture of 2.30 ml of trifluoroacetic acid and 0.23 ml of anisole.

After 45 minutes at room temperature, addition of 15 ml of ether gives a hard white precipitate which is filtered off, washed with ether and dried in vacuo over sodium hydroxide. The product was dissolved in 4 ml of water and the acetate form of “IRA4O”
Pass through an ion exchanger containing By freeze-drying the aqueous solution, a soft white powder 0.3777 is obtained. In system B, the chromatography was uniformly single with RfB: 0.07. PH5. Low voltage electrophoresis in barbiturate buffer of O (MillpOrePhOrOslld)
system) indicates that the product contains traces of impurities that can be removed by column chromatography on carboxymethyl cellulose.

112D -Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-PrO-Val-Gly-L
ys-Lys-Lys-Lys-NH2 acetate (Aq.
) Synthesis of BOc-D-Ser-Met-N2H33 (
0.055 V (0.155 mmol) was added at -20°C, followed by 0.021 ml (0.05 mmol) of isoamyl nitrite.
．． 160 mmol) is added.

After stirring for 10 minutes at -15°C, the solution was mixed with 0.065 ml (0.465 mmol) of triethylamine. and neutralize it. To this solution, add Glu(γ-0BUt)-HiS-Ph in 1 ml of dimethylformamide.
e-Arg-Trp-Gly-Lys(BOc)-Pr
A solution of O-Val-Gly-0H0.200y (0.146 mmol) is added at -15<0>C. 4 Place the solution in the refrigerator.

After 30 minutes it showed a pH of 5.5, so a further 0.022 ml (0.160 mmol) of triethylamine was added.

After storage in the refrigerator overnight, the precipitate of triethylammonium chloride that forms is separated and washed with 2×0.5 ml of dimethylformamide. 2ml of solution
, and then added dropwise to 20 ml of ethyl acetate. Separate the precipitate that forms and add 2×2 m of ethyl acetate.
1 and air dry. Weight: 0.191f (78% of theoretical amount), melting point: 193°C (gas generation), (
α)...-37.43, C-0.36 (in methanol),
Chromatography uniformly single with RfA: 0.46 and RfB: 0.70. 0.110 mmol of hydrochloric acid gas in 0.037 ml of ethyl acetate, H-Lys(BOc)-L
ys(BOc)Lys(BOc)-Lys(BOc)-
NH2O. lO3y (0.111 mmol), N-hydroxysuccinimide 0.230y (0.200 mmol) and N-N1-dicyclohexylcarbodiimide 0
．． 226y (0.110 mmol) was dissolved in (1) 0.1817 (
0.107 mmol).

After one week at room temperature, the precipitate of N-N1-dicyclohexylurea that forms is separated off. The solution is evaporated to a volume of 2 ml and then added dropwise to 15 ml of ethyl acetate while stirring with a magnetic stirrer. The slightly colored precipitate that forms is separated off and washed with a total volume of 10 ml of ethyl acetate. - After recrystallization from water-saturated n-butanol, 0.2477 of substance (87% of theory) is obtained. Melting point: 205℃ (gas generation), (α) M-20.8
:, C-0.36 (in dimethylformamide), RfA
-0.590(c) D-Ser-Met-Glu-H
is-Phe-Arg-Trp-Gly-Lys-Pr
O-Val-Gly-Lys-Lys-Lys-Lys
-NH2, acetate (Aq.) ([I) 0.0614y(
0,023 mmol) to trifluoroacetic acid 0.40
ml and 0.04 ml of anisole.

After 40 minutes at room temperature, addition of 5 ml of ester gives a hard white precipitate which is filtered off, washed with ether and dried under vacuum over sodium hydroxide. The product was dissolved in 2 ml of water and the acetic acid form of “IRA4OO
Pass through an ion exchanger containing ゜゛. Lyophilization of the aqueous solution yields 0.0497 material. Chromatography is carried out on a MerchDC-Fertigplatten Kiessel gel F254 in the following solvent system.

A: n-butanol: acetic acid: water 4:1:1 B: n-butanol lipyridine/acetic acid: water 15:10:3:6 Main embodiments of the present invention are as follows.

The peptide produced according to the present invention and the third international standard (The
ThirdTnternatiOnalStandar
The results of a comparative test regarding biological effects with d) are shown below. The table shows the effect of ACTH peptide by subcutaneous injection in dexamethasone flocked mice.

From the above table, it can be seen that the biological activity of the peptide according to the present invention by subcutaneous injection is surprisingly high, reaching 2801E or higher.

This is a very valuable property, especially in view of the short peptide chains. Figures 1 and 2 are graphs showing the relationship between the dose of ACTH peptide and the amount of corticosterone in 100ml of plasma, and Figure 1 shows the relationship between the peptide obtained in Example 2 and the third international standard. Figure 2 shows a comparison between the peptide obtained in Example 1 and the third international standard.

[Brief explanation of drawings]

Both FIGS. 1 and 2 are graphs showing the relationship between the dose of ACTH peptide and the amount of corticosterone in 100 ml of plasma.

Claims (1)

[Claims] 1 General formula ▲ Numerical formula, chemical formula, table, etc. ▼ (In the formula, But^t is a tertiary butyl group, Boc is a tertiary butyloxycarbonyl group, and -D-Ser-Met)
There are peptides with the general formula ▲mathematical formula, chemical formula, table, etc. There are general formulas ▲ mathematical formulas, chemical formulas, tables, etc. ▼ (where X is a benzyloxycarbonyl group or D -Ser-Met, and Y has the above meaning.