DE19703801A1 - New aza-bi:cyclic carboxylate ester di:peptide analogues - Google Patents

Abstract

Dipeptide analogues of formula (I) are new. R1 = CH=CH, NH or S; R2 = H, OH, halogen or 1-4C alkoxy; R3 = H or halogen; or R2+R3 = benzoid system optionally substituted by R6; R4 = amino protecting group; R5 = 1-4C alkyl or 7-11C aralkyl; R6 = OH, COOH, SH or Br; and n = 0 - 4.

Description

The present invention relates to new dipeptide templates, processes for their preparation and their use for incorporation into peptide sequences. The templates according to the invention are equally suitable for solid and liquid phase synthesis and correspond to the general formula I:

wherein
R ₁ -CH = CH-, NH, S;
R ₂ H, OH, halogen, OC ₁ -C ₄ alkyl;
R ₃ H, halogen or together with R ₂ a benzoid system, which in turn can be substituted by a functional group R ₆ ;
R _{4 is} a protective group suitable for protecting an amino group;
R ₅ -C ₁ -C ₄ alkyl, -C ₇ -C ₁₁ aralkyl
R ₆ -OH, -COOH, SH, Br;
n 0, 1, 2, 3, 4
can mean.

Compounds of the general formula I are preferred in which
R ₁ -CH = CH-
R ₂ H, OH, OCH ₃ , F, Cl;
R ₃ H, F, Cl, or together with R ₂ a benzoid system;
R ₄ trichloroethyloylcarbonyl, 9-fluorenylmethoxycarbonyl;
R ₅ OCH ₃ , O-benzyl;
R ₆ -OH;
n 0.1
can mean.

The dipeptide templates according to the invention can be used instead of the corresponding ones natural amino acids in known linear and / or cyclic peptide sequences to be built in.

Underivatized peptides can be in solution in a variety of conformations that are in a dynamic equilibrium. By installation Conformative fixations are induced by templates in such peptides.

Such a conformative fixation opens up a promising approach to more potent, longer effective and more selective peptide analogs. This is guaranteed if exactly the conformation is adopted, which preferred binds to a receptor. - On the other hand, such a conformative Change in signal transduction at the corresponding receptor is inhibited, which can result in competitive antagonism.

Furthermore, the incorporation of templates can lead to enzyme inhibitors that are necessary for therapeutic purposes are extremely promising.

The production of dipeptide templates is inherently state of the art known [D. Ben-Ishai, R. Mc Murray, Tetrahedron 42 (2), 439-450 (1987); F. Esser A. Carpy, H. Briem, H. Köppen, K.H. Pook, Int. J. Peptide Prot. Res., 45, 540-546 (1995)].

In addition, a large number of protective groups are also from the prior art Protection of the N-terminal end as well as the carboxyl group of peptides is known [Römpp, chemistry lexicon, publishing house Thieme, Stuttgart 1993 as well as cit. Lit; T.W. Greene, Protective Groups in Organic Chemistry, J Wiley and Sons, New York 1991].

The object of the present invention is therefore primarily in the diastereoselective construction of dipeptide templates, d. h .: in addition to one existing stereo center in the cyclization reaction according to the invention second stereo center generated in a defined configuration.  

Another object of the present invention is in particular To provide protecting groups for diastereoselective cyclization and stereoconservative deblocking are suitable.

Another object of the present invention is to create new ones To provide dipeptide templates that are suitable for use in the field of combinatorial chemistry are suitable.

The object of the invention is solved u. a. through an acid catalyzed Cyclization reaction and by dipeptide template of the general formula I. Die Acids used in this reaction are strong acids such as. B. sulfuric acid, Trifluoroacetic acid, methanesulfonic acid etc.

Preferred dipeptide templates according to the invention for Teoc-protected peptides are embodied in particular by [3] -benzapines and tetrahydroisoquinolines of the general formula II:

in the
R ₂ H, F, Cl, OH, OCH ₃ ;
R ₃ H, F, Cl, R ₆ ;
R ₆ -OH, -COOH, SH, Br;
n 0 and 1
can mean.

Such compounds are to be understood as derivatives of the peptides from glycine and phenylglycine or from glycine and phenylalanine. If the substituent R ₆ on the aromatic is a hydroxyl group, this is conceivable as an anchor on a carboxypolystyrene resin. This makes it possible to use the templates shown for combinatorial chemistry.

Z-protected dipeptide templates of the general formula III can also be produced, which are derived from the amino acids glycine and tryptophan:

in the
R ₁ NH or S;
R ₆ H or OH
can mean.

The installation of a functional group R ₆ - such as. B. a phenolic hydroxyl group - on the indole ring also allows later anchoring to a solid phase for the use of such templates in combinatorial chemistry.

According to the invention, the amino and carboxyl protecting groups are selected so that that on the one hand they are stable under the strongly acidic cyclization conditions on the other hand, under mild conditions, regioselective and stereoconservative can be split off. Examples of the amino protecting groups are Trihalogenethyloxycarbonyl, such as. B. 2,2,2-trichloroethyloxycarbonyl (Teoc) or the Z protecting group (benzyloxycarbonyl). Examples of carboxyl protecting groups are Alkyl esters, such as methyl or ethyl esters or aralkyl esters.  

The carboxyl protecting group is deprotected by enzymatic Hydrolysis of the corresponding aryl or aryl alkyl esters using a suitable one Enzyme, for example subtilisin.

The N-terminal ends can be eliminated by β-elimination with base metals such as e.g. B. zinc, for the coupling in the liquid phase deblock; the release of the Amino group of the Z-protected dipeptide template is done by hydrogenolysis. Likewise, by further reaction with Fmoc chloride, the templates for the Accessible in solid phase synthesis.

In addition, the exchange of the protective groups used is possible, e.g. B. Trichloroethoxycarbonyl (Teoc) versus 9-fluorenylmethoxycarbonyl (Fmoc). To this Wise is becoming a versatile way of incorporating such templates into peptides opened.

The C-terminal deblocking of the ester using enzymes - such as. B. preferably - Subtilisin is applicable to all derivatives of aromatic amino acids (D- or L-amino acids, e.g. As phenylalanine, tryptophan, tyrosine, phenylglycine), as well the N-terminal Teoc-protected derivatives using base metals such as B. Zinc. Z-ge Protected derivatives must be released by hydrogenolysis.

As stated at the outset, the present invention relates to the Dipeptide templates per se Process for their production.

The synthesis of protected - or Teoc-protected - peptides per se is from the Well known in the art. - Accordingly, the Create the template according to the invention as follows:

First, trichloroethanol, sodium cyanate (NaOCN) and trifluoroacetic acid (TFA) the trichloroethoxycarbonyl (Teoc) amide (1) is obtained.  

The reaction of the intermediate 1 thus obtained with Glyoxylic acid monohydrate - for example with azeotropic water separation Toluene under reflux - gives the bis (Teoc-amino) acetic acid 2.

According to the invention, this can be coupled with various amino acids in the form of C ₁ -C ₄ alkyl esters, preferably protected as methyl esters, which results in type 3 products.

For the condensation of the bis (Teoc-amino) acetic acid derivative 2 with Amino acid methyl esters can be those known per se from the prior art Methods of carbonyl activation, which are known from the prior art in the Peptide synthesis are known to be applied.

According to the invention, 2- (1H-benzotriazol-1 yl) -1,1,3,3- tetramethyluronium tetrafluoroborate (TBTU) applied at pH 8.5. Through their Aminolysis with an amino acid ester forms the pseudodipeptide derivative 3 in high yield.

Cyclization step

In the subsequent cyclization step, intermediate 3 is cyclized in the presence of strong acids, particularly preferably organic sulfonic acid derivatives and particularly preferably in the presence of methanesulfonic acid.

The formation of the six-membered ring (n = 0) almost exclusively results in the Configuration in which the hydrogen atoms are trans-constant to one another; at A seven-ring (n = 1) exclusively forms the cis configuration. The The stereocenter already contained in the dipeptide precursor remains at one Receive reaction management.

The tasks mentioned above are achieved by the inventive Dipeptide template and the production processes described in the examples solved. Different, different configurations of the method and the Possible uses of this template are known to those skilled in the art present description can be seen. However, it is expressly pointed out noted that the examples and the description associated with them only provided for the purpose of explanation and description and not as Limitation of the invention are to be seen.

1. Use of the template (4) for the liquid phase peptide synthesis (using the example of the Leu-enkephalinamide derivative) 1.1. Release of the carboxylic acid

The C-terminal end of the dipeptide template 4 obtained according to the invention is protected in the form of an alkyl ester or aralkyl ester - preferably in the form of a methyl ester. This can be released stereoconservatively with commercially available enzymes to the carboxylic acid. The proteases and esterases required for this are known from the prior art - e.g. B .:
Pork liver esterase, α-chymotrypsin, subtilisin, trypsin, alkalase, lipase, papain, protease N.

The enzyme subtilisin - preferred according to the invention - enables hydrolysis even with poorly water-soluble compounds. Acts as a mediator In the present case, for example, the solvent dimethylformamide (DMF), the up to a volume ratio of 60% DMF 140% potassium chloride solution (0.1 mol / l) the ester easily at a temperature of 37 ° C within four hours hydrolyzed. However, the use of any other suitable one Solution broker possible.  

The short notation for amino acids is used below. Template 5 corresponds accordingly

1.2. Use of the free carboxylic acid

The free carboxylic acid 5 can be coupled with conventional methods (TBTU) to N-terminal free amino acids or all other primary amines, e.g. B. Leucinamide. After this coupling step, it is advantageous or necessary to carry out preparative column chromatography (e.g. ethyl acetate / methanol 8: 2)

According to the invention, tripeptide template 6 is obtained in a preparative manner in this way.

1.3. Release of the amine

The Teoc-protected end of template 6 can then be released with zinc powder in 1 mol / l potassium hydrogenphosphate / tetrahydrofuran (KH ₂ PO ₄ / THF). The pH can be maintained at 6 by 1 molar hydrochloric acid. The conversion to amine 7 can be monitored by TLC control.

The reaction mixture is saturated, for example Sodium bicarbonate made alkaline, saturated with sodium chloride and three Extracted once with ethyl acetate. After drying the organic phase with Sodium sulfate and evaporation of the solvent in vacuo remains a solid Residue. An NMR spectrum of this residue shows that the Teoc group has been removed, but all other structural elements of the template have been removed were preserved.

1.4. Further implementation of the deprotected amine

The deprotected amine 7 is directly reacted with a protected dipeptide by TBTU. The Boc-protected leu-enkephalinamide derivative 8 is thus obtained.

The subsequent elimination of the Boc protective group, for example with 4 N HCl / dioxane solution and subsequent evaporation of the solvent in vacuo, makes the leu-enkephalinamide derivative 9 accessible.

2. Use of template (4) for solid phase peptide synthesis on Example of two dermorphine analogues

• - If the tetrahydoisoquinoline template 4 (n = 0) can be used as in 2.2.1. and described.
• - If [3] -Benzazepine 4 (n = 1) is shown, the dipeptide template cannot protected immediately after the Teoc protection group Fmoc has been split off (cf. Point 2.2!). In this case, the carboxylic acid must first be released enzymatically will. Only after coupling this carboxylic acid with the N-terminus of another Amino acid, the Teoc group can be removed (1.2.2.).

2.1. Use of the tetrahydroisoquinoline derivatives 2.1.1. Amine release and protection

The Teoc-protected dipeptide template 4 (n = 0) is made by zinc - as at the beginning described - deblocked to 10.

With 10% molar excess 9-fluorenylmethoxycarbonyl chloride (Fmoc-Clorid) the free amine 10 using triethylamine to Fmoc-protected Implement connection 11.  

2.1.2. Release of the carboxylic acid

As under 1.1. The Fmoc-protected dipeptide template 11, in short, can also be described

release with the help of the enzyme subtilisin to carboxylic acid 12.

This template is for conventional solid phase peptide synthesis Methods can be used. Thus, z. B. a heptapeptide Dermorphin analogue, build up.

2.2. Use of the [3] benzazepine derivatives

The [3] benzazepine 7 (n = 1) cannot exist as an ester or as a carboxylic acid if it is subsequently to be released by means of zinc at the N-terminus. A diketopiperazine 13 is formed in both cases:

In this case, the ester of dipeptide template 4 must first be enzymatic (see above) to be split into carboxylic acid 8.

In a further step, the next amino acid is coupled with TBTU. In the example of a dermorphine analog to be produced (see above), this is the as Benzyl ester-protected amino acid proline. N-terminal is the tripeptide obtained 17 Teoc protected.

For use in solid phase synthesis, this peptide - as under 2.1.1. described - unblocked at the N-terminus, then protected again with FmocCl be (18).

The next step is the hydrogenolytic removal of the benzylic carboxylic acid protection.

This Fmoc-protected tripeptide template can then be used for the Use solid phase peptide synthesis.

This allows a dermorphine analog to be built, e.g. B.

3.1. Representation of the Z-protected template

Two equivalent benzyl carbamates can be made with one equivalent Glyoxylic acid monohydrate and catalytic amounts of para-toluenesulfonic acid under azeotropic water separation by chloroform up to Implement benzyloxycarbonylaminoacetic acid 20. This is with the TBTU Amino group of an amino acid, here using the example of d-tryptophan, can be coupled.

Implementation of this pseudodipeptide 18 in Trifluoroacetic acid / trifluoroacetic anhydride at 120 ° C gives in 35% yield a Z-Gly-trp-OMe dipeptide template 19.

3.2. Implementation of the Z-protected template

The template 22 can be analogous to the methods described above Unblock enzymatically to carboxylic acid 20, it is then with conventional ones Methods of carbonyl activation can be coupled to amines.

In this example it is coupled to phenylalanine dimethylamide.

The N-terminus must be released by hydrogenolysis.

The resulting amine can be coupled conventionally to other carboxylic acids be, here on cyclohexylglycylcarboxylic acid.  

4. Use of the Z- and Teoc-protected template in the combinatorial chemistry

Does the aromatic of the amino acid carry a hydroxy substituent (a phenolic Group), this can act as an "anchor" for applications of these compounds in the combinatorial chemistry.

The peptidomimetics 7 (n = 0; 1) are Teoc- amidic, the 22 mimetics Z-protected. The C-terminus is protected as a methyl ester.

The templates that can be used for combinatorial chemistry should be expedient Fmoc and as tert. Protected butyl ester.

The methyl esters of all compounds according to the invention can be prepared using the Enzyme enzymatically and stereoconservatively cleaved to the carboxylic acid (see 1.1.).

The tert. Butyl ester can be prepared, for example, by removing the product the enzymatic ester cleavage in dimethylacetamide, one equivalent Benzyltriethylammonium chloride (BTEAC), 26 times excess potassium carbonate and 48 times excess tert. Allows butyl bromide to react at 55 ° C for 24 h.

The protective group of the templates obtained must be replaced by the Fmoc group will.

In the case of Z-protected compounds, the amine release takes place through Hydrogenolysis, in Teoc-protected with the help of zinc (see 2.1.1.). The Fmoc group can be obtained using Fmoc chloride and triethylamine in dimethylformamide introduce.

In all reactions (esterification and Fmoc introduction) the remains phenolic hydroxy group obtained.

This can serve as an anchor to a carboxystyrene resin.

For the purpose of anchoring, the phenolic hydroxy group is through Dicyclohexylcarbodiimide and pyridine in dimethylformamide with the carboxyl group a resin reacted.  

Now the Fmoc protective group (with sec. Amines, e.g. B. morpholine) or the tert. Butyl ester (hydrogen chloride / dioxane) to be split.

The liberated amine can be acylated or alkylated, from the liberated Carboxylic acid can be produced by carbonyl activation amides.  

At the end of the reaction, the phenolic hydroxy group is replaced by one equivalent Sodium methylate in THF released again. The phenolic is obtained Hydroxy function back.

In this way, many organic compounds can be represented in which the Templates 7 and 12 are installed.

Examples 1. Illustration of the β, β, β-trichloroethoxycarboxyamide

75 g (4.8 ml) (0.05 mol) of trichloroethanol are mixed with 6.5 g (0.1 mol) Sodium cyanate and 6 ml trifluoroacetic acid in 120 ml toluene over a Stirred for 3 hours. Then 250 ml of water are added, whereupon the organic phase is separated off and after drying over Sodium sulfate and filtering off the drying agent is concentrated. Here can 5.64 g (50%. Th.) Of the title compound as a crystalline substance with a melting range of 63-65 ° C can be obtained.

2. Preparation of bis (trichloroethoxycarbonyl) aminoacetic acid

18.29 g of β, β, β-trichloroethoxycarboxamide (83% = 0.078 mol) are mixed with 3.6 g (0.039 mol) glyoxylic acid monohydrate in the presence of 0.5 g p- Toluene sulfonic acid monohydrate in 150 ml of toluene on a water separator Reflux temperature heated until no more water is separated.

3. Coupling reaction with L-phenylalanine methyl ester

6 g (0.017 mol) of bis (trichloroethoxycarboxamide) aminoacetic acid 3.61 g (0.017 mol) L-phenylalanine methyl ester hydrochloride and 5.91 g (0.018 mol) 2- (1H-Benzotriazol-1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) are dissolved in 180 ml of dimethylformamide. In the solution with 4.93 ml Triethylamine set a pH of 8.5 and it is at 2 hours Stirred room temperature. Then it is saturated with water Sodium bicarbonate solution stirred, the coupling product precipitates, which is filtered off, washed and dried.

4. Cyclization step

2.5 g (4.2 mmol) from the above reaction Coupling product are at room temperature in 65 mol methanesulfonic acid dissolved and over a period of about 12 hours at room temperature Brought reaction. The reaction solution is then dripped into ice water. The  aqueous phase is exhaustively extracted with ethyl acetate. After drying the combined extracts, filtering off the drying agent and Concentrations in a vacuum remain after chromatographic purification Ethyl acetate on aluminum oxide, after concentration of the eluate and Drying 1.34 g (80% of theory) of the cyclization product in vacuo.

5. Stereoselective ester cleavage

0.13 g (3.0 mmol) of the ester resulting from the cyclization step are in a mixture of 10 ml of dimethylformamide and 20 ml of 0.1 N. Potassium chloride solution entered, mixed with 50 mg subtilisin and at one Temperature of 37 ° C over a period of 4 hours. Doing so the pH value by repeatedly potting 0.1 N sodium hydroxide solution kept at 8.5. After cooling the solution, acidifying with 1 N Hydrochloric acid, salting out with common salt, the reaction mixture Exhaustively extracted ethyl acetate, the combined extracts after Drying concentrated and the remaining residue from ethyl acetate crystallized.

6. Spin-off of the Teoc group

1.48 g (3.7 mmol) of the carboxylic acid obtained from step 5 are in 50 ml Dissolved tetrahydrofuran, with 8.5 ml of 1 molar potassium dihydrogen phosphate Solution and 2.4 g (37 mmol) of zinc dust and added over a period of 3 Hours stirred. It is about every 30 min. in the reaction solution with 2 N HCl adjusted a pH of 4. Then the reaction solution with aqueous sodium bicarbonate solution made alkaline with table salt saturated and finally extracted with ethyl acetate. The United Extracts are made after drying and filtering off the desiccant concentrated in vacuo.

In this way, 0.63 g of the deprotected product in a yield of 77% of theory obtained purely by thin layer chromatography will.  

7. The Boc protecting group is split off

Boc Tyr Gly GlyPhe Len-NH ₂

→ HTyrGlyGlyPhe-Len-NH ₂

0.2 g (0.3 mmol) of the pentapeptide are dissolved in 5 ml of a 4N solution of HCl Dissolved dioxane and stirred for one hour at room temperature in vacuo concentrated and mixed with diethyl ether until the reaction product precipitates. Man receives the cleavage product in quantitative yield.

8. Ester cleavage (to 1.1 / 3.2)

1.00g (2.5 mmol) substrate are dissolved in 15 ml dimethylformamide and with 30 ml of a 0.1 molar potassium chloride solution are added. The reaction mixture is then heated to 37 ° C and then with 50 mg of the enzyme Subtilisin added. The reaction mixture is left over a period of 4 React for hours at 37 ° C, the pH with 0.2 N sodium hydroxide solution Value is kept at 8. After processing - as in step 5 described - 0.74 g (75% of theory) of the desired dipeptide obtained with a free carboxyl group.

9. Bisbenzyloxycarbamoylaminoacetic acid

20 g (0.13 mol) benzyl carbamate, 6.1 g (0.066 mol) glycolic acid monohydrate and 0.25 g (0.0013 mol) of p-toluenesulfonic acid are dissolved in 200 ml of chloroform and on the water separator for a period of 5 1/2 hours Reflux temperature heated. After the azeotropic distillation has ended with 50 Molecular sieve (2 Å) added.

Claims (7)

1. Dipeptide template of the general formula I
wherein
R ₁ -CH = CH-, NH, S;
R ₂ H, OH, halogen, OC ₁ -C ₄ alkyl;
R ₃ H, halogen or together with R ₂ a benzoid system, which in turn can be substituted by a functional group R ₆ ;
R _{4 is} a protective group suitable for protecting an amino group;
R ₅ -C ₁ -C ₄ alkyl, -C ₇ -C ₁₁ aralkyl
R ₆ -OH, -COOH, SH, Br;
n 0, 1, 2, 3, 4
can mean. 2. Dipeptide template according to claim 1, characterized in that
R ₁ -CH = CH-;
R ₂ H, OH, OCH ₃ , F, Cl;
R ₃ H, F, Cl, or together with R ₂ a benzoid system;
R ₄ trichloroethyloycarbonyl, 9-fluorenylmethoxycarbonyl;
R ₅ OCH ₃ , O-benzyl;
R ₆ -OH;
n 0.1
can mean. 3. dipeptide template according to claim 1 or 2 according to the general formula II,
characterized in that
R ₂ H, F, Cl, OH, OCH ₃ ;
R ₃ H, F, chlorine;
n 0 and 1
can mean. 4. Dipeptide template according to claim 1 or 2 according to the general formula III
characterized in that
R ₁ NH or S;
R ₆ H or OH
can mean. 5. A process for the preparation of dipeptide templates of the general formula I, characterized in that
Trichloroethanol, sodium cyanate and trifluoroacetic acid which trichloroethoxycarbonylamide (1) produces,
this in the subsequent reaction step with glyoxylic acid monohydrate under azeotropic conditions to bis (triethoxycarbonylamino) acetic acid (2)
and uses this to produce the pseudodipeptide derivative 3 with an amino acid alkyl ester
this then cyclized in the presence of a strong acid
6. Use of dipeptide templates of the general formula (I) in the Peptide synthesis. 7. Use of dipeptide templates of the general formula (I) in the combinatorial chemistry.