JPH03167199A - Novel heptapeptide - Google Patents
Novel heptapeptideInfo
- Publication number
- JPH03167199A JPH03167199A JP1305476A JP30547689A JPH03167199A JP H03167199 A JPH03167199 A JP H03167199A JP 1305476 A JP1305476 A JP 1305476A JP 30547689 A JP30547689 A JP 30547689A JP H03167199 A JPH03167199 A JP H03167199A
- Authority
- JP
- Japan
- Prior art keywords
- enkephalin
- present
- fraction
- acid
- compound
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 150000003839 salts Chemical class 0.000 claims description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 abstract description 21
- 241000283690 Bos taurus Species 0.000 abstract description 9
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 6
- 239000000463 material Substances 0.000 abstract description 5
- 230000000202 analgesic effect Effects 0.000 abstract description 4
- 239000002532 enzyme inhibitor Substances 0.000 abstract description 4
- BQJCRHHNABKAKU-KBQPJGBKSA-N morphine Chemical compound O([C@H]1[C@H](C=C[C@H]23)O)C4=C5[C@@]12CCN(C)[C@@H]3CC5=CC=C4O BQJCRHHNABKAKU-KBQPJGBKSA-N 0.000 abstract description 4
- 238000002360 preparation method Methods 0.000 abstract description 3
- 238000004440 column chromatography Methods 0.000 abstract description 2
- 229960005181 morphine Drugs 0.000 abstract description 2
- 239000002671 adjuvant Substances 0.000 abstract 1
- 239000000730 antalgic agent Substances 0.000 abstract 1
- 229940125532 enzyme inhibitor Drugs 0.000 abstract 1
- 238000001641 gel filtration chromatography Methods 0.000 abstract 1
- 108090000765 processed proteins & peptides Proteins 0.000 abstract 1
- 230000003014 reinforcing effect Effects 0.000 abstract 1
- 150000001875 compounds Chemical class 0.000 description 15
- UUUHXMGGBIUAPW-UHFFFAOYSA-N 1-[1-[2-[[5-amino-2-[[1-[5-(diaminomethylideneamino)-2-[[1-[3-(1h-indol-3-yl)-2-[(5-oxopyrrolidine-2-carbonyl)amino]propanoyl]pyrrolidine-2-carbonyl]amino]pentanoyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-3-methylpentanoyl]pyrrolidine-2-carbon Chemical compound C1CCC(C(=O)N2C(CCC2)C(O)=O)N1C(=O)C(C(C)CC)NC(=O)C(CCC(N)=O)NC(=O)C1CCCN1C(=O)C(CCCN=C(N)N)NC(=O)C1CCCN1C(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)C1CCC(=O)N1 UUUHXMGGBIUAPW-UHFFFAOYSA-N 0.000 description 13
- 102000004270 Peptidyl-Dipeptidase A Human genes 0.000 description 13
- 108090000882 Peptidyl-Dipeptidase A Proteins 0.000 description 13
- 239000000126 substance Substances 0.000 description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 238000000034 method Methods 0.000 description 12
- 230000002401 inhibitory effect Effects 0.000 description 10
- 210000000278 spinal cord Anatomy 0.000 description 10
- 230000000694 effects Effects 0.000 description 9
- 239000000843 powder Substances 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 8
- 108010005324 enkephalin degrading enzyme Proteins 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 108090000915 Aminopeptidases Proteins 0.000 description 6
- 102000004400 Aminopeptidases Human genes 0.000 description 6
- URLZCHNOLZSCCA-VABKMULXSA-N Leu-enkephalin Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)CNC(=O)CNC(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=CC=C1 URLZCHNOLZSCCA-VABKMULXSA-N 0.000 description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 239000002904 solvent Substances 0.000 description 6
- 101000930822 Giardia intestinalis Dipeptidyl-peptidase 4 Proteins 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 4
- 238000010828 elution Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 239000003112 inhibitor Substances 0.000 description 4
- 229930014626 natural product Natural products 0.000 description 4
- 239000000741 silica gel Substances 0.000 description 4
- 229910002027 silica gel Inorganic materials 0.000 description 4
- 229960001866 silicon dioxide Drugs 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 230000000593 degrading effect Effects 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 238000012856 packing Methods 0.000 description 3
- 230000036407 pain Effects 0.000 description 3
- 239000005541 ACE inhibitor Substances 0.000 description 2
- DKPFZGUDAPQIHT-UHFFFAOYSA-N Butyl acetate Natural products CCCCOC(C)=O DKPFZGUDAPQIHT-UHFFFAOYSA-N 0.000 description 2
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 230000036592 analgesia Effects 0.000 description 2
- 229940044094 angiotensin-converting-enzyme inhibitor Drugs 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 229920001429 chelating resin Polymers 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000012847 fine chemical Substances 0.000 description 2
- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 1
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 238000003277 amino acid sequence analysis Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000001530 fumaric acid Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000001630 malic acid Substances 0.000 description 1
- 235000011090 malic acid Nutrition 0.000 description 1
- -1 marvin M Chemical compound 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 239000012046 mixed solvent Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 239000008177 pharmaceutical agent Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 235000011056 potassium acetate Nutrition 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- GAPYKZAARZMMGP-UHFFFAOYSA-N pyridin-1-ium;acetate Chemical compound CC(O)=O.C1=CC=NC=C1 GAPYKZAARZMMGP-UHFFFAOYSA-N 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は医薬殊にエンケファリン分解酵素産害剤として
有用紅新規へブタペブチド又はそＣ塩に関する。DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a novel hebutapebutide or its C salt useful as a pharmaceutical agent, particularly as an enkephalin-degrading enzyme production inhibitor.
（発明が解決しようとする課題及び課題を解汐するため
の手段）
本発明者らは，従来より生埋活性を有する世分子の生体
成分の探索を目的として，種々の４体成分を単離してス
クリー二／グを行ってきた今回ウ７脊髄から抽出された
物質につきスクリーニングを進めてきた結果，エンケフ
ァり／分帛１．
３，
酵素阻害活性を有する物質が存在することをつきとめ，
該物質の化学構造を確認したところ，従来文献未載のヘ
プタペプチドであることを知見して本発明を完成させる
に，至った。(Problems to be Solved by the Invention and Means for Solving the Problems) The present inventors have isolated various four-body components for the purpose of searching for biocomponents of molecules that have bioburial activity. As a result of screening the substances extracted from the spinal cord, Enkephali/Branch 1. 3. Find out that there is a substance that has enzyme inhibitory activity,
When the chemical structure of the substance was confirmed, it was discovered that it was a heptapeptide, which had not been previously described in any literature, leading to the completion of the present invention.
本発明の親規ヘブタペプチドは下記式（Ｉ）で示される
。The parent heptapeptide of the present invention is represented by the following formula (I).
Ｌｅｕ−Ｖａｌ −’Ｖａｌ −Ｔｙｒ −Ｐｒｏ −
Ｔｒｐ−Ｔｈｒ （Ｉ）すなわち，本発明は式（Ｉ）
で示されるヘブタペブチド又はその塩を発明の構或とし
，その提供を目的とする。Leu-Val-'Val-Tyr-Pro-
Trp-Thr (I) That is, the present invention provides formula (I)
The present invention constitutes and aims to provide hebutapebutide or a salt thereof shown in the following.
本発明化合物（Ｉ）の塩としては，塩酸，硫酸，硝酸，
リン酸ｋどの鉱酸，酢酸，シュウ酸，クエン酸，リンゴ
酸，フマール酸， マｖインＭ，コハク酸，酒石酸など
の有機酸との酸付加塩が挙げられる。Salts of the compound (I) of the present invention include hydrochloric acid, sulfuric acid, nitric acid,
Examples include acid addition salts with mineral acids such as phosphoric acid, acetic acid, oxalic acid, citric acid, malic acid, fumaric acid, marvin M, succinic acid, and tartaric acid.
また，本発明化合物（Ｉ）には，光学異性体が存在する
。本発明にはこれら異性体の単離されたものあるいはそ
の混合物も包含される。中でも工ｙケファリ／分解酵素
阻害剤としては特に天然型のものが好適である。Further, the compound (I) of the present invention has optical isomers. The present invention includes isolated forms of these isomers or mixtures thereof. Among these, natural type inhibitors are particularly suitable as cephalic acid/degrading enzyme inhibitors.
（製造法）
本発明化合物（Ｉ）は，ウシ脊髄より単離・精製するこ
とによって純粋物として得ることができるが，常法のベ
プチド合成法を適用することによって合成することも可
能である。(Production method) Compound (I) of the present invention can be obtained as a pure product by isolating and purifying it from bovine spinal cord, but it can also be synthesized by applying a conventional peptide synthesis method.
また，塩は化合物（【）を通常の造塩反応に付して製造
することができる。In addition, a salt can be produced by subjecting the compound () to a normal salt-forming reaction.
本発明化合物（Ｉ）はウシ脊髄から初めて抽出，分離，
精製されたものであるが，本発明化合物（１）が含まれ
ている天然物であればいずれも本発明化合物（ｒ）の抽
出原料とすることができ，必ずしもウシ脊髄のみに限定
されるものではない。The compound (I) of the present invention was extracted for the first time from bovine spinal cord, isolated,
Although it is a purified product, any natural product that contains the compound (1) of the present invention can be used as a raw material for extracting the compound (r) of the present invention, and is not necessarily limited to bovine spinal cord. isn't it.
天然物から本発明化合物（Ｉ）を得るには，通常天然物
から抽出し，単離・精製する手段が適用される。In order to obtain the compound (I) of the present invention from a natural product, methods of extraction, isolation and purification from the natural product are usually applied.
抽出は，本発明化合物（Ｉ）を溶解抽出可能な溶媒が用
いられ，このような溶媒としては例えば水，メタノール
，エタノール，グロパノール，イングロパノール，ｎ−
ブタノール紅どのアルコール類，ジメチルスルホキシド
やこれらの混合溶媒が好適である。For extraction, a solvent capable of dissolving and extracting the compound (I) of the present invention is used, such as water, methanol, ethanol, gropanol, ingropanol, n-
Alcohols such as butanol, dimethyl sulfoxide, and mixed solvents thereof are suitable.
特に，ウシ脊髄からの抽出は，ウシ脊髄をミンチにし，
ホモジナイズした細断物を用いるのが望ましい。In particular, extraction from bovine spinal cord is performed by mincing the bovine spinal cord and
It is preferable to use homogenized shredded material.
天然物からの単離・精製は，抽出後種々の吸着剤に対す
る吸着親和性の差，種々の溶剤に対する溶解性あるいは
溶解度の差，２種の混り合わない液相聞における分配の
差，分子の大きさに基づく溶出速度の差，溶液からの析
出性あるいは析出速度の差などの理化学的性質の差を利
用する手段を適用して行なわれる。これらの方法は，必
要に応じて単独に用いられ，あるいは任意の順序に組合
せ，また反覆して適用される。Isolation and purification from natural products involves the following factors: differences in adsorption affinity for various adsorbents after extraction, differences in solubility or solubility in various solvents, differences in distribution between two immiscible liquid phases, and molecular This is done by applying means that take advantage of differences in physical and chemical properties, such as differences in elution rate based on the size of the particles, and differences in the ability to precipitate from a solution or the rate of precipitation. These methods can be used alone, combined in any order, or repeatedly applied as needed.
特にウシ脊髄から，本発明化合物な単離・精製するには
，アンジオテンシン変換酵素阻害活性（アンジオテンシ
ン変換酵素はエンケファリンを分解する酵素でもある）
及びエンケ７アリン分解酵素阻害活性を指標としながら
，ＱＤＳ（山村化学社製），アンバーライト■（Ａｍｂ
ｅｒｌ ｉｔｅ ）Ｘ，ＳＤ−４（米国ローム・アンド
・ノ・−ス社製）などをカラム充填剤とするカラムクロ
マドグラフィー，エスピーセファデックス（ ＳＰ−Ｓ
ｅｐｈｅｄｅｘ）Ｃ−２５ （ファルマシア ファイ
ンケミカル社製），バイオゲル（Ｂｉｏｇｅｌ） Ｐ−
２ （バイオラド社製）々とをカラム充填剤とするゲル
Ｐ過，シリカゲル（Ｓｉｌｉｃａｇｅｌ） Ｃ−２００
（和光純薬社！Ｒ），カプセルパック（Ｃａｐｃｅｌ
ｌｐａｋ） Ｃ １８ ？．Ｃどをカラム充填剤とする
高速液体クロマトグラフィーなどに付して実施するのが
有利である。In particular, in order to isolate and purify the compound of the present invention from bovine spinal cord, it is necessary to have angiotensin-converting enzyme inhibitory activity (angiotensin-converting enzyme is also an enzyme that degrades enkephalin).
QDS (manufactured by Yamamura Chemical Co., Ltd.), Amberlite■ (Amb
Column chromatography using column packing materials such as erlite)
ephedex) C-25 (manufactured by Pharmacia Fine Chemicals), Biogel P-
2 (manufactured by Bio-Rad) as a column packing material, silica gel (Silicagel) C-200
(Wako Pure Chemical Industries!R), Capsule Pack (Capcel
lpak) C18? ．． It is advantageous to carry out the process by high performance liquid chromatography using C or the like as a column packing material.
（理化学的性質と同定）
ウシ脊髄より上記の方法によって得られた本発明化合物
（Ｉ）の埋化学的性質は以下の通りである。(Physical and chemical properties and identification) The chemical properties of the compound (I) of the present invention obtained from bovine spinal cord by the above method are as follows.
（！） 外観 白色粉末 溶剤に対する溶解性 水，メタノールおよびジメチルスルホキシドに可溶。(!) exterior white powder Solubility in solvents Soluble in water, methanol and dimethyl sulfoxide.
Ｒｆ値［薄層クロマトグラフィー：シリカ｛２）
（３）
？ル（メルク社製）］
Ｒｒ Ｏ．４８ ［展開溶媒；ｎ−ブタノール：酢酸ブ
チル：酢酸：水（容量
比４：１：１：１コ
に単一スポット。Rf value [thin layer chromatography: silica {2) (3) ? (manufactured by Merck & Co.)] Rr O. 48 [Developing solvent; n-butanol:butyl acetate:acetic acid:water (single spot in volume ratio 4:1:1:1).
（４）呈色反応 陽性
エード蒸気 陽性
ニンヒドリン 陽性
ライドンースミス 陽性
エーリッヒ 陽性
（ベプチドで，トリプトファン含有）
（５） 質量分析（Ｆ’ＡＢ−ＭＡＳＳ）第１図参照
。 ｍ／ｚ ＝８７７（Ｍ”＋１ ）これらの分析結果
と， Ｂ／Ｅリンクドスキャン測定による質量分析及
びア■ノ酸Ｎ一末端配列分析に基づいてこの化合物を以
下の構造を有するヘプタペプチドと同定した。(4) Color reaction Positive ade vapor Positive ninhydrin Positive Lydon-Smith Positive Ehrlich Positive (Veptide, containing tryptophan) (5) Mass spectrometry (F'AB-MASS) See Figure 1. m/z = 877 (M''+1) Based on these analysis results, mass spectrometry by B/E linked scan measurement and amino acid N-terminal sequence analysis, this compound was identified as a heptapeptide having the following structure. Identified.
なお，アミノ酸Ｎ一末端配列分析は気相プロテイン シ
ークエンサー（米国ＡＢＩ社ｉ ４７０Ａ−１２ＯＡ）
を用いて，エドマン分解によるＮ末端アミノ酸配列分析
を行った。The amino acid N-terminal sequence analysis was performed using a gas phase protein sequencer (ABI i 470A-12OA, USA).
N-terminal amino acid sequence analysis was performed using Edman degradation.
Ｈ−Ｌ−Ｌｅｕ−Ｌ−Ｖａｔ −Ｌ−Ｖａｌ −Ｌ−Ｔ
ｙｒＬ−Ｐｒｏ−Ｔｒｐ−Ｌ−Ｔｈｒ −ＯＨ（発明の
効果）
本発明化合物（Ｉ）及びその塩は，エンケファリン分酸
酵素の活性を阻害する作用を有し，エンケファリン分解
酵素阻害剤として有用である。H-L-Leu-L-Vat -L-Val -L-T
yrL-Pro-Trp-L-Thr -OH (Effects of the Invention) The compound (I) of the present invention and its salts have the effect of inhibiting the activity of enkephalin-degrading enzyme, and are useful as enkephalin-degrading enzyme inhibitors. .
エンケファリン分解酵素阻害剤は，鎮痛作用に関与する
内因性物質（オピオイド）のエンケファリンを分解する
酵素［エンケフアリン分解酵素：該酵素にはアミノペプ
チダーゼ（ＡＰ）ジペプチジルアミノペプチダーゼ（Ｄ
ＤＰ ；ＤＤＰ−ＡおよびＤＤＰ−８の２種類），アン
ジオテンシン変換酵素（ＡＣＥ） ，エンケファリンー
ジペプチジルカルポキシベブチダーゼがある］の活性を
阻害するので，エンケファリンが低レベルである疼痛患
者の疼痛を緩解するための鎮痛剤としてあるいは針鎮痛
，モルヒネ鎮痛の鎮痛作用を増強するための補助薬とし
て用いられる。Enkephalin-degrading enzyme inhibitors are enzymes that degrade enkephalin, an endogenous substance (opioid) involved in analgesic effects [enkephalin-degrading enzyme: the enzyme contains aminopeptidase (AP), dipeptidyl aminopeptidase (D
DP (two types, DDP-A and DDP-8), angiotensin converting enzyme (ACE), and enkephalin-dipeptidylcarpoxybebutidase], thereby reducing pain in pain patients with low enkephalin levels. It is used as an analgesic to relieve pain, or as an adjunct to enhance the analgesic effect of needle analgesia or morphine analgesia.
本発明化合物（Ｉ）の薬埋活性は，以下の方法によって
確認された。The potency of the compound (I) of the present invention was confirmed by the following method.
実験方法
（１１ オピオイド分解酵素の調製
サル脳の膜結合オビオイド分解酵素の調製は， Ｇｏｒ
ｅｎｓｔｅｉｎ， Ｃ＆Ｓｙｎｄｅｒ， Ｓ．Ｈ．らの
方法［ＬｉｆｅＳａ，， ２５， ２０６５ （１９７
９） ］ に準じて部分精製シた。Experimental method (11. Preparation of opioid degrading enzyme) Preparation of monkey brain membrane-bound ovioid degrading enzyme was performed using Gor.
enstein, C & Synder, S. H. [LifeSa, 25, 2065 (197
9)].
（２） エンケフアリ７分解酵素阻害活性の測定本発
明化合物のエンケファリン分解酵素阻害活性の測定は，
いずれも公知の方法により測定した。′
す々わち，
アミノペプチダーゼ（ＡＰ） ：
Ｍ．Ｓｈｉｍａｍｕｒａ， Ｈａｚａｔｏ ＆ Ｔ．Ｋ
ａｔａｙａｍａらの方法［ＢＢＡ， １７５６， ２２
３−２２９ （１９８３） ］ジペプチジルアミノベプ
チダーゼ（ＤＰＰ） ：Ｔ．Ｈａｚａｔｏ， Ｔ．Ｋａ
ｔａｙａｍａ ＆ Ｔ＋Ｙａｍａｍｏｔｏらの方法［Ｂ
．Ｂ， Ｒ．Ｃ．， １０５（２）， ４７０−４７５
（１９８２） ］アンジオテンシン変換酵素（ＡＣＥ）
：Ｈａｚａｔｏ Ｔ＋＆ Ｋａｓｅ， Ｒ，らの方法
［Ｂ， Ｂ， Ｒ， Ｃ．． １３９． ５２−５５（
１９８６）コに準ずる方法により行った。(2) Measurement of enkephalin heptalytic enzyme inhibitory activity Measurement of the enkephalin degrading enzyme inhibitory activity of the compound of the present invention is as follows:
All were measured by known methods. ' Aminopeptidase (AP): M. Shimamura, Hazato & T. K
The method of Atayama et al. [BBA, 1756, 22
3-229 (1983)] dipeptidyl aminopeptidase (DPP): T. Hazato, T. Ka
tayama & T+Yamamoto et al.'s method [B
．． B, R. C. , 105(2), 470-475
(1982)] Angiotensin converting enzyme (ACE)
: Hazato T+ & Kase, R, et al.'s method [B, B, R, C. ．． 139. 52-55 (
1986).
実験結果
上記構造特定がなされたヘプタペプチドのＡＰ，ＤＰＰ
， ＡＣＥに対する阻害活性の測定結果を以下に示す。AP, DPP of the heptapeptide whose structure was identified as above as a result of the experiment
, the measurement results of the inhibitory activity against ACE are shown below.
なお，表中のＩＣ５ｏの数値は，各エンケファリン分解
酵素の活性を５０％阻止するヘプタペブチドの濃度（μ
ｇ／ｍＺ）を示す。The IC5o value in the table is the concentration of heptapebutide that inhibits the activity of each enkephalin degrading enzyme by 50% (μ
g/mZ).
表１ （実施例） 以下に実施例を掲記し，本発明を更に詳細に説明する。Table 1 (Example) EXAMPLES The present invention will be explained in more detail with reference to Examples below.
なお，中間単離物質の薬埋活性は前記の方法で測定した
結果である。Note that the drug immobilization activity of the intermediate isolated substance is the result of measurement using the method described above.
実施例
（＋１ 新鮮なウシ脊髄４０ｋｇは屠殺所の芝浦臓器
（株）より購入し，直ちに４℃，生埋食塩水で血液など
を取り除き，実験に使用する当日まで−８０℃に保存し
た。実験当日，脊髄を解凍し，電気調理器（ナショナル
ＭＫ−Ｋ７０）でミンチした。その後，３倍のＩＭ酢酸
を加えポリトロン（ Ｐｏｌｙｔｒｏｎ）でホモジェナ
イズしＡＣＥ阻害物質を抽出した。Example (+1) 40 kg of fresh bovine spinal cord was purchased from Shibaura Organs Co., Ltd., a slaughterhouse, immediately kept at 4°C, blood etc. removed with raw saline solution, and stored at -80°C until the day it was used for the experiment.Experiment On the same day, the spinal cord was thawed and minced using an electric cooker (National MK-K70). Thereafter, 3 times as much IM acetic acid was added and homogenized using a Polytron to extract the ACE inhibitor.
更に，４℃氷室でＦ遇し（東洋／ｌ６２），上清（ １
２０ ｔ ）をＯＤＳカラム（ ４．４ Ｘ １３ｃ
ｍ， 山村化学６０／２００メッシ．）に付し，充分
量の蒸留水で担体を洗い， ８０％メタノールでＡＣ
Ｅ阻害物質を溶出し減圧乾固した。粗粉末として２．１
ｇを得た。Furthermore, the supernatant (1
20t) to an ODS column (4.4 x 13c)
m, Yamamura Kagaku 60/200 Messi. ), wash the carrier with a sufficient amount of distilled water, and wash it with AC with 80% methanol.
The E inhibitor was eluted and dried under reduced pressure. 2.1 as coarse powder
I got g.
（２） 粗粉末２．１ｇを蒸留水１ｔに溶解し，蒸留
水で平衡化されたアンバーライト（Ａｍｂｅｒｌｉｔｅ
）ＸＡＤ −４カラム（４．４ Ｘ １３ｅｍ）ｌ
米国ローム・アンド・ハース社）に付し，水＜５００１
ＩＩｌ）と８０％メタノール（５００ ｍｌ ）のグラ
ジエント（Ｇｒａｄｉｅｎｔ）をした。(2) 2.1 g of coarse powder was dissolved in 1 t of distilled water, and Amberlite was equilibrated with distilled water.
)XAD-4 column (4.4 x 13em)l
Attached to Rohm & Haas (USA), Wednesday <5001
A gradient of 80% methanol (500 ml) was created.
生埋活性物質は分画（Ｆｒ） ３０〜４８にＡＣＥの阻
害活性ピークとして観測された。なお，１分画１０？ｌ
とした。The bioactive substance was observed as an ACE inhibitory activity peak in fractions (Fr) 30 to 48. Furthermore, 1 fraction is 10? l
And so.
活性ピークを減圧乾固し，粉末として８７７ｆｆｌｇ得
た。The active peak was dried under reduced pressure to obtain 877fflg of powder.
（３）粉末８８７ ｒＩｌｌ＋をＩＭ酢酸４４０ ｒｎ
ｌに溶かし，ＩＭ酢酸で平衡化したエスピーセファデツ
クス（ｓｐ−Ｓｅｐｈａｄｅｘ） Ｃ − ２５カラム
［３．４Ｘ１２ｃｍ，ファルマシアファインヶ■カル（
Ｐｈａｒｍａｃｉａ ｆｉｎｅ ｃｈｅｍｉｃａｌｓ
）］に付した。活性物質の溶出はＩＭ酢酸（４００ ｍ
ｌ ）とＩＭ酢酸−ピリジン（ ４００ ｍｌ ）のグ
ラジエント（Ｇｒａｄｉｅｎｔ）をした。１分画１０ｍ
＃とじた。ＡＣＥ阻害活性は分画（Ｆｒ） ３２〜４２
， 分画（Ｆｒ） ４３ 〜５６，分画（Ｆｒ） ５
７〜７４にピーク観測された。それぞれの活性ピークを
減圧乾固し粉末とした。(3) Powder 887 rIll+ to IM acetic acid 440 rn
Sp-Sephadex C-25 column [3.4 x 12 cm, Pharmacia Fine Cal (
Pharmacia fine chemicals
)]. Elution of the active substance was performed using IM acetic acid (400 m
1) and IM acetic acid-pyridine (400 ml). 1 fraction 10m
# Closed. ACE inhibitory activity is in fraction (Fr) 32-42
, Fraction (Fr) 43 to 56, Fraction (Fr) 5
A peak was observed between 7 and 74. Each active peak was dried under reduced pressure to form a powder.
分画（Ｆｒ） ３２〜４２のピーク−１は ２９５ ＋
Ｔｌｇ （ＡＣＥＩＣ５０３．６μｇ）得た。さらに，
分画（Ｆｒ） ４３〜５６のピーク■は１４２ ｍｇ
（ＡＣＥＩＣ，０．９μｇ） ，分画（Ｆｒ）５７〜７
４のビークー■は，■（ＡＣＥ ＩＣ，。１．８■）を
それぞ１れ得た。Fraction (Fr) 32-42 peak-1 is 295 +
Tlg (ACEIC503.6 μg) was obtained. moreover,
Fraction (Fr) 43-56 peak ■ is 142 mg
(ACEIC, 0.9 μg), fraction (Fr) 57-7
4 Beku ■ obtained 1 ■ (ACE IC, .1.8 ■) each.
分画（Ｆｒ） ３２〜４２のピークには，エンケファリ
ン代謝酵素アミノペプチダーゼ（ＡＰ）に対して■Ｃ５
０８μｇ，とジペプチジルアミノベプチダーゼ（ＤＰＰ
）に対してＩＣ５６１８μｇとそれぞれ阻害活性を示し
た。The peaks of fractions (Fr) 32 to 42 contain ■C5 for enkephalin metabolizing enzyme aminopeptidase (AP).
08 μg, and dipeptidyl aminopeptidase (DPP)
) showed an inhibitory activity of IC5618 μg.
（４）そこで，ビークＩの粉末に含まれている活性物質
を各種カラム操作で単離することを試みた。(4) Therefore, we attempted to isolate the active substance contained in Beak I powder using various column operations.
ビークＩの粉末２９５ ｆｒａｇをシリカゲル（Ｓｉｌ
ｉｃａｇｅｌ）Ｃ − ２００カラム（２．９ Ｘ ７
．５ ａｍ ，和光純薬），展開溶媒ｎ−プタノー／Ｌ
／：酢酸プチル：酢酸：水（４：８：１：ｌｖ／ｖ）で
平衡化，１分画２ｍ４で溶出した。ＡＣＥ阻害活性ピー
クは分画（Ｆｒ） ２５〜３７１ ０．７ ＭＥ （
Ｉ Ｃ＋ｏ ２８μｇ）と分画（Ｆ’ｒ） ４３”６２
１４ ｒｒ＠ （ ＩＣ＋ｏ１，８μｇ）と２つ観測
された。Beak I powder 295 frag is mixed with silica gel (Sil
icagel) C-200 column (2.9 x 7
．． 5 am, Wako Pure Chemical Industries), developing solvent n-ptanol/L
/: butyl acetate:acetic acid:water (4:8:1:lv/v), and elution was carried out in 1 fraction of 2m4. The ACE inhibitory activity peak was in the fraction (Fr) 25-371 0.7 ME (
I C+o 28μg) and fraction (F'r) 43"62
Two samples were observed: 14 rr@(IC+o1, 8 μg).
（５）更に，分画（Ｆｒ） ４３〜６２ １４ｍｇを高
速液体クロマトグラフィ−（ＨＰＬＣ）で以下のように
精製した。(5) Furthermore, 14 mg of fractions (Fr) 43 to 62 was purified by high performance liquid chromatography (HPLC) as follows.
分画（Ｆｒ） ４３〜６２ １４ｍｇを高速液体クロマ
トグラフィー（ＨＰＬＣ， ＧＨ，Ｓｏ） ［カラム：
カプセルバック（ Ｃａｐｃｓｌｌｐａｋ） ＣＡＢ，
ＳＧ １２０， ４．６ Ｘ １５ｍｍ，溶媒：バッ
ファ−Ａ（１％クエン酸水溶液，５％酢酸カリウム）ト
，ハッファ一Ｂ（バツファ−Ａ４０％アセトニトリル）
］を用いてＡＣＥ阻害物質を精製した。溶出時間２２分
に活性ピークが観測された。Fraction (Fr) 43-62 14 mg was subjected to high performance liquid chromatography (HPLC, GH, So) [Column:
Capsule back (Capcsllpak) CAB,
SG 120, 4.6 x 15mm, Solvent: Buffer A (1% citric acid aqueous solution, 5% potassium acetate), Huffer B (Buffer A 40% acetonitrile)
] The ACE inhibitor was purified using the following method. An activity peak was observed at an elution time of 22 minutes.
検出器は紫外線吸光光度計（Ｕｖ２５４）を用いた。An ultraviolet absorption photometer (Uv254) was used as a detector.
活性分画を減圧乾固しゲルＦ過し，脱塩をした。The active fraction was dried under reduced pressure, filtered through Gel F, and desalted.
（６１ Ｈ Ｐ Ｌ Ｃで精製したＡＣｇ阻害物質な
ノくイオゲル（ Ｂｉｏｇｅｌ） Ｐ − ２カラム［
ＩＸ５３ｃｍ，バイオーラド（ Ｂｉｏ−Ｒａｄ） ］
で脱塩をした。ＡＣＥ活性は分画（Ｆｒ） ５３〜５４
に活性ピークが観測された。１分画０．５ ｍｌとした
。分画（Ｆｒ） ５３〜５４の活性ピークを減圧乾固す
ると１．３ ５ ｍｇの白色粉末を得た。(Biogel P-2 column [ACg inhibitor purified by 61 HPLC]
IX53cm, Bio-Rad]
I desalinated it. ACE activity is fraction (Fr) 53-54
An activity peak was observed. One fraction was 0.5 ml. The active peak of fractions (Fr) 53 to 54 was dried under reduced pressure to obtain 1.35 mg of white powder.
エンケファリン代謝酵素に対する阻害スペクトルは Ａ
Ｐ ＩＣ５０３．３μｇ／ ｌｍｌ， ＤＰ Ｐ ＩＣ
＋ｏ ８μｇ，ＡＣＥ１．２μｇを示した。The inhibition spectrum for enkephalin metabolic enzymes is A
P IC503.3μg/lml, DP P IC
It showed +o 8 μg and ACE 1.2 μg.
本物質の理化学的性質及び同定結果は前記のとおりであ
る。The physicochemical properties and identification results of this substance are as described above.
第ｔ図は実施例により得られたヘプタベプチドのマスス
ペクトルを示す。FIG. t shows the mass spectrum of heptabeptide obtained in Example.
Claims (1)
ｈｒで示される新規ヘプタペプチド又はその塩。[Claims] 1. The following formula Leu-Val-Val-Tyr-Pro-Trp-T
A novel heptapeptide represented by hr or a salt thereof.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1305476A JPH03167199A (en) | 1989-11-24 | 1989-11-24 | Novel heptapeptide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1305476A JPH03167199A (en) | 1989-11-24 | 1989-11-24 | Novel heptapeptide |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH03167199A true JPH03167199A (en) | 1991-07-19 |
Family
ID=17945617
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1305476A Pending JPH03167199A (en) | 1989-11-24 | 1989-11-24 | Novel heptapeptide |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH03167199A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997000890A1 (en) * | 1995-06-23 | 1997-01-09 | Hankyu-Kyoei Bussan Co., Ltd. | Peptide that inhibits blood triglyceride level rise and blood triglyceride level rise inhibitor containing said peptide as active ingredient |
| JP2000095794A (en) * | 1998-09-22 | 2000-04-04 | Itoham Foods Inc | Inhibitor for enkephalin decomposition enzyme |
| US6046168A (en) * | 1993-03-24 | 2000-04-04 | Hankyu-Kyoei Bussan Co., Ltd. | Peptide inhibits blood triglyceride level |
| CN108949880A (en) * | 2018-08-03 | 2018-12-07 | 吉林大学 | A kind of preparation method and applications of Medulla Bovis seu Bubali peptide |
-
1989
- 1989-11-24 JP JP1305476A patent/JPH03167199A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6046168A (en) * | 1993-03-24 | 2000-04-04 | Hankyu-Kyoei Bussan Co., Ltd. | Peptide inhibits blood triglyceride level |
| WO1997000890A1 (en) * | 1995-06-23 | 1997-01-09 | Hankyu-Kyoei Bussan Co., Ltd. | Peptide that inhibits blood triglyceride level rise and blood triglyceride level rise inhibitor containing said peptide as active ingredient |
| AU708938B2 (en) * | 1995-06-23 | 1999-08-19 | MG Pharma, Inc. | A peptide inhibiting elevations of triglyceride levels in blood and an agent for inhibiting elevations of triglyceride levels in blood comprising the peptide as an active component |
| EP0838474A4 (en) * | 1995-06-23 | 2001-04-18 | Hankyu Kyoei Bussan Co Ltd | Peptide that inhibits blood triglyceride level rise and blood triglyceride level rise inhibitor containing said peptide as active ingredient |
| JP2000095794A (en) * | 1998-09-22 | 2000-04-04 | Itoham Foods Inc | Inhibitor for enkephalin decomposition enzyme |
| CN108949880A (en) * | 2018-08-03 | 2018-12-07 | 吉林大学 | A kind of preparation method and applications of Medulla Bovis seu Bubali peptide |
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