PL154068B1 - The method of peptide derivative manufacture - Google Patents

Description

THE REPUBLIC PATENT DESCRIPTION 154 © 6

POLAND

Additional patent to patent no. Pending: 87 10 12 (P. 281585)

Int. Cl. ⁵ C07K7 / 26

OFFICE

PATENT

RP

Priority: 87 08 17 Switzerland

Application announced: 88 09 01

Patent specification published: 1992 02 28 ntuu

6 4 1 HA

Inventor -

The holder of the patent: SandozA. G.,

Basel (Switzerland)

Method for the production of peptide alcohols

The subject of the invention is a process for the preparation of peptide alcohols having two alcohol groups or one alcohol group and one thiol group at the C-terminal end of the peptide chain. This method is particularly suitable for the preparation of peptide alcohols containing the C-terminal radical of threoninol, serinol or cysteinol.

Solid-phase peptide synthesis has proved to be a very fast and advantageous method of producing polypeptides and has therefore become a widely used method. As is known, the amino acid is first bound by its carboxyl group to form an ester or amide group at the hydroxyl or amino group of the insoluble synthetic resin; the further amino acids are then attached thereto in the desired order and finally the complete polypeptide is cleaved from the resin support.

This synthesis proceeds without problems for common polypeptides with C-terminal amino acids. Polypeptide alcohols containing an amino alcohol at the C-terminus in place of an amino acid, however, are not so easily bound to the -OH or -NH2-containing carrier resin and / or cleaved again at the end of the synthesis.

As possible, the methods of producing peptide alcohols carried out in the solid phase have so far been proposed:

a) Simple preparation of a suitable C-terminal amino acid containing the polypeptide (as an ester of a resin bearing OH groups) and subsequent reductive cleavage with borohydrides, simultaneously converting the carboxyl group to an alcohol functional group. (See U.S. Patent No. 4,254,023 (4).

b) attachment of the terminal amino alcohol as an ether to the hydroxymethyl resin with carbonyldiimidazole and sufficient cleavage after peptide synthesis using the HCl / TFA or HBr / TFA system [Kun-hwa Hsieh and GR Marshall, ACS National Meeting, New Orleans, 21-25 March 1977].

A disadvantage of the described processes, however, is the drastic cleavage conditions.

It has now been found that cleavage of the peptide from the resin with simultaneous formation

The C-terminal peptide alcohol takes place under mild conditions if the C-terminal amino alcohol is attached to the resin via an acetal bond.

154 068

According to the invention, the production ¹ of a peptide alcohol, optionally still containing protecting groups, takes place by acid hydrolysis of the acetal from the (protected) peptide alcohol and from a resin containing formylphenyl radicals.

Schematically, this reaction is shown in Scheme 1, in which ^ is an insoluble synthetic resin radical, Z is a direct bond or a radical linking the resin with a acetalized formylphenyl group, X is oxygen or sulfur, Ri is hydrogen or methyl, and Y is a radical containing optionally alcohol protecting groups peptide, wherein the (acetalized) -CHO group occupies the -m or -p position relative to the Zj radical. For simplicity, formulas 2 and 3 of Scheme 1 have only drawn one substituting group on the resin; it is understood, however, that numerous such groups are bound to a single polymer molecule of the resin. The cleavage of the peptide alcohol from the resin by hydrolysis of the acetal group takes place, as mentioned above, under acidic conditions, for example with dilute trifluoroacetic acid. This hydrolysis can be carried out at room temperature.

In the case where Z represents a direct bond in formula 1, the acetal-loaded phenyl radicals are directly bonded to the polymer radical and belong to the polymer. Examples of such compounds of formula 2 are acetals of a formylated polystyrene resin (in formula 2 the symbol® then represents a polyethylene chain). When Z is a radical, such radical contains a group which is formed by reaction of a reactive group bonded directly or indirectly to the polymer with another reactive group bonded directly or indirectly to a (acetalized) formylphenyl group. This radical Z can, for example, be reproduced by the formula 4, in which Qi is a radical of a reactive group bound to a polymer, Q2 is a radical of a reactive group bound to a (acetalized) formylphenyl group, D is a radical linking the Qi group to the polymer, E is a radical binding the Q2 group with a (acetylated) formylphenyl group, apiq are each independently 0 or 1.

The group Qi - Q2 is preferably an ester or an amide group, especially a carbonamide group. Preferably the symbol Q is NH, the symbol Q2 and CO.

For example, D and E are independently of each other alkylene or alkyleneoxy radicals having 1-5 carbon atoms. Examples of compounds of formula II in which Z is the radical of formula IV are those in which the moiety R-D-Qi is a radical of an aminomethylated polystyrene resin and the moiety of formula V is the radical of formula VI in which R is hydrogen or methyl, and m is the number 0 or 1, with the acetal group again in the -m or -p position. So in this case, Z is a group of the formula -CH2-NH-CO-CH- (O) m-, and® is polystyrene. The radical of formula 6 is preferably a group of formula 7.

Instead of aminomethylated polystyrene, other polymers can also be used, especially polymers with free -NH2 groups, e.g. polyacrylamides having aminoethyl groups.

As mentioned above, the acetalized formylphenyl group is preferably attached to the polymer via an amide bond. This ensures that the binding of the acetalized formylphenyl radical to the resin is stable during the synthesis of the polypeptide and during cleavage, and that the cleavage, as desired, takes place in the acetal bond, so that on the one hand a peptide alcohol is produced and, on the other hand, the formylphenyl radical remains with resin. If desired, the peptide alcohol can be moved further away from the resin by the incorporation of so-called spacers (space groups) between the reactive groups of the polymer (especially amino groups) and the reactive groups of the acetalized formylphenyl derivative (especially carboxyl groups). This may be advantageous for certain reactions on the peptide alcohol prior to cleavage (e.g. oxidation of cysteine radicals). In this case, the radical D or E in formula IV additionally contains this spacer, and Qi or Q2 is the reactive radical of this spacer.

For example, an ω-aminocarboxylic acid such as ε-aminocaproic acid can be used as the spacer.

In the special case of using aminomethyl polystyrene, the radical of formula 6 and ε-aminocaproic acid as spacers, the symbol Z represents the group of formula 8.

Compounds of Formula 2 can be prepared from a starting compound of Formula 9 by methods practiced in solid phase technology.

In formula 9, A represents an amino-protecting group and the acetal group occupies the -m or -p position relative to the Z radical. For this purpose, the protecting group is first cleaved off

A, then this free amino group is reacted with the next N-protecting amino acid, etc., until all amino acids are attached to the resin in the order corresponding to the desired peptide alcohol.

As the protecting group for the amine in the amino acids to be used or in the amino alcohol, it is necessary to choose those groups which are cleaved under non-acidic conditions, since hydrolysis of the acetal group takes place under acidic conditions. As such amine protecting groups, e.g. CF3CO- or FMOC- (9-fluorophenylmethyloxycarbonyl) can be used. These protecting groups are basically cleaved by the known art of peptide chemistry.

Only the protecting group in the last amino acid attached can be acid labile, and then it is cleaved simultaneously with the release of the peptide alcohol from the resin. In this case, for example, the -Boc group should be used.

KOH, piperidine or also NaBH4, for example, can be used as bases.

The synthesis of a polypeptide chain also takes place in a known manner from a peptide radical with a free amino group and from an amino acid with a free or activated carboxyl group. Water-binding agents can also be added. Thus, the reaction can be carried out, for example, in the presence of the addition of hydroxybenzotriazole and dicyclohexylcarbodiimide.

The compounds of formula 9 can be prepared by reacting either a / a resin of formula 3 containing aldehyde groups in which the -CHO group is -m or -p to Z is reacted with an N-protected amino alcohol of formula HX- CHRi-CH (NHA} -CH2 <OH or with an activated form of this compound or b) a resin derivative of formula @ - (D) p-Q'i is reacted with a compound of formula 10 in which the acetal group occupies the position - m or -p with respect to the radical Q'2- (E) q-, and Q'i and Q * are two reactive groups that can react with each other to form a Qi-Q2 bridge.

Acetalization (method a) is carried out in a known manner with acid as catalyst. As acid, for example, p-toluenesulfonic acid or trifluoromethanesulfonic acid can be used.

Instead of the free alcohol, for example, a trimethylsilyl derivative can be used.

Process b / (e.g. esterification or especially amidation) also takes place in a known manner, e.g. by reacting a reactive derivative of a carboxylic acid with a polymer having an -OH or -NH2 group.

Compounds of formula 10 are prepared by acetalizing a compound of formula 11 with a compound of formula HX-CHR-i-CH (NHA) -CH2OH. This acetalization is as described for method a) above.

During the synthesis and before cleavage of the peptide alcohol from the resin, it is also possible to carry out reactions on the peptide alcohol radical, for example removal of protecting groups, e.g. sulfur protecting groups, or oxidation of cysteine radicals. These reactions can of course also take place in solution after cleavage of the (protected) peptide alcohol. Compounds of formula 2, 9 and 10 are new.

All peptide alcohols, in particular pharmacologically active peptide alcohols which contain two alcohol groups or one alcohol group and one thiol group at the C-terminus, can easily be produced by the process according to the invention.

The examples below illustrate the invention.

Example I. Preparation of a somatostatin derivative of the formula H-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-ol. / Preparation of an anchor acetal (N-Cf3-CO-threoninol acetal with p-formylphenoxy acetic acid)

105 g (1.0 mol) of L-threoninol are placed in 200 ml of methanol, purging with nitrogen. To the resulting clear solution at 0 ° C, 200 ml of methyl triyluoroacetate in 250 ml of methanol are added dropwise. In this case, the internal temperature is kept at around 10 ° C. by means of an ice bath. After 1.5 hours, no more threoninol was found in the reaction solution. After evaporation at 40 ° C, a white crystalline residue is obtained. It is dissolved at 70 ° C in 200 ml of ethyl acetate and precipitated with 100 ml of hexane. Next

Cool to 0 ° C, wash with hexane, and dry at room temperature. There is obtained N-trifluoroacetyl-threoninol.

50.3 g (0.25 mol) of the product thus obtained were dissolved: in 1.25 liters of tetrahydrofuran under a nitrogen atmosphere, and 75 ml of trimethylchlorosilate were added dropwise at room temperature. After that, a mixture of 70 ml of triethylamine and 250 ml of tetrahydrofuran is added. A white suspension forms which is stirred at room temperature for 4 hours. It is then filtered and the filtrate is evaporated at 40 ° C.

An oily substance is obtained. It is dissolved in 1.5 l of methylene chloride and 90.4 g of p-formyl-phenoxyacetic acid are added in portions at room temperature. A total of 9 ml of the trimethylsilyl ester of trifluoromethanesulfonic acid are then added in portions. The mixture is stirred at room temperature for 24 hours, then filtered, and the residue is thoroughly washed with methylene chloride. This filtrate was evaporated at 40 ° C to give a residual orange-red colored gummy product. The orange-red colored product is chromatographed on silica iron, eluting with ethyl acetate. After evaporation of the desired fractions, the above-mentioned acetal is obtained. Purity by liquid pressure chromatography 97%.

2 / Protected octapeptide synthesis, resin oxidation and cleavage.

17.2 g of aminomethylated polystyrene [0.7% by weight nitrogen, corresponding to 0.50 mmol / g], is suspended in 80 ml of methylene chloride / dimethylformamide 4: 1. 4.17 g of the final product from step 1 /, 1.6 g of hydroxybenzotriazole and 4.0 g of dicyclohexylcarbodiimtdu are successively added to the total. The Kaiser test is negative after 2 hours' stirring at room temperature. Filtration and washing are performed. The washed resin is suspended in 100 ml of tetrahydrofuran / methanol 3: 1 and 10.4 g of sodium borohydride are added portionwise. The mixture is stirred for 6 hours at room temperature, filtered and washed. This resin is resuspended in methylene chloride / dimethylformamide and 5.57 g of FMOC-Cys / S-tertiary-Bu / OH, 1.74 g of hydroxybenzotriazole (HOBT) and

3.6 g of dicyclohexylcarbodiimide (DCCI). After cleavage of grnp-FMOC with piperidine (2X20 minutes), N-FMOC-protected amino acids Thr-OH, Lys (BOC) -OH, D-Trp-OH, Phe-OH, Cys (S-III-rz .-Bu) OH and D-Phe-OH with the HOBT / DCCI system. Finally, an FMOC-pelleted octapeptide-resin system is obtained. Saturation 0.26 mmol / g.

The resin was suspended in 100 ml of trifluoroethanol / methylene chloride 1: 1 and mixed with 50 ml of tributylphosphine and then stirred for 70 hours at room temperature. It is then filtered, washed and mixed with 100 ml of a 1: 1 mixture of tetrahydrofuran and 1 M ammonium acetate solution. 1.1 ml of 30% aqueous hydrogen peroxide solution are added thereto, and the mixture is stirred at room temperature for 24 hours. It is then washed, mixed with a mixture of 20 ml of trifluoroacetic acid, 80 ml of methylene chloride, 10 ml of water and 2 ml of thioanisole, and stirred for 2 hours, then filtered and washed with trifluoroacetic acid and methylene chloride. 200 ml of diethyl ether are added to the filtrate, the precipitate is filtered off, dissolved in an aqueous buffer solution, deionized by treatment with Duolift and lyophilized as acetate.

The title compound is obtained as the acetic acid salt.

Example II. Preparation of N ^c ^ (α - glucosio) 1-4 (-deoxyfrurocoseIoy-SMS.

393 g of the resin bound octapeptide were produced by the method set out in the example. Cysteine protecting groups were removed by reduction. The resin bound peptide was oxidized to the cyclic octapeptide with hydrogen peroxide in a tetrahydrofuran / water mixture.

After washing with tetrahydrofuran and then with DMF, the peptide resin was shaken in 3600 ml of a DMF / AcOH mixture (8: 1). The suspension was treated with 527 g of D (+) -maltose as monohydrate. The mixture was heated to 60 ° C and stirred for 18 hours at this temperature.

The mixture was cooled and the peptide resin was filtered off and washed successively with DMF and methanol followed by methylene chloride. The peptide was cleaved from the resin for 1 hour with a mixture of 2900 ml of methylene chloride and 716 ml of trifluoroacetic acid with a trace of water.

154 068

The filtrate was then stirred, mixed with 597 g of sodium carbonate, stirred for 30 minutes and filtered. The residue was washed with methylene chloride and methanol. The filtrate was concentrated to dryness. Demineralised using a column packed with unfunctional polystyrene such as Duolite or a reversed phase HPLC material such as silica gel treated with silicone and containing long chain fatty alcohol groups (e.g. Labomatic, Switzerland, Brand HB-SIL-18-20 -100). Pure title compound was obtained.

Claims (5)

Patent claims Method for the production of a peptide alcohol which at the C-end of the peptide chain contains two alcohol groups or one alcohol group and one thiol group, characterized in that the peptide alcohol is cleaved by acid hydrolysis from the resin containing formylphenyl residues to which the alcohol is attached peptide via an acetal bond to the C-terminal alcohol group or alcohol and thiol groups. 2. The method according to p. 2. The process of claim 1, wherein in the preparation of a peptide alcohol of formula I in which Y-CO is a peptide residue, Ri is hydrogen or methyl, X is 0 or S, the resin with an attached peptide alcohol, represented by formula 2, is hydrolyzed under acidic conditions. where @ is the residue of an insoluble synthetic resin, and Z is a direct bond or residue connecting the resin to the acetalized formylphenyl group, wherein the acetalized CHO group is in the m- or p- position relative to the Z radical, and several acetalized compounds are linked to the polymeric resin molecule formyl groups. ³ . ^S COP ^b wedto ^g claim. 2, characterized ^{y y} m t, t ^o apply that ^Y in the formula ^Ce 2, ^k tórym (® is a residue of polystyrene, and Z is a direct bond. 4. The method according to p. The process of claim 2, wherein X is oxygen and Ri is CH3. ^ CHR _r XH Y-CO-NH-CH CHO ch ₂ oh Formula 1 Formula 3 - (^ / _p -Q _r Q ₂ - / E / ^ą Formula 4 Formula 2 ΐ ¹ X-CH Q2 ' ^E CH-NH-CO-Y O-CH2 Pattern- 5 / Y-CH CH 2 4 CH-NH-CO-Y -c-αH- / o / _m - ^ f ^ o-ch ₂ OR Pattern 6 and ¹ X-CH 'CH-NH-CO-Y O-CH2 Formula 7 -CH2-NH-CO- / CH2 / 5 -NH-CO-CH- / O / _m Formula 8 CH-NH-A Formula 9 ί ¹ X-CH CH 2 CHH-NH-A Formula 10 ^ = ^ C ' ^CH ° Q2- ^{/ D} / 9AQ / Formula 11 © -z ^ ^Χ 0-θζ X-CH CH J 1 CH-NH-CO-Y Formula 2 CHfR ₁ -XH and ¹ * Y-CO-NH-CH-CH2OH Formula 3 Formula 1 Scheme 1 Department of Publishing of the UP RP. Circulation 100 copies Price: PLN 3,000