GB2217329A - Heptapeptide - Google Patents
Heptapeptide Download PDFInfo
- Publication number
- GB2217329A GB2217329A GB8902426A GB8902426A GB2217329A GB 2217329 A GB2217329 A GB 2217329A GB 8902426 A GB8902426 A GB 8902426A GB 8902426 A GB8902426 A GB 8902426A GB 2217329 A GB2217329 A GB 2217329A
- Authority
- GB
- United Kingdom
- Prior art keywords
- prolyl
- boc
- heptapeptide
- pharmaceutically acceptable
- dcm
- 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.)
- Withdrawn
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a heptapeptide of the formula: L-lysyl-L-prolyl-trans-4-hydroxy-L-prolyl-L-seryl-L-tryptophyl-L- isoleucyl-L-prolinamide and further provides its pharmaceutically acceptable salt. The compound shows growth promoting activity in animals and its solid-phase synthesis is also described.
Description
TITLE
HEPTAPEPTIDE
DESCRIPTION
The invention relates to a new heptapeptide, to its pharmaceutically acceptable salts, to a process for its preparation or for preparation of its pharmaceutically acceptable salts, and to pharmaceutical compositions containing it or its pharmaceutically acceptable salts.
In this Specification, the abbreviations used for designating amino acids and protecting groups are based on recommendations of the IUPAC-IUB Commission on Biochemical
Nomenclature (see Eur. J. Biochem. 138, 9-37; 1984). In particular, the following abbreviations are used throughout the text; Boc, t-butyloxycarbonyl; Bzl, benzyl; C1Z, 2-chlorobenzyloxycarbonyl; DCM, dichloromethane; DIEA, diisopropylethylamine; DMF, dimethylformamide; HF, hydrogen fluoride; Hyp, trans-4-hydroxy-L-proline;
MBHA, 4-methylbenzhydrylamine; RP-HPLC, reversed-phase high-performance liquid chromatography; TFA, trifluoroacetic acid.
The invention provides a heptapeptide having the following structure.
L-lysyl-L-prolyl-trans-4-hydroxy-L-prolyl-L-seryl-L -t ryptophyl-L- isoleucyl-L-prol inamide and further provides its pharmaceutically acceptable salts.
The heptapeptide of the invention may be synthesised by solid phase methodology on a 4-methylbenzhydrylamine resin using an automated synthesizer and commercially available amino acid derivatives. This process is within the scope of the invention.
In a preferred process, the protected heptapeptide is assembled stepwise on an MBHA resin derived from a polystyrene-l%-divinylbenzene copolymer. N a -Boc-amino acids are used throughout the synthesis. Two of the functional groups not involved in peptide bond formation are protected by suitable protecting groups; the hydroxyl of serine by benzyl ESer(Bzl); the amino group of lysine by 2-chlorobenzyloxycarbonyl (Lys(ClZ)J. The other functional groups present in the side-chain of the reacting amino acid derivatives, i.e. the indole ring of tryptophan and the hydroxyl of trans-4-hydroxy-proline, are left unprotected.The two side-chain protecting groups are stable to the acidolytic conditions used for removing the a-amino protecting groups (Boc) at each step of the synthesis, are not split off under coupling conditions and are easily removed after completion of the synthesis.
Couplings are accomplished by the symmetrical anhydride method in DCM or DMF. All the Boc-amino acid anhydrides are coupled in a 2-fold excess. The first Boc-amino acid is attached to the solid support through a double coupling procedure; all the subsequent amino acids are coupled once to the growing chain, except Boc-Pro-OH and
Boc-Lys(ClZ), which are introduced through another double coupling procedure. The comprehensive procedure consists essentially of Boc-deprotection with SO TFA in DCM, containing 2.Ss ethanedithiol and 2.5k anisole as scavengers towards electrophilic alkylation and oxidative degradation of tryptophan, followed by neutralization with 10% DIEA in DMF or DCM and coupling of the Boc-amino acid symmetrical anhydride.Final side-chain deprotection and cleavage of the peptide from the resin is accomplished by treatment with HF: anisole: dimethylsulphide 10:1:1 by volume. The peptide is then extracted from the resin with aqueous acetic acid and purified by RP-HPLC.
The following Example illustrates the invention.
Notes:
The RF value was determined on pre-coated plates of silica gel 60 F254 (Merck), layer thickness 0.25 mm, length 20 cm, using n-butanol:acetic acid:water 4:1:1 by volume as a development system ('Merck' is a trade mark). The product was characterized by its mobility relative to Trp. High voltage paper electrophoresis is carried out with a
Pherograph-Original-Frankfurt Type 64 apparatus at pH 1.2 (formic acid:acetic acid:water 123:100:777 by volume), and at pH 5.8 (pyridine:acetic acid:water 450:50:4500 by volume).The product was characterized by its mobility at pH 1.2 relative to Glu (E1 2) and at pH 5.8 relative to
His (ES.8) Analytical RP-HPLC was performed with a Hewlett Packard
Mod. 1084B apparatus on a Lichrosorb Hibar (Merck, see above) RP-18 column, 250 x 4 mm I.D., particle diameter S > , using the following system: eluent A= KH2PO4 20 mM, pH 3.5: acetonitrile 9:1; eluent B=KH2PO4 20 mM, pH 3.5: acetonitrile 3:7; isocratic elution with 10% B for 1 min, then linear gradient from 10 to 90% B in 20 min; flow rate=l ml/min; detection wavelength = 210 nm.
EXAMPLE L-Lysyl-L-prolyl-trans-4-hydroxy-L-prolyl-L-servl-L-trvpto- phyl-L-isoleucyl-L-prolinamide.
Step 1
Preparation of L-lysyl(2-chlorobenzyloxycarbonyl)-L-prolyl- trans-4-hydroxy-L-prolyl-L-seryl (benzyl) -L-tryptophvl-L- -isoleucyl-L-prolyl-4-methylbenzhydrylamine resin (I).
1.04 g of MBHA resin hydrochloride (0.48 mmol/g) was placed in the reaction vessel, swollen by vortexing in DCM (3 x 0.5 min) and then submitted twice to the following coupling protocol:
PROTOCOL A
Step Reagents and Operations 1 DMF (1 x 0.5 min) 2 10% DIEA in DMF (1 x 0.5 min) 3 DMF (5 x 0.5 min) 4 1 mmol Boc-amino acid symmetrical anhydride
in DMF (1 x 32 min) 5 DMF (1 x 0.5 min) 6 DCM (5 x 0.5 min)
Four of the subsequent amino acids (namely Boc-Ile-OH,
Boc-Trp-OH, Boc-Ser(Bzl)-OH and Boc-Hyp-OH) were successively added according to the single coupling procedure illustrated in Protocol B::
PROTOCOL B
Step Reagents and Operations 1 33k TFA (containing 2.5s ethanedithiol + 2.52 anisole)
in DCM (1 x 80 sec) 2 50Z TFA (containing 2.5%
ethanedithiol + 2.5k anisole)
in DCM (1 x 18.5 min) 3 DCM (3 x 0.5 min) 4 10k DIEA in DMF (2 x 1 min) 5 DMF (5 x 0.5 min) 6 1 mmol Boc-amino acid symmetrical
anhydride in DMF (1 x 26 min) 7 DCM (5 x 0.5 min)
The two remaining amino acids (namely, Boc-Pro-OH and
Boc-Lys(ClZ)-OH) were attached according to a double coupling procedure consisting in the application of
Protocol B followed by Protocol A.After the last synthetic cycle, the Boc-heptapeptidyl-resin was deprotected according to steps 1 and 3 of Protocol B, neutralized with 10% DIEA in DCM (2 x 1 min) and washed with DCM (5 x 0.5 min). After drying product I under reduced pressure, a weight gain of 338 mg was obtained with respect to the starting resin.
Step 2 L-Lysyl-L-prolyl-t rans-4-hyd roxy-L-prolyl-L-se ryl-L-t rypto- -phyl-L-isoleucyl-L-prolinamide. 2 CFaCOOH.
1.3 g peptidyl resin I was stirred with a mixture of HF (20 ml), anisole (2 ml) and dimethylsulphide (2 ml) for 2 hours at 0 0C. Then the HF and dimethylsulphide were evaporated off under reduced pressure and anisole was removed with diethyl ether (1 x 100 ml | + I x 50 ml). The peptide thus cleaved and deprotected was extracted with 30k acetic acid (4 x 50 ml), and the combined extracts were lyophilized to give 258 mg of crude product. Purification was achieved by
RP-HPLC on a c-18 column running a gradient from 20* to 40% of eluent B (0.05% TFA in acetonitrile:water=7:3) in eluent
A (0.05k TFA in water) over 20 min. The collected fractions were checked by analytical HPLC, and those showing a purity greater than 99.5% were pooled; acetonitrile was removed under reduced pressure and the aqueous portion lyophilized. Yield: 140 mg. Amino acid analysis (after hydrolysis with mercaptoethanesulphonic acid): Ser 0.92 (1); Ile 1.00 (1); Lys 1.04 (1); Trp 0.90 (1); Hyp 1.01 (1); Pro 2.03 (2). Rf= 0.1 Trp; E1 .2= 1 Glu; E5.8= 0.78 His; HPLC retention time = 9.4 min.
BIOLOGICAL ACTIVITY
The compound of the invention possesses interesting growth promoting activity in animals as indicated by the in vivo in vitro test system on the protein synthesis of liver tissue as described by K. Kämmerer and A. Dey-Hazra in Veterinr-Medizinische Nachrichten, 99-112 (1980).
Accordingly, the invention further provides a pharmaceutical composition comprising the heptapeptide:
L-lysyl-L-prolyl-trans-4-hydroxy-L-prolyl-L-seryl-L -t ryptophyl-L- isoleucyl-L-prol inamide or a pharmaceutically acceptable salt thereof in admixture with a pharmaceutically acceptable diluent or carrier.
Claims (3)
1. A heptapeptide having the following formula:
L-lysyl-L-prolyl-trans-4-hydroxy-L-prolyl-L-seryl -L-tryptophyl-L-isoleucyl-L-prolinamide or a pharmaceutically acceptable salt thereof.
2. A pharmaceutical composition comprising the heptapeptide of claim 1 or a pharmaceutically acceptable salt thereof in admixture with a pharmaceutically acceptable diluent or carrier.
3. A process for the preparation of a heptapeptide as defined in claim 1, the process being substantially as herein described in the Example.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB888809184A GB8809184D0 (en) | 1988-04-19 | 1988-04-19 | Heptapeptide |
Publications (2)
Publication Number | Publication Date |
---|---|
GB8902426D0 GB8902426D0 (en) | 1989-03-22 |
GB2217329A true GB2217329A (en) | 1989-10-25 |
Family
ID=10635419
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
GB888809184A Pending GB8809184D0 (en) | 1988-04-19 | 1988-04-19 | Heptapeptide |
GB8902426A Withdrawn GB2217329A (en) | 1988-04-19 | 1989-02-03 | Heptapeptide |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
GB888809184A Pending GB8809184D0 (en) | 1988-04-19 | 1988-04-19 | Heptapeptide |
Country Status (1)
Country | Link |
---|---|
GB (2) | GB8809184D0 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004074312A2 (en) * | 2003-02-05 | 2004-09-02 | University Of Ulster | Tryptophyllin peptides and uses thereof |
-
1988
- 1988-04-19 GB GB888809184A patent/GB8809184D0/en active Pending
-
1989
- 1989-02-03 GB GB8902426A patent/GB2217329A/en not_active Withdrawn
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004074312A2 (en) * | 2003-02-05 | 2004-09-02 | University Of Ulster | Tryptophyllin peptides and uses thereof |
WO2004074312A3 (en) * | 2003-02-05 | 2005-01-06 | Univ Ulster | Tryptophyllin peptides and uses thereof |
Also Published As
Publication number | Publication date |
---|---|
GB8902426D0 (en) | 1989-03-22 |
GB8809184D0 (en) | 1988-05-25 |
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Legal Events
Date | Code | Title | Description |
---|---|---|---|
WAP | Application withdrawn, taken to be withdrawn or refused ** after publication under section 16(1) |