EP0434722A1 - New pentapeptide and a process for the preparation thereof - Google Patents

New pentapeptide and a process for the preparation thereof

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Publication number
EP0434722A1
EP0434722A1 EP89910214A EP89910214A EP0434722A1 EP 0434722 A1 EP0434722 A1 EP 0434722A1 EP 89910214 A EP89910214 A EP 89910214A EP 89910214 A EP89910214 A EP 89910214A EP 0434722 A1 EP0434722 A1 EP 0434722A1
Authority
EP
European Patent Office
Prior art keywords
pentapeptide
peptide
formula
boc
ser
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
Application number
EP89910214A
Other languages
German (de)
French (fr)
Inventor
Jan Erik Paulsen
Karl-Ludvig Reichelt
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nycomed Bioreg AS
Original Assignee
Hafslund Nycomed Bioreg AS
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Hafslund Nycomed Bioreg AS filed Critical Hafslund Nycomed Bioreg AS
Publication of EP0434722A1 publication Critical patent/EP0434722A1/en
Pending legal-status Critical Current

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    • 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
    • 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
    • C07K7/067—Hemoregulatory peptides based on sequence Glp-Glu-Asp-Cys-Lys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a new pentapeptide which exhibits an inhibitory effect on DNA synthesis and on the mitotic rate in regenerating liver as well as on DNA synthesis and growth of a malignant hepatoma cell-line in vitro and in vivo.
  • the pentapeptide may be used in the treatment of hepatic diseases in mammals, particularly humans, having undesired cell growth, as in hepatic cancer.
  • the peptide also affects normal liver cells and may accordingly also be used in other situations in which it is desirable to control the growth of the liver cells.
  • pentapeptides with a cell growth inhibitory activity there are previously known pentapeptides with a cell growth inhibitory activity, as described in WO 87/00180 (PCT/NO86/00041) .
  • the pentapeptides described therein inhibit the cell proliferation in squamous epithelia, and are therefore suitable for treatment of undesired cell growth in the epidermis
  • this activity is selective, and the previously known pentapeptides are therefore not equally active with respect to inhibiting undesired cell growth in other organs.
  • Z is CH 2 or CO
  • Y is H or OH
  • X 1 , X 2 and X 3 are each independantly OH or NH 2 , provided that
  • X 2 and X 3 are not both NH 2 .
  • the compounds may be prepared in any manner that is suitabl for the preparation of peptides.
  • the peptide will normally exist as a protected derivative, and the last step or the last steps of the process will be removal of the protective groups (from amino, amido, hydroxyl and/or carboxyl) .
  • a particularly suitable process is the socalled solid phase method which is considered suitable for the preparation of oligopeptides and their analogues in a rapid manner and with good yields. In this method the growing peptide chain is kept attached to a solid polymer support, and the synthesis starts by binding the C-terminal amino acid to the polymer.
  • the most common amino acids attached to a polymer support are today commercially available.
  • next amino acid is then coupled to this polymer-bound amino acid by a repeated cycle with deprotection, washing and coupling.
  • the entire peptide is built up in a polymer-bound form, and when the entire building up has been finished, the final product is split off from the polymer by means of a suitable reagent. Simultaneously or afterwards remaining protective groups are also removed.
  • the peptide chain starts with the C-terminal amino acid. All side chains except the oc-amino group are usually protected with suitable protectin groups stable under the conditions used during the entire synthesis.
  • the ⁇ -amino group is either free to react or semi- protected by an easily removable organic or inorganic salt.
  • To a solution of this C-terminal amino acid (or peptide) the next amino acid to be coupled to the growing peptide chain is added with stirring. All reactive side chains of said next amino acid except the ⁇ -carboxy group are usually protected by suitable protecting groups stable under the conditions used during the entire synthesis.
  • the ⁇ -carboxy group is either preactivated in any suitable way or ready for in situ activation by any suitable method for coupling to the free amino group of the C-terminal amino acid (or peptide) . If the C-terminal amino acid (or peptide) is se iprotected by an organic or • inorganic salt, one equivalent of a suitable base is added to provide the free ⁇ -amino function.
  • any suitable activation method can be used, such as DCC, EEDQ, mixed anhydride, BOP, azide etc.
  • the preactivated reagents may be symmetrical anhydrides, active esters etc.
  • Both preactivation and in situ activated carboxylic acid couplings may be assisted by certain chemical compounds added to speed the reaction or to supress/prevent racemization.
  • Such additives may be such compounds as hydroxybenzotriazole, N- hydroxy-succinimide etc.
  • the resulting peptide is isolated from the reaction mixture by any suitable procedure such as liquid liquid extraction or precipitation.
  • the crude product is usually used without further purification, and purity/identity tests are usually done by TLC.
  • the N-terminal ⁇ -amino group of the new peptide is deprotected by a reagent which is able to remove selectively and quantitatively the temporary N-protecting group, leaving the other (side chain) protecting groups intact.
  • This protected peptide with a free ⁇ -amino group is now the C-terminal part of the growing peptide chain, and the entire procedure is repeated with addition of the next amino acid to be added to the N-terminal of the growing peptide chain. This procedure is repeated until a protected derivative of the entire peptide is obtained.
  • the free peptide is then prepared by treating the protected derivative of the peptide with reagents able to split off all permanent protecting groups. Examples of such reactions/reagent are:
  • Liquid HF for very acid stable protecting groups Liquid HF for very acid stable protecting groups.
  • amino acid units that may be part of the pentapeptide, counted from the C-terminal end are the following amino acid units:
  • amino acids are used in their L-form, except alanine which may also suitably be used in its D-form.
  • the following table illustrates how compound A inhibited the regenerative increase of liver weight in mice after 70% hepatectomy.
  • Compound A was injected daily i.p. at 12oo a.m. in saline. Controls were treated with saline only.
  • this peptide also inhibits DNA synthesis and proliferation of H ⁇ C ⁇ , a clonal strain from a Morris transplantable hepatoma in vitro.
  • Peptide B reduces the total number of rat hepatoma cells in vitro to about 70% of the controls 72 hours after the peptide addition. This will appear from the following table:
  • Peptide B reduces the weight of the lung (lung + metastases to about 40% of the controls. The reduction of the tumor mass is even greater when the weight of the normal lung is subtracted.
  • the new pentapeptides of formula I are particularly suitable in the treatment of hepatic cancer, and can be incorporated in common formulations for parenteral administration. Suitable doses will depend on the actual peptide, the patient and the administration route and composition form. However, the doses used in the above examples indicate relevant doses.
  • the peptides were prepared using the solid phase method according to Merrifield. A known amount of a resin support was placed in a reaction vessel and mixed with 150 ml of the following solvents in the given order. All washings took place for 1 minute if nothing else is stated. All the amino acids used were in the L-configuration. Asn was ⁇ -Asn.
  • Boc-Glu(OcHex) is used instead of Boc-Gln(Xan) .
  • Boc-Glu(OcHex) is used in place of Boc-Gln(Xan) .
  • the peptide attached to the resin was placed in a Kel-F- vessel and cooled to 0°C.
  • Anisole was added as scavenger, and liquid hydrogen fluoride was introduced into the system and mixe with the resin for 45 minutes. Hydrogen fluoride was evaporated under vacuum after a standard procedure prescribed by the supplier.
  • the resin was extracted with ether to remove scavenger and lipophilic byproducts.
  • the crude peptide was extracted into acetic acid and water.
  • the column was pretreated with buffer A which was 0.1% TFA, using 500-1000 ml.
  • the peptide was detected by TLC. It was run on HPLC, and the purest fractions were collected and lyophilized.
  • EEDQ N-ethyloxycarbonyl-2-ethyloxy)-l-dihydroquinoline
  • BOP Benzotriazol-l-yl-oxy-tris-(dimethylamino)phosphonium- hexafluorophosphate
  • the above pentapeptide is prepared in solution by the active ester method with minimum purification of the protected intermediates.
  • Boc-Ser(Bzl)-OSu (1.2 eq.) dissolved in a minimum of DMF is added dropwise to a stirred solution of Asp(OBzl) 2 (1 eq.) in a minimum of DMF.
  • the reaction is monitored by TLC/Ninhydrin. Negative Ninhydrin test indicates total consumption of the amine compound.
  • the crude A is dissolved in icecold CH 2 C1 2 and diluted with an equal volume of TFA.
  • the reaction is monitored by TLC. After 30 min. the solvent is evaporated n vacuo. the residue is resuspended in CH 2 C1 2 and evaporated to dryness in vacuo.
  • the crude product is used without further purification.
  • Boc-Glv-Ser ( Bzl ) -ASP ( OBzl ) 2 (C) Boc-Gly-OSu (1.2 eq.) dissolved in a minimum of DMF is added dropwise to a stirred solution of B (1 eq. ) and NEM (1 eq.) in a minimum of DMF. The reaction is monitored with TLC/Ninhydrin. After 2h, further 0.2 eq. of Boc-Gly-OSu is added, and stirring is continued over night.
  • the crude product is used without further purification.
  • the crude product E is treated with an icecold solution of CH 2 C1 2 /TFA 1:1 for 30 min. TLC indicates full consumption of E.
  • the solvent is evaporated m vacuo; the residue is redissolved in MeOH and evaporated to dryness in vacuo.
  • the crude F is used without further purification.
  • the purified product G is dissolved in MeOH, and 10% Pd/C and ammonium formate (5 eq. ) are added. The reaction is monitored by TLC, and after 45 min. the starting material is totally consumed and TLC is UV 254 negative. The catalyst is removed by filtration, the solvent is evaporated in vacuo. Ammonium formate is removed by lyophilization, and the crude product is purified and identified as compound 4.
  • NEM N-ethyl morpholine

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  • General Chemical & Material Sciences (AREA)
  • Public Health (AREA)
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  • Pharmacology & Pharmacy (AREA)
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  • Engineering & Computer Science (AREA)
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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract

New pentapeptide with the general formula <CHEM> wherein Z is CH2 or CO, Y is H or OH, and X<1>, X<2> and X<3> are each independantly OH or NH2, provided that X<2> and X<3> are not both NH2. The pentapeptide can be used as active component in compositions. The preparation of the pentapeptide is described.

Description

NEW PENTAPEPTIDE AND A PROCESS FOR THE PREPARATION THEREOF
The present invention relates to a new pentapeptide which exhibits an inhibitory effect on DNA synthesis and on the mitotic rate in regenerating liver as well as on DNA synthesis and growth of a malignant hepatoma cell-line in vitro and in vivo.
Thus, the pentapeptide may be used in the treatment of hepatic diseases in mammals, particularly humans, having undesired cell growth, as in hepatic cancer. The peptide also affects normal liver cells and may accordingly also be used in other situations in which it is desirable to control the growth of the liver cells.
There are previously known pentapeptides with a cell growth inhibitory activity, as described in WO 87/00180 (PCT/NO86/00041) . The pentapeptides described therein inhibit the cell proliferation in squamous epithelia, and are therefore suitable for treatment of undesired cell growth in the epidermis However, this activity is selective, and the previously known pentapeptides are therefore not equally active with respect to inhibiting undesired cell growth in other organs.
In an article by Jan Erik Paulsen, Karl-L. Reichelt and Anne K. Petersen in Virchows Arch B (1987) 54:152-154, with the title "Purification and characterization of a growth inhibitory hepatic peptide, A preliminary note" it is described that a pentapeptide isolated from mouse liver seems to inhibit DNA synthesis and the mitotic rate in regenerating mouse liver. It was found that after partial hepatectomy the DNA synthesis and the mitotic rate were strongly reduced after treatment with a peptide isolated from mouse liver compared with the controls.
Except from the fact that there is probably a -question of a pentapeptide with N-terminal pyroglutamate, nothing is mentioned with respect to which amino acids are included and the sequence thereof. After extensive testing we have found several new pentapeptides with a fully identified structure.
According to the invention there are now provided new pentapeptides of the general formula COX1
OX~
wherein
Z is CH2 or CO,
Y is H or OH, and
X1, X2 and X3 are each independantly OH or NH2, provided that
X2 and X3 are not both NH2.
The compounds may be prepared in any manner that is suitabl for the preparation of peptides. At a late stage of its preparation, the peptide will normally exist as a protected derivative, and the last step or the last steps of the process will be removal of the protective groups (from amino, amido, hydroxyl and/or carboxyl) . A particularly suitable process is the socalled solid phase method which is considered suitable for the preparation of oligopeptides and their analogues in a rapid manner and with good yields. In this method the growing peptide chain is kept attached to a solid polymer support, and the synthesis starts by binding the C-terminal amino acid to the polymer. The most common amino acids attached to a polymer support are today commercially available. The next amino acid is then coupled to this polymer-bound amino acid by a repeated cycle with deprotection, washing and coupling. In this manner the entire peptide is built up in a polymer-bound form, and when the entire building up has been finished, the final product is split off from the polymer by means of a suitable reagent. Simultaneously or afterwards remaining protective groups are also removed.
In liquid phase peptide synthesis, the peptide chain starts with the C-terminal amino acid. All side chains except the oc-amino group are usually protected with suitable protectin groups stable under the conditions used during the entire synthesis. The α-amino group is either free to react or semi- protected by an easily removable organic or inorganic salt. To a solution of this C-terminal amino acid (or peptide) the next amino acid to be coupled to the growing peptide chain is added with stirring. All reactive side chains of said next amino acid except the α-carboxy group are usually protected by suitable protecting groups stable under the conditions used during the entire synthesis. The α-carboxy group is either preactivated in any suitable way or ready for in situ activation by any suitable method for coupling to the free amino group of the C-terminal amino acid (or peptide) . If the C-terminal amino acid (or peptide) is se iprotected by an organic or • inorganic salt, one equivalent of a suitable base is added to provide the free α-amino function.
For in situ activation, any suitable activation method can be used, such as DCC, EEDQ, mixed anhydride, BOP, azide etc.
The preactivated reagents may be symmetrical anhydrides, active esters etc.
Both preactivation and in situ activated carboxylic acid couplings may be assisted by certain chemical compounds added to speed the reaction or to supress/prevent racemization. Such additives may be such compounds as hydroxybenzotriazole, N- hydroxy-succinimide etc.
The resulting peptide is isolated from the reaction mixture by any suitable procedure such as liquid liquid extraction or precipitation. The crude product is usually used without further purification, and purity/identity tests are usually done by TLC. The N-terminal α-amino group of the new peptide is deprotected by a reagent which is able to remove selectively and quantitatively the temporary N-protecting group, leaving the other (side chain) protecting groups intact.
This protected peptide with a free α-amino group is now the C-terminal part of the growing peptide chain, and the entire procedure is repeated with addition of the next amino acid to be added to the N-terminal of the growing peptide chain. This procedure is repeated until a protected derivative of the entire peptide is obtained.
The free peptide is then prepared by treating the protected derivative of the peptide with reagents able to split off all permanent protecting groups. Examples of such reactions/reagent are:
TFA for medium acid stable protecting groups
Hydrogenolysis over palladium/carbon for benzylic protectin groups
Liquid HF for very acid stable protecting groups.
Thus, the amino acid units that may be part of the pentapeptide, counted from the C-terminal end, are the following
Generally, all the amino acids are used in their L-form, except alanine which may also suitably be used in its D-form.
Two of the compounds of formula I have been subjected to further tests in vitro and in vivo. These compounds are:
A: pGlu-Gln-Gly-Ser-β-Asn
(Z = CO, Y - OH, X1 = NH2, X2 = NH2, X3 = OH) B: pGlu-Glu-Gly-Ser-β-Asn
(Z = CO, Y = OH, X1 = OH, X2 = NH2, X3 = OH)
(all amino acids in L-configuration) .
The following table illustrates how compound A inhibited the regenerative increase of liver weight in mice after 70% hepatectomy. Compound A was injected daily i.p. at 12oo a.m. in saline. Controls were treated with saline only.
It was further found that this peptide also inhibits DNA synthesis and proliferation of H^C^, a clonal strain from a Morris transplantable hepatoma in vitro.
Compound B was given to mice subjected to 70% hepatectomy, and the mitotic rate was measured 5 hours after the administration. It was then found that the mitotic rate was about 40% of the mitotic rate of the controls as it will appear from the following table:
Peptide Mitotic rate pmole per animal % of controls
0 (control) 100 1 52 ± 9 < 0.0025 10 39 ± 11 < 0.0005 100 37 ± 9 < 0.0005
N = 6 ± SD
Four hours after treatment with the same peptide B, it was also found that the mitotic rate was reduced to about 50% of the rate in the controls 48 hours after CC14 intoxication. Peptide Mitotic rate pmole per animal % of controls
0 (control) 100
1 90 ± 13
10 49 ± 10 < 0.025
100 74 ± 12
N = 3 ± SD
Peptide B reduces the total number of rat hepatoma cells in vitro to about 70% of the controls 72 hours after the peptide addition. This will appear from the following table:
Peptide concentration Total number of cells log M % of controls
0 (control) 100 ■14 79 ± 6 < 0. 005 ■11 71 ± 5 < 0. 0025 - 8 80 ± 7 < 0. 025
N = 6 ± SD
To test the activity of compound B on the malignant hepatoma cell line also in vivo four groups of each 8 animals were used, and a predetermined number of malign cells were injected i.v. , and the animals were then treated by using three different concentrations of compound B.
In this experiment the metastasis spreading to the lungs was measured, and a great reduction could be observed in the treated groups.
Experimental
Immediately after the injection of peptide B, Buffalo female rats, 30 days old, were injected with 104 rat hepatoma cells (MH^Ci) in a tail vein. The rats then received the peptide i.p. three times per week during the experiment (a total of 12 injections) . Four weeks after the experiment started, the lungs containing metastases from the hepatoma cell injection, were weighed (wet weight) .
Results
Peptide B reduces the weight of the lung (lung + metastases to about 40% of the controls. The reduction of the tumor mass is even greater when the weight of the normal lung is subtracted The tests are illustrated in the following table:
Peptide B Weight of lung + metastases pmole per animal % of controls p
< 0.01
< 0.005
< 0.0005
When the weight of the normal lung tissue is taken into consideration and is subtracted from the weights found, the weight of the tumor is found as it will appear from the following table, calculated as % of control:
Peptide B, pmole per animal Tumor weight, % of control
2 45
20 43
200 29
In view of the above described properties, the new pentapeptides of formula I are particularly suitable in the treatment of hepatic cancer, and can be incorporated in common formulations for parenteral administration. Suitable doses will depend on the actual peptide, the patient and the administration route and composition form. However, the doses used in the above examples indicate relevant doses.
The preparation of the peptides of formula I is further illustrated in the following:
The peptides were prepared using the solid phase method according to Merrifield. A known amount of a resin support was placed in a reaction vessel and mixed with 150 ml of the following solvents in the given order. All washings took place for 1 minute if nothing else is stated. All the amino acids used were in the L-configuration. Asn was β-Asn.
1. Methylene chloride - three times
2. 40% trifluoroacetic acid in methylene chloride
3. 40% trifluoroacetic acid in methylene chloride - 30 minutes - once
4. Methylene chloride - once
5. Ethanol - once
6. Methylene chloride - twice
7. 10% triethylamine in methylene chloride - once
8. 10% triethylamine in methylene chloride - 10 minutes - once
9. Methylene chloride - three times 10. Coupling with the amino acid.
Each amino acid was coupled in turn, starting with the carboxyl terminal amino acid. Equal equivalents of the Boc amino acid and the DCC were added in excess based on the resin used. After each coupling the resin was examined by the Kaiser-test to ascertain that the reaction was complete. Deprotection was carried out with 40% TFA, and this was also controlled by the Kaiser-test. The synthesis was considered complete when the last amino acid was successfully coupled. Starting materials used in the synthesis of pGlu-Gln-Gly-Ser-As
1. para-methyl-benzhydrilamine resin
2. Boc-Asn-α-O-benzyl
3. Boc-Ser (Bzl)
4. Boc-Gly
5. Boc-Gln (Xan)
6. p-Glu
In the synthesis of pGlu-Glu-Gly-Ser-Asn, Boc-Glu(OcHex) is used instead of Boc-Gln(Xan) .
The starting materials used in the synthesis of pGlu-Gln-Gly-Ser-Asp:
1. Boc-Asp(OBzl)-resin
2. Boc-Ser (Bzl)
3. Boc-Gly
4. Boc-Gln (Xan)
5. pGlu
For the preparation of pGlu-Glu-Gly-Ser-Asp, Boc-Glu(OcHex) is used in place of Boc-Gln(Xan) .
HF-decomposition:
The peptide attached to the resin was placed in a Kel-F- vessel and cooled to 0°C. Anisole was added as scavenger, and liquid hydrogen fluoride was introduced into the system and mixe with the resin for 45 minutes. Hydrogen fluoride was evaporated under vacuum after a standard procedure prescribed by the supplier. The resin was extracted with ether to remove scavenger and lipophilic byproducts. The crude peptide was extracted into acetic acid and water.
Purification:
Purification of the above peptides was carried out as follows: 1. An open glass column was packed with C-18 resin washed with acetonitrile.
2. The column was pretreated with buffer A which was 0.1% TFA, using 500-1000 ml.
3. The peptide was dissolved and added to the top of the column.
4. A linear gradient was started from 0% B-buffer to 100% B in 40 minutes, buffer B being 60% acetonitrile in A.
5. The peptide was detected by TLC. It was run on HPLC, and the purest fractions were collected and lyophilized.
Explanations:
DCC = dicyclohexylcarbodiimide Bzl = benzyl Xan = xanthyl TFA = trifluoroacetic acid OcHex = cyclohexyloxy Boc = t-butoxycarbonyl
EEDQ = N-ethyloxycarbonyl-2-ethyloxy)-l-dihydroquinoline BOP = Benzotriazol-l-yl-oxy-tris-(dimethylamino)phosphonium- hexafluorophosphate
Analysis:
1) Analysis of pGlu-Gln-Gly-Ser-Asn m.w. 515.44
Thin layer chromatography:
Silica Fm (nBuOH:Pyr:HOAc:H20 15:15:3:12) o-tolidine
Results: Main spot with a distinct lower spot and a negligible upper spot. f = 0.28
Silica 1:1:1:1 (nBuOH:EtOAc:HOAc:H20) o-tolidine
Results: Major spot with a negligible upper spot and a negligible lower spot. Rf = 0.22
Electrophoresis:
Whatman 3MM, pH 3.5 (Pyr:Acetone) ; 1500V. 1 h. o-tolidine Results: Major spot migrates towards the anode with a negligib lower spot.
Rf = 0.25 with picric acid as reference.
Amino acid analysis:
% of peptide 91,1 %
Theory Found
Asp 1 1.09
Ser 1 0.92
Glu 2 2.01
Gly 1 0.98
2) Analysis of pGlu-Glu-Gly-Ser-Asn m.w. 516.42
Thin layer chromatography:
Silica gel Fm (nBuOH:Pyr:HOAc:H20, 15:15:3:12) o-tolidine
Silica gel 1:1:1:1 (nBuOH:EtOAc:HOAc:H 0) o-tolidine
Results: Fm: Major spot with a negligible lower spot
Rf = 0.44
1:1:1:1: Major spot with a negligible lower spot.
Rf = 0.39
Electrophoresis:
Whatman 3MM, pH 1.9 (HCOOH:Acetone) ; 1000V. 1 h. o-tolidine
Results: Single spot migrating towards the anode
Rf = 0.40 with picric acid as reference.
Amino acid analysis: % of peptide 90.1%
3) Analysis of pGlu-Gln-Gly-Ser-Asp m.w. 516.19 Thin layer chro atography:
Silica Fm (nBuOH:Pyr:HOAc:H20, 15:15:3:12) o-tolidine
Results: Major spot with a faint upper spot and a negligible lower spot. R2 = 0.27
Silicagel 1:1:1:1 (nBuOH:EtOAc:HOAc:H20) o-tolidine
Results: Major spot with a negligible upper spot and a negligible lower spot. R2 = 0.32
Amino acid analysis: % of peptide 87.4%
4) Analysis of pGlu-Glu-Gly-Ser-Asp m.w. 517.18
Thin layer chromatography:
Silica Fm (nBuOH:Pyr:HOAc:H20, 15:15:3:12) o-tolidine
Results: Major spot with a negligible upper spot and a negligible lower spot. Rf = 0.22
Silica 1:1:1:1 (nBuOH:EtOAc:HOAc:H20) o-tolidine
Results: Major spot with a negligible lower spot.
Rf = 0.34
Amino acid analysis: % of peptide 90.7%
The above pentapeptide is prepared in solution by the active ester method with minimum purification of the protected intermediates.
The reactants
Asp(OBzl)2
Boc-Ser(Bzl)-OSu
Boc-Gly-OSu
CbZ'-pGlu-OSu are all commercially available compounds.
Boc-Ser(Bzl)-ASP(OBzl) ' (A)
Boc-Ser(Bzl)-OSu (1.2 eq.) dissolved in a minimum of DMF is added dropwise to a stirred solution of Asp(OBzl)2 (1 eq.) in a minimum of DMF. The reaction is monitored by TLC/Ninhydrin. Negative Ninhydrin test indicates total consumption of the amine compound.
The solvent is evaporated in vacuo: the residue is dissolved in CH2C12 and extracted with:
0.05N H2S04 (2x)
1M NaHC03 (2x)
Brine (lx)
H20 (lx)
After drying over MgS04, filtering and evaporation of solvent .in vacuo, the residue is dissolved in a minimum of CH2C12 and triturated with ether/pet.ether and stored at +4°C over night. The precipitate is collected and washed with cold ether/pet.ether and dried in vacuo. Crude yield: 95%.
Ser(Bzl)-Asp(OBzl)2*TFA (B)
The crude A is dissolved in icecold CH2C12 and diluted with an equal volume of TFA. The reaction is monitored by TLC. After 30 min. the solvent is evaporated n vacuo. the residue is resuspended in CH2C12 and evaporated to dryness in vacuo. The crude product is used without further purification.
Boc-Glv-Ser(Bzl) -ASP (OBzl) 2 (C) Boc-Gly-OSu (1.2 eq.) dissolved in a minimum of DMF is added dropwise to a stirred solution of B (1 eq. ) and NEM (1 eq.) in a minimum of DMF. The reaction is monitored with TLC/Ninhydrin. After 2h, further 0.2 eq. of Boc-Gly-OSu is added, and stirring is continued over night.
The reaction mixture is now Ninhydrin negative on TLC, and the crude product is worked up with the same procedure as described for A. After trituration with ether/pet.ether, the crude product is used without further purification. Crude yield: 85%.
Glv-SerrBzl)-Asp(OBzl)2*TFA (D)
The crude product C is treated with an icecold solution of CH2C12/TFA 1:1 for 30 min. TLC indicates full consumption of C. The solvent is evaporated in vacuo: the residue is re- dissolved in MeOH and evaporated to dryness in vacuo. Crude D is used without further purification.
Boc-Glu(Υ-OBzl)-Glv-Ser(Bzl)-AS (OBzl)2 (E)
Boc-Glu(Υ-OBzl)-OSu (1.3 eq.) dissolved in a minimum of
DMF is added to a stirred solution of D (1 eq.) and NEM (1 eq.) in a minimum of DMF. The reaction is monitored by TLC/Ninhydrin. The solvent is evaporated in vacuo, and the crude product is worked up with the procedure described for A.
Crude yield: 90%.
The crude product is used without further purification.
Glu(Υ-OBzl)Glv-Ser(Bzl)-ASP(OBzl)2*TFA (F)
The crude product E is treated with an icecold solution of CH2C12/TFA 1:1 for 30 min. TLC indicates full consumption of E. The solvent is evaporated m vacuo; the residue is redissolved in MeOH and evaporated to dryness in vacuo. The crude F is used without further purification.
Cbz-pGlu-Glu(Υ-OBzl)-Glv-Ser(Bzl -ASPfOBzl)2 (G)
Cbz-pGlu-OSu (1.3 eq.) dissolved in a minimum of DMF is added dropwise to a solution of F (1 eq.) and NEM (1 eq. ) in a minimum of DMF. The reaction is monitored by TLC/Ninhydrin, and stirring is continued over night.
The solvent is evaporated in vacuo. and the crude product is worked up as described for A. The crude product is purified by flash chromatography with CHCl3/MeOH as solvent. Total yield from A: 33%.
pGlu-Glu-Glv-Ser-Asp (H)
The purified product G is dissolved in MeOH, and 10% Pd/C and ammonium formate (5 eq. ) are added. The reaction is monitored by TLC, and after 45 min. the starting material is totally consumed and TLC is UV254 negative. The catalyst is removed by filtration, the solvent is evaporated in vacuo. Ammonium formate is removed by lyophilization, and the crude product is purified and identified as compound 4.
Additional explanations:
DMF = dimethylformamide
Cbz - carbobenzoxy
Su = succinimido
NEM = N-ethyl morpholine

Claims

Patent Claims
1. New pentapeptide, characterized in that it has the formula:
wherein
Z is CH2 or CO,
Y is H or OH, and
X1, X2 and X3 are each independantly OH or NH2 , provided that
X2 and X3 are not both NH2.
2. Pentapeptide according to claim 1, characterized in that Z is CO, Y is OH, X1 is OH, X2 is NH2 and X3 is OH.
3. Pentapeptide according to claim 1, characterized in that Z is CO, Y is OH, X1 is OH, X2 is OH and X3 is OH.
4. Composition for inhibiting uncontrolled cell growth in liver, characterized in that it as the active component comprises a compound of the formula
X3
wherein
Z is CH2 or CO,
Y is H or OH, and
X1, X2 and X3 are each independantly OH or NH2 , provided that
X2 and X3 are not both NH2.
5. Composition according to claim 4, characterized in that it comprises a compound of formula I wherein Z is CO, Y is OH, X1 is OH, X2 is NH2 and X3 is OH.
6. Composition according to claim 4, characterized in that it comprises a compound of formula I wherein Z is CO, Y is OH, X1 is OH, X2 is OH and X3 is OH.
7. Method for inhibiting uncontrolled growth of hepatic cells, characterized by administration of a pentapeptide of the formula
CO-NH- COX3
wherein
Z is CH2 or CO,
Y is H or OH, and
X1, X2 and X3 are each independantly OH or NH2 , provided that
X2 and X3 are not both NH2.
8. Method according to claim 7, characterized in that the pentapeptide has formula I wherein Z is CO, Y is OH, X1 is OH, X2 is OH or NH2 and X3 is OH.
9. Process for the preparation of a pentapeptide of the formula
OX3
wherein Z is CH2 or CO,
Y is H or OH, and
X1, X2 and X3 are each independantly OH or NH2 , provided that
X2 and X3 are not both NH2, characterized in that a protected derivative thereof is deprotected.
10. Process according to claim 9, characterized in that the pentapeptide, optionally in a protected form, is built up by solid phase peptide synthesis wherein the C-terminal amino acid is first attached to a solid support and the following amino acids are then coupled thereto in the desired sequence, and the protective groups are removed.
EP89910214A 1988-09-13 1989-09-12 New pentapeptide and a process for the preparation thereof Pending EP0434722A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
NO884054A NO884054D0 (en) 1988-09-13 1988-09-13 NEW PENTAPEPTIME AND PROCEDURE FOR PREPARING THEREOF.
NO884054 1988-09-13

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Publication Number Publication Date
EP0434722A1 true EP0434722A1 (en) 1991-07-03

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JP (1) JPH04501710A (en)
KR (1) KR900701830A (en)
CN (1) CN1041158A (en)
AT (1) ATE102215T1 (en)
AU (1) AU623687B2 (en)
DE (1) DE68913411T2 (en)
DK (1) DK43891A (en)
ES (1) ES2062024T3 (en)
FI (1) FI911219A0 (en)
HU (2) HU895790D0 (en)
LV (1) LV10289B (en)
MD (1) MD503C2 (en)
MY (1) MY104912A (en)
NO (2) NO884054D0 (en)
NZ (1) NZ230625A (en)
RU (1) RU2010799C1 (en)
WO (1) WO1990002754A1 (en)
ZA (1) ZA896944B (en)

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US5620957A (en) * 1989-07-14 1997-04-15 Smithkline Beecham Corporation Hemoregulatory peptides
TW222280B (en) * 1991-11-26 1994-04-11 Smithkline Beecham Corp
IL104323A0 (en) * 1992-01-10 1993-05-13 Smithkline Beecham Corp Hemoregulatory peptides
RU2297239C1 (en) * 2006-05-30 2007-04-20 Общество С Ограниченной Ответственностью "Сиа Пептайдс" Peptide stimulating regeneration of liver tissue, pharmaceutical composition based on thereof and method for its using

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US2591748A (en) 1949-12-23 1952-04-08 Hercules Powder Co Ltd Amphoteric cellulose derivatives
FR2110268B1 (en) 1970-10-07 1976-06-04 Minnesota Mining & Mfg
US4237253A (en) 1977-04-21 1980-12-02 L'oreal Copolymers, their process of preparation, and cosmetic compounds containing them
NO860752L (en) * 1985-06-26 1986-12-29 Bio Tech As PENTAPEPTIDES WITH CELL GROWTH REGULATORY EFFECTS AND PROCEDURES FOR PRODUCING THEREOF.
GB8626539D0 (en) * 1986-11-06 1986-12-10 Nycomed As Peptide compounds

Non-Patent Citations (1)

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Title
See references of WO9002754A1 *

Also Published As

Publication number Publication date
NO884054D0 (en) 1988-09-13
HU206373B (en) 1992-10-28
LV10289A (en) 1994-10-20
MY104912A (en) 1994-06-30
EP0360481A1 (en) 1990-03-28
DE68913411T2 (en) 1994-06-23
FI911219A0 (en) 1991-03-12
DK43891D0 (en) 1991-03-12
ES2062024T3 (en) 1994-12-16
DK43891A (en) 1991-03-12
NO910933L (en) 1991-03-08
HUT58766A (en) 1992-03-30
MD503B1 (en) 1996-03-29
ZA896944B (en) 1990-06-27
LV10289B (en) 1995-04-20
CN1041158A (en) 1990-04-11
MD503C2 (en) 1996-07-31
HU895790D0 (en) 1991-11-28
AU4224089A (en) 1990-04-02
RU2010799C1 (en) 1994-04-15
ATE102215T1 (en) 1994-03-15
EP0360481B1 (en) 1994-03-02
JPH04501710A (en) 1992-03-26
AU623687B2 (en) 1992-05-21
DE68913411D1 (en) 1994-04-07
NO910933D0 (en) 1991-03-08
KR900701830A (en) 1990-12-04
NZ230625A (en) 1991-06-25
WO1990002754A1 (en) 1990-03-22

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