SI8811579A - Process in solid phase for preparing peptidalcohols - Google Patents

Description

Solid phase process for the preparation of peptidyl alcohols

Field of the Invention (MPK C 07 K)

The invention relates to the field of peptide chemistry and relates to a process for the preparation of peptidyl alcohols carrying two alcohol groups or one alcohol and one thiol group in the solid phase (Solid-Phase) at the C-terminal end of the peptide chain. The process is particularly suitable for the preparation of peptidyl alcohols containing C-terminated threoninol, serinol or cysteinol residues, which are of particular use in the pharmaceutical industry.

A technical problem

There is a need to prepare in a technologically advanced and prudent manner in the solid phase of peptidyl alcohol while avoiding drastic reaction conditions.

The state of the art

The synthesis of peptides in the solid phase has proven to be a particularly fast and advantageous process for the preparation of polypeptides and has therefore become a generally common method.

As is known, they first bind the amino acid to its carboxylic group upon formation of an ester or amide group to a hydroxyl or amino group of an insoluble artificial resin; further amino acids are then added to it in the desired sequence and finally the complete pelipeptide is cleaved from the carrier resin.

This synthesis takes place for normal C-terminal amino acid polypeptides without problems. However, polypeptidal alcohols carrying the C-terminus instead of the amino acid of the amino-alcohol may not be as easily bound to the carrier resins bearing the OH or NH ₂ groups and / or cleaved at the end of the synthesis.

J <ot possible solid-phase processes for the preparation of peptidyl alcohols have been proposed so far:

a) the usual preparation of a suitable polypeptide containing at the C-terminus, an amino acid (as ester of a group-bearing resin)

OH) and subsequent reductive cleavage with boron hydrides, converting the carboxyl group to an alcoholic function.

(US Patents 4,254,023 / 4).

b) addition of terminal amino alcohol as ether to hydroxymethyl resin with carbonyldiimidazole and finally, after peptide synthesis, vaccination with HC1 / TFA or HBr / TFA (Kun-hwa Hsieh and GR Marshall, ACS National Meeting, New Orleans, March 21, 1977) .

These methods, however, are common to drastic cleavage conditions. Description of solution to a technical problem with implementation examples

We have now found that the cleavage of the peptide from the resin occurs at the same time that C-terminal peptidyl alcohol is formed under mild conditions by coupling the C-terminal amino alcohol with the resin via an acetal bond.

According to the invention, peptidyl alcohol is prepared which

- ί'ά also bears a protecting group, optionally acidic acetal from (protected) peptidyl alcohol and resin, formylphenyl residues.

The reaction can be shown schematically as follows:

X-CH ^ o-chJ

CH-NH-CO-Υ (I)

hydrolysis bearing

CHR. ^-XH i I + y-co-nh-ch-ch ₂ oh (II) where (P) stands for the residue of an insoluble artificial resin,

Z for a direct bond or residue linking the resin to an (acetalized) formylphenyl group,

X for 0 or S,

R.j for hydrogen or methyl and

Y for a peptidyl alcohol residue bearing a protecting group, as appropriate, wherein the (acetalized) CHO group is relative to the Z residue in the m- or p-position. For the sake of simplicity, only one substitution group was drawn in formulas I and II of the above scheme per resin; however, it should be clear that a large number of such groups are attached to one molecule of the sraol polymer. The cleavage of the peptidyl alcohol from the resin by hydrolysis of the acetal group is carried out as mentioned above under acidic conditions, e.g. with dilute trifluoroacetic acid. This hydrolysis can be carried out at room temperature.

If it stands in Formula I

By direct bonding, the phenyl residues bearing the acetal groups are bonded directly to the polymer residue and belong to the polymer. Examples of such compounds of formula I are acetals of formulated polystyrene resins (in formula I stands (P) then for the polyethylene chain).

If Z stands for a residue, this residue contains a group formed by the metabolism of a reactive group which is directly or indirectly bound to the polymer with another reactive group which is directly or indirectly bound to the (acetalized) formylphenyl group. The residue Z can be given e.g. with the following formula III

III in which = polymer residue-bound reactive groups are

Q ₂ = residue on the (acetalized) formylphenyl group of the bound reactive group,

D = the residue that binds the group to the polymer,

E = a residue linking the group Q ₂ to the (acetalized) formylphenyl group, and p and q independently of each other o or 1.

Preferably the group is an ester or amide group, especially a carbonamide group. It preferably stands for NH and Q ₂ for CO.

D and E stand e.g. independently of alkylene- or alkynyloxy moieties having 1 to 5 carbon atoms. Examples of such compounds of formula I, in which Z stands for the residue of formula III, are those which contain (?) -D-O ^ for the residue of an aminomethylated polystyrene resin and a residue of ^K 1

I

X-CH

CH ^X CH-NH-CO-Y for

An O-CH ^ radical of formula IV

R, 1

X-CH ^

H ^X CH-NH-CO-Y \ X

0-CH3

-C-CH- (O)

IV

O R where is

R = hydrogen or methyl and m χ 0 or 1, wherein the acetal group is again in the m- or p-position.

In this case Z stands for

-CH -NH-CO-CH- (O) - and ₍ m

R (?) For polystyrene.

Preferably the residue IV is for / X-CH 1

-CO-CH.-oYo>'CH ^X CH-NH-CO-Y ² \ o-ch ₂

Other polymers can be used in place of aminomethylated polystyrene, especially those with free NH ₂ groups, e.g. polyacrylamides bearing aminoethyl groups.

As mentioned above, the acetylated formylphenyl group is bonded to the polymer preferably via an amide bond.

This ensures that the binding of the acetylated formylphenyl residue to the resin is stable during the synthesis of the polypeptide and during cleavage and that cleavage occurs as desired on the acetal bond so that peptidyl alcohol is formed on one side and the resin remains on the resin .

If desired, the incorporation of so-called spacers between the reactive groups of the polymer (especially amino groups) and the reactive groups of the acetylated formylphenyl derivative (especially the carboxyl groups) can move the peptidyl alcohol further than the resin. This may be advantageous in certain reactions to peptidyl alcohol prior to cleavage (eg oxidation of cysteine residues). In this case, the residue D or E in Formula III further comprises spacer and Q ₁ or Q _{2 is the} reactive spacer residue. As spacers we can use e.g. ου -aminocarboxylZ V not acids such as L-aminocapronic acid. In the specific case of using aminomethylated polystyrene, a residue of formula IV and E-aminocapronic acid, as spacer, Z is

-CH -NH-CO- (CH) -NH-CO-CH- (O) ^with -4 ->, m

R

The compounds of formula I can be prepared by methods that are customary in the solid phase technique, starting from the compound of formula V

Tl ^ X-CH

CH ^ CH-NH-A

O / cr $ \ \ / O-CH, in which A stands for the protecting group of the amino function and the acetal group is located with respect to the residue Z in the m- or p-position.

To this end, cleave protecting group A and then metabolize the free amino group with the following N-protecting one amino acid, etc., until all the amino acids have been adhered to the resin in a sequence corresponding to the desired peptidic alcohol.

As protecting groups for the amino in the amino acids used or. in amino alcohol, it is necessary to select those which cleave under non-acidic conditions because under acidic conditions the hydrolysis of the acetal group is carried out. As such, amino protecting groups may be used e.g. the group CF ^ CO or FMOC (9-fluoro nyl-methyloxycarbonyl). These protecting groups are cleaved in the manner usual in peptide chemistry in a basic environment.

Only the protecting group of the last-added amino acid can be acid-labile and then cleave from the resin simultaneously with the release of peptidyl alcohol. In such a case, for example, the Boc group should be used.

As bases we can use e.g. KOH, piperidine or also NaBHjj.

Also, synthesis of the polypeptide chain is carried out in a manner known per se from a peptide residue having a free amino group and amino acids having a free or activated carboxylic group. Water binding agents can also be added. Thus, a reaction can be carried out e.g. with the addition of hydroxybenzotriazole and dicyclohexylcarbodiimide.

The compounds of formula V can be prepared by either

a) a resin of formula II bearing aldehyde groups, © -Z-0 ^Cli9

II in which the CHO group is located with respect to the Z substituent in the m- or p-position, is reacted with an N-protected amino alcohol of the formula HX-CHR ₁ -CH (NHA) -CH ₂ OH, or with the activated form of this compound, or

b) a resin derivative of the formula © - (d) -q ₁ p

is reacted with a compound of formula / h

VI

X-CH

T \ ^v q \ _ / O-CH

CH-NH-A

VI in which the acetal group is located with respect to the residue Q ₂ ~ (E) q in the m- or p-positions and Q 'and Q ₂ stand for two reactive groups that can react with each other when the bridge is formed

M? '

Acetalization (process a) is carried out in the usual way with acid as a catalyst. As acid can be used e.g. p-toluenesulfonic acid or trifluoromethanesulfonic acid.

Instead of free alcohol we can use e.g.

trimethylsilyl derivative.

Procedure b) (e.g. esterification or in particular amidation) is also carried out in the usual manner, e.g. by reaction of a reactive carboxylic acid derivative with a polymer bearing the OH or NH _{2 group} .

The compounds of formula VI are prepared by acetalizing the compound of formula with the compound of formula

HX-CHR ₁ CH (NHA) -CH ₂ 0H. This acetalization is carried out as described above for process a).

During the synthesis and before the cleavage of the peptidyl alcohol from the resin, reactions can also be carried out on the peptial alcohol residue, e.g. removal of protecting groups, e.g. S-protecting groups, or oxidation of cysteine residues. Of course, these reactions can also be carried out after the cleavage of the (protected) peptidyl alcohol in solution. The compounds of formula I, V and VI are novel and also belong to the invention.

The method of the invention can be prepared in a simple manner by all, and in particular pharmacologically active peptidyl alcohols, which

- 9 contain at the C-terminus two alcohol groups or one alcohol and one thiol group.

The invention illustrates the following example which relates to the preparation of a somatostatin derivative of the formula

I: i

H-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-ol 4

EXAMPLE:

j}

Preparation of H-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-ol

1) Preparation of the anchoracetate (N-CF ^ CO-threoninol-acetal p-formylphenoxy-acetic acid

105 g (1.0 mol) of L-threoninol are presented by flushing with nitrogen in 200 acre of methanol. A solution of 200 ml of trifluoroacetic acid methyl ester in 250 ml of methanol was added dropwise to the resulting clear solution. The ice bath is maintained at an internal temperature of about 10 ° C. After 1.5 hours, threoninol can no longer be detected in the reaction solution * Evaporation at 40 ° C gives a white crystalline residue. This was dissolved at 70 ° C in 200 ml of acetic acid ethyl ester and precipitated with 100 ml of hexane. It is then cooled to 0 ° C, washed with hexane and dried at room temperature. N-trifluoroacetyltreoninol is obtained.

50.3 g (0.25 mol) of the product obtained are dissolved in nitrogen in 1.25 liters of tetrahydrofuran and 75 ml of trimethylchlorosilane are added dropwise at room temperature. A mixture of 70 ml of triethylamine and 250 ml of tetrahydrofuran was then added immediately. A white suspension is formed, which is stirred for 4 hours at room temperature, then filtered and the filtrate is evaporated at 40 ° C. An oily substance is obtained. This was dissolved in 1.5 liters of methylene chloride and 90.4 g of p-formyl-phenoxyacetic acid were added at room temperature. A total of 9 ml of trimethylsilyl ester of trifluoromethanesulfonic acid are then added in portions. The mixture was stirred at room temperature for 24 hours, then filtered and the residue was washed thoroughly with methylene chloride. The filtrate was evaporated at 4QC to give an orange-red resinous product as a residue. The orange-red product was chromatographed over silica gel. Elution with acetic acid ethyl ester. Evaporation of the desired fraction gave the aforementioned acetal. Purity with HPLC 97%.

2) Synthesis of protected octapeptide ₂ _ resin oxidation and cleavage

17.2 g of aminomethylated polystyrene (0.7 wt% N, equivalent to 0.50 mmol / g) was suspended in 80 ml of methylene chloride / dimethylformamide 4: 1. To this was added progressively 4.17 g of the final product from step 1), 1.6 g of hydroxybenztriazole and 4.0 g of dicyclohexylcarbodiimide. After stirring at room temperature for 2 hours, the Kaiser test was negative. Filter and wash. The washed resin was suspended in 100 acres of tetrahydrofuran / methanol 3: 1 and 10.4 g of sodium borohydride was added in portions. The mixture was stirred at room temperature for 6 hours, filtered and washed. The resin was resuspended in methylene chloride / dimethylforraamide and added 5.57. g FM0C-Cys (S-t-Bu) 0H, 1.74 g hydroxybenztriazole (HOBT) and 3.6 g dicyclohexylcarbodiimide (DCCI). After cleavage of the FMOC protecting group with piperidine (2 x 20 min), the further N-FMOC protected amino acids Thr-OH, Lys (BOC) -OH, D-Trp-OH, Phe-OH, Cys (St- Bu) OH and D-Phe-OH. Finally, the octapeptide resin is obtained with the FMOC.

0.26 mmol / g added.

Suspend this resin in 100 ml of trifluoroethanol / methylene chloride 1: 1 and add 50 ml of tributylphosphine. Stirred for 70 hours at room temperature. It is then filtered, washed and added

100 acre mixture of 1: 1 tetrahydrofuran / 1 molar solution of ammonium acetate. To this was added 1.1 ml of 30% aqueous hydrogen peroxide and stirred at room temperature for 24 hours. The mixture was then washed and added 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. It is filtered and washed with trifluoroacetic acid and methylene chloride. 200 ml of diethyl ether was added to the filtrate, filtered from the isolated, dissolved in aqueous buffer, desalinated by Duolite treatment and lyophilized as acetate.

The title compound is obtained as an acetic acid salt.

Claims (8)

1. Solid-phase process for the preparation of peptidyl alcohols, optionally bearing protecting groups, and bearing at the C-terminal end of the peptide chain two alcohol groups or one alcohol and one thiol group, preferably of the formula • CHRi-ΧΗ Y-C0-NH-CH CH ₂ 0H in which it stands Y for a peptidyl alcohol residue which, if appropriate, bears protecting groups, R ^ for hydrogen or methyl and X for O or S, characterized in that from a resin containing formylphenyl residues to which a (protected) peptidyl alcohol is attached via an acetal group with C-terminal alcohol groups or. an alcohol and thiol group, preferably of a peptide alcohol-bearing resin of formula I I ¹ ®- ^z ~ <O? \ 25 -NH-CO-Y '-' o-chI '' in which they stand (P) for the residue of an insoluble artificial resin, and Z for a direct bond or residue linking the resin to an acetylated formylphenyl group, wherein the acetalized CHO group relative to the Z residue is in the m- or p-position, and several acetylated formylphenyl groups are attached to one resin polymer molecule, under acidic conditions we break off this (protected) peptidal alcohol. The solid phase process according to claim 1, characterized in that it is in the formula I (p) -Z-4jo ^ f for a polystyrene residue. The solid phase method according to claim 1, characterized in that it is in formula i Z for the group of formula III - (D) _P - Q. ₁ - C _fe - (E) _q - III in which the residue of the reactive group bound to the polymer is the residue of the reactive group bound to the acetalized formylphenyl group, D is the residue that associates the group Q with the polymer, E is a residue linking the group to the acetalized formylphenyl group, and p and q are independently 0 or 1. A solid phase process according to claim 1 or 3, characterized in that .in the formula I Z is a group -CH "-NH-C0-CH- (O) 2 | m R in which R stands for H or CH2, m for 0 or 1 and (P) for a polystyrene residue. 5. The solid phase process according to claim 1, characterized in that it is in formula IX for 0 and R ₁ for CHg. Solid-phase process according to claim 1, characterized in that the incorporation of the spacer between the reactive polymer groups and the reactive group of the acetalized formylphenyl derivative moves the peptidyl alcohol further than the resin. The solid phase process according to claim 3, characterized in that it contains a residue D or E in formula III of spacer, preferably -aminocarboxylic acid, and that and Q _{2 is a} reactive spacer residue. The solid phase process according to claim 1, characterized in that it is in the formula -Z- for polyacrylamides bearing the -NH ^ free groups.