JPS63141996A - Novel active peptide - Google Patents

Abstract

NEW MATERIAL:L-prolyl-L-tyrosyl-L-proline and its acid addition salt. USE:A hypotensor. It has angiotensinase inhibiting activity and low toxicity and is expected to be useful as a medicine or health food, etc., for depressing the blood pressure level. PREPARATION:Amino acid are introduced into a desired sequence e.g. by a peptide synthesis comprising a proper combination of the protection, condensation and deprotection of NH2 group or COOH group of amino acid.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a novel active peptide, specifically a novel active peptide that has angiotensin converting enzyme inhibitory activity and is expected to be useful in the treatment or prevention of hypertension. Regarding new peptides.

(Prior Art) It is well known that the renin-angiotensin system is deeply involved in the onset of hypertension. The renin-angiotensin system contains angiotensin converting enzyme that is involved in blood pressure regulation, and this enzyme is involved in increasing blood pressure by converting angiotensin I to angiotensin n, which has a strong vascular wall smooth muscle contraction effect. Therefore, inhibiting this enzyme activity: ni:yo1)
It is possible to prevent an increase in blood pressure (lower blood pressure).

Various substances have already been discovered as activity inhibitors of angiotensin converting enzyme, such as D-2-methyl-3-mercaptopropanoyl-
Due to its high inhibitory activity, proline (-ship name: cabtopril) is currently in practical use as an oral antihypertensive agent. Further, as natural products or inhibitors derived from natural products, snake venom peptides and their analogues, peptides obtained by tryptic decomposition of Ushiyama casein, and the like are known.

Among these, natural products or inhibitors derived from natural products are expected to be safer antihypertensive agents, whereas synthetic products still have problems in terms of toxicity and side effects. However, casein-derived inhibitory peptides are being considered for practical use as antihypertensive agents because they are expected to be advantageous in terms of cost as well as safety and efficacy.

(Purpose of the Invention) In view of the usefulness of the above-mentioned casein-derived inhibitory peptides, the present inventors searched for those having effective angiotensin converting enzyme inhibitory activity from among their related peptides or partial structures, and found the following: As a result of conducting intensive research in order to expand the range of options for applying this group of peptides to antihypertensive networks and to further increase the possibility of their practical application, we discovered that the tripeptide described below satisfies these purposes, The present invention has now been completed.

(Structure of the Invention) That is, the present invention is a novel active peptide, 1-prolyl-1-tyrosyl-1-proline, and an acid addition salt thereof.

Preferred examples of the acid addition salt include pharmaceutically acceptable salts, such as hydrochloride, sulfate, succinate, citrate, and tartrate.

The peptide of the present invention is a partial structure of bovine casein,
It can also be obtained by enzymatic hydrolysis of the protein,
More advantageously, an example of a synthetic method will be shown using an organic chemical synthesis procedure, in which all amino acids are in the L form,
Furthermore, the abbreviations of amino acid protecting groups represent the following residues.

Boc: tertiarybutyloxycarbonyl (tr
et-Butyloxy-carbonyl) group Bzl
: Benzyl group (1) Boc-Tyr (Bz ri-OR and H-Pro
-OBzl is subjected to a condensation reaction to form Boc-Tyr (B2 +
)-Pro-OBz I was synthesized, and I(-Tyr (B21)-Pr) was synthesized by unblocking reaction.

0th order to get -0Bzl, then this)[-Tyr(Bzl)
-Pro-OBz IJrcI3oc-Pro -O
Boc-Pro-Tyr (Bz
l) -Pro-OBz I was synthesized, and the target II-Pro-'ryr-Pr was obtained by the same unblocking reaction.

get.

In the above reaction, the reaction conditions etc. can be the same as in the case of ordinary peptide synthesis.

Vepti r4 of the present invention. shows a strong inhibitory effect on angiotensin converting enzyme, and has low toxicity, so it can be expected to be useful when applied to medicines or health foods for the purpose of lowering blood pressure.

The results of activity tests showing the usefulness of the peptides of the present invention are listed below.

[Angiotensin converting enzyme inhibitory activity] (1) Test sample (a) obtained in the example below) (-pro-Tyr-
Pro-OH (peptide of the present invention) (b) Captopril (
Control) (2) Test method (i) Preparation of angiotensin converting enzyme solution (ACE solution) 5 g of rabbit lang acetone powder (manufactured by Sigma)
was dissolved in 50 ml of O, l M borate buffer (pH 8,3) and 40.0 OO x g.

The mixture was centrifuged for 40 minutes, and the supernatant was further diluted 5 times with the above buffer to obtain an angiotensin converting enzyme solution.

(ii) Put 0.03 ml of the activity measurement sample into a test tube, and add 0.1 ml of the above enzyme solution.
After incubating at 37°C for 10 minutes, 25 (lul of hyplylu, one histidyl, one leucine) [Aldrich Chemikaf 1 (^1dri) was added as a substrate.
ch Ches, Co,) 5J,, Jl final concentration 5mM
, containing 300mM NaCl], 37
The reaction was carried out at ℃ for 30 minutes. Then, IN salt #0.25m
1 was added to stop the reaction, 1.5 ml of ethyl acetate was added, and the mixture was stirred vigorously for 15 seconds. After that, 3.5
The mixture was centrifuged at 00 rpm for 15 minutes, and 1 ml of the ethyl acetate layer was collected. The ethyl acetate layer was heated at 120° C. for 30 minutes to remove the solvent. After removing the solvent, 2 ml of distilled water was added, and the absorption of the extracted hyperlic acid (absorbance at 228 nm) was measured, which was taken as the enzyme activity. In addition, the absorbance at 228 nm when no inhibitor is included under these conditions is o,
It's soooo.

The inhibition rate was calculated from the following formula, pc:

=÷ − Inhibition rate − A−B/AXl! loO% A: Absorbance at 228 nm without inhibitor (0,
500) B: Absorbance at 228 nm with addition of inhibitor and
The inhibitory concentration when the inhibition rate is 50% is defined as ID50.

(c) Test results The test results are shown in Table 1.

As is clear from the above results in Table 1, the peptide of the present invention has an inhibitory effect on angiotensin converting enzyme, and can be expected to exert an effective hypotensive effect in vivo.

Furthermore, the peptide of the present invention has extremely low toxicity, with an acute toxicity value (LD50) of 3 g/kg or more when administered orally to mice. Therefore, we can expect this to happen.

When the peptide of the present invention is used as a medicine for the treatment or prevention of hypertension, the peptide may be used in a pharmaceutically acceptable carrier (excipient, lubricant, binder, coloring agent, flavoring agent, flavoring agent, etc.). preparations for oral administration, such as tablets, capsules, troches, powders,
It is administered orally in the form of fine granules, granules, etc., or intravenously in the form of an injection.

On the other hand, when used as a health food, it may be in the form of an oral preparation similar to the above, or as a solid or liquid food or luxury item (e.g. sweets, powdered tea, alcoholic drinks, sports drinks, etc.). It may be in the form of
The dosage is generally in the range of 80 mg to 4 g/day of body weight for adult males, and an appropriate amount is selected from this range depending on the purpose of administration (feeding), etc.

Next, the present invention will be explained by examples.

Example 1 (1) H-Tyr (Bz l) -1i';:F
Synthesis of '0-OBzl H-Pro-OBzl・MCI 2.42g (101
11101), BOC-Tyr(Bzl)-
0H3,71g (10gmol) and l-hydroxybenzotriazole (IIOB t) 1.35
g (10m layer of dimethylformamide CDMP)
1.4 ml of triethylamine and 2.06 g of dicyclohexylcarposiimide were added to this WI solution under water cooling at 0°C, and then stirred overnight while maintaining the temperature at 5°C.

The generated dicyclohexylurea was filtered off, the filtrate was concentrated to dryness, and the residue was dissolved in ethyl acetate. This solution was thoroughly washed with a 10% aqueous citric acid solution, a 4% aqueous sodium bicarbonate solution, and then water, and dehydrated and dried over anhydrous sodium sulfate. After filtering off anhydrous sodium sulfate, the filtrate was concentrated under reduced pressure, and the residue was crystallized from ethyl acetate.

This crystal (Boc-Tyr (Bz 1)-Pr) was left for 0 minutes. Next, the reaction mixture was concentrated under reduced pressure, and the residue was washed twice with ether. The ether was distilled off and t(-Ty
r (Bz I)-Pro-OBz; was obtained as trifluoroacetate [yield: 10.52 g, (lamol)
, Properties: pale yellow powder].

(2) Boc-Pro-Tyr (Bz 1)-Pro
Synthesis of -OH H-Tyr (Bz I)-Pro-OBz I ・Trifluoroacetate 0.52 g (lamol), B
.

c-Pro-OH0,22g (1ms+ol) and ■0BtO,14g (lamol) in DMF4
0.15 ml of triethylamine and 0.21 g of dicyclohexylcarposiimide were added to this solution under water cooling at 0°C, and the mixture was stirred overnight while being maintained at 5°C.

The generated dicyclohexylurea was filtered off, the filtrate was concentrated to dryness, and the residue was dissolved in ethyl acetate. This solution was mixed with 10% citric acid aqueous solution, water, 4% sodium bicarbonate aqueous solution 1. ; Next, thoroughly wash with water and 30% water, dehydrate and dry with anhydrous sodium sulfate and water;
.

It was dry. After filtering off the anhydrous sodium sulfate, the filtrate was compressed under reduced pressure to obtain a residue.

This residue (Boc-Pro-Tyr (Bz 1)-P
ro-OBz I) was dissolved in a mixture of 18 ml of methanol, 79 ml of dioxa, and 1.5 ml of IN NaOH, left at room temperature for 5 hours, water was added and washed with ether, and then cation exchange resin (BIO-RAD A
G50W-X8).

The resin was removed by filtration, and liquid a was concentrated to dryness under reduced pressure to form B.
oc-Pro-Tyr (Bz 1)-Pro-OH was obtained as a white powder [Yield: 1 t0.18 g (0,
33sn+ol) ).

(3) Synthesis of H-Pro-Tyr-Pro-OH
-Pro-Tyr (Bz I)-Pr.

0,080 g (0,15 mmol) of -0H was dissolved in a mixture of 1 ml of acetic acid saturated with hydrogen bromide and 1 ml of anisole, and after being left at room temperature for 1 hour, it was concentrated under reduced pressure. Washed with.

;S3. - Next, this residue was subjected to high performance liquid chromatography (column: Radial PAk) CI m
+ Eluent Niacetonitrile 10.1% Trifluoroacetic acid - 60/40. Elution rate: 2ml/5in),
The active fraction was collected and dried under reduced pressure to obtain H-Pro-Tyr-
Pro-Off purified product 0°0027 g (0,00
8usol) was obtained as a colorless powder.

The physical property values and analysis results of the peptide thus obtained were as follows.

◎Specific rotation [α], dew 5-93° (C-0,1, H*O) ◎Amino acid analysis (hydrolysis in 6NHC1 for 24 hours) Tyr 1.00 (11, P r o 1. 7
＋21◎ Thin layer chromatography [silica gel plate, developing solution; upper layer of butanol:acetic acid:water (4:1:5), UV absorption and ninhydrin color development], Rr value 0.29, same sub-base, same Kanebo Co., Ltd. company

Claims (1)

[Claims] L-prolyl-L-tyrosyl-L-proline and its acid addition salts.