NO172895B - PEPTID AMINOAL CYLAMIDS, THEIR USE FOR PREPARATION OF PEPTIDES, AND A PROCEDURE FOR THE PREPARATION OF SUCH PEPTIDES - Google Patents

Abstract

Compounds of the formula I <IMAGE> in which A is hydrogen or an amino protective group, B is an amino acid residue, X is alkylene or aralkylene, Y<1>, Y<2>, Y<3> and Y<4> are identical or different and are hydrogen, methyl, methoxy or nitro, V is hydrogen or a carboxyl protective group, W is -[CH2]n- or -O-[CH2]n-, m is 0 or 1, n is 0 to 6 and p is 0 to 5, are prepared as described and used in the solid-phase synthesis.

Description

The present invention relates to peptide-aminoalkylamides, their use for the production of peptides and a method for the production of these peptides.

The introduction of an aminoalkylamide at the C-terminal end of a biologically active peptide has in some cases had a positive effect on the metabolic stability and the effect (EP-A-179 332). In the preparation of the thus modified peptides, the classical fragment coupling in solution was used.

In the solid-phase synthesis of peptides (compare Patchornik, Cohen, "Perspectives in Peptide Chemistry", pages 118-128 (Karger, Basel 1981)) the reactive chains are often not grafted directly onto the synthetic resin body, but are connected with so-called spacers or links to the carrier. From the literature (for example Atherton, Sheppard, "Perspectives in Peptide Chemistry", pages 101-117 (Karger, Basel 1981)) reagents for introducing such spacers (so-called "linkage agents"), which have the formulas VI , VII and VII.

New "linkage agents" have now been found which make it possible to build up C-terminals with aminoalkylamide- or hydrazide-modified peptides directly by means of solid-phase synthesis.

According to this, the present invention relates to new compounds in the form of peptide aminoalkyl amides and these are characterized by formula I

there

A means hydrogen or 9-fluorenylmethyloxycarbonyl

B means phenylamine or alanine

X means (C1-<C>12)-<a>alkylene

V means hydrogen, phenylcarbonylmethyl or (C^_6)alkyl W means -O-[CH2]n-,

n means an integer from 1 to 6 and

p means an integer from 0 to 5.

Functional groups in the side chains of amino acid residues may be protected. Suitable protecting groups are described by Hubbuch, Kontakte (Merck) 1979, No. 3, pages 14-23 and by Bullesbach, Kontakte (Merck) 1980, No. 1, pages 23-35. Preferred are such groups which are stable against bases and weak acids and which can be cleaved off with the help of strong acids.

The alkyl may be straight or branched.

The compounds of formula I can be prepared by

a) a compound of formula II

there

R means a nucleophilic releasable leaving group and

V and W have the above-mentioned meaning, are reacted with a compound of formula III where A means 9-fluoromethylmethyloxycarbonyl and B, X, p have the above-mentioned meaning, and that in the formed protected compound of formula I, one or both protecting groups A and/or V is optionally split off to form the free NH2 and/or CO2H group(s), or

b) a compound of formula I, where A means hydrogen and B, X, v, W, n and p have the meaning indicated above, is reacted

with a compound of formula IV

A-[B]5_p-OH (IV),

where A, B and p have the meaning given above but A does not mean hydrogen, or their active ester, halide or azide, and, if V is different from hydrogen, optionally cleaves a carboxyl protecting group V to form a carboxyl group.

A nucleophilic releasable cleavable group R is, for example, halogen such as chlorine, bromine and iodine or activated aryloxy such as p-nitrophenoxy.

The reaction of a compound of formula II with a compound III is preferably carried out in an aprotic solvent, such as THF, DMF, CHC13 or CH2C12, preferably in the presence of a base such as a tertiary amine, for example ethyltriisopropylamine, triethylamine or pyridine, in which the addition of an acylation catalyst such as DMAP, HOObt or HOBt works advantageously, at a temperature between 0°C and the boiling point of the reaction mixture, preferably between 0°C and 40°C.

The compounds of formula I (A = hydrogen) are reacted with a compound of formula IV, their active ester, halide or azide, preferably in an organic solvent such as DMF, advantageously in the presence of a base such as a tert.-amine, at a temperature between -10°C and the boiling point of the reaction mixture, preferably at room temperature. Suitable activators are, for example, the ONSu, OBt, OObt and p-nitrophenoxy compounds. Preferred halogen derivatives are the chlorides. Pyridinium perchlorate can be added to improve solubility.

The compounds of formula II are prepared, for example, by reacting esters of formula IX

where W and V have the above-mentioned meaning, however, V does not mean hydrogen, with phosgene or phosgene derivatives such as for example chloroformate nitrophenyl ester, in an aprotic, polar solvent, for example THF or DMF, in mixture with a tert. base, for example a tert.amine such as pyridine, preferably in a 1:1 ratio at a temperature between -40°C and room temperature, preferably between -20°C and 0°C. The invention further relates to the use of a compound of formula I where V means hydrogen and A does not mean hydrogen, in solid-phase synthesis of a compound of formula V

where P means a peptide residue of q < p+1 α-amino acids and X and p have the meaning indicated above.

Finally, the invention relates to a process for the production of a peptide of formula I where P, X and p have the above meaning in solid phase synthesis, and this method is characterized by connecting a compound of formula I where A does not mean hydrogen and V means hydrogen , to a resin, cleaves the protecting group A, stepwise connects to Q-p, with 9-fluorenylmethyloxycarbonyl temporarily protected α-amino acids, optionally in the form of activated derivatives and, after completion of construction, sets free the peptide of formula V by treatment with a medium to strong acid from the resin, whereby simultaneously or subsequently by suitable precautions, temporarily introduced side chain protecting groups are cleaved off.

If to prevent side reactions or necessary for the synthesis of particular peptides, the functional groups in the side chains of amino acids are also protected (see for example T.W. Green "Protective Groups in Organic Syntheses", New York, John Wiley & Sons, 1981), whereby Arg(Tos), Arg(Mts), Arg(Mtr), Asp(OBzl), Asp(OBut), Cys(4-MeBzl), Cys(Acm), Cys(SBut), Glu( OBzl), Glu(OBut), His(Tos), His(Fmoc), His(Dnp), His(Trt), Lys(Cl-2), Lys(Boc), Met(O), Ser(Bzl), Ser(But), Thr(Bzl), ThrfBu<*>).

The resins used as carriers are commercially available. BHA and MBHA resins are preferred.

The cleavage of peptides of formula V then takes place by treatment with medium to strong acids usually used in peptide syntheses (for example trifluoroacetic acid, HF), in which the urethane protecting groups contained in the spacer are cleaved.

As a coupling reagent for the compound of formula I (V = H) and the further amino acid derivatives, all possible activation reagents used in peptide syntheses can be used, see for example Houben-Weyl, "Methoden der organischen Chemie", volume 15/2, but especially carbodiimides which for for example N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide or N-ethyl-N'-(3-dimethylaminopropyl)carbadiimide. The coupling can thereby be carried out directly by the addition of an amino acid derivative with an activating reagent and optionally an additive that suppresses the racemization, such as for example 1-hydroxy-benzotriazole (HOBt) (W. KSnig, R. Geiger, "Chem. Ber." 103, 708 (1970 )) or 3-hydroxy-4-oxo-3,4-dihydrobenzotriazine (HOObt) (W. Konig, R. Geiger, "Chem. Ber." 103, 2054 (1970) to the resin or else the preactivation of the amino acid derivative take place separately as symmetrical anhydride or HOBt or HOObt ester and the solution of the aquitated species in a suitable solvent has to the coupling-capable peptide resin.

The coupling or activation of the compound of formula I (V = H) and the amino acid derivatives with one of the above-mentioned activation reagents can be carried out in dimethylformamide or methylene chloride or a mixture of both. The activated amino acid derivative is usually used in a 1.5- to 4-fold excess. In cases where an incomplete coupling occurs, the coupling reaction is repeated, without previously carrying out the unblocking of the α-amino group of the peptide resin necessary for the coupling of the next amino acid.

The consequent progression of the coupling reaction can be investigated using the ninhydrin reaction, as discussed for example by E. Kaiser et al. in "Anal. Biochem." 34'595 (1970). The synthesis can also be automated, for example with a peptide synthesizer Model 430 A from fa. Applied Biosystems, in that either the synthesis program prescribed by the device manufacturer can be used or also set up by the user himself. The latter is used in particular when using amino acid derivatives protected with the Fmoc group.

When separating the peptide amides from the resin with hydrogen fluoride or trifluoroacetic acid, substances such as phenol, cresol, thiocresol, thioanisole, anisole, ethanedithiol, dimethyl sulphide, ethyl methyl sulphide or a mixture of two or more of these aids are usually added as cation traps. The trifluoroacetic acid can therefore also be used diluted with suitable solvents, such as, for example, methylene chloride.

Abbreviations used:

The invention will be explained in more detail with the help of some examples.

Example 1: 4-Hydroxymethylphenoxyacetic acid methyl ester

18.2 g of 4-hydroxymethylphenoxyacetic acid are dissolved together with 17.1 ml of N,N-dilsopropylethylamine and 50 ml of DMF and then added to the stirred solution of 6.1 ml of methyl iodide. Thereby, the mixture heats up slightly. After 3 hours the reaction is complete. The solvent is removed. The residue is taken up in ether and extracted once with 0.5N hydrochloric acid. The aqueous phase is extracted a further 3 times with ether, the combined ether phases are washed with aqueous sodium bicarbonate solution and evaporated. The residue is dissolved in vinegar and filtered over a short column of silica gel. The slightly yellowish oil formed after evaporation crystallized on standing.

NMR and mass spectrum agree with the given structure.

Example 2:

9.8 g of 4-hydroxymethylphenoxyacetic acid methyl ester are dissolved in 200 ml of dry CH2CI2, then 10.1 g of chloroformic acid p-nitrophenyl ester and 7 ml of triethylamine are added. The mixture is boiled for approx. 6 hours under reflux until the product is completely converted. A suspension of 15.5 g of Fmoc-NH-(CH2)4-NH2 (prepared by reacting Boc-NH-(CH2)4~NH2 with Fmoc-ONSu • and subsequent Boc cleavage) in 100 ml of dry CH2C12 is then added as well as a further 7 ml of triethylamine and the mixture is boiled under reflux. After completion of the reaction, the solvent is removed, the residue is digested with ether and suctioned off. The filter residue is washed with aqueous IN Na2C03 solution and then with warm water and dried in a desiccator under high vacuum.

Melting point 122-124°C, NMR and mass spectrum agree with the given structure.

Example 3:

5.2 g of the ester formed according to example 2 is suspended in 100 ml of methanol and 6 equivalents of an aqueous 1N NaOH are added. After completion of the reaction, the pH is adjusted to 3 with aqueous IN HC1 and methanol is removed. The precipitate is sucked off, washed with a little H2O and then digested in ether and sucked off again.

Melting point from 196°C (decomposition), NMR and mass spectrum are in accordance with the given structure.

Example 4:

1.5 g of the product formed according to example 3 is suspended in 50 ml of dry DMF. Then, 0.9 g of pyridinium perchlorate (to improve solubility), as well as 2.6 g of Fmoc-Phe-00bt and 0.5 ml of triethylamine are added in order. The mixture is stirred at room temperature. After completion of the reaction

the solvent is removed and the residue is distributed between vinegar H2O. The water phase is extracted again with vinegar and the combined organic phases are dried and evaporated. The residue is digested with a little CHCl3 and sucked off. The filter residue is then washed with a little ether and dried.

Melting point from 140°C (decomposition), NMR and mass spectrum are in accordance with the given structure.

1.4 g of the Fmoc-phenylalanine spacer acid formed according to example 4 is dissolved together with 350 mg of HOBt in 40 ml of dry DMF and made into 3.66 g of 4-methylbenzhydrylamine resin (Nova Biochem, charge 0.4 mmol/g). It is then mixed with 0.6 ml of diisopropylcarbodiimide and allowed to react while constantly mixing. After completion of the reaction, suction is taken off, washed with DMF, isopropanol, CH2CI2 and tert-butyl methyl ether and dried in a high vacuum. Charge according to elemental analysis (N determination): 0.3 mmol/g.

Example 6: Synthesis of [des-Tyr<24>, des-Arg<23>]-r-atriopeptin III-(4-amino)butylamide

The peptide synthesis takes place on 1 g of the above-mentioned resin using Fmoc-amino acid OOBt esters with an automatic peptide synthesizer Model 430A from fa. Applied Biosystems and self-modified synthesis programs.

For this, 1 mmol of the corresponding amino acid derivative is used in the cartridges supplied by the manufacturer, Fmoc-Arg(Mtr )-OH, Fmoc-Asn-OH and Fmoc-Glri-OH are weighed together with 1.5 mmol of HOBt in the cartridge. The pre-activation of these amino acids takes place directly in the cartridge by dissolving in 4 ml of DMF and adding 2 ml of a 0.55 M solution of diisopropylcarbodiimide in DMF. The HOObt ester is dissolved in 6 ml of DMF and then pumped similarly to the in situ preactivated amino acids arginine, asparagine and glutamine onto the resin previously deblocked with 20% piperidine in DMF. The in situ activated amino acids are double linked.

After completion of synthesis, the peptide-butylamide is cleaved from the resin while simultaneously removing the side chain protecting groups with trifluoroacetic acid, which contains thioanisole and m-cresol as cation scavengers. The residue formed for the removal of trifluoroacetic acid is digested several times with acetic acid and centrifuged. The remaining crude peptide is treated to remove the cysteine protecting groups with tributylphosphine and trifluoroethanol. After removing the solvent, digest again with vinegar and centrifuge. The reduced crude peptide is oxidized at once with iodine in 8056 µg aqueous acetic acid, the ^-excess is removed with ascorbic acid and the reaction mixture is desalted after evaporation to a small volume on "Sephadex" G25 with aqueous 1N acetic acid. The fractions containing the pure peptide are combined and freeze-dried.

According to amino acid analysis, the peptide corresponds to the stated formula in the amino acid composition.

Example 7: 4-Hydroxymethylphenoxyacetic acid phenacyl ester.

182 g of 4-hydroxymethylphenoxyacetic acid and 199 g of o-bromoacetophenol are dissolved in 600 ml of dry DMF and then quickly added dropwise at 0"C 138 ml of triethylamine. It is allowed to come to room temperature and stirred overnight. The DMF solution is poured into 3.5 1 water and the aqueous phase is extracted with ethyl acetate. The organic phase is washed with water, dried over sodium sulfate and evaporated. During evaporation, the product precipitates. It is sucked off, washed with ethyl acetate:n-hexane 1:1 and dried under high vacuum.

Melting point: 94-95°C, NMR is consistent with the given structure.

Example 8: 30 g of 4-hydroxymethylphenoxyacetic acid phenacyl ester are dissolved under protective gas in 500 ml of a mixture of THF:pyridine 1:1 and cooled to -20°C. Then 21 g of chloroformic acid p-nitrophenyl ester dissolved in 100 ml of THF are added dropwise. After stirring for 30 minutes at this temperature, the whole is allowed to warm to 0°C and the mixture is stirred into 1 1 of a half-saturated aqueous NaCl solution at 0°C and stirred for 30 minutes. The precipitate is suctioned off, washed with ice water and expelled after drying with n-hexane.

Melting point: 142-145°C, NMR is consistent with the given structure.

Example 9:

9.3 g of the compound prepared in example 8, 12.25 g of Fmoc-Phe-NH-(CH2)g-NH2-trifluoroacetate and 3.26 g of HOObt have as a solid in a flask, then poured over with a mixture of 2 .58 g of methyldiisopropylamine in 100 ml of dry DMF. The mixture is then stirred for a further 3.5 hours at 40°C and then stirred into 500 ml of half-saturated aqueous NaCl solution. The formed precipitate is suctioned off, washed with ice water and expelled after drying with ether/ethyl acetate.

Melting point: 147-150°C, NMR and MS agree with the given formula.

The following compounds (examples 10 to 14) are prepared analogously to example 9:

Example 10:

Melting point 144-147°C, NMR and MS correspond to the given formula.

Example 11:

Melting point: 179-181°C, NMR and MS correspond to the given formula.

Melting point 144-145°C, NMR and MS correspond to the given formula.

Example 13:

Melting point 172-175°C, NMR corresponds to the given formula.

Example 14:

Melting point 165-166°C, NMR corresponds to the given formula.

suspended in a mixture of 150 ml of glacial acetic acid and 50 ml of dichloromethane and mixed in portions with 12 g of zinc powder which had previously been activated by washing with 1N HCl and dry ethanol. After a few minutes, the suspension thickens and

difficult to stir under weak heating. A further 80 ml of glacial acetic acid and 50 ml of dichloromethane are therefore added and the whole is stirred further overnight. It is then sucked from over a clear-bed filter and washed with glacial acetic acid and dichloromethane. The filtrate is evaporated, the remaining oil is taken up in a little dichloromethane and stirred with ethyl acetate and ether. The precipitated product is sucked off and dried under high vacuum.

Melting point: from 160°C during decomposition, NMR and MS are in accordance with the given formula.

Following the method described in example 15, the compounds in examples 16 to 18 are also prepared:

Example 16:

Melting point: from 150°C during decomposition, NMR and MS in accordance with the given formula.

Example 17:

Melting point: from 160°C during cleavage, NMR and MS in accordance with the given formula.

Example 18:

Melting point: from 154°C during decomposition, NMR and MS in accordance with the given formula.

Example 19:

The production proceeded analogously to example 2.

Melting point: 115-118°C, NMR and MS agree with the given formula.

Example 20:

was produced according to the method mentioned in example 3. Melting point 184-187°C under decomposition, NMR and MS agree with the given formula.

Example 21:

The production took place analogously to example 4.

Melting point: from 120°C under decomposition, NMR and MS agree with the given formula.

Claims (3)

1. Compound, characterized in that it has formula I there A means hydrogen or 9-fluorenylmethyloxycarbonyl B means phenylamine or alanine X means (C 1 -C 1 -C 8 )-alkylene V means hydrogen, phenylcarbonylmethyl or (C1_^,)alkyl W means -O-[CH2]n-, n means an integer from 1 to 6 and p means an integer from 0 to 5. 2. Use of a compound of formula I according to claim 1, where V means hydrogen and A does not mean hydrogen, in the solid-phase synthesis of compounds of formula V where P means a peptide residue of q < p+1 α-amino acids and X, and p has the meaning stated in claim 1. 3. Process for the production of a peptide of formula V, in which P, X, m and p have the meaning specified in claim 2, by solid-phase synthesis, characterized by connecting a compound of formula I where A does not mean hydrogen and V means hydrogen, to a resin, cleaves the protecting group A, stepwise connects to q-p, with 9-fluorenylmethyloxycarbonyl temporarily protected α-amino acids, optionally in the form of activated derivatives and, after completion of construction, sets free the peptide of formula V by treatment with a medium to strong acid from the resin, whereby simultaneously or subsequently by suitable precautions, temporarily introduced side chain protecting groups are cleaved off.