GB2097404A

GB2097404A - A process for the preparation of lysine-tryptophan oligopeptides and of their physiologically-useful derivatives and salts

Info

Publication number: GB2097404A
Application number: GB8211276A
Authority: GB
Original assignee: Individual
Current assignee: Individual
Priority date: 1981-04-23
Filing date: 1982-04-19
Publication date: 1982-11-03
Also published as: DK179682A; CH657861A5; IT1151728B; FR2504525A1; ES501583A0; IT8220834A0; FR2504525B1; ES8202788A1; BE892932A; NL8201649A; SE8202414L; GB2097404B; DE3213501A1

Abstract

A process for the preparation of ologopeptides constituted by lysinyl and tryptophanyl residues characterised by the following general formula (1): (Lys)a(Trp)b 1 wherein Lys represents a lysinyl residue, Trp represents a tryptophanyl residue, a>o and b>o with the sole condition that when the peptide link, -CONH-, has originated through the amino group of the lysine, that the amino group be that in the alpha position to the carboxyl which gives rise to the formation of said link is carried out in three stages. Firstly, a compound of general formula 2 is reacted with a compound of general formula 3, <IMAGE> in which R1 and R2 independently represent beta -methylenidole or epsilon - aminobutyl with the -NH2 group protected or any residue of any peptide sequence (Lys)c-(Trp)d (wherein c and/or d>o) with the condition that at least one of R1 and R2 is a residue of beta -methylenidole or protected epsilon -aminobutyl, and R3 is an alkyl aryl or arylalkyl group, whereby to obtain the compound of the general formula 4 shown below: <IMAGE> Secondly, the o-nitrosulphenyl group is eliminated and replaced by a hydrogen atom. Thirdly, the resultant amino-ester is treated to remove any amine-protecting groups and is then optionally saponified, followed by neutralization, to give the free peptide 1. Physiologically useful derivatives or salts may then be formed therefrom.

Description

SPECIFICATION A process for the preparation of lysine-tryptophan oligopeptides and of their physiologicallyuseful derivatives and salts This invention refers to a process for the preparation of oligopeptides constituted by lysinyl and tryptophanyl residues characterised by the following general formula (1): (Lis) - (Trpl) wherein Lis represents a lysinyl residue, Trp represents a tryptophanyl residue, a > o and b > o; said general formula being understood to cover all possible combinations of sequences that can be formed with these aminoacids of both the dseries and the / series or with all the possible sequential combinations possible of the d series and of the / series, with the sole condition that when the peptide link. --CONHH-, has originated through the amino group of the lysine, that the amino group be in the a position to the carboxyl which gives rise to the formation of said link.

The invention also extends to the physiologically-useful derivatives and salts of those compounds of general formula 1 and especially to those that facilitate possible therapeutic action.

The process according to the present invention is characterised by the three following stages: Stage 1: Reaction of a compound of general formula 2 with a salt of a compound of formula 3, preferably a hydrochloride or a p- toluenesulphonate in an inert aprotic solvent such as tetrahydrofuran, acetonitrile, N,N-dimethylformamide or mixture of these solvents, in the presence of an aprotic base such as triethylamine, N-methylmorpholine, 2,6-lutidine, etc., for 1 to 2 hours.

where R, and R2 independently represent a /3-methylenindole

or E-aminobutyl group where the E-amino group is protected by a group P (PHN-CH2)3-CH2-) which represents any normal amine-protecting group of aminoacid and protein chemistry, such as substituted carbobenzyloxy, carbotert-butyloxy, trytyl, tosyl, etc., or any residue of any peptide sequence (Lis)c(Trp)d (wherein c > o and/or d > o), with the sole condition that at least one of R1 and R2 is a residue of p-methylenindole or protected E-aminobuWl, and R3 represents an alkyl, aryl or arylalkyl group.

Thus a compound of general formula 4 is obtained without the occurrence of any racemization at all:

wherein R1, R2 and R3 are as noted above.

Compounds of general formula 2 may be obtained by the reaction of o-nitrophenylsulphenyl chloride with Leuch anhydrides (N-carboxy anhydrides of a-aminoacids or peptides) according to the procedure described by H. R. Kricheldorf,Angew. Chem 85, 86 (1973), Chem. Ber. 107 3553 (1974) and R. Kataki, J. Org. Chem. 40 (19), 2697 (1975).

Stage 2: Reaction under gentle conditions of the compound of general formula 4 with any of the reagents known in the literature for the elimination of the o-nitrosulphenyl group, such as thioles in nonaqueous inert solvents, hydrogen halides in dilute dioxane solution at low temperatures, tAO , etc.

When hydrogen halides are used the minimum reaction time must be employed (the reaction is generally instantaneous) and the process of isolating the product must be rapidly proceeded with due reasons of the reactivity of o-nitrosulphenyl chlbride in an acid medium towards the residues of tryptophan present in the peptide.

In this way the compound of general formula 5, is obtained in the form of the hydrohalide if HCI or HBr/dioxane is used as reactant:

where R1, R2 and Rs have the meaning noted previously.

Stage 3: Treatment of the compound of general formula 5, obtained in Stage 2 under an atmosphere of nitrogen or in the presence of an antioxidant, with any of the reactants habitually used in peptide and aminoacid chemistry for the elimination of amine-protecting groups, such as hydrogen on various catalysts in suitable solvents, hydrogen halides in acetic acid, etc.Thus an ester derived from the product of general formula 1 is obtained which by means of treatment with a base in an inert solvent in accord with the normal methods of organic chemistry for ester saponification followed by the neutralization of the previous alkaline solution, preferably by passing said solution through an acid ionexchange column, leads to the free peptide comprising the amioacids lysine and tryptophan and of the following general formula 1: (Lis), -- (Trp), The following methyl ester, 1-Lis-1-TrpOMe, derived from the compound of general formula 1, in which a = b = 1, has already been previously described in its mono- and di-hydrochloride form by M.

Szekerke, J. Erchegyi and J. Sagi Acta chim. Acad. Sci. Hung. 80. 193 (1 974), nevertheless, the procedure described for its synthesis is different.

The yields obtained in all the reactions are good ( > 90%), no racemisation at all occurring.

The following examples illustrating the process to which this invention refers are not to be considered to be limiting. In all of same the following abbreviations are utilized: NPS, NCA and Z, which have those meanings indicated below, these abbreviations being those that are universally and habitually used in aminoacid and peptide chemistry to indicate the groups that they represent and which are widely known to specialists in the field.

NPS = o-nitrophenylsulphenyl NCA = a-N-carboxy-anhydride Z = Carbobenzyloxy EXAMPLE 1 HCl. 1-Lis-1-TrpOMe 1. NPS-E-Z-1-Lis-1-TrpOme 38.29 of methyl-1-tryptophanate hydrochloride is suspended in 300ml of dry tetrahydrofuran and then 21 ml of triethylamine is added, the triethylamine hydrochloride formed then being filtered off. To the filtrate is added 72.6g NPS-E-Z-1-Lis-NCS and the mixture is agitated for two hours. The solution is evaporated under reduced pressure, bath temperature not exceeding 350C, leaving a residue which is dissolved in 600ml of ethyl acetate, washed successively with 5% aqueous tartaric acid solution, 5% sodium bicarbonate and water.The organic phase is dried over sodium sulphate, and evaporated in vacuo to ieave a thick syrup which is used in the following stage and whose identity and structure are determined by spectroscopy and analysis.

NMH (dimethylsulphoxide -d6, ) 7-8.5 (m, 14.5 of indol, 5 group Z, 4NPS): 5.1(5.2 CH2 group Z);3A(5.3, CO2 CH3) Analysis: Calculated for C32H35N507S: C, 60.65%; H, 5.56%; N, 11.05%; S, 5.04% Found C, 61.03%; H, 5.89% N, 10.88% S, 5.29% 2. HCL. E-Z-1-Lis-1-TrpOMe 88.3g of the NPS E-Z-1-Lis-1-TrpOMe obtained previously is dissolved in 350ml of a 1 N solution of hydrogen chloride dissolved in dioxane cooled to -50C. The solution is then evaporated to dryness under reduced pressure to give a gummy residue which solidifies on treatment with benzene and agitation and whose structure is determined by spectroscopy and analysis NMR (dimethylsulphoxide-d6, ) 5.1 (S, 2CH2 group Z); 3.5 (S, 3, CO2CH3).

Analysis: Calculated for C26H33N405C1.2H20: C, 56.47%; H, 6.69% N, 10.13%; CI, 6.42% Found C, 56.28%; H, 6.90%; N, 9.97%; Cl, 6.68% 3. HCL. 1-Lis-1-TrpOMe A solution of 8g HCI. E-Z-1.Lis.TrpOMe, obtained from the previous stage, in 40ml acetic acid saturated with hydrogen chloride is heated at 1 000C for 1 hour under a nitrogen atmosphere. The solution is evaporated to dryness in vacuo to give a syrup which is washed with acetone and which then takes on a gummy consistency.Although crystallisation was not possible, its identity was confirmed based on spectroscopy and it showed analogous rotation powers as described in the bibliography; [a] = +8 (C = 1.5 H20); [ai bibliography = +6.2 (C = 1.1 H20).

EXAMPLE 2 HCI. 1-Lis-1-Lis-1-TrpOMe 1. NPS-E-Z-1-Lis-E-Z-1-Lis-1-TrpOMe 62g of HCI. E-Z-1-Lis-1-TrpOMe obtained from the above example, is suspended in 600ml dry tetrahydrofuran and 1 6.8my triethylamine is added. The precipitate formed is filtered off and to the filtrate is added 50.4g NPS-E-Z-1-Lis-NCA. The mixture is agitated for 2 hours at room temperature. The solution is evaporated to dryness under reduced pressure and the residue is dissolved in ethyl acetate (2 litres), and washed successively with aqueous solutions of tartaric acid 5% and sodium bicarbonate 5%, and water.

The organic phase is dried over sodium sulphate and the ethyl acetate solution once dry is evaporated to dryness in vacuo, to give a gummy residue solidified by treatment with hexane to give an amorphous solid characterised by NMR spectroscopy and analysis: NMR (dimethylsulphoxide -d6, ) 7-8.5 (m, 19; 5 indol, 1 OZ groups, 4NPS); 5,1 (5.4, CH2 Z groups); 3.5 (S, 3, CO2 CH3).

Analysis: Calculated for C46H53H7010S: C, 61.67%; H, 5.92%; N, 10.94%; S, 3.57% Found C, 61.48% H, 6.16% N, 10.69%; S, 3.72% 2. HCI E-Z-1-Lis-E-Z-1-Lis-1-TrpOMe NPS-E-Z-1-Lis-E-Z-1-Lis-1-TrpOMe obtained from the previous stage (102 g) is dissolved in a 1 N solution of hydrogen chloride in dioxane cooled to -50C. The solution is then evaporated to dryness under reduced pressure to give a thick syrup which is solidified by the addition of ethyl ether. The solid is washed with ether and crystallized from tetrahydrofuran-ethyl ether, mp. 120-121 .

Analysis: Calculated for C40H51N608CI.2H20; C, 58.93%; H, 6.70% N, 10.31%; CI, 4.35% Found C, 58.59%; H, 6.31% N, 10.27%; Cl, 4.72% 3. HCl. 1-Lis-1-Lis-1-TrpOMe HCI. E-Z-1-Lis-E-Z-1-Lis-1-TrpOMe, obtained from the previous stage, in a solution in acetic acid saturated with hydrogen chloride (300 ml) is heated for one hour at 1 000C under a nitrogen atmosphere. It is then evaporated to dryness in vacuo giving a residue which is washed by agitating with acetone until it acquires an amorphous solid texture, which, although it could not be crystallised, was identified by spectroscopy and analysis.NMR (dimethylsulphoxide-d6-ô), 1.1-1.9 (m, 12, CH2 lysines not adjacent to the amine group), 3.50 (S, 3, CO2 CH3) 6.87-7.90 (m, s, tryptophan).

Analysis Calculated for C24H39N604CI: C, 56.41%; H, 7.64%; N, 16.45%; Cl, 6.95% Found C, 56.73%; H, 7.28% N, 16.09%; Cl, 70.2% EXAMPLE 3 1 -Lis- 1 -Lis- 1-Lis- 1 -TrpOMe and 1 -Lis- 1 -Lis-1 -Lis-TrpOH 1. NPS--Z-I-Lis-E-Z-I -Lis-E-Z-l-TrpOMe HCI. E-Z-1-Lis--Z-1-Lis-1-TrpOMe obtained as per Example 2 (79g) was suspended in dry tetrahydrofuran (600ml), and triethylamine (14.6 ml) was added. The precipitate formed is filtered off and NPS-E-Z-1-Lis-NCA (46 g) is added to the filtrate.The mixture is agitated for 1 2 hours at room temperature, when it is then evaporated in vacuo giving a thick syrup; it is solidified with ethyl acetate to give a yellow solid which is crystallised from ethyl acetate, mp. 172--1740.

NMR (Dimethylsulphoxide-d6, b) 7-8.5 (m, 24: 5 indole, 15 group Z, 4 NPS); 5.1 (S, 6, CH2 Z groups); 3.4 (S, 3, CO2 CH3).

Analysis: Calculated for C60H72Ng013S: C, 62,18%; H, 6.22%; N. 10.88% Found C, 62.42%; H, 5.93%; N. 10.58% 2. H Cl. E-Z- 1-Lis-#-Z-1-Lis-#-Z-1-Lis-1-TrpOMe NPS-E-Z- I-Lis-E-Z- I-Lis-E-Z-l -Lis-l -TrpOMe from the previous stage (107 g) is dissolved in a 1N solution of hydrogen chloride in dioxane cooled to -50C. Then it is then evaporated to dryness under reduced pressure, bath temperature not exceeding 35cC, to give a solid residue which is repeatedly washed with ethyl either. A product is obtained which is recrystallised from tetrahydrofuran-ethyl ether, mp.170--171 .

Analysis Calculated for C54H69N011Cl.2H20: C, 60.20%; H, 6.78% N, 10.40%; CI, 3.30% Found C, 60.54%; H, 6.48% N, 10.76%; Cl, 3.45% 3. HCI. 1-Lis-1-Lis- 1 -Lis-TrpOMe and 1-Lis-1-Lis-1-Lis-TrpOH A solution of HCI. #-Z-1-Lis-#-Z-1-Lis-#-Z-1-Lis-1-TrpOMe obtained from the previous stage (80g) in acetic acid saturated with hydrogen chloride (400ml) was heated at 1 000C for 1 hour under a nitrogen atmosphere. It was then evaporated to dryness in vacuo to give a gummy residue which on being washed and agitated with acetone acquires the consistency of an amorphous solid.

Although it could not be crystallised it was identified as HCI. 1-Lis-1-Lis-1-Lis-TrpOMe by analysis and spectroscopy. NMR (dimethylsulphoxide-d6) S 1, 1-1.9 (m, 18, CH2 lysines not adjacent to the amine group) 3.52 (S, 3, CO2 CH3), 6, 85-7.90 (m, 5, tryptophan).

Analysis Calculated for C30H4,N805CI: C, 57.28%; H, 6.52%; N, 17.82%; Cl, 5.64% Found C, 57.49%; H, 6.78%; N, 17.51%; Cl, 5.82% Then this product is dissolved in ethanol (575 ml) and 4N sodium hydroxide solution (80 ml) is slowly added while agitating. The mixture is agitated for 121 hours at room temperature, evaporated to dryness in vacuo, to give a residue which is then dissolved in water and neutralized by passing through an acid ion-exchange column, IR-1 20. The dilute aqueous solution from the column is evaporated to dryness in vacuo to give a solid which recrystallizes from methanol-acetone, mp. 187--188 , and which corresponds to the free tetrapeptide 1 -Lis- 1 -Lis- 1 -Lis-TrpOH.

NMR (dimethylsulfphoxide-d6) S 1.1-1.9 (m, 18 CH2 lysines not adjacent to the NH2 group), 6.85-7.90 (m, 5, tryptophan).

Analysis: Calculated for C29H38N805: C, 60.20%; H, 6.57%; N, 19.37% Found C, 59.97%: H, 6.53%; N, 19.08%

Claims

1. A process for the preparations of lysine-tryptophan oligopeptides or their physiologically-useful derivatives and salts, corresponding to the following general formula 1: (Li5)a - (Trp) wherein Lis represents a lysinyl residue, Trp represents a tryptophanyl residue, a > o and b > o; comprising all the possible combinations of sequences that can be formed with lysine and tryptophan from the d and/or / series, with the sole condition that when the peptide linkage -- CONH -- originates from an amine group of a lysine residue, that amine group is in the a-position to the carboxyl group that participates in the formation of said linkage; the process comprising effecting the three following reaction stages: Stage 1 Reaction of the compound of general formula 2 with a salt of a compound of general formula 3 in the presence of an aprotic base and in a suitabie inert solvent:

in which R, and R2 independently represent p-methylenidole or -aminobutyl with the -NH2 group protected or any residue of any peptide sequence (Lis)c(Trp)d (wherein c and/or d > o) with the condition that at least one of R, and R is a residue of p-methylenidole or protected E-aminobutyl, and R3 1 2 is an alkyl aryl or arylalkyl group, whereby to obtain the compound of the general formula 4 shown below:

Stage 2:Reaction of the compound of general formfula 4 obtained from the previous stage to eliminate the o-nitrosulphenyl group and obtain a compound of the general formula 5:

or an acid addition salt thereof, where R, R2 and R3 have the same meaning as previously, Stage 3: Reaction of the compound of general formula 5 or its salt obtained from the previous stage to remove any amine-protecting groups, whereby an ester derived from the product of general formula 1 is obtained which through optional saponification by an aqueous or aqueous-alcoholic solution of a base, followed by neutralization-of the resulting mixture, leads to the free peptide of the following general formula 1 which comprises any of those possible peptide combinations previously mentioned.

(L15)a - (Trp) 1 and optionally forming a physiologically useful derivative or salt therefrom.

2. A proces for the preparation of lysine-tryptophan oligopeptides or of their physiologically-useful derivatives and salts according to Claim 1, characterised by the reaction corresponding to Stage 1 being effected in tetrahydrofuran at room temperature.

3. A process for the preparation of lysine-tryptophan oligopeptides or of their physiologicallyuseful derivatives and salts, according to Claim 1 or Claim 2, characterised by the aprotic base used in Stage 1 being triethylamine.

4. A process for the preparation of lysine-tryptophan oligopeptides or of their physiologicallyuseful derivatives and salts according to any one of the preceding claims, characterised by the reaction corresponding to Stage 2 being effected with a dilute solution of an acid in an appropriate inert solvent.

5. A process for the preparation of lysine-tryptophan oligopeptides or of their physiologicallyuseful derivatives and salts, according to claim 4, characterised by the reaction corresponding to Stage 2 being effected with 1 N hydrogen chloride in dioxane at a temperature of 00+50C.

6. A process for the preparation of lysine-tryptophan oligopeptides or of their physiologicallyuseful derivatives and salts, according to any one of the preceding claims, characterised by the protective group on the terminal amino group of the lysine being carbobenzyloxy.

7. A process for the preparation of lysine-tryptophan oligopeptides or of their physiologicallyuseful derivatives and salts, according to any one of the preceding claims, characterised by the reaction to remove an amine-protecting group corresponding to Stage 3 being effected with a solution of a hydracid in acetic acid or with hydrogen in acetic acid in the presence of a catalyst.

8. A process for the preparation of lysine-tryptophan oligopeptides or of their physiologicallyuseful derivatives and salts, according to to Claim 7, characterised by an elimination reaction of a carbobenzyloxy group corresponding to Stage 3 being effected in a saturated solution of hydrogen chloride in acetic acid at 80-1 000C for a period of 30 to 60 minutes.

9. A process for the preparation of lysine-tryptophan oligopeptides or of their physiologicallyuseful derivatives and salts, according to any one of the preceding claims, characterised by the saponification reaction corresponding to Stage 3 being effected with an alkaline or alkaline earth hydroxide in an aqueous-alcoholic medium.

10. A process for the preparation of lysine-tryptophan oligopeptides or of their physiologicallyuseful derivatives and salts, substantially as herein described with reference to any one of the specific examples.

11. Lysine-tryptophan oligopeptides or their physiologically-useful derivatives or salts when prepared by the process according to any of the preceding claims.

12. The compound 1-Lis-1-Lis-1-TrpOMe hydrochloride.

13. The compound 1-Lis-1-Lis-1-Lis-1-TrpOMe hydrochloride.

14. The compound 1-Lis-1-Lis-1 -Lis- 1 -TrpOH.