JPH01226898A - Novel peptide and hypotensive agent containing said peptide - Google Patents

Abstract

NEW MATERIAL:A peptide (salt) expressed by formulas I-XIII. USE:A hypotensive agent. PREPARATION:For example, proline having the amino group protected with 9-fluorenylmethoxycarbonyl group is bound to a p-alkoxybenzyl alcohol resin in the presence of a condensing agent to remove the protecting group. Amino acids having protected alpha-amino group and functional group in the side chain are linked in the presence of a condensing agent according to the amino acid sequence of a peptide and the alpha-amino protecting group is removed. The above- mentioned operation is repeated to synthesize a peptide chain. The resin having the two peptide chains linked thereto is subsequently suspended in a solvent and treated with trifluoroacetic acid to eliminate the peptide from the resin. After removing all the protecting groups, the peptide is purified by ion exchange chromatography, reversed phase liquid chromatography, etc., to afford the aimed peptide expressed by the formula.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel antihypertensive agent containing peptide 2.

Conventional technology In recent years, enzymes for hexacrystalline proteins such as milk casein have been added to opioid 4-peptide (1), C-breath absorption promoting peptide, and annootensin-mutant enzyme inhibitor peptide (2-terminal).

It has been reported that there are various types of medicines that can be used for burial, and the prize is only 5F! It is thought that these peptides not only have nutritional benefits, but also exhibit OT ability, which is involved in the regulation of the body as an extrinsic factor. has not been clarified so far.

References (1): 1) V, Brantl, H,T
eschemacher, As1(enahen
, and F* Lottspach, Hop
p@-8@yl@rsZ, Physiol, Chem,
360.1211 (1979).

□ -＼5ノ2) S, Loukas, D, Varoueha
, C. Zloudrou.

R, A, 5 treaty and W, A, 1e*
, Bloch@m1stry , Emi.

4567 (1983).

(2): S, Maruyama, K, N
akagomi, N., Tomizuka.

and ) i, 5uzuki, Agric, B
i ol, Chem, , 49 (5) at\-
However, conventional pharmaceuticals often have side effects such as J+3 and have safety issues, making the development of highly safe drugs with fewer side effects an expensive task. . When using the above-mentioned food protein Pegnato T- from Teiyumai as a medicine, it is difficult to obtain a highly safe product because of its origin or because it is a food that we consume on a daily basis. Thinking further, it is possible to obtain safer products by using human milk casein instead of milk casein, which is a foreign protein for humans, and by finding pharmaceutical peptides from this. Mouth ■Ura Demeru. However, it is difficult to obtain human milk casein quantitatively using domestically produced methods.However, depending on the substrate specificity of the #element used, the reaction conditions, etc., the resulting peptides may be converted into proteins and the target peptides may be extracted. Not limited to.

The problem to be solved is the development of short-chain peptides that have passed the medical regulations mentioned above.

Means to Solve the Problem In order to solve the above-mentioned problem, the present inventors conducted intensive and careful research, and as a result, the present inventors thoroughly investigated the peptide fragments in casein using humans whose structure was known, and found the following. The present invention was completed by successfully synthesizing a peptide (i-) and discovering that it is useful as an antihypertensive agent.
The present invention is an antihypertensive agent containing a novel peptide represented by any of the following structural formulas and a salt of 7, and at least -St as an active ingredient thereof.

Lys-Thr-Ala-Pr.

Thr-Alt-Pr.

Mat-Tyr-Tyr Tyr-Ala-Asn-Pro-Ala-Mal-V
al-Arg-Pr.

Alt-Aan-Pro-Ala-Val-Yal-A
rg-Pr.

Asn('ro-Alt-Val-Val-Arg-P
r.

Pro-Ala-Mal-Val-Arg-Pr.

Ala-Val-Val-Arg-Pr.

Val-Val-Arg-Pr.

Pro-Asn-8@r-)1ts-Pr.

5et-Hls-Pr.

Pro-Thr-Pro-Al t-Pr.

Pro-Alt-Pr.

Salt form #h sweet, its salts include hydrochloric acid 1, bromide water A salt, hydrogen iodide 11 salt, sulfate, inorganic acid salt 2 such as phosphate, acetate, trifluoroacetic acid Examples include organic acid salts such as salt, citrate, maleic acid salt, fumaric acid salt, tartaric acid salt, milk salt, methanesulfone dtJE, and P-) luenesulfone salt.Also contained in antihypertensive agents. In some cases, it takes the form of a pharmaceutically acceptable salt.

Amino riN constituting the peptide is preferably L-integrated because it occurs naturally.

The present invention (!tide) is a solid-phase method commonly used for peptide synthesis, and the reactive force equivalent to 1 t of 7 fragments of 2 halves divided by the injection of pegylated M is 1 t.
= t-, and a raw material having a reactive aba] group corresponding to the local 7 fragments was treated with noaclohexylcarmetzimide ff1t, and the condensate formed was preserved.
When the k group is used, it can be produced by removing the seven disclaimers.

In this reaction process, officials who should not be involved in the reaction are protected by Bao Kenji. Examples of the protective group for the amino group include pennoloxycarbonyl, t-bunaloxycarbonyl, P-bioxycarbonyl, 9-fluorenylmethyloxycarbonyl, and the like. . , C's cargoquine is thalolmetal afDi7, oxy-' f le (i (fat,
p-Flucoki 7 pennor alcohol resin is attached to the carrier.

The cell reaction is carried out in the presence of a binding agent such as nocyclohexylcalmetzimide.

When the Tsumugi reaction is completed, the bonding group is removed and the bond between the C-terminus of the pegylated resin and the resin is cut off.

In addition, the Kuraki pegged of the present invention is manufactured according to a conventional method. For example, ion A; F, Hong chromatography,
3! E, phase liquid chromatography, affinity chromatography, etc.

The active ingredient of the antihypertensive agent of the present invention - [1 Used in PEGTE 1]
7t is the administration of that salt &! For sweetfish, it is administered orally.

Either parenteral administration or direct BljV-3 administration may be used,
Oral administration is preferred. 4: The dosage of the salt of Peptide 19-7 of the invention varies depending on the type of compound, administration method, patient's symptoms and age, etc., but the dosage is 0.001 to 1000 Da per dose, preferably 0.00 Da. 01-109 1-3 times per day. The salt of the present invention is usually mixed with a pharmaceutical carrier and administered in the form of a specific preparation. As a pharmaceutical carrier, a substance that is widely used in the pharmaceutical field and that does not react with Kaede is used for the Vegted"!7t of the present invention.
Ru. Ingredients include 1, for example milk saccharide, pudox sugar, mannitol,
r Kistrin, Cyclodextrino, De7gun, A Kinuta,
Aluminum modified magnesium, sweet acid aluminum silicate, crystalline cellulose, sodium cardaki 7-methylcellulose, hydroxyno-a bildungsten, carboxymethyl cellulose urzocum, i:t/9. m*fat, methylcellulose, gelatin, gum arabic, hydroxide 7
f lobil cellulose, titma hydroxyglobil cellulose, hydroxypropyl methyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, @'Xk chickadee cough.

Magnenium stearin, Merck, tragacanth, bentonite, vegum, carboxyvinyl poly-r-,
Natan, sorbitan fatty acid ester, sodium lauryl sulfate, glycerin, fatty acid glycerin ester, Fflg lanoli/, glycerogelatin, polysorbate, macaco/I/, amon, wax, liquid,
Examples include narafin, white petrolatum, fluorocarbon x:y, nonionic surfactants, fa-pyrendalicol, water, and the like. Dosage forms include tablets, capsules, granules, powders, 70g tablets, and cloudy tablets.

Examples include base preparations, ointments, creams, glues, pastes, inhalants, injections, etc. Based on the trial decision, these preparations are subject to
I'm sorry. In addition, it may be in the form of a liquid preparation, which is dissolved or suspended in water or other suitable medium at the time of use. The preparations and granules may be coated by a well-known method. In the case of VC, the -27" tide maku of the present invention may be prepared by soaking and scaling the -27" kaede of the present invention in water, but it is not necessary. gE due to the cost
, it can also be commonly understood as a glucose bath solution.
You may also make a self-cutting hole in the buffer or storage shell.

In these preparations, Vegted 17t of the present invention is a salt thereof.
It can be present in a proportion of 0.2% or more, preferably 0.5 to 70%. These preparations also.

Other components of therapeutic value may also include gold.

$! , example Hereinafter, the four inventions will be specifically explained using examples.

Note that the abbreviations used in this specification have the following meanings.

Ala alanine (hereinafter referred to as Ai) rjlRrz are all L+).

Arg Alinine Asn Asnoya Line Asp Asnon Lazen Sword Gin Glutamine Glu glutamine relatives G17 glycine H1m1m Histi Gin isoleucine Leu Loinno Lys lysine M@tmethionine ph/phenylalanine Pro Glolin S@r Serin Thr threonine Trp) Ligtofan Tyr tyrosine TLC4J-Chromatography Example I Lys-Thr-Ala-Pr.

Fmoc-Pro side 17 (krnoc-Pro O
p-alkoxybennoralcohol resin) in which H is introduced at a rate of 0.53 mmol/g of fallen fat) 3029t
The mixture was suspended in 20 ml of DMF C) for 30 minutes in a M. rrifl.

Frnoc-Pro @ 13vtal1 haiku friend.

This time, the following Frnoe amm cycle was applied.

a) Shake for 1 minute (once) in Dh'W 20 nt.

b) Boil for 3 minutes in 5096 pi (Ritz-DMF melt a201111j).

c) 50% H < 17 J-DMF bath solution 20
111j Chute IO Bunkangi Toshi leaves the Frnoc group.

d) 4 times'elc# in DMF' 201117.

6) Wash once with Isoglono or Nol 20 #11.

Here, the Kaiser method [E, Kaiser at
al, , Anal.

Blochem, 34,595 (1970),
Make sure Fmoc & is completely removed and if
If it is incomplete, repeat the above removal cycle. l,
If completely removed, then subject to the motion table cycle shown below.

f) The Fonoch cycle was obtained fl-
Swelling was carried out by drilling twice with H-Pro side fat gold DMF 20 d.

g) Fmoe-Ala-OH (149”?, 0.4
8t Add DMF bath water (20mt) of HOBE (78cm, 0.58mmol) and stir for 1 minute.

Add 0.52817 t of acrohexyl lugoimide methylene chloride bath solution and stir for 70 minutes.

1) Wash twice with DMF'20aJ.

j) Iso-10-9-nol 2 (washed twice with IJ).

Here, use Kaiaor'rE to check whether the condensation is photon or not, and if it is incomplete, repeat the above condensation cycle.

When using Fmgc-Pro-resin, use the following method as the F'moc & removal cycle here.k) Shake for 1 minute in 20μ DMF (once).

1) 0.2% Hyalinoy-DMF bath? ei
, Take an image for 10 minutes in 20 Ill.

Pro) 0.2% Hype IJ Non-DMF bath! L2
During 01j, image for 10 minutes and remove FrflOO&n) Wash 4 times with DMF 201LI.

O) Ingropanol 2 oMLt and 1 round a purification.

Here, it was confirmed that the Fmoc pickle was completely removed by the Kal@r method. g) Slug t″Fm
oe-Thr(Bu)-(subjected to the condensation cycle performed by JH. According to Mr. Imagari, F+noe product removal cycle and Fcnoc amino acid sardine Boc.

The combination cycle was repeated to perform a desorption step from the combined resin.

That is, wash twice with 20M methylene chloride, and wash with yl chloride.
fv:y(10au)-7-1-7-#(2,00rn
l) - Suspended in a mixed solution of thiophenol (0,661117), then trifluoroic acid rR(2Qau)-methylene tetrachloride (2,3211Lt) was added, and the mixture was shaken for 1 hour. The resin was filtered, and the resulting f'L filtrate was condensed under reduced pressure.
By adding ether and filtering, the resulting white powder is subjected to reverse phase liquid chromatography to separate the desired Lys-Thr-Alt-Pro fraction gold, and the resulting eluted fraction is concentrated to dryness. Then, after adding distilled water and repeating concentration to dryness several times, it is dissolved in a small quantity of distilled water and freeze-dried. In this way, the elliptical Lys-Thr-Al
Obtained a-Prof. After removing some gold from the treetop proboscis and conducting elemental analysis, we found that the molecular formula was Cl8H3, N506.2C.
F, C00H-N20 analysis value C, 39,74H,
5,60N10.33, calculated value C,39,94N5
．． 64 N10.59.

Mano, I conducted molecular measurements using the FAR Jidai analyzer.
/z: 416 (M” 10H), which corresponded directly to *1.

Furthermore, when it was hydrolyzed with 6N-HCt71 bath solution and subjected to amino quasi-analysis, Thr O, 95, Pro 1.0
2, Alm 1.02.

The ratio of Lysl and UO and n also agrees with the theory of 1 and is 9.

Therefore, it is confirmed that the desired peptide has been synthesized and its purity is confirmed by thin-layer chromatography and reversed-phase plate chromatography.

The following (gutedo) was synthesized by performing the same reactions and treatments as above.

Example 14 Activity Test The salt of Peptide Makuv1 of the present invention has an anonootensin converting enzyme inhibitory effect. The enzyme inhibitory effect will be explained below.

225 μl of 10 m
Mp-hydroxybenzoylglycyl-L-hisnanolume-leucine, 2.5mM 4-7 minoantipyrine,
3 units/Ill Hyglycase (0.7MN aCL
Say 0.12 1 t 11 Yakubi Sou's mochi liquid Ll) Kuchie 3
After incubating at 7°C for 3 minutes, about 70 milliunits of rabbit lung annootensin' and xenobiotics were injected to start the reaction.
Insulated at 37℃ for 0 minutes, 750μ! 3mM gDTA
, 0.2%) Rydon X-100,6,5m
Add M sodium periodate solution to the mouth to stop the reaction.

Then, keep it warm for 3 minutes to develop a color.The reaction bath solution was subjected to colorimetric constant charging at a wavelength of 5 U 5 nm using water as a control.

As an IC5° container for the sample when the inhibition rate is 50%.

IfLt-fit for some of the peptides of the present invention is shown in Table 1. Based on the M values in the above table, the peptides of the present invention have an inhibitory effect on annotensin.

Effects of the Invention From the above results, the novel peptide of the present invention is useful as an antihypertensive agent, and the present invention is important in the pharmaceutical industry.

Claims (1)

[Claims] 1. A peptide represented by any of the following structural formulas and a salt thereof. [Gene sequence is available] 2. An antihypertensive agent containing a peptide represented by one of the structural formulas below or a pharmaceutically acceptable salt thereof as an active ingredient. [There is a gene sequence]