HU199786B - Process for producing peptide derivatives suitable for treating high blood pressure - Google Patents

Abstract

31 Methods are claimed for preparing aminoacylthio-alkanoyl amino acid derivs. of formula (I) - R-A-S-(CH2)n-CHR1-CO-R2 (I) - (where R is H, t-butyloxycarbonyl, cyclopentenecarbonyl-L-lysyl, pyro-L-glutamyl-L-lysyl, L-lysyl, L-arginyl, pyro-L-glutamyl or R', where R' is formyl, acetyl, propionyl, butyryl, phenylacetyl, phenylpropionyl, benzoyl or cyclopentanecarbonyl; A is D- or L-Phe, -Ala, -Trp, -Tyr-, -Gly, -Lle, -Leu, -His or -Val; R1 is H or Me; R2 is L-proline, L-2,4-dehydroproline, DL-3, 4-dehydroproline, L-3-hydroxyproline, L-4-hydroxyproline, L-thiazolidine-4-carboxylic acid or L-5-oxoproline; n = 0 or 1, provided that R1= Me when n= 0 and that the CH2CHR1 gp. is in the D configuration when n= 1 and R1 = Me). - Typical methods include (a) reacting R-A with an aryl or alkyl thiol in the presence of a coupling agent, opt. deprotecting the product and reacting it with an R' coupling cpd., reacting the product with MHS (M= H or alkali metal) to form R-A-SH, reacting this with acrylic or methacrylic acid to form R-A-S-CH2-CHR1-COOH (II), opt. resolving this to isolate the isomer with CH2CHR1 in the D configuration, and coupling the product with R2; (b) reacting R-A with pentachlorophenol, pentafluorophenol or N-hydroxysuccinimide, opt. deprotecting the product and reacting it with a R' coupling cpd., reacting the product with HS(CH2)nCHR1COOH, opt. resolving the product and coupling with R2; etc.

Description

This invention relates to novel peptide derivatives for use in the treatment of hypertension.

The angiotensin converting enzyme (ACE, also known as peptidyl dipeptide hydrolase) plays a central role in the physiology of hypertension. This enzyme is capable of being called angiotensin I and

Convert the (amino acid sequence) decapeptide of the H-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-OH sequence to angiotensin egy, an octapeptide, by cleaving the penultimate peptide bond of angiotensin I. Removes the His-Leu unit from the C-terminal.

In the present description, certain chemical groups are designated as follows:

ACE = angiotensin converting enzyme

Ac = acetyl

Lower = L-alanine

Arg = L-arginine

Asp = L-aspartic acid

Boc = tert-butoxycarbonyl

Bpoc = 2- (4-biphenylyl) -2-propoxycarbonyl

Bz = benzoyl

Cbo = benzyloxycarbonyl

CpCO = cyclopentylcarbonyl

Ddz = N-α, α-dimethyl-3,5-dimethoxybenzyloxycarbonyl

EtCO = propanoyl

Gly = glycine

Glu = L-glutamic acid

Glu = pyro-L-glutamic acid (L-5-oxo-proline)

His = L-histidine

Hip - Hippuric Acid (Benzoyl-Glycine)

Ile = L-isoleucine

Leu = L-leucine

Phe = L-phenylalanine

Ppoc = 2-phenylisopropoxycarbonyl

Pro = L-proline

Δ Pro = L-3,4-dehydro-proline

Ser = L-serine

Trp = L-tryptophan

Tyr = L-tyrosine

Val = L Select

Angiotensin I is produced by the renin, an endopeptidase enzyme in the kidney, other tissues and plasma that acts on the serum α-2 globulin.

Blood pressure is affected by certain peptides present in the blood. One of these, angiotensin II, is a powerful agent (which increases blood pressure). Another peptide, bradykinin, is a monapeptide of the sequence H-Arg-Pro-Pro-Gly-Phe-SerPro-Phe-Arg-OH, a potent antihypertensive agent.

In addition to its direct effect on raising blood pressure, angiotensin II also stimulates the release of aldosterone, which in turn raises blood pressure by retaining extracellular salts and fluids. A normal amount of angiotensin II is present in normal human blood. However, patients with hypertension of renal origin have higher blood levels.

Both normal and hypertensive people have higher levels of angiotensin converting enzyme activity than would be needed to maintain measured levels of 2 angiotensin Π. However, it has been found that treating patients with hypertension with inhibitors of the angiotensin converting enzyme can achieve significant reductions in blood pressure [Gavras, I., et al., New Engl. J. Med., 291, 817 (1974)].

The angiotensin converting enzyme is a peptidyl dipeptide hydrolase enzyme. It catalyzes the hydrolysis of the penultimate peptide bond of the acylated tripeptide and the longer polypeptide containing a large number of unprotected carboxyl groups. The angiotensin-converting enzyme hydrolytically cleaves the penultimate peptide bond of the C-terminus to form a dipeptide and a residue.

The reactivity of the enzyme is highly dependent on the substrate (material to be converted). There is at least one type of peptide bond in which the proline nitrogen atom is involved which is not cleaved at all by the enzyme. The apparent Michaelis constant (Km) for each substrate may vary by several orders of magnitude. The rate characteristics of enzyme-catalyzed reactions are generally discussed in Lehninger, A., Biochemistry, Worth Publishers, Inc., New York, 1970, 153-157. side. Many peptides, referred to as inhibitors of the enzymatic conversion of angiotensin I to angiotensin Π, are in fact substrates with a Michaelis constant (Km) lower than that of angiotensin I.

Such peptides are more preferably referred to as competitive substrates. Such competitive substrates include bradykinin and BPPsa (also designated SQ20475), a peptide isolated from snake venom, the latter having the sequence Glu-Lys-Trp-Ala-Pro.

Angiotensin Π is a powerful pressor (antihypertensive) agent. In addition to acting directly on the pressor, angiotensin II also stimulates the release of aldosterone, which in turn raises blood pressure by retaining extracellular salts and fluids. A normal amount of angiotensin II is present in normal human blood. However, patients with hypertension of renal origin have higher blood levels. In both normal and hypertensive patients, the level of angiotensin converting enzyme in the blood is higher than would be needed to maintain the level of angiotensin II observed. However, it has been found that treating patients with hypertension with inhibitors of the angiotensin converting enzyme can achieve a significant reduction in blood pressure [I. Gavras et al., New Engl., J. Med., 297, 817 (1974).

Recognizing the role of angiotensin-converting enzymes in the pathogenesis (development) of hypertension, they have begun to search for inhibitors of this enzyme that could be used to treat hypertension. See, e.g., U.S. Patent Nos. 3,891,616, 3,947,575, 4,052,511. and 4,053,651. U.S. Pat. The reactivity of the angiotensin converting enzyme is highly dependent on the substrate (the substance to be converted). Many peptides, called inhibitors of the enzymatic conversion of angiotensin I to angiotensin ΙΙ, are essentially substrates having a Michaelis constant (Km) less than angiotensin

HU 199786 Β

1 year Such peptides are more preferably referred to as competitive substrates.

Ondetti et al., U.S. Patent No. 3,832,337, published August 1974

27) have shown that many synthetic peptide derivatives inhibit the angiotensin converting enzyme. One such potent inhibitor that exhibits a strong biological effect upon oral administration is D-3-mercapto2-methylpropanoyl-L-proline (designated SQ14225), see Ondetti et al., U.S. Patent No. 4,046,889. , published September 6, 1977) and DW Cushman et al., Biochemistry 16, 5484 (1977) and M. Ondetti et al., Science 196, 441 (1977). The inhibitor of SQ14225 has been reported to have an Iso value of 2.3 x 10 M. The L50 value reported by Cushman et al., Supra, is the concentration of inhibitor that inhibits the enzyme by 50% under standard assay conditions at a substrate concentration lower than the Michaelis constant above. It is obvious that the I50 values can only be directly compared if all the factors that may influence the reaction are kept constant. Such factors include, for example, the source of the enzyme, the purity of the enzyme, the substrate used and its concentration, and the composition of the buffer solution used for the assay. All I50 values given herein are determined by the same assay system, using the same enzyme (angiotensin converting enzyme isolated from human urine) and approximately 1/2 Km of substrate and are therefore comparable.

The concept of the mode of action of SQ 14225 is based on a model of active sites for the angiotensin converting enzyme, which is based on the analogue of a more well-known related enzyme, carboxypeptidase A. It has been suggested that the active site has a cationic center that binds to the carboxyl end group of the substrate, and a site that can bind the C-terminal amino acid side chain and establish a particularly strong bond with the terminal proline heterocycle. A similar site for the penultimate amino acid binding was also assumed; according to published data, the steric (spatial) requirements are very stringent, since the D-form of the inhibitor is significantly more effective than its stereoisomer or the 3-methyl or unsubstituted analogue. It is believed that the mercapto group of the inhibitor, which is thought to be attached to the active site near the catalytic center, plays a central role in the inactivation of the enzyme when attached to the zinc atom, which is known to be important for catalytic activity. When the mercapto group is substituted, for example with a methyl group, or an S-acetyl derivative, they significantly reduce the activity of the inhibitor. See D. W. Cushman et al., Biochemistry, supra.

The mechanism of inhibition of SQ14225 and its analogs by the angiotensin converting enzyme is difficult to study because these compounds are not normally stable. For example, it has been observed that a freshly prepared aqueous solution of SQ 14225 at 1 mg / ml causes a significant loss of activity at a pH of 8 and a further loss of activity at a further stand. It is believed that the decrease in activity results from the dimerization of the compound SQ14225 by the mercapto end groups, which results in the formation of a disulfide which has virtually no inhibitory effect. Since the free mercapto group is highly reactive and readily polar, it can be oxidized to acidic groups such as sulfones or sulfoxides; it is also conceivable that the decreased activity of aqueous solutions of SQ 14225 under in vitro conditions is in part due to one or more of these oxidation reactions, which give rise to sulfones or sulfoxides which do not effectively inhibit the angiotensin-converting enzyme. .

Reports of clinical trials of SQ14225 are known; some of them refer to the compound as "Captopril," indicating that the product is sufficiently stable in most gastrointestinal systems to effectively inhibit the angiotensin converting enzyme when administered orally. However, it is not clear from these reports that there were patients for whom SQ14225 was essentially ineffective. Because of the high reactivity of the free mercapto group, SQ 14225 can readily form mixed disulfides with serum, cellular proteins, peptides, or other free mercapto group substances in the gastrointestinal tract, as well as form dimers and undergo oxidative degradation. A mixed disulphide antigen with a protein can also act and indeed allergic reactions have occasionally been observed in clinical trials. See Gavras et al., New England J. Med., 298, 991 (1978). If SQ14225 produces disulfides and oxidation degradation products, they may at best be inactive. Thus, it is anticipated that the dose-effect relationships of SQ14225 may vary depending on the route of administration and may also vary from patient to patient. In addition, undesirable side effects may occur in at least some patients and it may be difficult to maintain effective concentrations of the inhibitor in the body.

Thioester derivatives are generally known to be highly reactive and the thioester bond can be easily hydrolyzed to a mercapto group and a carboxyl group. Therefore, thioesters are often used as active esters when acylation is desired under mild conditions. Ondetti et al., In the patents cited above, use such groups as protecting groups, such as acetylmercapto. It is believed that thiosester-type intermediates also occur in the biosynthesis of certain cyclic peptides, such as thyrocidin and gramicidin S. See F. Lipmann, Accounts Chem. 6, 361 (1973).

Ondetti et al., Nos. 4,046,889, 4,052,511, 4,053,651, 4,133,715. and 4,154,840. U.S. Pat. Of the described analogues of SQ 14225, zeol 3

HU 199786 Compounds in which the 5-membered hetero ring of proline is replaced by a 4- or 6-membered ring. The inhibitory efficacy of these analogs relative to SQ 14225 has not been reported.

When D-proline is incorporated into the molecule instead of L-proline, the inhibitory properties of the 3-mercaptopropanoylamino acids are greatly reduced (D. W. Cushman et al., Supra).

The role of the amino acid to the left of the sulfur atom of thioester derivatives has not yet been determined. It is believed that this amino acid serves as an additional recognition site for the enzyme. If this assumption were true, then a compound containing a particular amino acid moiety at this site would be expected to be the best inhibitor. However, the inventors have found that such compounds containing different amino acid units are effective and that hydroxyproline, proline, L and D, L-3,4-dehydroproline, thiazolidine-4-carboxylic acid and L-5-oxoproline derivatives are both effective antihypertensive agents and potent inhibitors of the angiotensin converting enzyme.

The present invention relates to a process of formula (I) wherein R is hydrogen, tert-butoxycarbonyl, cyclopentanecarbonyl-L-lysyl, pyro-L-glutamyl, or R ', wherein R' is formyl, propanod, butanoyl, phenylacetyl, phenylpropanod, benzoyl or cyclopentanecarbonyl;

A is L-fend-aland, D-fend-aland, DL-fend-aland, L-aland, D-alanyl, L-tryptophyl, D-tryptophyl, L-tyrosyl, glycyl , L-valyl, D-valde, L-leucyl, D-leucyl or L-isoleucyl, whose α-amino group is amide bonded to R;

R 1 is hydrogen or methyl;

R ₂ is L-proline or L-3,4-dehydroproline, the imino group of which is in an imide bond with the attached carbond group;

n is 0 or 1 with the proviso that when n is 0, R 1 is methyl; the -CH ₂ -CH (R 1) - group has the D configuration; and their pharmaceutically acceptable salts with base forms, which process comprises a number of process variants. The compounds of the invention inhibit the angiotensin-converting enzyme and, when administered orally (orally), are effective in reducing pathologically high blood pressure.

The compounds of formula I form salts with inorganic and organic bases. Such salts include ammonium, alkali metal, alkaline earth metal salts, organic base salts, amino acid salts and the like. An example of an alkali metal salt is the sodium or potassium salt. Alkaline earth metal salts include calcium or magnesium salts. Organic base salts include, for example, benzathine, N-methyl, D-glucamine or hydrabamine salts. An example of a salt-forming amino acid is arginine or lysine. Sodium, potassium or lysine salts are preferred.

The salts of the present invention are prepared by reacting the free acid of formula (I) with an equivalent amount of a base providing the desired cation, preferably in a solvent or solvent mixture in which the salt formed is insoluble or in water and then freezing the water. removed by drying.

The compounds of the present invention and their salts inhibit the conversion of the angiotensin I decapeptide to angiotensin II and are thus useful in reducing or controlling hypertension related to the action of angiotensin. The renin enzyme produces angiotensin I as a result of its action on the plasma pseudoglobulin. Angiotensin I is converted to angiotensin II by the angiotensin converting enzyme. The latter has been shown to cause different types of hypertension in mammals such as rats and dogs. The novel salts of the present invention affect the angiotensinogen-angiotensin I-angiotensin Π process by inhibiting the angiotensin converting enzyme and thereby reducing or eliminating the formation of angiotensin II. Thus, angiotensin-dependent hypertension can be controlled or eliminated by the addition of salts of the compounds of formula I according to the invention. Administration of the active compounds of the invention in a single dose, or preferably four or two times daily, in divided doses of 0.1 to 100 mg / kg / day, preferably 150 mg / kg / day, is suitable according to S.L. Engel, T.R. Schaeffer, Μ. H. Waugh and B. Rubin, Proc. Soc Exp Bioi. Med., 143, 483 (1973)] for the reduction of hypertension. The active compounds of the present invention are preferably administered orally but may also be administered parenterally, for example subcutaneously, intramuscularly, intravenously or intraperitoneally.

The salts of the present invention may be used in the preparation of preparations such as tablets, capsules or elixirs for oral administration, or for the preparation of sterile solutions or suspensions for parenteral administration. For this purpose, 10-500 mg of a salt of Formula I may be formulated with a pharmaceutically acceptable excipient, carrier, binder, preservative, stabilizer, flavoring agent, and the like. when mixed, it is converted into a generally accepted dosage form in medicine. In these formulations, the active ingredient is present in an amount such that the dosage ranges indicated above are preferred.

Suitable carriers for the preparation of tablets, capsules and the like are, for example; excipients such as tragacanth, acacia, corn starch or gelatin, fillers such as dicalcium phosphate, disintegrating agents such as corn starch, potato starch, alginic acid and the like, lubricants such as magnesium stearate, sweeteners, lactose or sucrose, gaulteria oil or cherry. In the manufacture of a capsule, it may also contain a liquid carrier such as an oil. Other agents, such as tablets coating agents or agents to modify the physical state of the dosage unit, may also be used. For example, tablets may be coated with shellac, sugar, or both. A syrup or elixir may contain the active ingredient, sucrose as a sweetening agent, methyl and propylparaben as a preservative, coloring agents and flavoring agents such as cherry or orange flavor.

Sterile injectable preparations may be prepared by conventional methods of formulation. Thus, the active ingredient is contained in a carrier, such as water for injection, a natural vegetable oil, for example

It is dissolved or suspended in sesame oil, cocoa oil, peanut oil, cotton oil, etc. or in a synthetic fat carrier such as ethyl oleate. If necessary, preservatives may be added to the composition.

For the sake of simplicity, all embodiments of the process of the invention are exemplified by the preparation of a compound of formula I wherein R is benzoyl, A is phenylalanyl, R1 is methyl, R2 is L-proline, and n is 1, i.e. the model compound is N "- [3N" -benzoyl-phenyl-alanyl-meacapto] -2-D-methyl-propanoyl] -L-proline. It will be apparent to those skilled in the art that, if desired, other groups may be substituted for the groups used in the examples to illustrate process variants. Thus, for example, L-3,4-dehydroproline may be used in place of Lproline in all of the following embodiments.

Similarly, any of the other amino acids listed above can be used in place of the amino acid moiety A instead of phenylalanine. Any additional desired compound may be prepared with similar substitutions; deviations from this are given separately.

In some embodiments described below, N ^is -R3-thiophenylalanine, wherein R3 is hydrogen, Bz, Boc or Ddz, reacted with methacrylic acid or its ester or 3-bromo-2-methylpropionic acid to give 3-N. "-R 3 -phenylalanyl mercapto-2-methylpropionic acid or an ester thereof. Subsequently, before coupling with L-proline, the compound is resolved to give 3-N '-R 3 -phenylalanyl-mercapto-2D-methylpropionic acid or ester. Alternatively, in ^the N-R4-phenylalanine, or an active ester thereof, wherein R4 is Bz-, Bpoc-, Ddzcsoport reaction with 3-mercapto-2-methylpropionic acid, or an ester of ^{3-phenyl-N-R4} alanyl mercapto-2-methylpropionic acid or an ester thereof. Then, before being connected to the L- proline compound is resolved to give ^the 3-N-phenyl-alanilmerkapto -R4-D-2-methylpropionic acid or ester. In some other embodiments, the 3-R5-mercapto-2-methylpropionic acid or ester wherein R5 is Cbo, acetyl or amino is first resolved to give the 3-R5-mercapto-2-D-methylpropionic acid or ester. and then reacting it with RA-OH.

The 3-benzoylmercapto-2-D-methylpropionic acid from which the compounds of formula I are prepared according to the process of the invention is commercially available (Chemical Dynamics Corp., 3001 Hadley Road, South Plainfield, New Jersey).

In the following embodiments, any conventional coupling methods other than those described herein may be used. Examples of such coupling methods are the mixed anhydride method and the following reagents: dicyclohexylcarbodiimide, azide, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, N-isobutoxycarbonyl-2-isobutoxy, 1,2-dihydroquinoline, symmetric anhydride, Woodward's K-reagent (N-ethyl-5-phenylisoxazolium-3'-sulfonate) and carbonyldiimidazole. See Methoden dér Organischen Chemie (Houben-Weyl), XV. Volume II. Volume 1, page 1974.

As above, where a tert-butoxycarbonyl protecting group is used in the disclosed embodiment, it may be replaced by any other acid-sensitive protecting group, such as

2- (4-biphenylyl) -2-propoxycarbonyl (Bpoc), 2-phenylisopropoxycarbonyl (Ppoc), benzyloxycarbonyl (Cbo), or other acid-sensitive Naralkoxycarbonyl protecting group. The deprotection may be carried out by conventional methods, for example with trifluoroacetic acid in anisole, hydrochloric acid in acetic acid, cold trifluoromethanesulfonic acid or methanolic ammonia.

See Methoden der Organischen Chemie (Houben-Weyl), XV. Volume I, page 376 (1974).

Method (a)

In a preferred embodiment of the invention ^the N -t-butoxycarbonyl-phenylalanine (Boc-Phe) by a conventional condensing agent, in an anhydrous medium with thiophenol to give ^the N -t-butoxycarbonyl phenylalanine thiophenyl ester. For this purpose, ethyl acetate is used as the solvent and the mixed anhydride method is employed.

In the second step of this procedure, the Boc protecting group is removed by reacting the product of step 1 with trifluoroacetic acid and anisole. The product is then reacted with hydrochloric acid in ethanol to give the hydrochloride salt of phenylalanine thiophenyl ester. (Hydrochloric or hydrobromic acid dissolved in acetic acid or other organic solvents, and methanesulfonic acid or substituted methanesulfonic acid may also be used to deprotect the protecting group). The salt prepared as described above, then ethyl acetate and water, sodium carbonate is reacted with benzoyl chloride in the presence of ^the thus obtained N-benzoyl-phenylalanine-thiophenyl ester.

In step 3, the product of step 2) is reacted with sodium hydrogen sulfide in ethanol under nitrogen to give N 'benzoylthiophenylalanine (Bz-Phe-SH). This step is a known method [Bull. Chem. Soc. Jap. 38, 320 (1965)].

In another preferred embodiment of this embodiment, the ester prepared in 1) is reacted with sodium hydrogen sulfide as described in step 3 to produce Boc-Phe-SH. Boc-PheSH can also be prepared by coupling Boc-Phe with hydrogen sulfide by the mixed anhydride method (see J. Am. Chem. Soc. 74, 4726 (1952)). From this intermediate, benzoylthiophenylalanine (Bz-Phe-SH) is prepared by a similar procedure to that described in step 2).

In a further preferred embodiment of the present process, Bz-Phe-SH can also be prepared by reacting phenylalanine with benzoyl chloride in the presence of sodium hydroxide. The resulting product is then coupled with the mixed anhydride method, hydrogen sulfide [J. Biol. Chem. 138, 627 (1941)].

In step 4, the thioic acid prepared as described above is heated in toluene with methacrylic acid to give 3- (N 'benzoylphenyl) v ^: ' mercapto) -2-methylpropionic acid.

Then, the resultant product is resolved in step 5) to give ^N - (3-benzoyl-phenylalanyl-mercapto) -25

HU 199786 Β

D-methylpropionic acid is obtained. Resolution is accomplished by crystallization, liquid chromatography, countercurrent partitioning, ion exchange or the use of resolving agents. Suitable resolving agents are, for example, 3-nitro-D-tyrosine methyl ester or hydrazide, d (+) - α-methylbenzylamine or 1- (-) - α-methylbenzylamine, quinine, (-) - ephedrine or N, N-dicyclohexylamine.

In step 6, the acid obtained in the above manner is coupled with L-proline tert-butyl ester (dicyclohexylcarbodiimide) or by the mixed anhydride method. such as ^N - [3- ^{(N-benzoyl-phenylalanyl-mercapto)} -2-D-methyl propanoilj-L-proline tert.butyl ester. This ester was dissolved in anisole and trifluoroacetic acid mixture to give N '- [3- ^{(N-benzoyl-phenylalanyl-mercapto)} -2-D-methyl-propanoyl] -L-proline obtained. Alternatively, the product of Step 5 may be coupled with hydroxysuccinimide using dicyclohexylcarbodiimide or by the mixed anhydride method followed by reacting the product with L-proline.

The above-described method ^of N - [3- ^(N -benzoyl phenylalanyl-mercapto) -2-D-methylpropanoyl] -L-proline preferred preparation, because the yield is generally high and the product is easy to separate the crystalline state.

The above method is also advantageous because the L, D or DL forms of the phenylalanine derivatives can be prepared simply and in pure form. Thus, when Boc-L-Phe or Boc-DPhe is used as starting material, Ν ^α - [3- (Ν ^α -benzoyl-L-phenylalanyl mercapto) -2-D-methylpropanoyl] -L proline or N '- [3- ^{(N-benzoyl-D-phenylalanyl-mercapto)} -2-D-methylpropanoyl] -L-proline. Starting from a mixture of Boc-L-Phe and Boc-D-Phe, the product is obtained in DL form.

Using acrylic acid instead of methacrylic acid in step 4 above gives the corresponding 3-mercaptopropionic acid derivative.

If step 0009898 is replaced with Boc-Phe in step 1) above. The corresponding compounds of the general formula R-A-OH, prepared as described in European Patent Applications, are used, and

Steps 1) and 3) -6), the corresponding compounds containing the desired R and A groups are obtained.

Method b

In another preferred embodiment of the invention, 3-benzoylmercapto-2-D-methylpropionic acid is reacted with 2-methylpropene in the presence of sulfuric acid to give 3-benzoylmercapto-2-D-methylpropionic acid tert-butyl- ester.

In step 2, the benzoyl group is cleaved according to standard procedures to give 3-mercapto-2-D-methylpropionic acid tert-butyl ester. Common reagents for cleaving the benzoyl group include, for example, ammonium hydroxide, methanolic ammonia, and methanolic sodium methylate solution.

Step 3) 2) coupling ^the product of step N-benzoyl-phenylalanine (Bz-Phe), the active ester method to give ^the 3-N-benzyl-phenylalanyl-mercapto-2D-methylpropionic acid tert butyl ester is obtained. As the active ester, N-hydroxysuccinimide ester, p-nitrophenyl ester, pentachlorophenyl ester and the like can be used.

In process variant, according to another preferred embodiment of step 2) was coupled ^{to the} product of N-benzoyl-phenylalanine, under conditions in which the racemization is only slight; Coupling with, for example, the azide, dicyclohexylcarbodiimide, in the presence of a reagent that suppresses racemization, e.g., in the presence of 1-hydroxybenzotriazole, and coupling with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline . such as 3 N to give ^the benzoyl-phenyl-alanilmerkapto-2-D-methyl-propionic acid tert.-butyl ester.

In step 4) the product obtained in step 3) cleavage of the tert-butyl ester group is a conventional method, to give ^{3-N-benzoyl-phenyl} alanilmerkapto-2-D-methyl-propionic acid.

In step 5, the product of step 4 is coupled with N-hydroxysuccinimide using dicyclohexylcarbodiimide or the mixed anhydride method to give the N-hydroxysuccinimide ester. This ester is then reacted with L-proline to convert N '- [3- (N ^is benzoylphenylalanyl mercapto) -2-D-methylpropanoyl] L-proline. The active ester may also be prepared by using other suitable esterifying agents instead of N-hydroxysuccinimide. Alternatively, the product of Step 4 may be coupled with L-proline tert-butyl ester using dicyclohexylcarbodiimide or the mixed anhydride method to give the corresponding product tert-butyl ester. The tert-butyl ester group is then cleaved in a manner known per se.

When Bz-L-Phe, Bz-D-Phe or Bz-D, L-Phe is used as starting material, N ^a - [3- (N ^a benzoyl-L-phenylalanyl) - is used. mercapto) -2-D-methylpropanoyl] -Lprolint, ^Ν α - ^[3-Ν α - benzoyl-D-phenylalanyl-mercaptopropyl) 2-D-methyl-propanoilj-L-proline or ^N - [ 3- (N '-benzoyl-D, L-phenyl-alanyl-mercapto) -2-D-methyl-propanoyl] -L-proline is obtained. The pure L, D or D, form of Bz-Phe can be prepared by the methods described herein.

When using the corresponding R-A-OH compounds of the general formula R-A-OH prepared in the manner described in European Patent Application 0009898 instead of Bz-Phe used in Step 3), the corresponding R and A compounds are carried out. products.

(c) method

According to a third preferred embodiment of the process of the invention, process (c) is carried out first with 3-mercapto-2-D-methylpropionic acid tert-butyl in the presence of N "-Bpoc-Phe-t-hydroxybenzotriazole with dicyclohexylcarbodiimide or by the mixed anhydride method. ester (prepared as described in steps 1 and 2 of process variant b)) ^{to give} 3Na-Bpoc-phenyl-alanyl-mercapto-2-D-methyl-propionic acid tert-butyl ester.

This ester obtained from step 1) in step 2) is deprotected to give ^3-N-phenyl alanilmerkapto-2-D-methyl-propionic acid tert.-butyl ester.

In Step 3, the product of Step 2 is treated with a benzoyl halide such as benzoyl chloride or benzoyl bromide or an ester of benzoic acid such as benzotriazolyl, ρ-nitrophenyl, ο-nitrophenyl, 5-nor-61

GB 199,786 Β bomet-2,3-dikarboximidil-, pentaklór- coupled phenyl or pentafluorophenyl ester, and N, such as ^{3-benzoyl-phenylalanyl-mercapto-2-D-methyl-propionic} acid tert.-butyl ester.

In Step 4, the tert-butyl ester group is cleaved as described in Method B, Step 4.

In step 5, the product of step 4 is coupled with L-proline or L-proline tert-butyl ester as described in method b), step 5), N ^a - [3- (N ^a benzoylphenylalanyl mercapto) -2- D-methylpropanoyl] -L-proline is prepared.

Instead of the phenylalanine used in Step 1, any other desired A-OH compound can be used. Similarly, in step 2) any other desired R group may be used instead of the benzoyl group.

(d) method

According to a fourth preferred embodiment of the process according to the invention first of N "-t-butoxycarbonyl-phenylalanine was coupled with thiophenol in the same manner as described in method a) -lépésében 1) to give ^N -tert-butoxycarbonyl-phenylalanine-thiophenyl ester we get.

In step 2) of this ester under a nitrogen atmosphere, reaction in ethanol as solvent, with sodium hydrogen sulfide, affords the corresponding thioacid of ^the N -t-butoxycarbonylbiphenyl-thiophenyl-alanine. The thioacid can also be prepared by ^the N -t-butoxycarbonyl-phenylalanine was reacted as described in method a) with hydrogen sulfide.

In step 3, the thioic acid is heated in toluene with methacrylic acid to give 3- (N '-tert-butoxycarbonylphenylalanylmercapto) -2-methylpropionic acid.

In step 4, the product of step 3 is resolved as described in step 5 of method a).

In step 5, the product of step 4 is deprotected in a mixture of trifluoroacetic acid and anisole. The resulting product is then reacted with benzoyl chloride

3- ^{(N-benzoyl-phenyl-alanyl-mercapto)} -2-D-methyl-propionic acid. Is prepared as described in Step [3- ^{(N-benzoyl-phenylalanyl-meikapto)} -2-D-methylpropanoyl] -L-proline A) Method 6) - N ^a.

In another preferred embodiment of this embodiment, the product of Step 4 may be coupled with L-proline tert-butyl ester, dicyclohexyl calbodiimide, or the mixed anhydride method. The resulting product is then deprotected with trifluoroacetic acid and anisole and reacted with benzoyl chloride to give the desired compound.

In a further preferred embodiment of this embodiment, the product of Step 4 is coupled using dicyclohexylcarbodiimide or the mixed anhydride method, N-hydroxysuccinimide. The product is then reacted with L-proline, deprotected and the free compound is reacted with benzoyl chloride as described above to give the desired product.

In a further preferred embodiment of this embodiment, the product of step 2) is deprotected with acetic acid in hydrochloric acid to give the hydrochloride salt of Phe-SH. This salt is then heated with methacrylic acid as described in step 3) and resolved as described in step 4). The product obtained is reacted with benzoyl chloride in the presence of sodium carbonate to give the desired compound.

In step 3, using acrylic acid instead of methacrylic acid, the corresponding 3-mercaptopropionic acid derivative is obtained. In step 1, any other desired compound of formula R-A-OH, as defined above, can be used instead of Boc-Phe. If the desired R or A group is to be incorporated, it is not necessary to remove the R protecting group according to step 5).

(e) method

In a fifth preferred embodiment of the process of the invention, the benzoyl group is cleaved from 3-benzoylmercapto-2-D-methylpropionic acid in a manner known per se.

In Step 2, the product of Step 1 is obtained by Method b)

The active ester was coupled with N "-Boc-Phe as described in step 3) to give 3-N" -Boc-phenylalanyl-mercapto-2-D-methyl-propionic acid.

The product obtained in Step 2) is then deprotected in a mixture of trifluoroacetic acid and anisole to give the trifluoroacetic acid salt of 3-phenylalanylmercapto-2-D-methylpropionic acid.

In Step 4, the product of Step 3 is reacted with benzoyl chloride in sodium bicarbonate solution.

^{3-N-benzoyl-phenyl} alanyl-mercapto-2-D-methyl-propionic acid.

In step 5) given in the Method B) 5) using ^N - [3- (N, prepared from ^the benzoyl-phenylalanyl-mercapto) -2-dimethylcarbamoyl propanoilj- L-proline.

In step 2, any other desired A-OH compound can be incorporated in place of phenylalanine. Similarly, the product of step 3 may be coupled with any other R-containing reagent in step 4 instead of benzoyl chloride.

(f) method

According to a seventh preferred embodiment of the invention, 3-mercapto-2-D-methylpropionic acid is a

In the same manner as in step 3 of method b), the active ester method is coupled with N®-Bpoc-Phe to give 3-N '-Bpocphenylalanyl mercapto-2-D-methylpropionic acid.

In step 2, the product of step 1 is coupled with LPro-tert-butyl ester in the presence of 1-hydroxybenzotriazole using dicyclohexylcarbodiimide or by the mixed anhydride method to yield N '- [3 (N ^a -Bpoc-phenyl) (alanyl mercapto) -2-D-methylpropanoyl] -L-proline tert-butyl ester is obtained.

Using diluted in step 3) of TFA cleaved from Bpoc protecting group, to give the ^N - [3-phenyl-alanyl-mercapto-2-D-methylpropanoyl] -L-proline tert.butyl ester, trifluoroacetic acid salt obtained.

In Step 4, the product of Step 3 is obtained by Method e)

Reaction with benzoyl chloride as described in Step 4) affords Ν ^α - [3- (Ν ^α -benzoylphenylalanyl mercapto) -2-D-methylpropanoyl] -L-proline tert-butyl ester.

In step 5) in a known manner is cleaved from the t-butyl ester group to give ^the N - [3- (N "-benzoyl-mercapto-phenyl alanyl) -2-D-methylpropanoyl] -L-proline.

In step 1, any other desired A-OH compound can be used in place of phenylalanine. Similarly, the product of step 3 may be coupled with any other reagent containing the desired R group in step 4 instead of benzoyl chloride.

HU 199786 Β

(g) method

According to a seventh preferred embodiment of the process of the invention, Boc-Phe is first reacted with pentachlorophenol by the mixed anhydride method to obtain N 'tert-butoxycarbonylphenylalanine pentachlorophenyl ester (Boc-Phe-OPcp). Alternatively, pentachlorophenol or N-hydroxysuccinimide may be used in the reaction in place of pentachlorophenol.

In step 2, the ester obtained above is reacted with acetic acid in hydrochloric acid or hydrobromic acid to give the hydrochloric or hydrobromic salt of the phenylalanine pentachlorophenyl ester.

In step (3), the salt prepared in step (2) is treated with benzoyl chloride in a mixture of aqueous sodium hydrogen carbonate and ethyl acetate to give Bz-Phe-pentachlorophenyl ester (Bz-PheOPcp).

In step 4, Bz-Phe-OPcp is coupled with 3-mercapto-2-methylpropionic acid to give 3- (N ^a -benzoylphenylalanyl-meacapto) -2-methylpropionic acid, the coupling alone by a known method.

In step 5, the above product is resolved with a resolving agent as defined above.

In step 6) of the resolved product is coupled with L-proline t-butyl ester, namely the application or the mixed anhydride method preferably dicyclohexylcarbodiimide to give ^the N - [3- (N "-benzoyl-phenylalanyl-mercapto) -2-D-methylpropanoyl] -L-proline tert-butyl ester is obtained.

This ester is finally treated with anisole and trifluoroacetic acid to give the desired product.

Using 3-mercaptopropionic acid or 2-mercaptopropionic acid instead of 3-mercapto-2-methylpropionic acid in Step 4 gives the corresponding 3-thiopropionic acid or 2-thiopropionic acid derivatives. In step 1, any other desired R-A-OH compound can be used in place of Boc-Phe. If a compound containing the desired R and A groups is prepared, then it is not necessary to perform steps 2) and 3).

(h) method

According to an eighth preferred embodiment of the process of the invention ^the N -benzoyl-phenylalanine (BzPhe) method, the mixed anhydride is reacted with hydrogen sulfide to give N "- benzoyl-thio-phenylalanine (BzPhe-SH) was prepared.

In Step 2, Bz-Phe-SH is reacted with methacrylic acid as described in Method 4, Step 4.

The product of step 2) is then resolved as described in step 5 of method a). In step 4, the resolved product is coupled with L-proline tert-butyl ester and then, in a mixture of trifluoroacetic acid and anisole, is deprotected as described in method a), step 6). According to a further preferred embodiment of this embodiment of the invention a

The product of Step 3 may be coupled using dicyclohexylcarbodiimide or the mixed anhydride method with N-hydroxysuccinimide and then reacted with L-proline.

In step 1, any other desired R-A-OH compound can be used instead of Bz-Phe. In step 2, using acrylic acid instead of methacrylic acid, the corresponding 3-mercaptopropionic acid derivative is obtained.

(i) method

According to a ninth preferred embodiment of the invention, the methacrylic acid is first coupled with N-hydroxysuccinimide and then reacted with L-proline to give 2-methylpropenoyl-L-proline.

In another preferred embodiment of this embodiment, the methacrylic acid chloride is reacted with L-proline to form 2-methylpropenoyl-L-proline.

In a further preferred embodiment of Step 1, the methacrylic acid is coupled to the L-proline tert-butyl ester by the mixed anhydride method, and the resulting 2-methylpropenoyl-L-proline tert-butyl ester is protected with trifluoro cleavage with acetic acid to give 2-methylpropenoyl-L-proline.

In Step 2, 2-methylpropenoyl-L-proline prepared by one of the above methods is heated in toluene N ^with benzoylthiophenylalanine (Bz-Phe-SH).

In another preferred embodiment, 2-methylpropenoyl-L-proline tert-butyl ester may be used in place of 2-methylpropenoyl-L-proline in step 2 above. The product obtained with this reagent is then treated with trifluoroacetic acid to give the desired compound.

Acrylic acid may be used instead of methacrylic acid, or acrylic acid chloride may be used instead of methacrylic acid chloride, so that in step 1)

A 3-mercaptopropionic acid derivative is obtained. The use of another desired R-A-SH compound in step 2 instead of Bz-Phe-SH results in the final product containing the appropriate R and A groups.

(j) method

According to another preferred embodiment of the process of the invention, phenylalanine is reacted with benzoyl chloride in sodium hydroxide solution to form N 'benzoylphenylalanine (Bz-Phe) [J. Biol. Chem. 138, 627629 (1941)].

In step 2, thioacetic acid is reacted with methacrylic acid to give 3-acetylmercapto-2-methylpropionic acid. This acid is then dissolved in a mixture of methanol and ammonia to give 3-mercapto-2-methylpropionic acid.

In Step 3, the Bz-Phe prepared in Step 1 is reacted with the excess N-hydroxysuccinimide in your mixed anhydride method. The product obtained is reacted with the 3-mercapto-2-methylpropionic acid prepared in Step 2 to give 3- (N '-benzoylphenylalanyl mercapto) -2-methylpropionic acid.

In Step 4, the product of Step 3 is resolved by methods known per se.

In step 5, the resolved product of step 4 is reacted with N-hydroxysuccinimide by the mixed anhydride method. The product is then reacted with L-proline to give the desired product. In another preferred embodiment of the present embodiment, the tert-butyl ester of this amino acid may be used in step 5 instead of L-proline. If this is the case, the product must be deprotected, for example with trifluoroacetic acid in the presence of anisole.

Using acrylic acid instead of methacrylic acid in step 2 of the present embodiment provides the corresponding 3-mercaptopropionic acid derivative. If, in step 3), 2-mercapto-propionic acid is used instead of 3-mercapto-2-methylpropionic acid, the corresponding

HU 199786 Β

A 2-mercaptopropionic acid derivative is obtained. The use of another desired R-AOH compound in step 3 instead of Bz-Phe results in products containing the appropriate R and A groups.

k) method

The invention according to a further preferred embodiment of ^the N-benzoyl-phenyl-alanine (Bz-Phe) módszenei the mixed anhydride is reacted with thiophenol to give N "- benzoyl-phenylalanine obtained thiophenyl ester.

In a further preferred embodiment, Boc-Phe can be used in step 1 instead of Bz-Phe. In this case, after step 1), the Boc protecting group must be cleaved and the product reacted with benzoyl chloride; to give the ^{N-benzoyl-phenylalanine-thiophenyl} ester.

In another preferred embodiment, N-hydroxysuccinimide may be used in the mixed anhydride reaction of step 1).

In step 2), the product of step 1) is obtained by method a)

As described in 3) above is reacted with sodium hydrogen sulfide to give ^the N-benzoyl-thiophenyl alanine.

In a further preferred embodiment, can be reacted with 1) the product of step in an alkaline medium with hydrogen sulfide to give to give ^the N-benzoyl-thiophenyl alanine. See Naturwissenschaften, 40, 242-243 (1953).

In step 3) of step 2) The product is reacted with an excess of methacrylic acid, to give 3- ^{(N-benzoyl-phenylalanyl-mercapto)} - to give 2-methyl-propionic acid,

In Step 4, the product of Step 3 is resolved in a manner known per se.

In step 5, the resolved product is first reacted with N-hydroxysuccinimide and then reacted with L-proline or its tert-butyl ester as described in step 5 of method f).

In step 3, acrylic acid can be used instead of methacrylic acid to give the 3-mercaptopropionic acid derivatives. The use of another desired R-A-OH compound in step 1 instead of Bz-Phe results in the final product containing the desired R and A groups.

l) method

The invention in a further preferred version of first Boc-Phe was coupled to thiophenol as described) method step 1) to yield ^the N -t-butoxycarbonyl phenylalanine thiophenyl.

The thiophenyl ester is then reacted in an alkaline medium with sodium hydrogen sulfide or hydrogen sulfide according to method b) step 2) to give BocPhe-SH.

In Step 3, the product of Step 2 is reacted with excess methacrylic acid to give 3- (N '-tert-butoxycarbonyl) phenylalanyl mercapto-2-methylpropionic acid according to Method 3, Step 3.

In step 4), the product of step 3) is resolved in a manner known per se, for example as described in step 5 of method a).

In step 5, the resolved product of step 4) is reacted with hydrochloric acid as a solvent in ethyl acetate. The product was reacted with benzoyl chloride in a biphasic mixture of ethyl acetate and water in the presence of sodium carbonate to give 3- (N ^a -benzoylphenylalanyl-mercapto) -2-methylpropionic acid.

In step 6, the product of step 5) is reacted with N-hydroxysuccinimide by the mixed anhydride method and then reacted with L-proline or L-proline tert-butyl ester as described in step j), step 5) to give the desired product. .

In step 3, using acrylic acid instead of methacrylic acid, the corresponding 3-mercaptopropionic acid derivative is obtained. The use of another desired R-A-OH compound in step 1 instead of Boc-Phe results in the final product containing the appropriate R and A groups; in this case, you do not need to perform step 5).

m) method

According to this preferred embodiment of the invention, phenylalanine is substituted with a benzoyl halide such as benzoyl chloride or benzoyl bromide or an active ester of benzoic acid such as N-succinimidyl, ρ-nitrophenyl, o-nitrophenyl, by reaction with benzotriazolyl, 5-norbomene-2,3-dicarboximidyl, pentachlorophenyl or pentafluorophenyl ester to form benzoylphenylalanine (Bz-Phe).

In step 2, Bz-Phe was obtained in step 1)

Coupled with 3-mercapto-2-methylpropionic acid to give 3- (N 'benzoyl-phenyl-alanyl-mercapto) -2-methyl-propionic acid.

In step 3, the product of step 2) is resolved in a manner known per se.

In Step 4, the resolved product is coupled with L-proline tert-butyl esters using dicyclohexylcarbodiimide or the mixed anhydride method to give Ν ^α- [3- (Ν ^α -benzoylphenylalanyl mercapto) -2-methylpropanoyl] L-proline tert-butyl ester is obtained. The ester is deprotected with trifluoroacetic acid and anisole to give the desired product.

In step 4, using 3-mercaptopropionic acid or 2-mercaptopropionic acid instead of 3-mercapto-2-methylpropionic acid, the corresponding 3-mercaptopropionic acid or 2-mercaptopropionic acid derivatives are obtained. In step 2, using another compound of formula R-A-OH instead of Bz-Phe, the final product containing the appropriate R and A groups is obtained.

n) method

In a further preferred embodiment of the process of the invention, Na,? -Dimethyl-3,5-dimethylbenzyloxycarbonylphenylalanine (Ddz-Phe) is coupled by the carbonyldiimidazole method with 3-mercapto-2-methylpropionic acid in the presence of triethanolamine. to give 3- (D-phenylalanyl-mercapto) -2-methyl-propionic acid. Other photosensitive (photosensitive) amine protecting groups such as 6-nitro-verryloxycarbonyl or 2-nitrobenzyloxycarbonyl may be used instead of the Ddz protecting group.

In step 2, the product of step 1) is resolved in a manner known per se.

Step 3) The resolved in step 2) The product was coupled using dicyclohexyl carbodiimide or the mixed anhydride method, L-proline t-butyl ester, to give ^the N - [3- (Na Ddz-phenylalanine-mercapto) -2-methylpropanoyl] -L-proline tert-butyl ester is obtained.

HU 199786 Β

In step 4) the product obtained in Step 3) was deprotected by irradiation, and thus ^the N - obtain [3-phenyl-alanyl-mercapto-2-methylpropanoyl] -L-proline tert.butyl ester.

In step 5), the product of step 4) is obtained by method m)

It is reacted with a reagent capable of introducing benzoyl reagent 1) was repeated as described to give ^the N - [3- ^(N -benzoyl phenylalanyl-mercapto) -2-methylpropanoyl] -L-proline t-butyl ester .

In step 6, the ester obtained in step 5) is deprotected with trifluoroacetic acid and anisole to give the desired final product. In step 1, 3-mercapto-propionic acid or 2-mercapto-propionic acid is used in place of 3-mer 'nto-2-methyl-propionic acid to give 3-mercapto-propionic acid or 2-mercapto-propionic acid derivatives. In step 1, any other desired AOH compound can be used in place of phenylalanine. Similarly, the product of step 4) may be coupled with another desired R-addition reagent in step 5 instead of the benzoyl-reactive reagents.

o) method

According to another preferred embodiment of the process of the invention, 2- (4-biphenylyl) -2-propoxycarbonyl-phenylalanine (Bpoc-Phe) by the carbonyldiimidazole method as described in step 1 of method n)

Coupled with 3-mercapto-2-methylpropionic acid. Instead of the Bpoc protecting group, 2-phenylisopropoxycarbonyl or any other acid-sensitive N-aralkoxycarbonyl protecting group can be used.

In Step 2, the 3- (N ^a -Bpocphenylalanyl mercapto) -2-methylpropionic acid obtained in Step 1 is resolved in a manner known per se.

In step 3, the product resolved in step 2) is coupled with L-prolyl tert-butyl ester as described in step 3 of method m) to give Ν ^α - [3- (Ν ^α Bpoc-phenylalanyl mercapto) -2 -methylpropanoyl] -L-proline tert-butyl ester.

Product of step 4) as described in step 3), the protecting group lahasítjuk triíluorecetsavval dichloromethane to give ^N - [3- (phenylalanyl-mercapto) -2-propanol) - L- proline tertiary butyl ester is obtained.

In step 5), the product of step 4) is obtained by Method N)

1) using a benzoyl reagent.

In the final step, the product of step 5) is deprotected in anisole with trifluoroacetic acid to give the desired product.

In step 1, using 3-mercaptopropionic acid or 2-mercaptopropionic acid instead of 3-mercapto-2-methylpropionic acid, the corresponding 3-mercaptopropionic acid or 2-mercaptopropionic acid derivatives are obtained. In step 1, using another desired A-OH compound instead of phenylalanine, the corresponding A-containing end product is obtained. Similarly, the product of Step 4 may be reacted with another reagent suitable for the introduction of the R group instead of the benzoyl addition reagents.

Step 5).

p) method

According to another preferred embodiment of the process of the invention, 3-acetylmercapto-2-methylpropionic acid is reacted with 2-methylpropene in the presence of sulfuric acid to give 3-acetylmercapto-2-methylpropionic acid tert-butyl ester. The acetyl group is then removed in an alkaline medium to give 3-mercapto-2-methylpropionic acid tert-butyl ester.

In Step 2, the product of Step 1 is reacted with N ^a benzoylphenylalanine (Bz-Phe). Any conventional method may be used for this coupling, except for the halides using methods FGY 3- ^{(N-benzoyl-phenylalanyl-mercapto)} -2-methyl-propionic acid tert.-butyl ester.

In Step 3, the product of Step 2 is deprotected with trifluoroacetic acid in anisole to afford

3- ^{(N-benzoyl-phenyl} alanyl-mercapto) -2-methyl-propionic acid.

In Step 4, the product of Step 3 is resolved in a manner known per se.

In step 5, the product resolved in step 4) is coupled with L-pro-tert-butyl ester and the resulting ester is deprotected with trifluoroacetic acid in anisole as described in method m, step 4.

In step 1, 3-acetylmercaptopropionic acid is substituted for 3-acetylmercapto-2-methylpropionic acid or

Using 2-acetyl mercapto-propionic acid is suitable

3-mercaptopropionic acid and 2-mercaptopropionic acid derivatives are obtained. The use of another desired R-A-OH compound in step 2 instead of Bz-Phe results in the final product containing the appropriate R and A groups.

(r) method

According to another preferred embodiment of the process of the invention, 3-acetylmercapto-2-methylpropionic acid is reacted with 2-methylpropene to give 3-acetylmercapto-2-methylpropionic acid tert-butyl ester. The acetyl group of this product is obtained by method p)

Removal as described in step 1) affords 3-mercapto-2-methylpropionic acid tert-butyl ester.

In step 2) the ester from step 1) coupling protected Bpoc, Ddz groups Ppoc or phenylalanine, ^to give ^the alanyl-phenyl-mercapto) -2-methylpropionic acid, tertiary butyl ester protected 3- (N .

In step 3, the ester obtained in step 2) is deprotected using, for example, a weak acid, to give 3-phenylalanyl-mercapto-2-methylpropionic acid tert-butyl ester. The Ddz protecting group may be removed by the irradiation method.

In step 4, the ester obtained in step 3 is benzoylated to give 3- (N '-benzoyl-phenyl-alanyl-mercapto) -2-methyl-propionic acid tert-butyl ester. Any of the reagents described in step 1 of Method n) can be used for benzoylation.

In step 5, the product of step 4 is deprotected with trifluoroacetic acid in anisole as described in step p), step 3, to give 3- (N ^a- benzoyl-phenyl-alanyl-mercapto) -2-methyl-propionic acid.

In step 6), the product of step 5) is resolved in a manner known per se.

In Step 7, the product of Step 6 is coupled with L-proline tert-butyl ester and the ester is deprotected with trifluoroacetic acid in anisole as described in Step 4 of Method m).

In the above steps of the present process, benzyl-101 may be substituted for the tertiary butyl ester group.

The ester group and the protecting group may also be cleaved in hydrobromic acid in acetic acid.

In step 1, using 3-acetylmercaptopropionic acid or 2acetyl mercaptopropionic acid instead of 3-acetylmercapto-2-methylpropionic acid, the corresponding

3-mercaptopropionic acid and 2-mercaptopropionic acid derivatives are obtained. In step 2, using any other desired A-OH compound instead of phenylalanine, the final product containing the desired A group is obtained.

Similarly, the product of step 3) may be coupled with another reagent suitable for the introduction of the desired R group in step 4 instead of the benzoyl addition reagent.

s) method

In a further preferred embodiment of the process of the invention, Ddz-Phe is coupled with p-nitrophenol by the dicyclohexylcarbodiimide method to give N 'Ddz-phenylalanine p-nitrophenyl ester. Bpoc, Ppoc or Bz may be used in place of the Dj protecting group. Alternatively, other active ester groups may be used instead of the p-nitrophenyl ester group, such as those described in step 1 of Method m). Subsequently, N- ^a- Ddz-phenylalanine p-nitrophenyl ester is reacted with sodium hydrogen sulfide to give D-thiophenylalanine.

In step 2), 1) the product of step is reacted with methacrylic acid 3- ^(N -Ddz-phenylalanyl-mercapto) -2-methyl-propionic acid.

In step 3, the acid obtained in step 2) is resolved in a manner known per se.

Step 4) The resolved product is coupled with L-proline tert.butyl ester as in step 3) as described n) the method in Step 3) to give ^the N - [3- (N "Ddz-phenyl-alanyl-mercapto) -2 -methylpropanoyl] -L-proline tert-butyl ester.

In step 5, the protecting group from the ester obtained in step 4 is irradiated by method n).

4) step as described, to give ^N - obtain [3-phenyl-alanilmerkapto-2-methylpropanoyl] -L-proline tert.butyl ester.

In step 6), the product of step 5) is obtained from one of the

benzoylated as described in m) method step 1) as reagent N) method step 5) to give ^the N - [3- ^{(N-benzoyl-phenyl} alanyl-mercapto) -2-methylpropanoyl] -L-proline, tertio butyl ester.

In step 7, the ester prepared in step 6 is deprotected with trifluoroacetic acid in anisole.

In step 2, using acrylic acid instead of methacrylic acid, the corresponding 3-mercaptopropionic acid derivative is obtained. The use of another desired A-OH compound in step 1 instead of phenylalanine yields the final product containing the corresponding A group. Similarly, the product of step 5) may be reacted with another R-addition reagent in step 6 instead of the benzoyl addition reagent.

(t) method

In another preferred embodiment of the process of the invention, methacrylic acid is coupled to the dicyclohexylcarbodiimide method with L-proline tert-butyl ester to give 2-methylpropenoyl-L-proline tert-butyl ester.

In step 2, Ddz-phenylalanine is coupled with p-nitrophenol by the dicyclohexylcarbodiimide method to give Ddz-phenylalanine p-nitrophenyl ester. This ester is then reacted with sodium bisulfide to give D-thiophenylalanine according to Method 1, Step 1).

In Step 3, the product obtained in Step 1 is a

2) reacting the product obtained in step to give N '- [3- ^(N -Ddz-phenylalanyl-mercapto) -2-methylpropanoyl] -L-proline tert.butyl ester.

Step 4) Step 3) The product of the deprotected as irradiation described n) method, step 4) to give ^N - [3-phenyl-alanyl-mercapto-2-methylpropanoyl] -L-proline tert butyl ester.

In Step 5, the product of Step 4 is reacted with one of the benzoylating reagents described in Step 1 of Method m) as described in Step 5 of Method n) to give Ν ^α- [3- (Ν ^α -benzoylphenylalanylmercapto) - 2-Methylpropanoyl] -L-proline tert-butyl ester is obtained.

In step 6, the ester prepared in step 5) is deprotected with trifluoroacetic acid in anisole.

In Step 7, the product of Step 6 is resolved in a manner known per se.

According to this process variant a further preferred embodiment of the step 3) may be used instead of ^the N Ddz-thiophenyl-alanine - benzoyl-thiophenyl-alanine, and we will not need to perform the steps 4) and 5).

In step 1, using acrylic acid instead of methacrylic acid, the corresponding propenoyl L-proline derivative is obtained. In step 2, using any other desired A-OH compound instead of phenylalanine, the final product containing the corresponding A group is obtained. Similarly, the product of Step 4 may be reacted with another reagent for the desired R group instead of the benzoyl addition reagent.

Step 5).

u) method

In a further preferred embodiment of the process according to the invention, 3-acetylmercapto-2-methylpropionic acid is preferably coupled to the L-proline tert-butyl ester by the dicyclohexylcarbidimide method, so that N ^a (3-acetylmercapto-2- methylpropanoyl) -L-proline tert-butyl ester

In Step 2, the product of Step 1 is dissolved in an alkaline solution, such as a mixture of methanol and ammonia, to give N '- (3-mercapto-2-methylpropanoyl) -L-proline tert-butyl ester.

In step 3, the product of step 2) is resolved, for example, using a metal chelate complex.

In step 4, the resolved product is coupled with N "benzoylphenylalanine (Bz-Phe) to give N ^a - [3- (N" benzoylphenylalanyl mercapto) -2-D-methylpropanoyl] L-proline tert-butyl ester is obtained.

In step 5, the product of step 4 is deprotected with trifluoroacetic acid in anisole as described in step 6 of method n).

In step 1, using 3-acetylmercaptopropionic acid or 2acetyl mercaptopropionic acid instead of 3-acetylmercapto-2-methylpropionic acid, the corresponding

3-mercaptopropionic acid or 2-mercaptopropionic acid derivatives are obtained. If, in step 4), another general R-A-OH image is desired instead of Bz-Phe

-111

HU 199786, the final product containing the appropriate R and A groups is obtained.

(v) method

In a further preferred embodiment of the process of the invention, the desired compounds are prepared by solid phase synthesis known per se. Enzymol., 32, 221 (1969)]. The solid phase may be an o-nitrobenzyl or chloromethylated resin.

In step 1, Boc-Pro is coupled to the resin. The proline is then deprotected with trifluoroacetic acid.

In step 2), proline bound to the resin obtained in step 1) 3- ^{(N-dicyclohexyl} carbodiimide method - coupled Benzoyl-phenylalanyl-mercapto) -2-D-methyl-propionic acid.

In step 3, the desired product is cleaved from the resin. If an o-nitrobenzyl resin is used, the cleavage is performed by irradiation. However, if a chloromethylated resin is used, the product is cleaved from the resin with hydrogen fluoride.

Step 2) 3- ^{(N-benzoyl-phenylalanyl-mercapto)} -2-D-methyl-propionic acid instead of another three-RAS-2-methyl- propionic acid, 2-or 3-propionic RAS RAS -propionic acid, the corresponding derivatives containing the desired R and A groups are obtained.

z) method

In a further preferred solid phase synthesis of the process according to the invention, Boc-Pro is coupled to a solid phase, such as an o-nitrobenzyl resin, and then deprotected as described in step 1) of method v) above.

In step 2, the resin-bound proline obtained in the same manner as in step 1 is coupled with 3-Cbo-mercapto-2-methylpropionic acid by the dicyclohexylcarbodiimide method. The protecting group is then deprotected in an alkaline solution such as methanol / ammonia.

In step 3, the product of step 2) is coupled with protected phenylalanine, the phenylalanine protecting group may be Boc, Bpoc, Ppoc, or any other acid-sensitive protecting group.

In Step 4, the product of Step 3 is deprotected with trifluoroacetic acid.

In step 5), the product of step 4) is obtained from one of the following

Method n) is coupled with the benzoyl addition reagent described in Step 3).

In step 6, N "- [3- (N" -benzoylphenylalanylmercapto) -2-methylpropanopyl] -L-proline is cleaved from the resin as described in step 3) of method v) above,

In step 7), the product obtained in the manner described in step 6 is resolved in a manner known per se to give the desired product.

If N 'benzoylphenylalanine is substituted for protected phenylalanine in step 3), steps 4) and 5) are not required.

In Step 2, using 3-acetylmercaptopropionic acid or 2acetyl mercaptopropionic acid instead of 3-acetylmercapto-2-methylpropionic acid

3-mercaptopropionic acid or 2-mercaptopropionic acid derivatives are obtained. The use of another desired A-OH compound 12 in step 3 instead of phenylalanine results in the final product containing the corresponding A group. Similarly, the product of step 4) may be coupled with another reagent suitable for the introduction of the desired R group in step 5 instead of the benzoyl addition reagent.

Method (aa)

According to another preferred embodiment of the process according to the invention, phenylalanine thiophenyl ester hydride is reacted with hydrogen sulfide in a slightly alkaline medium to give thiophenylalanine hydrochloride.

In step 2, the thiophenylalanine hydrochloride is reacted with methacrylic acid to give 3-phenylalanyl mercapto-2-methylpropionic acid hydrochloride.

In step 3, the product of step 2) is resolved in a manner known per se. The product resolved in step 4) from step 3) is one of methods m)

Reaction with a benzoyl-introducing reagent such as benzoic acid N-succinimidyl ester or benzoyl chloride as described in Step 1) to give 3- (N ^a -benzoyl-phenylalanyl-mercapto) -2-D-methylpropionic acid.

In step 5, the product of step 4) is coupled with L-proline tert-butyl ester and the protecting group is cleaved with trifluoroacetic acid in anisole as described in step 4 of method m).

If desired, the order of steps 3) and 4) may be reversed.

In step 2, using acrylic acid instead of methacrylic acid, the corresponding 3-mercaptopropionic acid derivative is obtained. The use of another desired A-OH compound in step 1 instead of phenylalanine yields the final product containing the corresponding A group. Similarly, the product of Step 3 may be reacted with another reagent suitable for the introduction of the desired R group instead of the benzoyl addition reagent.

(ab) method

In another preferred embodiment of the process of the invention, benzoylthiophenylalanine (Bz-Phe-SH)

Reaction with 3-bromo-2-methylpropionic acid gives 3- (N ^a -benzoylphenylalanyl mercapto) -2-methylpropionic acid. Alternatively, 3-chloro or 3-iodo-2-methylpropionic acid may be used instead of 3-bromo-2-methylpropionic acid.

In Step 2, the product of Step 1 is coupled with L-proline tert-butyl ester and the protecting group is cleaved with trifluoroacetic acid in anisole as described in Step 4 of Method m).

In step 1, replace 3-bromo-2-methylpropionic acid

Using 3-bromopropionic acid or 2-bromopropionic acid, the corresponding 3-mercaptopropionic acid or

Reference may be made to 2-mercaptopropionic acid derivatives. The use of another desired R-A-SH compound in step 1 instead of Bz-Phe-SH results in the final product containing the desired R and A groups.

method (ac)

According to another preferred embodiment of the process of the invention, 3-benzyloxycarbonyl-121 is reacted with benzyloxycarbonyl mercaptan (CboSH) with methacrylic acid.

HU 199786 Β mercapto-2-methylpropionic acid was prepared. Any other acid-sensitive amine protecting group may be used instead of the Cb protecting group.

In step 2, the product of step 1) is resolved in a manner known per se.

In step 3, the product resolved in step 2) is preferably coupled with Lproline tert-butyl ester using dicyclohexylcarbodiimide to give N '(3-benzyloxycarbonylmercapto-2-D-methylpropanoyl) L-proline. yields tert-butyl ester.

In step 4, the product of step 3 is deprotected with hydrogen bromide and trifluoroacetic acid in anisole to give 3-mecapto-2-D-methylpropanoyl-L-proline.

In step 5, the product of step 4) is preferably coupled by the active ester method to N-benzoylphenylalanine (Bz-Phe).

In step 1, using acrylic acid instead of methacrylic acid, the corresponding 3-mecapto-propionic acid derivative is obtained. In step 5, using any other desired compound of formula R-A-OH instead of Bz-Phe, the final product containing the appropriate R and A groups can be obtained.

method (ad)

According to another preferred embodiment of the process of the invention, thiophenylalanine hydrochloride is reacted with methacrylic acid to give 3-phenylalanyl mercapto-2-methylpropionic acid hydrochloride.

In step 2), 1) the product of step in the presence of triethanolamine is reacted with benzoyl chloride to give 3- ^{(N-benzoyl-phenyl-alanyl-mercapto)} -2-methyl-propionic acid.

The product of step 3) is resolved in a manner known per se.

In step 4, the product resolved in step 3 is coupled with L-proline tert-butyl ester and the product is deprotected with trifluoroacetic acid in anisole as described in step 4 of method m) to give the desired product.

In step 1, using acrylic acid instead of methacrylic acid, the corresponding 3-mercaptopropionic acid derivative is obtained. The use of another desired compound A-SH in step (1) instead of thiophenylalanine results in the final product containing the corresponding A group. Similarly, the product of step 1) may be reacted with another reagent suitable for the introduction of the desired R group in step 2 instead of the benzoyl addition reagent.

Method (ae)

In another preferred embodiment of the process of the invention, 3-acetylmercapto-2-methylpropionic acid is reacted with o-nitrobenzyl alcohol to give o-nitrobenzyl 3-acetylmercapto2-methylpropionic acid.

In step 2, the ester obtained in step 1) is resolved in a manner known per se.

In step 3, the product resolved in step 2) is deprotected by irradiation and thus

3-Acetylmercapto-2-D-methylpropionic acid is obtained.

In Step 4, the product of Step 3 is coupled with L-proline tert-butyl ester, preferably dicyclohexylcarbodiimide, to yield N '- (3-acetylmercapto-2-D-methylpropanoyl) -L-proline tertiary. butyl ester is obtained.

In step 5), 4) the product of step ammonia, methanol and anisole by treatment with an alkaline mixture of ^N - (3-mercapto-2-D-methyl propanoyl) -L-proline tert-butyl ester.

Step 5) is preferably the product of the active ester method, ^the coupling of N-benzoyl-phenylalanine (Bz-Phe) Step 6) to give N '- [3- ^{(N-benzoyl-phenylalanyl-mercaptopropyl)} -2- D-methylpropanoyl] -L-proline tert-butyl ester is obtained. Other protecting groups, such as the Boc or Cb protecting group, may be used to protect the amino group on the phenylalanine instead of the benzoyl group.

In step 7, the product of step 6 is deprotected with, for example, trifluoroacetic acid in anisole to give the desired final product.

In step 1, using 3-acetylmercaptopropionic acid or 2acetyl mercaptopropionic acid instead of 3-acetylmercapto-2-methylpropionic acid, the corresponding

3-mercaptopropionic acid or 2-mercaptopropionic acid derivatives are obtained.

In step 6, using another compound of formula R-A-OH instead of Bz-Phe, the final product containing the appropriate R and A groups can be obtained.

(af) method

In another preferred embodiment of the process of the invention, 3-amino-2-methylpropionSuvat is first resolved in a manner known per se, and thus

3-Amino-2-D-methylpropionic acid was prepared.

In step 2, the product of step 1 is reacted with sodium nitrite in the presence of hydrogen bromide to give 3-bromo-2-dimethylpropionic acid. In another preferred embodiment of this embodiment, the product of Step 1) can be converted to 3-chloro-2-D-methylpropionic acid with hydrochloric acid and nitric acid. In a further preferred embodiment, 3-hydroxy-2-methylpropionic acid can be reacted with phosphorus tribromide to give 3-bromo-2-methylpropionic acid.

In Step 3, the product of Step 2 is reacted with N 'benzoylthiophenylalanine (Bz-Phe-SH) to afford

3- ^(N -benzoyl phenylalanyl-mercapto) -2-D-methyl-propionic acid.

In step 4, the product of step 3 is coupled with L-proline tert-butyl ester and the product is deprotected as described in step 4 of method m), for example trifluoroacetic acid in anisole to give the desired final product.

In step 2, using 3-aminopropionic acid or 2-aminopropionic acid instead of 3-amino-2-D-methylpropionic acid, the corresponding 3-mercaptopropionic acid or 2-mercaptopropionic acid derivatives can be obtained. In step 3, using another compound of formula R-A-SH instead of Bz-Phe-SH, the final product containing the appropriate R and A groups can be obtained.

(ag) method

In another preferred embodiment of the process of the invention, 3-amino-2-methylpropionic acid is first resolved by method (f), in a manner known per se, to give 3-amino-2-D-methylpropionic acid.

In Step 2, the product of Step 1 is reacted with sodium nitrite in the presence of hydrogen bromide to give

-131

This gave 3-bromo-2-dimethylpropionic acid.

In Step 3, the product of Step 2 is reacted with thiobenzoic acid to give 3-benzoylmercapto-2-D-methylpropionic acid. In this reaction, thioacetic acid may be used instead of thiobenzoic acid.

In step 4, the product of step 3 is treated with an alkaline medium such as a mixture of methanol and ammonia to give 3-mercapto-2-D-methylpropionic acid.

In step 5) is reacted with ^{N-benzoyl-phenyl-alanine} (Bz-Phe), N-hydroxy szűkeinimiddel the mixed anhydride method and the product obtained by the above

as described in j) the method in Step 3) reacting the product obtained in this process variant 4), to provide 3- ^{(N-benzoyl-phenylalanyl-mercapto)} -2-D-methylpropionic acid.

In step 6, the product of step 5) is reacted with N-hydroxysuccinimide by the mixed anhydride method and the product obtained is reacted with L-proline according to step j) of method j) above to give the desired final product.

In step 2, using 3-aminopropionic acid or 2-aminopropionic acid instead of 3-amino-2-D-methylpropionic acid, the corresponding 3-mercaptopropionic acid or 2-mercaptopropionic acid derivatives are obtained. The use of another desired R-A-OH compound in step 5 instead of Bz-Phe results in the final product containing the desired R and A groups.

When it comes to the D-configuration of the methyl side chain of 3-mercapto-2-methylpropionic acid, it is equivalent to the configuration of the carbon atom bearing the methyl side chain of S.

The compounds of the invention form salts with various inorganic and organic bases; the preparation of salts is also within the scope of the invention. Examples of such salts are ammonium salts, alkali metal salts such as sodium and potassium salts (these are preferred), alkaline earth metal salts such as calcium and magnesium salts, and salts with organic bases such as dicyclohexyl. salts with -amine, benzathine, N-methyl-D-glucamine, hydrabamine, and salts with amino acids such as arginine, lysine and the like. Non-toxic pharmaceutically acceptable salts are preferred, although other salts may be used, for example, for the isolation and purification of products, as exemplified by the dicyclohexylamine salts.

Salts are prepared in a manner known per se by reacting the free acid form of the product with one or more equivalents of the appropriate base in a medium in which the salt is insoluble or insoluble, or by freezing water with water. removed by plowing. When a salt is reacted with an insoluble acid such as a cation exchange resin in the hydrogen form (e.g. a polystyrene sulfonic acid type resin such as Dowex 50) or an aqueous acid and then the mixture is treated with an organic solvent such as ethyl acetate, dichloromethane. extraction with methane or the like, the products may be obtained in the free acid form and, if desired, converted to another salt.

The invention is further illustrated by the following non-limiting examples. 14

For simplicity, the individual procedures of the N '- [3- ^{(N-benzoyl-phenyl} alanyl-mercapto) -2-D-methylpropanoyl] -L-proline are shown in production examples. In the examples, reagents are used in equivalent amounts. Yield values are between 1 and 42%.

Example 1

Method (a)

1st step

^N -t-butoxycarbonyl-L-phenylalanine methyl ester alanintiofenil

To a solution of 3.98 g of Boc-L-Phe in about 20 ml of double-distilled ethanol in an ice-dry ice-methanol cooling bath at -20 ° C is added 2.04 ml of N-ethylmorpholine, followed by 2.04 ml of chloroformate, and the mixture was stirred at -15 ° C for 3 minutes. 1.8 ml of thiophenol are then added and the reaction mixture is stirred at -15 ° C for 1.5 hours. The mixture was then allowed to slowly warm to room temperature and stirred at this temperature for a further 2 hours. Ethyl acetate (20 mL) was added and the mixture was cooled in an ice bath three times with cold water, three times with cold 1N citric acid, three times with cold saturated sodium chloride solution and then with cold 1N sodium hydrogen. wash three times with carbonate solution and finally with cold saturated sodium chloride solution three times. The ethyl acetate portion is then dried over anhydrous magnesium sulfate, the desiccant is filtered off and the solvent is distilled off on a rotary evaporator. The residue was crystallized from a mixture of benzene and petroleum ether to give 3.68 g of product as white needle crystals, m.p. 113.5-114.5 ° C. Infrared, NMR, elemental analysis, paper electrophoresis and TLC indicated the title compound.

Step 2

N ^a -Benzoyl-L-phenylalanine thiophenyl ester

1) To 2.0 ml of anisole is added 2 g of the product obtained in Step 1 and the solution is cooled in an ice-acetone cooling bath. To remove the N '-Boc protecting group, the mixture was stirred with 4 ml of anhydrous trifluoroacetic acid at room temperature for half an hour. The trifluoroacetic acid is then distilled off at 30 ° C on a rotary evaporator. Trifluoroacetic acid and anisole were distilled off at 30 [deg.] C. under high vacuum to give the product as a white crystalline solid.

2) To the above product is added 10 ml of 5.7 M hydrochloric acid in ethanol and the mixture is stirred for 10 minutes at room temperature. Anhydrous diethyl ether (15 mL) was added and the mixture was cooled in an ice bath for half an hour. The precipitated crystalline material was filtered off and washed several times with diethyl ether. The product was then dried overnight under reduced pressure with sodium hydroxide and phosphorus pentoxide to give 1.03 g of white crystals, m.p. 164.5165 ° C (dec.).

3) 1.12 g of the L-phenylalanine thiophenyl ester hydrochloride obtained above are suspended in 15 ml of ethyl acetate and 0.45 ml of benzoyl chloride is added under vigorous stirring at room temperature. thereafter

-141

A solution of 1.01 g of sodium carbonate in 10 ml of water is added dropwise and the mixture is stirred vigorously for another half hour. After cooling in an ice bath for a half hour, filtered to seven times with cold water, cold ethyl acetate followed .oss cr _w times, and finally with anhydrous ether twice, then reduced pronoun with sodium hydroxide and phosphorus pentoxide for several hours dried. this way, white, crystal!? ·! 32 g of product are obtained, m.p. 182-183 ° C. Infrared, NMR, elemental analysis, paper electrophoresis and thin layer chromatography indicated the title compound.

Step 3

^{N-Benzoyl-L-thio-phenylalanine}

1.01 g of sodium hydrogen sulfide in 25 ml of anhydrous

Nitrogen was bubbled at 35-40 ° C and 2.17 g of the product obtained in Step 2 was added over a quarter hour. At the end of the addition, the temperature of the reaction mixture drops to about 20 ° C. The mixture was then stirred vigorously at 20 ° C for 1.5 hours, and then the ethanol was distilled off at 32 ° C using a rotary evaporator. Water (30 mL) was added to the residue and the solution was acidified to pH 2 with 50% sulfuric acid in an ice bath. After leaving the mixture in an ice bath for half an hour, the precipitate was filtered off, washed several times with cold water and dried under reduced pressure overnight with sodium hydroxide and phosphorus pentoxide. The dried material was recrystallized from a mixture of benzene and hexane to give 1.45 g of product as white needles, m.p. 108.5-109.5 ° C. Infrared, NMR, elemental analysis, paper electrophoresis and TLC indicated the title compound.

Step 4

3- ^{(N-benzoyl-phenylalanyl-mercapto)} -2-methylpropionic acid

A mixture of 285 mg of the product obtained in Step 3 and 86.1 ml of methacrylic acid in 2 ml of toluene was refluxed until the reaction was complete by TLC and paper electrophoresis. The mixture was then allowed to stand overnight at room temperature and then the solvent was distilled off under high vacuum at 35 ° C using a rotary evaporator. The oily residue was crystallized from a mixture of benzene and n-hexane and then recrystallized to give 195 mg of product as a white crystalline solid, m.p. 120-122.5 ° C. Infrared and NMR spectra, elemental analysis, paper electrophoresis, and TLC showed the title compound.

Step 5

3- (N ^a -Ben: oil-L-phenyl-alanyl-mercapto) -2-D-methyl-propionic acid (resolution)

a) The product obtained in Step 4 is recrystallized several times from benzene to give the title compound. Recrystallization using the seed crystals obtained as described in steps (f) (l) to (3) of method (f) affords the title compound more rapidly and with better yields.

b) Kano * as described in Step 4; the product is dissolved in benzene, an equal volume of 1 L = _{10 L} of dicyclohexylamine is added and the mixture is stirred overnight. After standing overnight at 4 ° C, the precipitated crystals were filtered off, washed and dried under reduced pressure. This crystalline material was dissolved in a biphasic mixture of water and ethyl acetate, acidified to pH 2 with concentrated hydrochloric acid, cooled in an ice bath, and the ethyl acetate layer was washed with saturated sodium chloride solution and dried over magnesium sulfate. The drying set was filtered off and the solvent was distilled off from the filtrate on a rotary evaporator. The title compound is then crystallized from benzene.

Step 6

N ^a - [3- (N ^a -Benzoyl-L-phenyl-alanyl-mercapto) -2-D-methyl-propanes] -L-proline

a) 1) 2.5 mmol of the product obtained in step 5 are dissolved in 3 ml of dimethylformamide and 2.5 ml of dicyclohexylcarbodiimide in 1.5 ml of dimethylformamide are added in an ice-acetone cooling bath at 0 ° C. formamide solution. The mixture is stirred for 5 minutes at 0, then a solution of L-proline tert-butyl ester (2.5 mmol) in dimethylformamide (1.5 mL) is added. After stirring overnight at 4 ° C, ethyl acetate (10 mL) was added and the precipitate was filtered off and washed with ethyl acetate (20 mL). The combined ethyl acetate solutions were cooled and washed three times with saturated sodium chloride solution, once with cold 0.1N sodium bicarbonate solution, and finally three times with saturated sodium chloride solution, and dried over anhydrous magnesium sulfate. filtering the desiccant. The solvent was removed from the filtrate by rotary evaporation under reduced pressure to give a white solid.

2) The above residue is suspended in 4 ml of anisole, 8 ml of trifluoroacetic acid are added and the mixture is stirred at room temperature for 1.5 hours. Trifluoroacetic acid is then distilled off under high pressure under a rotary evaporator, the residue is dissolved in 1 ml of tetrahydrofuran and chromatographed. The fractions containing the desired product were combined, the solvent was distilled off, and the residue was dissolved in a little isopropanol and chromatographed again. From the fractions containing the desired product, the solvent is distilled off to give an oily product which is dissolved in tetrahydrofuran and the tetrahydrofuran is distilled under a stream of nitrogen. Traces of solvent are finally removed on a rotary evaporator under heavily reduced pressure.

b) To a solution of 2.5 mmol of the product obtained in Example 5 and 2.5 mmol of N-hydroxysuccinimide in dimethylformamide was added dropwise a cooled solution of 2.5 mmol of dicyclohexylcarbodiimide in dimethylformamide at 0 ° C. The reaction mixture was stirred for half an hour at 0 'C and then overnight at 4' C. The precipitated crystalline dicyclohexylurea was filtered off and extracted with ethyl acetate

-151

HU 199786 Β Wash. The filtrate was evaporated under reduced pressure and the residue was crystallized from benzene / hexane. The crystalline product is dissolved in cold tetrahydrofuran and a solution of 2.5 mmol of L-proline and 2.5 mmol of sodium bicarbonate in a mixture of terahydrofuran and water is added. The reaction mixture was stirred overnight at room temperature and then the tetrahydrofuran was distilled on a rotary evaporator at 35 ° C. Water was added to the residue, and the solution was adjusted to pH 9 by addition of solid sodium bicarbonate. The aqueous layer was then partitioned between ethyl acetate and cooled in an ice bath and acidified to pH 2 with 1N hydrochloric acid in the presence of ethyl acetate. The latter ethyl acetate layer was washed twice with cold water and twice with saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and the desiccant was filtered off. The filtrate was evaporated in a rotary evaporator and the residue was crystallized from diethyl ether / hexane. This affords the title compound.

Alternative synthesis of N ^a -Benzyl-L-thiophenylalanine

a) The Boc-L-phenylalanine thiophenyl ester is prepared as described in Step 1. The thio acid, Boc-L-thiophenylalanine, is prepared essentially as described in Step 3. Alternatively, this thioic acid may be prepared by reacting Boc-L-Phe with the mixed anhydride method, hydrogen sulfide, see J. Am. Chem. Soc. 74, 4726 (1952).

The thioic acid thus obtained is reacted with ethanolic hydrochloric acid and the product is reacted with benzoyl chloride or an active ester of benzoic acid,

As described in step 2.

b) ^{N-benzoyl-Phe} was prepared by the Phe was reacted with sodium hydroxide, chloride equivalent of [J. Bio. Chem. 138, 627 (1941)]. Then, the title compound was prepared according to ^the N-benzoyl-L-Phe can be reacted with the mixed anhydride coupling method, hydrogen sulfide substantially in the known method [J. Chem. Soc. 74, 4726 (1952)].

Alternative Synthesis of 3- (N ^a- Benzoyl-L-Phenyl Alanyl-Mercapto) -2-D-Methyl-Propionic Acid

Boc-L-thiophenylalanine is prepared as described in Methods b) 1) and 2). L-thiophenylalanine is prepared by mixing Boc-L-thiophenylalanine in acetic acid with hydrochloric acid at room temperature. Anhydrous diethyl ether was added and the mixture was cooled in an ice bath. The precipitate was filtered off and washed several times with diethyl ether and then dried under reduced pressure with sodium hydroxide and phosphorus pentoxide. The product thus obtained is reacted with methacrylic acid in the same manner as in Step 4 to give the hydrochloride salt of 3-L-phenylalanylmercapto-2-methylpropionic acid. This product can be carried forward in one of two ways: by first resolving it as described in step 5 and then reacting it with benzoyl chloride as described in step 2, point 3; or by first reacting with benzoyl chloride as described in step 2, step 3, and then resolving it.

Example 2

Method b

1st step

N is ^a thiophenyl ester ^of -Boc-L-Phenyl-atanyl

The title compound was prepared as described in Example 1, Step 1.

Step 2

N ^a -Boc-L-Thio-phenylalanine

The title compound is essentially the same as Example 1

Using the procedure described in Step 3, but starting with N, instead of ^the benzoyl-L-phenylalanine thiophenyl ester, the product is obtained as described in Step 1 of this method. The title compound may also be prepared by a known method, J. Am. Chem. Soc., 74, 4726 (1952).

Step 3

3- ^{(N-Boc-L-phenyl-alanyl-mercapto)} -2-methylpropionic acid

The title compound is essentially the same as Example 1

Step 4. Following the procedure and using N instead ^of benzoyl-L-phenylalanine thio prepared using the present method the product of Example 2, step.

Step 4

3- (N ^a -Boc-L-Phenylalanyl mercapto) -2-D-methylpropionic acid (Resolution)

Example 1 was used to obtain the title compound as described in Step 3 of this method

It is prepared by resolution of the resole as described in Step 5.

Step 5

N ^a - [3- (N ^a -Benzoyl-L-phenyl-alanyl-mercapto) -2-D-methyl-propanod] -L-proplin

a) The product obtained in the same manner as in Step 4, using trifluoroacetic acid in anisole, is essentially the

Treated as described in Example 1, Step 2, (1), the protecting group is cleaved. The product thus obtained is then reacted with benzoyl chloride as in Example 1, Step 2 (3) to give 3- (N 'benzoyl-L-phenylalanyl-mercapto) -2-D-methylpropionic acid. The product is then prepared according to the procedure described in Example 1, Step 6.

b) Using the procedure described in Example 1, Step 6 (a), starting from 3- (N ^a- benzoyl-L-phenyl-alanyl-mercapto) -2-D-methyl-propionic acid as starting material in Example 4 of this method. Using the product described in Step 1c, N '- (3-L-phenylalanyl-mercapto-2-D-methylpropanoyl) -L-proline is prepared.

This product is then reacted with benzoyl chloride essentially as described in Example 1, Step 2, Step 3 to give the title compound, the physical constants of which are listed in Table I (Compound 5).

Alternatively, the same procedure as described in point b) of Example 1, Step 6, and starting from 3- (N instead ^of benzoyl L-phenylalanyl-mercapto) -2D-methylpropionic acid of the present method, step 4 Using ^the product obtained as described, N ^a - [3- (N ^a -Boc-L-phenylalanyl mercapto) -2-D-methylpropanoyl] -2-proline is obtained. This product is deprotected as described in Example 1, Step 2 (1) and reacted with benzoyl chloride according to Example 1, Step 2 (3) to give the title compound.

-161

HU 199786 Β

Example 3

(c) method

1st step

To a solution of N ^a -Cbo-L-Phenylalanine pentachlorophenyl ester in mmole Cbo-L-Phe in about 60 ml of double-distilled ethyl acetate was added 15 mmoles of N-ethyl in an ice-dry ice-methanol cooling bath at -20 ° C. -morpholine followed by 15 mmol of chloroformic acid isobutyl ester followed by stirring for a few minutes at -15 ° C and then 15 mmol of pentachlorophenol. The reaction mixture is stirred for 1.5 hours at -15 ° C and then allowed to warm slowly to room temperature, and stirred at this temperature for a further 2 hours. Ethyl acetate (100 mL) was added, the mixture was cooled in an ice bath and the ethyl acetate layer was washed several times with cold water, cold saturated sodium chloride solution, cold 1N sodium bicarbonate, and finally cold saturated sodium chloride. solution. The ethyl acetate portion is then dried over anhydrous magnesium sulfate, the desiccant is filtered off and the solvent is distilled off on a rotary evaporator. The residue was crystallized from ethyl acetate to give the title compound, m.p.

153.5-154 ° C.

Step 2

L-phenylalanine-pentachlorophenyl ester hydrobromide

The product obtained in Step 1 was stirred with acetic acid hydrobromic acid at room temperature for 45 minutes. Anhydrous diethyl ether was added and the mixture was cooled in an ice bath. The precipitate was filtered off, washed several times with diethyl ether and dried overnight under reduced pressure with sodium hydroxide and phosphorus pentoxide. This gave the title compound, m.p. 178-180 ° C.

Step 3

N ^is -Benzoyl-L-phenylalanine pentachlorophenyl ester

By using the procedure described in Step 2 (3) of Example 1 and starting from the product obtained in Step 2 of this method instead of L-thiophenylalanine hydrochloride as starting material, m.p. 161-162 ° C. .

Step 4

3- (N ^a -Benzoyl-L-phenylalanyl-mercapto) -2-methyl-propionic acid was dissolved in 3 mmol of 3-mercapto-2-methyl-propionic acid in dioxane and 10 mmol was added to a neutralized solution of N-ethylmorpholine. A solution of the product obtained in Step 1b in double distilled dioxane was added and the reaction mixture was stirred at room temperature until complete by TLC. The solvent was distilled off under reduced pressure at 30 ° C and ethyl acetate was added to the residue. The mixture was cooled in an ice bath and the ethyl acetate layer was washed first with 0.1 N hydrochloric acid and then with saturated sodium chloride solution several times. It is then dried over anhydrous magnesium sulfate and the solvent is distilled off on a rotary evaporator to give the title compound.

Step 5

3- (N ^a -Benzoyl-L-phenyl-alanyl-mercapto) -2-D-methyl-propionic acid (Resolution)

The title compound was prepared as described in Example 1, Step 5.

Step 6

N ^a - [3- (N ^a -Benzoyl-L-phenyl-alanyl-mercapto) -2-D-methyl-propanod] -L-proline

The title compound was prepared according to Example 1, Step 6

(a).

Example 4

(d) method

1st step

N ^a -Benzoyl-DL-thiophenylalanine The title compound is prepared ^from equivalents of N ^a- benzoyl-D, L-Phe and hydrogen sulfide by the mixed anhydride method, see J. Am. Chem. Soc. 74, 4726 (1952)],

Step 2

3- ^{(N-Benzoyl-DL-phenylalanyl-mercapto)} -2-methylpropionic acid

Following the procedure of Example 1, Step 4, and using instead ^of N-benzoyl-L-alanine-thiophenyl compound is prepared the title compound using the present method the product of Example 1, step.

Step 3

3- (N ^a -Benzoyl-DL-phenyl-alanyl-mercapto) -2-D-methyl-propionic acid (resolution)

The title compound was prepared as described in Example 1, Step 5.

Step 4

N - (1S-N, N-benzoyl-DL-phenyl-alanyl-mercapto) - (N-D-methyl-propanoyl) -L-proline

By following the procedure described in Example 1, Step 6 and starting from the product of Step 3 of the present embodiment, the title compound is obtained.

Example 5

(e) method

1st step

2-methylpropanoyl-L-proline

1) To a solution of 5 mmol of methacrylic acid and 5 mmol of N-hydroxysuccinimide in dichloromethane was added dropwise a solution of 5 mmol of dicyclohexylcarbodiimide in dichloromethane at 0 ° C and the reaction mixture was heated at 0 ° C for half an hour and then overnight. Stir at 4 ° C. The crystalline dicyclohexylurea was filtered off and washed with ethyl acetate. The filtrate was evaporated under reduced pressure and the residue was crystallized from a mixture of benzene and hexane. The crystalline product is dissolved in cold tetrahydrofuran and a cooled solution of L-proline (5 mmol) and sodium bicarbonate (5 mmol) in tetrahydrofuran / water is added. After stirring overnight at room temperature, the tetrahydrofuran was distilled off at 35 ° C on a rotary evaporator. Water was added to the residue and the pH of the solution was solid sodium hydrogen 17

-171

HU 199786 Β carbonate is adjusted to pH 9. The mixture was partitioned between ethyl acetate and the aqueous was acidified to pH 2 with 1N hydrochloric acid in the presence of ethyl acetate, cooled in an ice bath. The latter portion of ethyl acetate was washed with cold water followed by saturated sodium chloride solution, dried over anhydrous magnesium sulfate, filtered and the solvent was distilled off on a rotary evaporator. The residue was crystallized from a mixture of diethyl ether and hexane to give the title compound.

2) To an aqueous solution of 2.5 mmol of L-proline and sodium bicarbonate in an ice bath is added 2.5 mmol of methacrylic acid chloride under vigorous stirring. After the addition, the mixture was stirred at room temperature for 2 hours and then partitioned between diethyl ether. The aqueous layer was then acidified with 1.0 N hydrochloric acid and extracted with ethyl acetate. The ethyl acetate solution was evaporated under reduced pressure to give the title compound.

3) To a solution of methacrylic acid (2.5 mmol) in double-distilled ethyl acetate in an ice-dry ice-methanol cooling bath at -20 ° C was added 2.5 mmol of N-ethylmorpholine followed by 2.5 mmol of chloroformic acid isobutyl ester. After stirring for a few minutes at -15 ° C, 2.5 mmol of L-proline tert-butyl ester was added and the reaction mixture was stirred for 1.5 hours at -15 ° C. Then allow to warm slowly to room temperature and stir at this temperature for a further 2 hours. Ethyl acetate was added and the mixture was cooled in an ice bath and the ethyl acetate portion was washed several times with cold water, then with cold 1N citric acid, then with cold saturated sodium chloride, and then cold 1N sodium bicarbonate. and finally with cold saturated sodium chloride solution. It is then dried over anhydrous magnesium sulfate, the desiccant is filtered off and the solvent is distilled off on a rotary evaporator. The residue is crystallized from a mixture of benzene and petroleum ether and the tert-butyl ester group is then essentially removed as described in Example 1, Step 6 (a).

Step 2

^N - [3- ^{(N-Benzoyl-L-phenylalanyl-mercapto)} -2-D-methyl- propanoyl] -L-proline

By following the procedure of Example 1, Step 4, starting from the product of Step 1 of the present process, instead of methacrylic acid, the 2-DL-methylpropanoyl derivative is prepared. This product was recrystallized from methanol / benzene / diethyl ether, or resolved by liquid chromatography to give the title compound. Alternatively, N '2-methylpropanoyl-L-proline tert-butyl ester may be used instead of the product of Step 1 as starting material. After resolution, the resolved product is deprotected as described in Step 1 to give the title compound.

Example 6 Method f)

1st step

N ^a -Benzoyl-L-phenylalanine

The title compound is prepared by reacting an equivalent amount of benzoyl chloride and L-phenyl18 alanine in a solution of sodium hydroxide in a known manner [J. Biol. Chem. 138, 627 (1941)].

Step 2

A mixture of 3-mercapto-2-methylpropionic acid (g) thioacetic acid and 40.7 g methacrylic acid was heated in a water bath for 1 hour and then allowed to stand at room temperature for 18 hours. After confirmation by NMR spectroscopy that the reaction was complete, the mixture was directly distilled under reduced pressure to give the desired 3-acetylmercapto-2-methylpropionic acid at 128.5-131 ° C. A portion (3.4 g) of this product was dissolved in a mixture of 10.5 ml of water and 6.4 ml of concentrated ammonium hydroxide solution, allowed to stand for 1 hour, diluted with water and the insolubles were filtered off. The filtrate was partitioned between ethyl acetate and the aqueous layer was acidified with concentrated hydrochloric acid, saturated with sodium chloride, and then extracted twice with ethyl acetate. The latter portions of ethyl acetate were washed with saturated sodium chloride solution and the solvent was distilled off to give the title compound.

Step 3

To a solution of 3- (N ^a -Benzoyl-L-phenylalanyl-mercapto) -2-methylpropanoic acid in mmol of N ^a benzoyl-L-Phe in double-distilled ethyl acetate was added ice-dry ice-methanol in a cooling bath at -20 ° C. N-ethylmorpholine, followed by isobutyl ester of chloroformate. After stirring for a few minutes at -15 ° C, 50 mmol of N-hydroxysuccinimide was added and the reaction mixture was stirred at -15 ° C for 1.5 hours. The mixture was allowed to slowly warm to room temperature, and then a solution of 3 mmol of 3-mercapto-2-methylpropionic acid neutralized with N-ethylmorpholine was added, and the reaction mixture was stirred until complete by TLC. Ethyl acetate was added and the mixture was acidified to pH 3 with 1N hydrochloric acid, cooled in an ice bath. The ethyl acetate layer was washed several times with cold water and then with saturated sodium chloride solution, dried over anhydrous magnesium sulfate, filtered and the solvent was distilled off on a rotary evaporator. The residue is crystallized from a mixture of benzene and petroleum ether to give the title compound.

Step 4

N ^a - [3- (N ^a -Benzoyl-L-phenyl-alanyl-mercapto) -2-D-methyl-propanoyl] -L-proline (resolution)

The title compound was prepared as described in Example 1, Step 5.

Step 5

N ^a -13- (N ^a -Benzoyl-L-phenyl-alanyl-mercapto) -2-D-methyl-propanoyl] -L-proline, a solution of the product obtained in Step 4 in double-distilled ethyl acetate was treated with ice-dry ice. - methanol in a cooling bath at -20 ° C, add 10 mmol N-ethylmorpholine followed by 10 mmol chloroformic acid isobutyl ester, and after stirring for a few minutes at -15 ° C add 10 mmol N-hydroxysuccinimide and

-181

The reaction mixture was stirred at -15 ° C for 1.5 hours. It is then allowed to slowly warm to room temperature and stirred at this temperature for a further 2 hours. Ethyl acetate was added and the mixture was cooled in an ice bath. The organic layer was washed several times with cold water, followed by cold 1N sodium bicarbonate solution and finally with cold saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and the desiccant was filtered off. After distilling off the solvent in a rotary evaporator and the residue was crystallized from benzene-petroleum ether gave 3- (N to give ^the benzoyl-L-phenylalanine alanilmerkapto-2-D-methyl-propionic acid N-hydroxysuccinimide ester. This The product is dissolved in tetrahydrofuran and a solution of L-proline in sodium hydrogen carbonate (1: 1, 10 mmol) in tetrahydrofuran and water is added, and the mixture is stirred at room temperature overnight, and ethyl acetate is added thereto. The ethyl acetate portion was cooled with cold water, followed by a cold 1N citric acid solution, followed by a cold saturated sodium chloride solution, followed by a cold 1N sodium bicarbonate solution, and finally a cold, saturated sodium chloride solution. solution, then dried over anhydrous magnesium sulfate, the desiccant is filtered off and the solvent is distilled off on a rotary evaporator and the residue was crystallized from a mixture of diethyl ether and hexane to give the title compound.

Example 7

1st step

N ^a -Benzoyl-L-phenylalanine thiophenyl ester

By following the procedure described in Example 1, Step 1, using benzoyl-L-phenylalanine instead of Boc-L-Phe, the title compound is obtained.

Step 2

^{N-Benzoyl-L-alanine-thiophenyl}

The title compound was prepared as described in Example 1, Step 3, starting from the product of Step 1 of the present embodiment.

Step 3

3- ^{(N-Benzoyl-L-phenylalanyl-mercapto)} -2-methylpropionic acid

The title compound is prepared according to the procedure described in Example 1, Step 4 and starting from the product obtained in Step 2 of the present embodiment.

Step 4

3- (N ^a -Benzoyl-L-fendanyl-mercapto) -2-D-methyl-propionic acid (Resolution)

The title compound was prepared as described in Example 1, Step 5.

Step 5

N ^a - [3- (N ^a -Benzoyl-L-fend-aldan-mercapto) -2-D-methylpropanoyl] -L-proline

The title compound was prepared starting from the product of Step 4 above and following the procedure of Example 11, Step 5.

Example 8 Method g)

1st step

N is ^a thiophendate ^of -Boc-L-Phenylalanine

The title compound was prepared as described in Example 1, Step 1.

Step 2

N ^a -Boc-L-Thio-phenylalanine

The title compound was prepared as described in Example 1, Step 3.

Step 3

3- ^{(N-Boc-L-phenylalanyl-mercapto)} -2-methylpropionic acid

The title compound was prepared as described in Example 4, Step 3.

Step 4

3- ^{(N-Boc-L-phenylalanyl-mercapto)} -2-D-methyl-propionic acid

The title compound was prepared as described in Example 1, Step 5.

Step 5

3- ^{(N-phenyl-benzo-Aland-mercapto)} -2-D-methyl-propionic acid

By following the procedure of Example 1, Step 2 and using the product of Step 4 of this embodiment, instead of the N '-Boc-L-phenylalanine thioester, the title compound is obtained.

Step 6

N ^a - [3- (N ^a -Benzoyl-L-phenyl-alanyl-mercapto) -2-D-methyl-propanod] -L-proline

The title compound is all. Example 5 was prepared as described in Step 5.

Example 9

(h) method

1st step

^{N-Benzoyl-L-alanine-fend}

The title compound is all. Example 1b.

Step 2

3- ^{(N-phenyl-benzofuran-L-alanyl-mercapto)} -2-propionic acid of methanesulfonyl

The title compound is all. Example 3 was prepared as described in Step 3. This method yields a slightly racemized product.

Step 3

3- (N ^a -Benzod-L-phenyl-alanyl-mercapto) -2-D-meth-propionic acid (Resolution)

The title compound was prepared as described in Example 1, Step 5.

Step 4

N ^a - [3- (N ^a -Benzod-L-fendaland mercapto) -2-D-meth-propanod] -L-proline

The title compound was prepared according to Example 1, Step 6

(a).

Example 10

(i) method

1st step

-191

HU 199786 Β

3- ^{(N-Phenyl-L--Ddz} Aland-mercapto) -2-propionic acid of methanesulfonyl

Ddz-L-Phe is prepared in a known manner [Liebigs Ann. Chem. 763, 162 (1972), or Hirsch, C., Peptide, 1972. Proc. of the 12th Eur. Pept. Symp., Hanson,

H. and Jakobke, H.D., Eds., North-Holland Publishers,

72, 1973]. To a solution of Ddz-L-Phe (10 mmol) in twice-distilled dioxane in a ice-dry ice-acetone cooled bath at -20 'C was added a solution of Ι, carbonyldiimidazole in dioxane and the mixture was stirred for 2 hours at -10' C. The above solution was then added to a cooled solution of 10 mmol of 3-mercapto-2-methylpropionic acid in dioxane and neutralized with N-ethylmorpholine. The mixture was stirred at -10 ° C until TLC showed complete reaction. The mixture was allowed to slowly warm to room temperature, the solvent was distilled off under reduced pressure at 30 ° C and the residue was dissolved in ethyl acetate. The solution was cooled in an ice bath, washed with 0.1 N hydrochloric acid, then brine, dried over anhydrous magnesium sulfate, and the solvent was distilled off on a rotary evaporator. This affords the title compound.

Step 2

3- ^{(N-Phenyl-L--Ddz} Aland-mercapto) -2-D of methanesulfonyl acid (resolution)

The title compound was prepared as described in Example 1, Step 5.

Step 3

^N - [3- ^(N-L---Ddz fend-Aland-mercapto) -2-D-methyl propane] -L-proline tert.butyl ester

The title compound is essentially the same as Example 1

As described in Step 6) of paragraph 1), and 3- (N instead of ^this benzoyl-L-phenylalanyl-mercapto) -2-dimethylcarbamoyl-propionic acid was prepared using the present process step 2 of the product.

Step 4

^N - [3- (L-alaninyl-mercapto-phenyl) -2-propanone of methanesulfonyl D] -L-proline tert.butyl ester

The product obtained as described in Step 3 above is dissolved in tetrahydrofuran and the solution is cooled to 20 ° C. The solution was filled into a vial and irradiated with 350 nm light for 5 hours. Subsequently, the tetrahydrofuran was distilled on a rotary evaporator to give the title compound.

Step 5

N ^a - [3- (N ^a -Benzoyl-L-phenyl-alanyl-mercapto) -2-D-methyl-propanod] -L-proline tert-butyl ester

The title compound is prepared essentially as described in Example 1, Step 2, Step 3, starting from the product of Step 4 of the present live version.

Step 6

N ^a - [3- (N ^a -Benzoyl-L-phenyl-lower mercapto) -2-D-methylpropanod] -L-protin

The title compound is prepared by cleaving the tert-butyl ester group from the product of Step 5 of this embodiment as described in Example 1, Step 6 (a) (2).

Example 11

(j) method

1st step

3- ^(N-L--Bpoc Fend-alanyl-mercapto) -2-methylpropionic acid

N -Bpoc ^the L-Phe can be prepared by a known method [Helv. Chim. Acta, 57, 614 (1968)]. Prepared according to the same procedure as described in Example 15, Step 1, and instead of L-Phe ^Ddz-N - using Bpoc-L-Phe was prepared.

Step 2

3- (N ^a -Bpoc-L-Fend-Aland mercapto) -2-D-methylpropionic acid (Resolution)

The title compound is essentially the same as Example 1

Prepared as described in Step 5.

Step 3

^N - [3- ^(N-L--Bpoc Fend-alanyl-mercapto) -2-D-methyl propane] -L-proline tert.butyl ester

Example 1 Step 6 (a) (1) Using the product of Step 2 of this embodiment instead of 3- (N ^a benzoyl-L-phenylalanylmercapto) -2-D-methylpropionic acid to give the title compound.

Step 4

^N - [3- (L-phenylalanyl-mercapto) -2-D of methanesulfonyl propanoyl] -L-proline tert.butyl ester budl

The product of Step 3 is dissolved in dichloromethane containing 0.5% trifluoroacetic acid and the solution is stirred for 10 minutes. Trifluoroacetic acid and dichloromethane were then distilled off on a rotary evaporator to give the title compound.

Step 5

N ^a - [3- (N ^a -Benzoyl-L-phenylmandan-mercapto) -2-D-methylpropanod] -L-proline tert-butyl ester

The title compound was obtained in Example 1, Step 2

2) and starting from the product of Step 4 of the present process variant.

Step 6

N ^a - [3- (N ^a -Benzoyl-L-phenyl-lower mercapto) -2-D-meth-propanod] -L-proline

The title compound is prepared by cleaving the tert-butyl ester group from the product of Step 5 as described in Example 1, Step 6 (a) (2).

Example 72

k) method

1st step

3-Mercapto-2-of methanesulfonyl-propionic acid tert.-butenedioic acid ester

To a solution of 2-methylpropene (0.56 g) in 1N sulfuric acid was added 0.62 g at -10 ° C.

3-acetylmercapto-2-methylpropionic acid, the mixture was stirred for half an hour and then allowed to warm to room temperature. This gives 3-acetylmercapto-2-methylpropionic acid tert-butyl ester. This product is dissolved in a mixture of ammonia and water, allowed to stand for 1 hour, diluted with water and

-201

HU 199786 Β insoluble matter is filtered off. The filtrate was partitioned between ethyl acetate and the aqueous portion was acidified with concentrated hydrochloric acid, saturated with sodium chloride and extracted twice with ethyl acetate. The latter ethyl acetate portions were washed with a saturated sodium chloride solution and the solvent was evaporated to give the title compound.

Step 2

3- (N ^a -Benzoyl-L-phenyl-alanyl-mercapto) -2-methyl-propionic acid tert-butyl ester

In the same manner as in Example 11, Step 3, and

Instead of 3-mercapto-2-methylpropionic acid, the title compound is obtained using the product of Step 1 of this embodiment.

Step 3

3- ^{(N-Benzoyl-L-phenylalanyl-mercapto)} -2-methylpropionic acid

The title compound is prepared by cleaving the tert-butyl ester group from the product of Step 2 as described in Example 1, Step 6 (a) (2).

Step 4

3- (N ^a -Benzoyl-L-phenyl-alanyl-mercapto) -2-D-methyl-propionic acid (Resolution)

The title compound was prepared as described in Example 1, Step 5.

Step 5

^N - [3- (Benzoyl-L-phenylalanyl-mercapto) -2-D-methyl- propanoyl] -L-proline

The title compound was prepared according to Example 1, Step 6

(a).

Example 13 Method l)

1st step

3-mercapto-2-methylpropionic acid tert-butyl ester

The title compound was prepared as described in Example 17, Step 1.

Step 2

3- (N ^a -Ppoc-L-Phenyl-alanyl-mercapio) -2-methyl-propionic acid tert-butyl ester

By using the procedure described in Example 15, Step 1, the title compound is replaced ^by Np-Pdoc-L-Phe (provided by Int. J. Peptide Protein Slit). 6, 111 (1974)] and is prepared using the product of Step 1 of the present process in place of 3-mercapto-2-methylpropionic acid.

Step 3

3-L-phenylalanyl-mercapto-2-methylpropionic acid tert-butyl ester

The product of Step 2 is dissolved in dichloromethane containing 0.5% trifluoroacetic acid and the solution is stirred for 10 minutes. The trifluoroacetic acid and dichloromethane were then distilled off on a rotary evaporator to give the title compound.

Step 4

3- (N ° -Benzoyl-L-phenyl-alanyl-mercapto) -2-methyl-propionic acid tert-butyl ester

The title compound is prepared by reacting the product of Step 3 with an equivalent amount of benzoyl chloride in a biphasic aqueous solution of ethyl acetate and sodium bicarbonate.

Step 5

3- ^{(N-Benzoyl-L-phenylalanyl-mercapto)} -2-methylpropionic acid

The title compound is prepared by cleaving the tert-butyl ester group from the product of Step 4 essentially as described in Example 1, Step 6 (a), (2).

Step 6

3- (N ^a -Benzoyl-L-phenyl-alanyl-mercapto) -2-D-methyl-propionic acid (Resolution)

The title compound was prepared as described in Example 1, Step 5.

Step 7

^N - [3- ^{(N-Benzoyl-L-phenylalanyl-mercapto)} -2-D-methyl- propanoyl] -L-proline

The title compound was prepared according to Example 1, Step 6

(a).

Example 14 Method m)

1st step

M solution of ^the -Ddz-L-thio-phenylalanine in mmoles of N "-Ddz-L-Phe dimethylformamide ice-acetone cooling bath at 0 ° C was added 5 mmoles of dicyclohexylcarbodiimide in dimethylformamide was added, the mixture was some After stirring for 1 min at 0 ° C, a solution of 5 mmol p-nitrophenol in dimethylformamide was added. The reaction mixture was stirred overnight at 4 ° C and the solvent was distilled off on a rotary evaporator. Ethyl acetate was added to the residue, the solution was filtered and the insolubles were washed with ethyl acetate. The filtrate was cooled, washed three times with cold 1N sodium bicarbonate solution and three times with saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and the desiccant was filtered off. The solvent was distilled off under reduced pressure on a rotary evaporator. Subsequently, essentially the same procedure as in Example 1, Step 3, and substituting the product obtained above for the benzoyl-L-phenylalanine thiophenyl ester, the title compound was obtained.

Step 2

3- ^(N -Ddz-L-phenylalanyl-mercapto) -2-methylpropionic acid

Using essentially the same procedure as described in Example 1, Step 4, and N ⁱⁿ place ^of benzoyl-L-alanine-thiophenyl compound is prepared the title compound using the present process the Step 1 product.

Step 3

3- ^(N -Ddz-L-phenylalanyl-mercapto) -2-D-methyl-propionic acid

The title compound was prepared as described in Example 1, Step 5.

-211

HU 199786 Β

Step 4

^N - [3- ^{(N-Phenyl-L--Ddz} Aland-mercapto) -2-D-methyl propane] -L-proline tert.butyl ester-butenedioic

The title compound is essentially the same as Example 1

Following the procedure of step 6) of paragraph 1), and 3- ^(N - instead of benzoyl-L-phenylalanyl-mercapto) -2D-methyl-propionic acid was prepared using the present process, Step 3 product.

Step 5

^N - [3- (L-alaninyl-mercapto-phenyl) -2-D-methylpropanal] -L-proline tert.butyl ester-butenedioic

The title compound was prepared as described in Example 15, Step 4.

Step 6

N ^a - [3- (N ^a -Benzoyl-L-fendanand-mercapto) -2 D-methylpropanod] -L-proline tert-butyl ester

The title compound is prepared by reacting the product of Step 5 with an equivalent amount of benzoyl chloride in a biphasic solution of ethyl acetate and sodium bicarbonate.

Step 7

N ^a - [3- (N ^a -Benzod-L-phenylalanyl-mercapto) -2-D-meth-propanod] -L-proline

The title compound is prepared by cleaving the tert-butyl ester group from the product of Step 6 as described in Example 1, Step 6 (a) (2).

Example 75

n) method

1st step

To a solution of 2-methylpropenoyl-L-proline tert-butyl ester in dimethylformamide (2 mmol) was added dropwise a solution of dicyclohexylcarbodiimide (2 mmol) in dimethylformamide at 0 ° C. and the reaction mixture was stirred for half an hour at the same temperature and then overnight at 4 ° C. The crystalline dicyclohexylurea was then filtered off and washed with ethyl acetate. The filtrate was evaporated under reduced pressure and the residue was crystallized from benzene / hexane to give the title compound.

Step 2

^{N-Thio--Ddz-L-alanine} fend

The title compound was prepared as described in Example 19, Step 1.

Step 3

N ^a - [3- (N ^a -Ddz-L-Phenylalanyl mercapto) -2-methylpropanod] -L-proline tert-butyl ester

By proceeding as described in Example 1, Step 4, instead of benzoyl-L-thiophenylalanine, the present process variant

Using the product of Step 2, the title compound is obtained.

Step 4

^N - [3- (L-alanyl-Fend-mercapto) -2-methylpropanoyl] -L-proline tert.butyl ester-butenedioic

The title compound was prepared as described in Example 19, Step 4.

Step 5

N ^a - [3- (N ^a -Benzoyl-L-phenyl-alanyl-mercapto) -2-methyl-propanod] -L-proline tert-butyl ester

The title compound was prepared as described in Example 19, Step 6.

Step 6

N ^a - [3- (N ^a Benzoyl-L-phenylmandan-mercapto) -2-methylpropanod] -L-proline

The title compound is prepared by cleaving the tert-butyl ester group from the product of Step 5 as described in Example 1, Step 6 (a) (2).

Step 7

N ^a - [3- (N ^a -Benzod-L-phenylaland mercapto) -2-D-methylpropanod] -L-proline (resolution)

The title compound was prepared as described in Example 10, Step 2.

Example 76

o) method

1st step

3-mercapto-2-acetate of methanesulfonyl propanoyl-L-proline t-butyl ester

The title compound was prepared as described in Example 20, Step 1, starting from 3-acetylmercapto-2-methylpropionic acid instead of methacrylic acid.

Step 2

3-Mercapto-2-methylpropanoyl-L-proline t-butyl ester

The product of Step 1 was dissolved in a mixture of methanol and concentrated ammonium hydroxide solution, allowed to stand for 1 hour, diluted with water, and the insolubles were filtered off. The filtrate is partitioned between ethyl acetate and the aqueous portion is acidified with concentrated hydrochloric acid, then saturated with sodium chloride and extracted twice with ethyl acetate. The latter ethyl acetate portions are washed with .

Step 3

3-Mercapto-2-D-methylpropanoyl-L-proline tert-butyl ester (resolution)

The product of Step 2 is resolved by liquid chromatography or using a metal chelate complex to give the title compound.

Step 4

N ^a - [3- (N ^a -Benzoyl-L-phenylmandan-mercapto) -2-D-methylpropanod} -L-proline tert-butyl ester

The title compound is substantially all. example

Step 3 was prepared using the product of Step 3 of the present process in place of 3-mercapto-2-methylpropionic acid.

Step 5

N ^a - [3- (N ^a -Benzoyl-L-phenyl-alanyl-mercapto) -2-D-methyl-propanod] -L-proline

-221

HU 199786 Β

The title compound is prepared by cleaving the tert-butyl ester group from the product of Step 4 as described in Example 1, Step 6 (a) (2).

Example 17 Method p)

In this variant of the process, a method known per se for solid phase peptide synthesis is used. See Advan for details on this method. Enzymol. 32, 221 (1969).

1st step

Coupling of L-Proline to the Solid Phase Resin

Following a conventional procedure, a solid phase resin containing 3-nitro-4-bromomethylbenzylamide groups in the presence of diisopropylethylamine is added to a solution of N '-Boc-L-Pro in ethyl acetate. The unreacted N '-Boc-L-Pro is then washed with ethyl acetate, methanol and then dichloromethane. A 25% solution of trifluoroacetic acid in dichloromethane containing 1 mg of indole per ml is then added to the resin-bound protected amino acid to remove the Boc protecting group. The resin is then washed with dichloromethane.

Step 2

Coupling of 3- (N ^a -Benzoyl-L-phenyl-alanyl-mercapto) -2-D-methyl-propionic acid to the product of Step 1

To the title compound and a 1: 1 mixture of dicyclohexylcarbodiimide in dimethylformamide / dichloromethane are added the resin obtained in Step 1. The title compound is coupled with proline in this step to yield N '- [3- (N ^is benzoyl-L-phenylalanyl-mercapto) -2-D-methylpropanoyl] -L bonded to the o-nitrobenzyl groups of the resin. -proline. Unreacted reagents were washed with ethanol followed by dichloromethane and the product dried under a stream of nitrogen.

Step 3

Cleavage of N ^a - [3- (N ^a -Benzoyl-L-phenylalanyl-mercapto) -2-D-methylpropanod] -L-proline from the resin

The product of Step 2 was irradiated with 350 nm light in methanol, see J. Am. Chem. Soc. 97, 1575 (1975). The title compound was dissolved from the resin with ethyl acetate. The solution was then distilled off from the solution on a rotary evaporator to give the title compound.

Example 18

(r) method

1st step

Coupling of L-proline to the solid phase resin

L-proline was coupled to the resin as described in Example 17, Step 1.

Step 2

Coupling of 3-Mercapto-2-methylpropionic acid with the product of Step 1

The title compound was coupled to the resin-bound L-proline by following the procedure described in Example 17, Step 2 and replacing 3- (N ^a- benzoyl-L-phenyl-alanyl-mercapto) -2-D-methyl-propionic acid. 3Cbo-2-methylpropionic acid was used. A solution of trifluoroacetic acid is then added to the resin to cleave the Cbo group to give the desired product. The resin was then washed with ethanol and then dichloromethane.

Step 3

Coupling of L-Feryl alanine to the product of Step 2

To a solution of N "-Boc-L-Phe and dicyclohexylcarbodiimide in a 1: 1 mixture of dimethylformamide and dichloromethane is added the product of Step 2, so that N" -Boc-L-Phe is added to the mercapto group of the resin bound compound. switch. The unreacted reagents were then washed with ethanol and dichloromethane. Then, in the resin with 25% dichloromethane trifluoroacetic acid solution was added, containing 1 mg of indole per ml A to yield Cleavage of the Boc protecting group, ^{N is} bound to the resin - (3-L-phenylalanyl (mercapto) -2-methylpropanoyl-L-proline is obtained. The excess trifluoroacetic acid was washed with dichloromethane.

Step 4

Benzoylation of the product of Step 3

To the product of Step 3 is added a solution of benzoyl chloride and an equivalent amount of triethanolamine in dimethylformamide. Thus, the benzoyl group is linked to the amino group of phenylalanine. The excess reagent was flushed with ethanol and dichloromethane, and thus the resin-bound ^N - [3- ^{(N-benzoyl-alanyl} Lfenil- mercaptomethyl) -2-methylpropanoyl] -L-proline.

Step 5

The ^N - [3- ^{(N-benzoyl-L-alanyl-fend} mercaptomethyl) -2-methylpropanoyl] -L-proline cleavage from the resin

The title compound was cleaved from the resin as described in Example 17, Step 3.

Step 6

N ^a - [3- (Benzoyl-L-phenyl-alanyl-mercapto) -2-D-methyl-propanod] -L-proline (Resolution)

The title compound is essentially Example 10

Prepared in the same manner as in Step 2.

Example 19

s) method

1st step

Thio-L-phenylalanine hydrochloride

L-Phenylalanine thiophenyl ester hydrochloride is prepared in a known manner [Liebigs Ann. Chem. 576, 104 (1952)].

The title compound is prepared essentially in a manner known per se, Chem. Wage. 87, 1093 (1954) and Naturwissenschaften 40, 242 (1953)].

Step 2

3- (L-alaninyl Fend-mercapto) -2-methylpropionic acid hydrochloride

By following the procedure described in Example 1, Step 4, using L-thiophenylalanine hydrochloride instead of N-benzoyl-L-thiophenylalanine, the title compound is obtained.

Step 3

3-L-Phenylalanyl-mercapto-2-D-meth-propionic acid (Resolution)

-231

HU 199786 Β

The product of Step 2 is essentially resolved as described in Example 1, Step 5, or by ion exchange chromatography.

Step 4

3- (N ^a -Benzoyl-L-phenyl-alanyl-mercapto) -2-D-methylpropionic acid was prepared in a mixture of the product obtained in Step 3 and 10 mmol of sodium bicarbonate in tetrahydrofuran and water, To the cold solution was added a solution of 10 mmol of benzoic acid N-hydroxysuccinimide ester in cold tetrahydrofuran and the reaction mixture was stirred overnight at room temperature. The tetrahydrofuran is then distilled on a rotary evaporator at 35 ° C, water is added to the residue and the pH of the aqueous mixture is adjusted with solid sodium bicarbonate. The aqueous layer was then partitioned between ethyl acetate and the aqueous layer was acidified to pH 2 with 1N hydrochloric acid in the presence of ethyl acetate, cooled in an ice bath. The latter ethyl acetate layer was washed twice with cold water and twice with saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and the desiccant was filtered off. The solvent was evaporated on a rotary evaporator and the residue was crystallized from diethyl ether / hexane to give the title compound.

Step 5

^N - [3- (N-benzo-L-phenylalanyl-mercapto) -2-D-methyl] -L-proline

The title compound was prepared according to Example 1, Step 6

(a).

Example 20

(t) method

1st step

3- (N ^a -Benzod-L-phenylalanyl-mercapto) -2-D-methylpropionic acid was dissolved in 1 N aqueous sodium hydroxide solution (3 mmol) and added to the solution in an ice bath. 15 mmol of N in ^{a solution of} benzoyl-thiophenylalanine and potassium carbonate in water. After stirring overnight at room temperature, the reaction mixture was acidified with concentrated hydrochloric acid, the aqueous portion was extracted with ethyl acetate, and the ethyl acetate portion was washed with water, dried, and the solvent was evaporated. The product thus obtained is then resolved as described in Example 1, Step 5 to give the title compound. Alternatively, 3-bromo-2-D-methylpropionic acid may be started in this step without the need for resolution of the product.

Step 2

N ^a - [3- (N ^a -Benzod-L-fendanyl-mercapto) -2-D-methylpropanod] -L-proline

The title compound was prepared according to Example 1, Step 6

(a).

Example 21

u) method

1st step

Benzyloxycarbonyl-3-mercapto-2-methylpropionic acid

In the same manner as in Example 1, Step 4, and

Instead ^of N-benzoyl-L-alanine-thiophenyl compound is prepared the title compound using tiohangyasav benzyl ester.

Step 2

-Benzyloxycarbonyl mercapto-2-D-methylpropionic acid (resolution)

The title compound is prepared by crystallizing the product of Step 1 as its salt or by liquid chromatography.

Step 3

N ^a - [3- (Benzyloxycarbonylmercapto) -2-D-methylpropanoyl] -L-proline tert-butyl ester

The title compound was prepared according to Example 1, Step 6

a) and using the product of Step 2 of the present process in place of 3- (N '-benzoyl-L-phenylalanyl-mercapto) -2-D-methylpropionic acid.

Step 4

N ^a - (3-Mercapto-2-D-methylpropanoyl) -L-proline

The product of Step 3 is suspended in anisole and stirred with acetic acid hydrobromic acid for half an hour at room temperature. Diethyl ether was then added and the mixture was kept at 0 ° C for 1 hour. The precipitate was filtered off and washed several times with anhydrous diethyl ether and then dried under reduced pressure with sodium hydroxide and phosphorus pentoxide.

Step 5

N ^a - [3- (N ^a -Benzoyl-L-phenyl-alanyl-mercapto) -2-D-methyl-propanod] -L-proline

The title compound is all. Example 3 was prepared using the product of Step 4 of the present embodiment instead of 3-mercapto-2-methylpropionic acid.

Example 22

(v) method

1st step

3-Phenyl-L-alaninyl-mercapto-2-methylpropionic acid

N '-Boc-L-Phe and an equivalent amount of hydrogen sulfide are coupled by the mixed anhydride method [J. Chem. Soc. 74, 4726 (1952)]. The Boc protecting group of N '-Boc-L-thiophenylalanine thus obtained is depicted in FIG. Example 2 is cleaved as described in step 2 (1). During the reaction, the trifluoroacetic acid and the anisole must be completely removed. Starting from the L-thiophenylalanine thus obtained and essentially following the procedure of Example 1, Step 4, the title compound is obtained.

Step 2

3- (N ^a -Benzoyl-L-phenyl-alanyl-mercapto) -2-methyl-propionic acid, The product obtained in Step 1 was prepared with a 4: 3 mixture of sodium bicarbonate and tetrahydrofuran / water (7: 3). slurry with vigorous stirring. 2 mmol of benzoyl chloride are then added at room temperature and the reaction mixture is stirred vigorously for an additional 1 hour.

-241

HU 199786 Β stir. Ethyl acetate was then added and the pH of the mixture was adjusted to 2 with 0 N HCl at 0 ° C. The ethyl acetate portion was washed with saturated sodium chloride solution, dried over magnesium sulfate, and the desiccant was filtered off. The solvent is then distilled off on a rotary evaporator and the residue is crystallized from a mixture of benzene and n-hexane and recrystallized to give a white crystalline product.

Step 3

3- (N ^a -Benzoyl-L-phenyl-alanyl-mercapto) -2-D-methyl-propionic acid (Resolution)

The title compound was prepared as described in Example 1, Step 5.

Step 4

^N - [3- (N ^-B dihydroxybenzoyl-L-phenyl-alanyl-mercapto) -2-D-methyl- propanoyl] -L-proline

The title compound was prepared according to Example 1, Step 6

(a).

Example 23 Method z

1st step

To a solution of 3-acetylmercapto-2-methylpropionic acid-o-nitrobenzyl ester in mmol of o-nitrobenzyl alcohol in 2-fluoro-1,3,5-trinitrobenzene was added 10 mmol of 3-acetylmercapto-2 with stirring at room temperature. methyl propionic acid in 2-fluoro-1,3,5-trinitrobenzene, the mixture was stirred for 1.5 hours and the solvent was distilled off on a rotary evaporator to give the title compound.

Step 2

O-Nitrobenzyl ester of 3-acetylmercapto-2-D-methylpropionic acid (resolution)

The product of Step 1 is resolved by liquid chromatography to give the title compound.

Step 3

3-acetyl-mercapto-2-D-methylpropanoic acid

The product of Step 2 is irradiated with 350 nm light to give the title compound (J. Am. Chem. Soc. 97, 1575 (1975)).

Step 4

^N - (3-acetyl-mercapto-2-D-methylpropanoyl) -L-proline tert-butyl ester

Using the procedure described in Example 1, Step 6 (a) (1), replacing 3- (N '-benzoyl-L-phenylalanyl-mercapto) -2-D-methyl-propionic acid from the product of Step 3 of this embodiment. starting from the title compound.

Step 5

^N - (3-mercapto-2-D-methylpropanoyl) -L-proline tert-butyl ester

The product of Step 4 was dissolved in a mixture of concentrated ammonia, methanol and anisole, kept at room temperature for 1 hour, and then the solvent was evaporated under reduced pressure. The residue was crystallized from a mixture of benzene and hexane to give the title compound.

Step 6

N ^a - [3- (N ^a -Benzoyl-L-phenyl-alanyl-mercapto) -2-D-methyl-propanoyl] -L-proline tert-butyl ester

By following the procedure of Example 11, Step 3, using the product of Step 5 of this embodiment, instead of 3-mercapto-2-D-methylpropanoyl-L-proine, the title compound is obtained.

Step 7

^N - [3- (N ° --Benzoil-L-phenylalanyl-mercapto) -2-D-methyl- propanoyl] -L-proline

From the product of Step 6, the tert-butyl ester group was cleaved according to Example 1, Step 6 (a) (2) to give the title compound.

Example 24 Method aa

1st step

3-Amino-2-D-methylpropionic acid (Resolution)

Starting from 3-amino-2-methylpropionic acid and using known methods [J. Biol. Chem., 236, 3283 (1961)] to give the title compound.

Step 2

-Bromo-2-D-methylpropionic acid

To a solution of the product of Step 1 in water at -5 ° C is added a small amount of sodium nitrite and 48% hydrogen bromide. The reaction mixture was stirred for half an hour and then allowed to warm slowly to room temperature. The solvent was distilled off on a rotary evaporator to give the title compound.

Step 3

3- ^{(N-Benzoyl-L-phenylalanyl-mercapto)} -2-D-methyl-propionic acid

The title compound was prepared as described in Example 25, Step 1.

Step 4

N ^a - [3- (N ^a -Benzoyl-L-phenyl-alanyl-mercapto) -2-D-methyl-propanoyl] -L-proline

The title compound was prepared according to Example 1, Step 6

(a).

Example 25 Method b)

1st step

3-Amino-2-D-methylpropionic acid (Resolution)

3-Amino-2-methylpropionic acid according to the known method [J. Biol. Chem. 236, 3283 (1961)] to give the title compound.

Step 2

3-Bromo-2-D-methyl-propionic acid

The title compound was prepared as described in Example 29, Step 2.

Step 3

-Benzoyl-mercapto-2-D-methyl-propionic acid

Starting from the product of Step 2 and essentially following the procedure described in Step 1 of this embodiment, the title compound is obtained.

-251

HU 199786 Β

Step 4

3-mercapto-2-D-methylpropanoic acid

Cleavage of the benzoyl group as described in Example 11, Step 2 gave the title compound.

Step 5

3- ^{(N-Benzoyl-L-phenylalanyl-mercapto)} -2-D-methyl-propionic acid

By following the procedure described in Example 11, Step 3, starting from the product of Step 4 of the present embodiment, the title compound is obtained.

Step 6

N ^a - (3- (N ^a -Benzoyl-L-phenyl-alanyl-mercapto) -2-D-methyl-propanoyl] -L-proline

The title compound was prepared as described in Example 11, Step 5.

Example 26 Method c)

1st step

3-Benzoyl-mercapto-2-D-methyl-propionic acid tert-butyl ester in a solution of 3-benzoyl-mercapto-2-D-methyl-propionic acid in dichloromethane containing 0.5 ml of concentrated sulfuric acid, isobutylene was added for 1 hour, the reaction vessel was sealed and the mixture was stirred for 16 hours. The mixture is partitioned between diethyl ether and the organic solvent is distilled off on a rotary evaporator and the residue is dried under high vacuum.

Step 2

3-mercapto-2-D-methyl-propionic acid tert.-butyl ester

The product of Step 1 is dissolved in methanolic ammonia solution under nitrogen. After 1 hour, the solvent was distilled off, the residue was dissolved in diethyl ether and the solution was washed with 0.1 N hydrochloric acid and then with saturated sodium chloride solution in an ice bath. The organic phase is then dried over anhydrous magnesium sulfate, the desiccant is filtered off and the solvent is distilled off on a rotary evaporator. The residue was distilled under reduced pressure to give the title compound.

Step 3

3- (N ^a -Benzoyl-L-phenyl-alanyl-mercapto) -2-D-methyl-propionic acid tert-butyl ester

a) 1) Preparation of the active ester A solution of N, ^a benzoyl-L-Phe in double-distilled ethyl acetate was cooled in an ice-dry ice-methanol bath to -20 ° C. [N "benzoylL-Phe" is disclosed in U.S. Pat. No. 128,953. See, for example, Carter et al., J. Biol. Chem. 138, 627 (1941)]. To the above solution was added 25 mmol of Netmorpholine, 25 mmol of isobutyl ester of chloroformate and the mixture was stirred at -15 ° C for a few minutes, followed by addition of 50 mmol of N-hydroxysuccinimide. The reaction mixture was stirred for 1.5 hours at -15 ° C.

2) Active ester coupling method

The above reaction mixture was allowed to slowly warm to room temperature and 25 mmol was added.

3-mercapto-2-D-methylpropionic acid tert-butyl ester prepared as described in Step 2. The mixture was then stirred until the reaction was complete according to TLC. Ethyl acetate was then added and the mixture was acidified to pH 3 with 1N hydrochloric acid, cooled in an ice bath. The ethyl acetate layer was washed several times with cold water and then with cold saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and the desiccant was filtered off. The solvent was evaporated on a rotary evaporator and the residue was crystallized from benzene / petroleum ether to give the title compound.

b) To 25.1 mmol of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline is added a solution of 25 mmol of N 'benzoyl-L-Phe in tetrahydrofuran followed by 25 mmol of 3-methcapto-2-D-methyl -propionic acid tert-butyl ester. Stir the reaction mixture until the reaction is complete according to TLC. The solvent is then distilled off on a rotary evaporator and the product is crystallized from a mixture of benzene and petroleum ether.

Step 4

3- ^{(N-Benzoyl-L-phenylalanyl-mercapto)} -2-D-methyl-propionic acid

The product of Step 3 is suspended in 4 ml of anisole, 8 ml of trifluoroacetic acid are added and the mixture is stirred at room temperature for 1.5 hours. The trifluoroacetic acid and the anisole were distilled off on a rotary evaporator and the residue was dried under high vacuum under sodium hydroxide and phosphorus pentoxide to give the title compound.

Step 5 iM ^a - [3- (N ^a -Benzyl-L-phenyl-alanyl-mercapto) -2-D-methyl-propanod} -L-proline

a) A cooled solution of 25 mmol of dicyclohexylcarbodiimide in dimethylformamide was added dropwise at 0 ° C to a solution of 25 mmol of the product obtained in Step 4 and 25 mmol of N-hydroxysuccinimide in dimethylformamide. The reaction mixture was then stirred for half an hour at 0 'C and then overnight at 4' C. The precipitated dicyclohexylurea was filtered off and washed with ethyl acetate. The filtrate was evaporated under reduced pressure and the residue was crystallized from benzene / hexane. The crystalline product was dissolved in cold tetrahydrofuran and a solution of 25 mmol of L-proline and 25 mmol of sodium bicarbonate in THF / water was added. The reaction mixture was stirred overnight at room temperature and then the tetrahydrofuran was distilled off at 35 ° C on a rotary evaporator. Water was added to the residue and the pH of the mixture was adjusted to 9 by addition of solid sodium bicarbonate. The aqueous phase is then partitioned between ethyl acetate and the aqueous phase is acidified to pH 2 with 1N hydrochloric acid in the presence of ethyl acetate, cooled in an ice bath. The latter portion of ethyl acetate was washed twice with cold water and then with saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and the desiccant was filtered off. The solvent is rotating

-261

The residue was crystallized from a mixture of diethyl ether and hexane to give the title compound.

b) 1) To a solution of 25 mmol of the product obtained in Step 4 in ethyl acetate was added a solution of 25 mmol of dicyclohexylcarbodiimide in ethyl acetate in a cooling bath at 0 ° C and stirred for 5 minutes at 0 ° C. and then a solution of L-proline tert-butyl ester (25 mmol) in ethyl acetate was added. After stirring overnight at 4 ° C, ethyl acetate (10 mL) was added and the insolubles were filtered off and washed with ethyl acetate (20 mL). The combined ethyl acetate portions were cooled and washed three times with saturated sodium chloride solution, once with cold 0.1N sodium bicarbonate solution, and three times with saturated sodium chloride solution. After drying over anhydrous magnesium sulfate, the desiccant is filtered off and the solvent is distilled off on a rotary evaporator under high vacuum to give a white residue.

2) The above residue is suspended in 4 ml of anisole, 8 ml of trifluoroacetic acid are added and the mixture is stirred at room temperature for 1.5 hours. Trifluoroacetic acid is then distilled off under high pressure under a rotary evaporator, the residue is dissolved in 1 ml of tetrahydrofuran and this solution is chromatographed. From the fractions containing the desired product, the solvent was distilled off, the residue was dissolved in a small amount of isopropanol and the solution was chromatographed again. The oily residue obtained from the fractions containing the desired product was dissolved in tetrahydrofuran and the tetrahydrofuran was distilled off under a stream of nitrogen. Solvent residues are removed on a rotary evaporator under heavily reduced pressure.

Example 27 Method d)

1st step

S-1N, 4-Boc-L-Phenylalanyl-mercapto-4-D-methyl-propionic acid tert-butyl ester

N ^a -Bpoc-L-Phe was prepared according to the method of Sieber et al., Helv. Chim. Acta 57, 614 (1968)]. The 3-mercapto-2-D-methylpropionic acid tert-butyl ester was prepared as described in Example 2, Steps 1 and 2. The title compound was prepared essentially as described in Example 2, Step 3 (a) using N "-Bpoc-LPhe instead of N" -Bz-L-Phe.

Step 2

3- (L-Phenylalanyl-mercapto) -2-D-methyl-propionic acid tert-butyl ester, trifluoroacetic acid salt

The product of Step 1 is dissolved in dichloromethane containing 0.5% trifluoroacetic acid and the solution is stirred for 10 minutes. Subsequently, dichloromethane and excess trifluoroacetic acid were distilled off on a rotary evaporator to give the title compound.

Step 3

3- (N ^a -Benzoyl-L-phenyl-alanyl-mercapto) -2-D-methyl-propionic acid tert-butyl ester, m.p. benzoyl chloride (15 mmol) was added followed by the dropwise addition of aqueous sodium bicarbonate (32 mmol) and the reaction mixture was stirred vigorously for another half hour. The reaction mixture was cooled in an ice bath for half an hour, extracted with ethyl acetate, and the ethyl acetate solution was cooled again, washed several times with water, 1N citric acid, then with saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and on a device. This affords the title compound.

Step 4

3- ^{(N-Benzoyl-L-phenylalanyl-mercapto)} -2-D-methyl-propionic acid

The product of Step 3 was cleaved by tert-butyl ester as described in Example 2, Step 4 to afford the title compound.

Step 5

N ^a -13- (N ^a -Benzoyl-L-phenyl-alanyl-mercapto) -2-D-methyl-propanod] -L-proline

The title compound was prepared as described in Example 2, Step 5.

Example 28 Method ae)

1st step

3-mercapto-2-D-methylpropanoic acid

The title compound was prepared as described in Example 2, Step 2 using 3-benzoylmercapto-2-D-methylpropionic acid instead of the tert-butyl ester used therein.

Step 2

3- ^{(N-Boc-L-phenylalanyl-mercapto)} -2-D-methyl-propionic acid

By essentially the same procedure as in Example 2, Step 3 (a), replacing N ^a -Bz-L-Phe with N ^a -BocL-Phe and 3-mercapto-2-D-methylpropionic acid tert-butyl ester. Instead, the title compound is prepared using the product of Step 1 of the present embodiment.

Step 3

3-L-Fend-Alanyl-mercapto-2-D-methyl-propionic acid, trifluoroacetic acid salt

The product of Step 2 was stirred with a mixture of 4 ml of anisole and 8 ml of trifluoroacetic acid at room temperature for 1.5 hours, then the anisole and excess trifluoroacetic acid were distilled off on a rotary evaporator to give the title compound.

Step 4

3- ^{(N-Benzoyl-L-phenylalanyl-mercapto)} -2-D-methyl [-propionic

By proceeding as described in Example 3, Step 3, and substituting the trifluoroacetic acid salt of 3-L-phenylalanyl-mercapto-2-D-methylpropionic acid tert-butyl ester, as described in Step 3 of this embodiment. yielding the title compound.

-271

HU 199786 Β

Step 5

N ^a - [3- (N ^a -Benzod-L-phenylalanyl-mercapto) -2-D-methylpropanod] -L-proline

The title compound was prepared as described in Example 2, Step 5.

Example 29 Method f)

1st step

3- ^(N -Bpoc-L-phenylalanyl-mercaptopurine) -2-D-methyl-propionic acid

The title compound was prepared as described in Example 3, Step 1, using 3-mercapto-2-D-methylpropionic acid (prepared as in Example 5, Step 1) instead of the tert-butyl ester used therein. live.

Step 2

N ^a - [3- (N ^a -Bpoc-L-Phenylalanyl-mercapto) -2-D-methylpropanod] -L-proline tert -butyl ester, m.p. A cooled solution of 10 mmol of dicyclohexylcarbodiimide and 10 mmol of 1-hydroxybenzotriazole in dimethylformamide was added dropwise to a mixture of 10 mmol of L-proline tert-butyl ester in dimethylformamide and the reaction mixture was stirred for half an hour at 0 ° C. and then overnight at 4 ° C. The precipitated crystalline dicyclohexylurea was filtered off and washed with ethyl acetate. The filtrate was evaporated under reduced pressure and the residue was crystallized from diethyl ether / hexane to give the title compound.

Step 3

^N - (3-L-alaninyl-mercapto) -2-D-metd- 35

-propanoyl-L-proline tert-butyl ester, trifluoroacetic acid salt

The title compound was obtained in the same manner as in Example 3, Step 2 instead of 3- (N ^a -Bpoc-L-phenylalanyl mercapto) -2-D-methylpropionic acid tert-butyl ester. using the product of Step 1 of the present process variant.

Step 4

^N - [3- ^(N-B ENZO L-phenylalanyl-mercapto) -2 - 45

-D-methylpropanod] -L-proline tert-butyl ester

By essentially the same procedure as in Example 3, Step 3, and substituting the trifluoroacetic acid salt of 3-L-phenylalanyl-mercapto-2-D-methylpropionic acid tert-butyl ester, the product of Step 3 of this embodiment is obtained. using the title compound

Step 5

N ^a - [3- (N ^a -Benzoyl-L-fendaland) -2-D-meth-propanoyl] -L-proline

The title compound was prepared as described in Example 2, Step 5 (b) (2).

Example 30 Method g)

Step 1 ^ - [d-1N 4 -Boc-L-Phenylalanyl-mercaptoyl-D-meth-propanod] -L-proline

Following the procedure described in point a) of Example 2, Step 5, and 3- (N, prepared as described in Example 5, Step 2 instead ^of benzoyl-L-phenylalanine-mercapto) -2D-methylpropionic acid 3- (N ^a 'Boc-L-Phenylalanyl-mercapto) -2-D-methylpropionic acid is obtained to give the title compound.

Step 2

^N - [3-L-phenylalanyl-mercapto-2-D-methyl-propanoyl] -L-proline, trifluoroacetate salt

The title compound was prepared as described in Example 5, Step 3.

Step 3

N ^a - [3- (N ^a -Benzoyl-L-phenylmandan-mercapto) -2-D-methylpropanod] -L-proline

Using the procedure described in Example 3, Step 3, and substituting the trifluoroacetic acid salt of 3-L-phenylalanyl-mercapto-2-D-methylpropionic acid tert-butyl ester for the procedure described in Step 2 of this embodiment. using the product to provide the title compound.

1-30. The following peptide derivatives were prepared by the methods described in Examples 1 to 5.

The following systems were used for thin layer chromatography:

1. methanol-chloroform (1: 1)

2. benzene-water-acetic acid (9: 1: 9)

3. Acetic acid-water-n-butanol (26: 24: 150)

4. n-Butanol-pyridine-acetic acid-water (15: 10: 3: 12)

5. chloroform-methanol-ammonium hydroxide (60:45:20)

6. n-butanol-acetic acid-ethyl acetate-water (1: 1: 1: 1)

7. Chloroform-methanol-acetic acid (2: 10.003)

8. n-butanol-acetic acid-water (4: 1: 1)

In the table, compounds of the formula R-A-SQ are listed, wherein SQ is mercapto- (2-D-methylpropanoyl) -L-proline.

Bz = benzyl

Ac = acetyl

EtCO = propanoyl

CpCO = cyclopentanecarbonyl

Table I

Compounds of formula R-A-SQ

Compound R sign THE TLC: Rf 1 2 3 4 5 6 Nuclear Magnetic Resonance Spectrum 7 8 60 MHz (delta) 1. Bz Gly 0.60 0.46 0.60 0.55 0.68 (CDCl 3): 1.15 (3H, broad d, CH 3), 1.6-2.4 (4H, m, CH ₂ ), 2.5-3.2 (3H, m, CH and CH ₂ ),

-281

HU 199786 Β

Vegyü- TLC: Rf Nuclear Magnetic Resonance Spectrum let R A 1 2 3 4 5 6 7 8 60 MHz (delta) sign

3.3-3.8 (2H, m, CH 2), 4.1-4.6 (1H, m, CH), 4.30 (2H, d, CH ₂ ), 7.2-8.1 ( 5H, m, aromatic H) and 10.10 (1H, s, COOH)

Second

Third

Bz Trp 0.70 0.68 0.59

Ac Phe 0.47 0.65 0.45

0.74 0.56 0.73 0.64 0.54 0.59

4. Boc Phe 0.60 0.82 0.68

5. Bz Phe (CDCl 3): 1.17 (3H, broad d,

CH 3), 1.75-2.35 (4H, m, CH ₂ )

1.91 (3H, s, _CH3), 2.55 to 3.25 (5H, m, CHésCH _2), 3.4-3.8 (2H, m, _CH2), 4.1 to 4, Δ (1H, m, CH) 4.6-5.0 (1H, m, CH), 6.63 (1H, d, NH), 6.82 (1H, s COOH) and 7.17 (5H, broad s, aromatic H).

0.56 0.74

6. Bz Alá

0.58 0.75

7. Bz Leu

0.58 0.82

8. EtCO Trp

9. CpCO Al 0.62 0.74

0.52 0.75

10. Ac Gly

0.48 0.42

11. H Gly

0.16 - 0.09

12. Boc Gly

0.63 0.70 (CDCl ₃ + CD ₃ OD): 1.19 (3H, broad d, CH 3),

1.5-2.4 (4H, m, CH ₂ ),

2.5 - 3.3 (5H, m, CH and CH ₂ )

3.3-3.8 (2H, m, _CH2), 4.0-4.4 (IH, m, 'CH), 4.8-5.2 (IH, m, CH), 7.23 (5H, s, aromatic H) δ

7.2-7.9 (5H, m, aromatic H) (CDCl3): 1.19 (3H, edge d, CH3), 1.48 (3H, d, CH3)

1.7-2.4 (4H, m, _CH2), 2.5-3.2 (3H, m, CHésCH _2), from 3.25 to 3, (2H, m, _CH2), 4.2 -4.7 (1H, m, CH) 4.89 (1H, q, CH), 7.06 (1H, d, NH),

7.3-8.0 (5H, m, aromatic H) and 10.21 (1H, s, COOH).

(CD 3 OD): 0.99 (6H, br d, CH 3), 1.22 (3H, br d, CH ₃ ),

1.5-2.4 (7H, m, CH and CH ₂ ), 2.5-3.15 (3H, m, CH and CH ₂ ), 3.4-3.9 (2H, m, CH ₂ ), 4.1-4.45 (1H, m, CH),

4.6- 5.0 (1H, m, CH) and

7.45 to 8.1 (5H, m, aromatic H) (CDCl? + CD 3 OD): 1.20 (3H, br d, CH _3), 1.38 (3H, d, CH3), l, 5-2 , 4 (12H, m, CH ₂ ),

2,4- 3,2 (4H, m, CH and _CH2);

3.3- 3.8 (2H, m, CH ₂ ) and 4.2-4.8 (2H, m, CH) (CD 3 OD): 1.20 (3H, broad d, CH 3), 1.9-2, 3 (4H, m, CH ₂ ),

2.00 (3H, s, CH ₃ ), 2.5-3.2 (3H, m, CH and CH ₂ ), 3.5-3.8 (2H, m, CH ₂ ), 4.04 ( 2H, s, CH ₂ ) and 4.2-4.7 (1H, m, CH) (CD 3 OD): 1.17 (3H, broad d, CH 3), 1.8-2.4 (4H, m, CH ₂ ),

2,4- 3,3 (3H, m, CH and _CH2);

4.11 (2H, s, CH ₂ ) and 4.3-4.7 (1H, m, CH (CDCl 3): 1.22 (3H, broad d, CH ₃ ), 1.43 (9H, s, CH ₃ ),)

1.7- 2.4 (4H, m, CH ₂ ), 2.6-3.2 (3H, m, CH and CH ₂ ), 3.4-3.8

-291

HU 199786 Β

Vegyü- TLC: Rf Nuclear Magnetic Resonance Spectrum let R A 1 2 3 4 5 6 7 8 60 MHz (delta)

sign

13. H Phe 0.52 0.51 14. CpCO-Lys Phe 0.16 0.52 0.58 15. <Glu Phe 0.43 0.63 0.48 0.75 16. CpCO Phe 0.48 0.73 0.62 0.72 17. EtCO Try

18. Bz Try

19. Bz-Phe-2-thioprop-0.58 0.78 0.59 0.59 -Panoyl-L-Pro (2H, m, CH ₂ ), 3.98 (2H, d, CH ₂ ), 4 , 4 to 4.7 (IH, m, CH) and 8.68 (lH, s, COOH) (CD ₃ OD): 1.02 (3H, t, CH3), 1.08 (3H, br d,

CH 3), 1.8-2.5 (4H, m, CH ₂ ), 2.20 (2H, q, CH ₂ ),

2.5-3.1 (3H, m, CH and _CH2), 3.1-3.9 (4H, m,

CH ₂ ), 4.1-1.5 (1H, m, CH), 4.7-5.0 (1H, m, CH) and 6.95-7.8 (5H, m, aromatic H) ( CD 3 OD): 1.10 (3H, br d, CH _3), 2.3 1,65- (4H, m, _CH2), 2.6-3.1 (3H, m, CH and _CH2); 3.2-3.7 (4H, m, CH ₂ ), 4.05-4.35 (1H, m, CH), 4.8-5.1 (1H, m, CH) and 6.88-. 7.82 (10H, m, aromatic H)

Example 31

Nu [3- (Na henzoil-alanyl-DL-phenyl-thio) -2-D-methylpropanoyl] -L-proline, sodium salt

Mixing with a solution of 234.3 mg (0.5 mmol) of N '- [3- (N' -benzoyl-DL-phenylalanylthio) -2-D-methylpropanoyl] -L-proline in 1 ml of anhydrous ethanol while adding 0.5 mM sodium bicarbonate (as a 1M solution). The solvent was evaporated at 35 ° C in a rotary evaporator. Fresh ethanol was added to the oily residue and the solvent was again removed on a rotary evaporator. This procedure is performed once more with absolute alcohol and then twice with benzene. The white residue is dried in a vacuum desiccator over phosphorus pentoxide. Recrystallization from 95% ethanol / benzene gave 135 mg (55.1%) of a white crystalline solid, m.p. 185-186 ° C.

(Softens at 150 'C.)

IR (KBr) 1398 and 1600 cm ^-1 (zwitterion) and 1682 cm ^-1 (thioester).

Thin layer chromatography on silica gel plate (with 3 different solvent systems) and paper electrophoresis at pH 2 and pH 5 showed a single spot. (Detection: short-wave UV light, development: phenazine methosulfate).

Elemental analysis according to the formula C ₂ 5 H _{2 7} N ₂ SNaOs.2H ₂ O:

Calculated: C, 57.02; H, 5.93; N, 5.32; S, 6.09. Found: C, 56.37; H, 5.59; N, 5.30; S, 5.99.

Example 32

N ^a - [3- (N ^a benzoyl-DL-phenylalanylthio) -2-D-methylpropanoyl] -L-proline potassium salt

The title potassium salt was prepared as described in Example 1 using 1 molar potassium bicarbonate solution instead of 1 molar sodium bicarbonate solution. Recrystallization gave 70 mg of a white solid, m.p. 132134 ° C.

Example 33

^N - [3- ^{(N-benzoyl-DL-phenylalanyl-thio)} -2-D-methylpropanoyl] -L-proline, L-lysine salt

A solution of 73.1 mg (0.5 mmol) of L-lysine free base in 0.3 mL of deionized water was added dropwise with stirring to 234.3 mg (0.5 mmol) of Ν ^α - [3- (Ν ^α benzoyl-DL-phenyl) -alanylthio) -2-D-methylpropanoyl] -Lproline in 1 ml of absolute ethanol. Removal of the solvent in a rotary evaporator gave a white residue. To this was added absolute ethanol again and the solvent was removed in a rotary evaporator. This procedure was repeated twice more with absolute ethanol and twice with benzene. After drying in a vacuum desiccator over phosphorus pentoxide, 0.292 g of product, m.p. 152-154 ° C, is obtained. Recrystallization from absolute ethanol, a few drops of water and benzene gave 141 mg of a white precipitate, m.p. 162.5163.5 ° C (softening point: 160 ° C).

IR (KBr): 1389 and 1620 cm- ¹ (zwitterion), 1641 cm- ¹ (carbonyl and amide), 1678 cm- ¹ (thioester).

Example 34

Determination of angiotensin converting enzyme activity

For most of the experiments described herein, enzyme activity was determined at pH 8.0 in 0.05 M Hepes buffer containing 0.1 M sodium chloride and 0.75 M sodium sulfate. Substrate benzoyl-Gly-His-Leu was used in a final concentration of 1.10 ^-4 moles (K _m = 2.10 ^{with 4} moles) cpm of ^[3 H] benzoyl-Gly-His-Leu about 130.000 (25 Ci / mmol). The enzyme solution was diluted with said buffer solution to the extent that 40 μΐ of the buffered enzyme solution hydrolyzed 13 percent of the substrate at 37 ° C for a quarter hour. The assay was performed by first holding 40 μΐ enzyme solution and 10 μΐ buffer solution or inhibitor dissolved in buffer solution for 5 minutes at 37 ° C. Subsequently, 50 μΐ substrate solution was added and

-301

HU 199786 was maintained at 37 ° C for 4 hours. The reaction was quenched by the addition of 1 mL of 0.1 M hydrochloric acid and 1 mL of ethyl acetate was added. Subsequently, the mixture was stirred on a rotary mixer and centrifuged briefly to separate the phases.

A 500 μΐ aliquot of the ethyl acetate solution was poured into a liquid scintillation cuvette containing 10 ml of Riafluor (trademarked by New England Nuclear Corporation, Boston, Massachusetts). To determine the I50 values, the activity of the enzyme, measured in the presence of different amounts of inhibitor, was compared with that of the enzyme in experiments without inhibitor. The percent inhibition was plotted against the concentration of inhibitor to determine the

The in vitro inhibitory activity of the compounds prepared as described above was measured by the assay method described in Example 34. The enzyme preparation is isolated from human urine and purified according to the method of JW Ryan et al., Tissue and Cell, 10, 555 (1978). The Iso values of some compounds are shown in the table below. The Iso value is the concentration of inhibitor that inhibits the enzyme by 50 percent in a standard assay system in which the substrate concentration is substantially lower than the Michaelis constant. In the experiment, no. compounds showed 1,6.10 ^'8M The ID₅₀ value.

Example 35

^N - besides [3- ^(N--benioil DL-phenylalanyl-thio) -2-D-methylpropanoyl] -L-proline, sodium salt pharmacological action by oral administration

Rats weighing 150-190 g were fasted overnight and anesthetized by intraperitoneal administration of 50-60 mg / kg pentobarbital. The animals were tracheal incised and ventilated mechanically. Angiotensin I was administered by injection into a femoral vein and a second cannula was inserted into the carotid artery for direct measurement of arterial blood pressure. To prevent coagulation, 1000 units of heparin was added to the femoral vein. Blood pressure was recorded via transducer via pen. The rats were administered 400 ng / kg angiotensin I dissolved in 20 μΐ 0.9% sodium chloride solution. This dose increased arterial blood pressure by 45 mmHg. After the rat's response to the administration of angiotensin I was determined, 10 pmol / kg of the above compound was administered via the gastric tube. The compound was dissolved in 0.15 mL of water and 10 μΐ of 1 N sodium bicarbonate solution. The effect of 400 ng / kg angiotensin I on arterial blood pressure was determined periodically. The results are given below:

Time after oral administration, minutes Change in blood pressure with 400 ng / kg angiotensin 1 (as a percentage of control) -5 100 (45mmHg) +5 82 10 33

Time after oral administration, minutes Change in blood pressure with angiotensin I 400 ng / kg (as a percentage of control) 15 24 20 27 30 27 40 22 50 24 60 27 90 36 120 44 150 62 180 60 210 71 240 73 percent. Example 36 Intravenous Activity of the N ^a - {3- (N ^a -Benzoyl-DL-phenylalanylthio) -2-D-methylpropanoyl] -L-Proline Potassium Salt The intravenous activity of the above compound was tested according to the experiment described above, with the difference that 1 μιηόΐ / kg was administered intravenously. The results are shown in the table below shows: After oral administration Change in blood pressure 400 ng / kg time, minutes by angiotensin I (as a percentage of control) -5 100 (54mmHg) +5 46 10 37 15 41 20 37 30 44 40 50 50 39 60 46 90 52 ' 120 59 percent.

Example 37

The following compounds were prepared by the procedures described above, which were characterized by physical constants and biological activity (Iso).

For TLC, the following solvent systems were used (run on silica gel plates):

Rf (1): I. system methanol-chloroform (1: 1 v / v)

Rf (2): 2. system: benzene-water-n-butanol (9: 1: 9)

Rf (3): 3. system acetic acid-water-n-butanol (26: 24: 150)

Rf (4): 4. system: n-butanol-pyridine-acetic acid-water (15: 10: 3: 12)

Rf (5): 5. system: n-butanol-acetic acid-ethyl acetate-water (1: 1: 1: 1)

Rf (6): 6. system: chloroform-methanol-acetic acid (2: 1: 0.003)

Rf (7): 7. system: n-butanol-acetic acid-water (4: 1: 1)

-311

HU 199786 Β

Rf (8): 8. system: ethyl acetate-acetic acid (38: 2).

The NMR spectrum was recorded at 60 MHz.

150: the concentration required to inhibit 50% of the angiotensin converting enzyme. The assay was performed by mixing a total volume of 1 ml with 0.6 ml borate buffer, 0.2 ml 17.5 mM benzoyl-Gly-His-Leu borate buffer, enzyme solution and additional borate buffer. incubated at 37 ° C for 1 min.

Borate buffer: 0.1 M boric acid and 0.8 M sodium chloride solution, and 0.1 M sodium carbonate and 0.8 M sodium chloride, and pH is set to 8.3.

The measurement was made at 228 nm based on UV absorption after the benzoyl-Gly was extracted with ethyl acetate.

N ^a - [3- (N ^a- benzoyl-L-phenylalanylthio) -2-D-methylpropanoyl] -L-proline [α] _D = -116.8 Ms = 468,

Rf (1) = 0.52

Rf (2) = 0.70

Rf (3) = 0.56

Rf (4) = 0.61

Rf (5) = 0.75

Rf (6) = 0.56

Rf (7) = 0.74

Nuclear Magnetic Resonance Spectrum (CDCl3 = CD3OD), delta:

1.19 (3H, broad, d), 1.5-2.4 (4H, m), 2.5-3.3 (5H, m), 3.3-3.8 (2H, m), 4.0-4.4 (1H, m), 4.8-5.2 (1H, m), 7.23 (5H, s), 7.2-7.9 (5H, tn).

^N - [3- (N "-tert-butyloxycarbonyl-L-phenylalanyl-thio) -2-D-methylpropanoyl] -L-proline

Í50 = 3.4 x ΙΟ ' ⁷

Rf (1) = 0.60

Rf (2) = 0.76; 0.82

Rf (3) = 0.68; 0.72.

^N - (3-L-phenylalanyl-D-thio-2-methylpropanoyl) -L-proline

Rf (1) = 0.51

Rf (4) = 0.52

Rf (5) = 0.51

Rf (8) = 0.1.

^N - [3- ^{(N-benzoyl-D-phenylalanyl-thio)} -2-D-methylpropanoyl] -L-proline [?] _D = -25.1

Rf (6) = 0.56

R _f (7) = 0.74

Nuclear Magnetic Resonance Spectrum (CDCl3 + CD3OD); delta:

1.19 (3H, broad, d), 1.5-2.4 (4H, m), 2.5-3.3 (5H, m), 3.3-3.8 (2H, m), 4.0-4.4 (1H, m), 4.8-5.2 (1H, m), 7.23 (5H, s), 7.2-7.9 (5H, m).

^N - [2- ^{(N-benzoyl-L-phenylalanyl-methylthio)} propanoyl] -L-proline

Rf (1) = 0.58

Rf (2) = 0.78

Rf (3) = 0.59

R _f (4) = 0.59.

^N - [2- ^{(N-benzoyl-L-phenylalanyl-methylthio)} propanoyl] -L-3,4-dehydro-proline in

Rf (2) = 0.73

Rf (3) = 0.59

Rf (4) = 0.615

Rf (5) = 0.73.

^N - [3- ^{(N-benzoyl-D-alanyl-thio)} -2-D-methylpropanoyl] -L-proline [?] D = -103.0

Rf (6) = 0.58

Rf (7) = 0.75

Nuclear Magnetic Resonance Spectrum (CDCl 3), δ

1.19 (br, d, 3H), 1.48 (3H, d), 1.7-2.4 (4H, m), 2.5-3.2 (3H, m), 3.25-3, Δ (2H, m), 4.2-4.7 (1H, m), 4.89 (1H, q), 7.06 (1H, d), 7.3-8.0 (5H, m) .

10.21 (1H, s).

N '- [3- ^{(N-benzoyl-D-tryptophyl-thio)} -2-D-methylpropanoyl] -L-proline [?] _D = -33.1

Rf (6) = 0.56

Rf (7) = 0.73.

Nuclear Magnetic Resonance Spectrum (CD3OD), delta:

1.10 (3H, broad, d), 1.65-2.3 (4H, m), 2.6-3.1 (3H, m), 3.2-3.7 (4H, m), 4.05-4.35 (1H, m), 4.8-5.1 (1H, m), 6.88-7.82 (10H, m).

N '- [3- (N' -benzoyl-D-leucylthio) -2-D-methylpropanoyl] - L-proline [α] _D = -59.0

Rf (6) = 0.58

Rf (7) = 0.82.

Nuclear Magnetic Resonance Spectrum (CD3OD), delta:

0.99 (6H, broad, d), 1.22 (3H, broad, d), 1.5-2.4 (7H, m), 2.5-3.15 (3H, m), 3, 4-3.9 (2H, m), 4.1-4.45 (1H, m), 4.6-5.0 (1H, m), 7.5-8.1 (5H, m).

^N - [3- ^{(N-tert-butyloxycarbonyl-L-alanyl-thio)} -2-D-methylpropanoyl] -L-proline [?] D = -62.5

Rf (6) = 0.59

Rf (7) = 0.73.

Nuclear Magnetic Resonance Spectrum (CDCl 3), δ

1.20 (3H, broad, d), 1.24 (9H, s), 1.39 (3H, d),

1.7-2.4 (4H, m), 2.5-3.3 (3H, m), 3.3-3.9 (2H, t),

4.1-4.9 (2H, m), 6.1-6.5 (1H, m), 7.85 (1H, s).

^N - (3-D-alanyl-D-thio-2-methylpropanoyl) -L-proline [?] _D = -105.4

Rf (6) = 0.25

Rf (7) = 0.11.

^N - [3- (N-acetyl-L-phenylalanyl-thio) -2-D-methylpropanoyl] - L-proline

Ms = 406

Rf (1) = 0.47

RfD (2) = 0.65

Rf (3) = 0.45

Rf (δ) = 0.64

Rf (6) = 0.54

Rf (7) = 0.59.

Nuclear Magnetic Resonance Spectrum (CDCl 3), δ

1.17 (3H, broad, d), 1.75-2.35 (4H, m), 1.91 (3H,

s), 2.55-3.25 (5H, m), 3.4-3.8 (2H, m), 4.M.6 (1H,

m), 4.6-5.0 (1H, m), 6.63 (1H, d), 6.82 (1H, s),

7.17 (5H, bs, s).

^N - [3- ^(N cyclopentane-carbonyl-L-phenylalanyl-thio) -2-D-methylpropanoyl] -L-proline

R f (1) = 0.48

R _f (2) = 0.73

Rf (3) = 0.62

Rf (δ) = 0.72.

^N - [3- ^(N -cyclopentanecarbonyl-L-lysyl-L-phenylalanyl-thio) -2-D-methylpropanoyl] -L-proline

Rf (3) = 0.16

Rf (4) = 0.52

Rf (5) = 1.58.

-321

HU 199786 Β

^N - [3- ^{(N-pyrid-L-glutamyl-L-phenylalanyl-thio)} -2-D-methylpropanoyl] -L-proline

Rf (1) = 0.43; 0.45 Rf (2) = 0.63 Rf (3) = 0.48 Rf (5) = 0.75.

N ^a - [2- (N ^a -acetyl-L-phenylalanylthio) -propanoyl] -L-proline [a] _D = -44.4 Rf (6) = 0.83.

N ^a - [3- (N ^a -acetyl-L-phenylalanylthio) -2-D-methylpropanoyl] -L-proline [a] _D = -51.9 Rf (6) = 0.54 Rf (7) = 0.59.

Nuclear Magnetic Resonance Spectrum (CDCl 3), δ:

1.17 (3H, broad, d), 1.75-2.35 (4H, m), 1.91 (3H,

s), 2.55-3.25 (5H, m), 3.4-3.8 (2H, m), 4.1-4.6 (1H, m), 4.6-5.0 ( 1H, m), 6.63 (1H, d), 6.82 (1H, s),

7.17 (5H, bs, s).

^N - [3- ^{(N-acetyl-D-alanyl-thio)} -2-D-methylpropanoyl] - L-proline, [(X] _D = -72.3.

^N - [3- ^(N propanoyl-D-alanyl-thio) -2-D-methylpropanoyl] -L-proline [<x] D = -62.0.

^N - [3- ^{(N-butanoyl-D-alanyl-thio)} -2-D-methylpropanoyl] -L-proline [<x] _D = -57.9.

N ^a - [3- (N ^a -cyclopentanecarbonyl-D-alanylthio) -2-D-methylpropanoyl] -L-proline [a] _D = -47.2 R 1 (6) = 0.62 Rf (7) = 0.74.

Nuclear Magnetic Resonance Spectrum (CDCl3 + CD3OD), delta:

1.20 (3H, broad, d), 1.38 (3H, d), 1.5-2.4 (12H,

m), 2.4-3.2 (4H, m), 3.3-3.8 (2H, m), 4.2-4.8 (2H, m).

N ^a - [3- (N ^a -propanoyl-D-tryptophylthio) -2-D-methylpropanoyl] -L-proline [<x] d = -28.6 Rf (6) = 0.52 Rf (7) = 0.75.

Nuclear Magnetic Resonance Spectrum (CD3OD), delta:

1.02 (3H, t), 1.08 (3H, broad, d), 1.8-2.5 (4H,

m), 2.20 (2H, q), 2.5-3.1 (3H, m), 3.1-3.5 (4H, m), 4, W, 5 (1H, m), 4 , 7-5.0 (1H, m), 6.95-7.8 (5H, m).

^N - [3- ^(N # -acetyl-D-leucyl-thio) -2-D-methylpropanoyl] -L-proline, [alpha] D = -29.5 Rf (6) = 0.55 Rf ( 7) = 0.59.

^N - [3- ^{(N-acetyl-D-valyl-thio)} -2-D-methylpropanoyl] -L-proline, [alpha] _D = -73.8.

^N - [3- (N '-acetyl-glycyl-thio) -2-D-methylpropanoyl] -L-proline

Ms = 316 [α] D = -51.0 R f (6) = 0.48 R _f (7) = 0.42.

Nuclear Magnetic Resonance Spectrum (CD3OD), delta:

1.20 (3H, broad, d), 1.9-2.3 (4H, m), 2.00 (3H,

s), 2.5-3.2 (3H, m), 4.04 (2H, s), 4.2-4.7 (1H, m). I50 = 4.0 x 10 '.

^N - [3- ^{(N-acetyl-L-isoleucyl-thio)} -2-D-methylpropanoyl] -L-proline [?] D = 97.8.

Nuclear Magnetic Resonance Spectrum (CDCl 3), δ

0.9 (3H, t), 0.95 (3H, d), 1.25 (3H, d), 1.7-2.5 (7H, m), 4.0-5.0 (2H, m), 6.3-6.8 (1H, m), 8.48 (1H, s).

^N - [3- ^{(N-acetyl-L-leucyl-thio)} -2-D-methylpropanoyl] -L-proline [?] _D = -151.3

Nuclear Magnetic Resonance Spectrum (CDCl 3), δ:

0.92 (6H, d), 1.21 (3H, d), 2.01 (3H, s), 1.4-4.0 (12H, m), 4.0-5.0 (2H, m), 6.0-6.6 (1H, m), 6.72 (1H, s).

N ^a - [3- (N ^a -acetyl-L-valylthio) -2-D-methylpropanoyl] -L-proline [a] _D = -153.5

Nuclear Magnetic Resonance Spectrum (CDCl 3), δ

0.90 (3H, d), 0.96 (3H, d), 1.23 (3H, d), 1.7-2.5 (4H, m), 2.08 (3H, s), 2 , 5-4.0 (6H, m), 4.1-5.0 (2H,

m), 6.58 (1H, d), 8.58 (1H, s).

^N - [3- ^{(N-acetyl-L-alanyl-thio)} -2-D-methylpropanoyl] -L-proline, [alpha] D = -151.7

Nuclear Magnetic Resonance Spectrum (CDCl 3), δ

1.20 (3H, d), 1.37 (3H, d), 2.01 (3H, s), 1.7-2.5 (4H, m), 2.5 ^ 1.0 (5H, m), 4.0-5.0 (2H, m), 6.5-7.3 (2H, m).

^N - [3- ^{(N-propanoyl-L-tryptophyl-thio)} -2-D-methylpropanoyl] -L-proline [a] o = -116.7

Nuclear Magnetic Resonance Spectrum (CDCl 3), δ

1.0 (3H, t), 1.21 (3H, d), 1.6-4.0 (13H, m),

4.0-5.3 (2H, m), 6.0-6.6 (1H, m), 6.8-7.8 (5H, m),

8.8-9.2 (2 H, m).

^N - [3- ^{(N-tert-butyloxycarbonyl-L-phenylalanyl-thio)} -2-D-methylpropanoyl] -L-proline

Rf (1) = 0.60 Rf (2) = 0.76; 0.82 Rf (3) = 0.72.

N '- [3- ^{(N-tert-butyloxycarbonyl-L-isoleucyl-thio)} -2-D-methylpropanoyl] -L-proline

R f (1) = 0.82 R _f (2) = 0.72 R f (3) = 0.78.

IR spectrum (CHQ3):

karlx> nyl band at 1643 and 1690-1740 cm ^-1 and NH band at 3445 cm ^-1 .

N '- [3- ^{(N-tert-butyloxycarbonyl-L-alanyl-thio)} -2-D-methylpropanoyl] -L-proline

Mp 143-145 ° C.

[<x] d = -153.8.

N ^a - [3- (N ^a -cyclopentanecarbonyl-L-phenylalanylthio) -2-D-methylpropanoyl] -L-proline

Rf (1) = 0.48 Rf (2) = 0.73 Rf (3) = 0.62 Rf (5) = 0.72.

^N - [3- ^(N cyclopentane-carbonyl-L-isoleucyl-thio) -2-D-methylpropanoyl] -L-proline [?] D = -157.6 Rf (6) = 0.54 Rf (7) = 0.80.

I50 = 1.2 χ ÍO- ⁷ .

Rf (1) = 0.65

-331

HU 199786 Β

Rf (2) = 0.76 Rf (3) = 0.70 IR (CHCl3):

carbonyl band 1645; 1675 and 1722 cm at wave number ¹ .

^N - [3- ^(N cyclopentane-carbonyl-L-alanyl-thio) -2-D-methyl-1-propanol 1] -L-proline

Ms = 384.

[α] D = -157.6.

Rf (6) = 0.63 Rf (7) = 0.72.

Nuclear Magnetic Resonance Spectrum (CDCl 3 + CD 3 OD) δ

1.20 (3H, broad, d), 1.38 (3H, d), 1.5-2.4 (12H,

m), 2.4-3.2 (4H, m), 3.3-3.8 (2H, m), 4.2-4.8 (2H, m).

I50 = 4.2 x IO ^7th

^N - (3-L-phenylalanyl-D-thio-2-methylpropanoyl) -L-proline

Rf (1) = 0.51 Rf (4) = 0.52 Rf (5) = 0.51 Rf (8) = 0.1.

^N - (3-glycyl-2-thio-D-methylpropanoyl) -L-proline, MS = 274 [?] D = -93.6.

Rf (6) = 0.16 Rf (7) = 0.09.

Nuclear Magnetic Resonance Spectrum (CD3OD), delta:

1.17 (3H, broad, d), 1.8-2.4 (4H, m), 2.4-3.3 (3H, m), 3.5-3.9 (2H, m), 4.11 (2H, s), 4.3 ^ 4.7 (1H, m).

I50 = 6.5 χ 10A

N '- (3-L-isoleucyl-D-thio-2-methylpropanoyl) -L-proline

Rf (1) = 0.42 Rf (2) = 0.35.

^N - [3- ^(N -cyclopentanecarbonyl-L-lysyl-L-phenylalanyl-thio) -2-D-methyl-propanoyl] -L-proline

Rf (3) = 0.16 Rf (4) = 0.52 Rf (5) = 1.58.

^N - [3- (pyro-L-glutamyl-L-phenylalanyl-thio) -2-D-methylpropanoyl] -L-proline

R _f (1) = 0.43; 0.45 Rf (2) = 0.63 Rf (3) = 0.48 Rf (4) = 0.75.

^N - [3- (N "-benzoyl-L-tryptophyl-thio) -2-D-methyl 1-propanol] -L-proline

Ms = 507.

Rf (2) = 0.70 Rf (3) = 0.68 Rf (4) = 0.59 Rf (5) = 0.74 Rf (6) = 0.56 Rf (7) = 0.73.

Nuclear Magnetic Resonance Spectrum (CD3OD), delta:

1.10 (3H, broad, d), 1.65-2.3 (4H, m), 2.6-3.1 (3H, m), 3.2-3.7 (4H, m), 4.05-4.35 (1H, m), 4.8-5.1 (1H, m), 6.88-7.82 (10H, m).

^N - [3- ^{(N-benzoyl-glycyl-thio)} -2-D-methylpropanoyl] -L-proline

Ms = 378 [α] D = -111.0 Rf (2) = 0.60

Rf (3) = 0.46

Rf (δ) = 0.60

Rf (6) = 0.55

Rf (7) = 0.68.

Nuclear Magnetic Resonance Spectrum (CDCl 3), δ

1.15 (3H, broad, d), 1.6-2.4 (4H, m), 2.5-3.2 (3H, m), 3.3-3.8 (2H, m), 4.1-4.6 (1H, m), 4.30 (2H, d), 7.2-8.1 (5H, m), 10.10 (1H, s).

I50 = 1.1 χ ίο ^-7 .

^N - [3- ^{(N-benzoyl-L-alanyl-thio)} -2-D-methylpropanoyl] -L-proline

Ms = 392.

Rf (6) = 0.58

Rf (7) = 0.75.

Nuclear Magnetic Resonance Spectrum (CDCl 3), δ

1.19 (6H, broad, d), 1.22 (3H, broad, d), 1.5-2.4 (7H, 3H, m), 3.4-3.9 (2H, m), 4.1-4.45 (1H, m),

4.6-5.0 (1H, m), 7.45-8.1 (5H, m).

N ^a - [2- (N '-benzoyl-L-phenyl-alanyl-thio) -propanoyl] -L-3,4-dehydro-proline

Rf (2) = 0.73

Rf (3) = 0.59

R _f (4) = 0.615

Rf (5) = 0.73.

^N - [3- ^{(N-tert-butoxycarbonyl-glycyl-thio)} -2-D-methylpropanoyl] -L-proline [<X] D = -101.7

Rf (6) = 0.56

Rf (7) = 0.63.

Nuclear Magnetic Resonance Spectrum (CDCl 3), δ

1.21 (3H, d), 1.35 (9H, s), 1.5-2.5 (4H, m),

2.5-3.9 (5H, m), 3.9-4.8 (3H, m), 6.5-7.4 (1H, m), 11.11 (1H, s).

Claims (7)

PATENT CLAIMS A process of the formula I wherein R is hydrogen, tert-butoxycarbonyl, cyclopentanecarbonyl-L-lysyl, pyro-L-glutamyl or R ', wherein R' is formyl, propanod, butanoyl, phenylacetyl, phenyl -propanoyl, benzoyl or cyclopentanecarbonyl; A is L-phenylalanyl, D-phenylalanyl, DL-phenylalanyl, L-Alanyl, D-Alanyl, L-Tryptophyl, D-Tryptophyl, L-Tyrosyl, Glycyl- , L-valyl, D-valyl, L-leucyl, D-leucyl or L-isoleucyl, the a-amino group of which is amide bonded to the R group; R 1 is hydrogen or methyl; R ₂ is L-proline or L-3,4-dehydroproline, the imino group of which is imide bonded to the carbonyl group to which it is attached; and n is 0 or 1 with the proviso that when n is 0 then R 1 is methyl; the -CH ₂ -CH (R 1) - group has the D configuration; and pharmaceutically acceptable base salts thereof a) (1) reacting a compound of formula RA-OH wherein R and A are as defined above with an aryl mercaptan or alkyl mercaptan in the presence of a condensing agent, provided that when R is R 'in the compound to be prepared, wherein R3 is R3, wherein R3 is the acid-sensitive amino protecting group known in peptide chemistry, and step (2) is performed, (2) is cleaved from the compound obtained in step (1) -341 And then reacting the resulting aryl or alkylthioester with a reagent suitable for introducing R ', followed by (3) reacting the compound obtained in steps (1) or (2) with an alkali metal hydrogen sulfide, or reagáltajuk hydrogen sulfide, (4) obtained in step (3) R-SH compound is reacted with acrylic acid or methacrylic acid, the RAS-CH ₂ - CH (R) -COOH, wherein R is hydrogen or methyl, to afford vagyülethez wherein formula with the proviso that when R 1 is methyl, step (5) must be carried out, followed by (5) resolution of the product of step (4) in a known manner to give the product wherein CH 2 -CH (CH 3) - is D- and finally (6) condensing the product of step (4) or (5) with an amino acid corresponding to R ₂ , or b) (1) reacting a compound of formula R3-A-OH wherein R3 is in the presence of a condensing agent with an aryl mercaptan or an alkyl mercaptan, followed by (2) reacting the compound obtained in step (1) with an alkali metal hydrogen reacting sulfide or hydrogen sulfide, then (3) A-R3-SH compound obtained in step (2) is reacted with acrylic acid or methacrylic acid to give AS _R3-CH2-CH (R) -COOH, a compound of formula wherein R₁ is hydrogen or methyl, with the proviso that when R 1 is hydrogen, step (4) does not have to be performed, (4) resolving the product of step (3) to give the product in which the -CH ₂ -CH (CH 3) - group has the D configuration then (5) condensing the product of step (3) or (4) with an amino acid corresponding to R ₂ , finally (6) deprotecting R 3 and then applying the product to one of the R 'groups. reacting with a reagent to give R'-AS-CH ₂ -CH (R 1) -COR ₂ , wherein in the product of this step R is R 'or (c) (1) a compound of formula RA-OH wherein R and A are reacted with the above - pentachlorophenol, pentafluorophenol or N-hydroxysuccinimide, provided that when R is R 'in the compound prepared in this step, then R is R3 in the compound of formula RA, and performing steps (2) and (3), (2) deprotecting R3 from the compound obtained in step (1), and (3) reacting the compound obtained in step (2) with a reagent suitable for introducing R ' then (4) reacting the compound obtained in steps (1) or (3) with a compound of the formula HS- (CH ₂ ) n -CH (R 1) -COOH, wherein n and R 1 are as defined above, provided that if n 1 is and R 1 is methyl step (5), (5) resolving the compound of step (4) in a known manner to give the product in which the CH ₂ -CH (CH 3) - group is in the D configuration, and finally (6) Or condensing the compound obtained in steps (4) or (5) with an amino acid corresponding to R _2; or d) (1) reacting a compound of formula RA-OH wherein R and A are as described above with hydrogen sulfide, then (2) reacting the compound of formula RA-SH obtained in step (1) with acrylic acid or methacrylic acid to give RAS-CH ₂ -CH (R 1) -COOH general The compound of Formula 10 is obtained wherein R 1 is hydrogen or methyl, provided that when R 1 is methyl, step (3), (3), the compound obtained in step (2) must be carried out in a known manner. Resolution 15 yields a product wherein the -CH ₂ CH (CH 3) - group is in the D configuration and (4) condensing the compound obtained in steps (2) or (3) with an amino acid corresponding to R ₂ , or E) (1) condensing an amino acid of the meaning given above with R ₂ with acrylic acid, methacrylic acid or a reactive derivative thereof, and then (2) reacting the H ₂ C = C (R 1) -CO-R ₂ substituent obtained in Step (1). wherein R 1 is hydrogen or methyl, is reacted with a compound of formula RA-SH, wherein R and A are as defined above, with the proviso that if R 1 is methyl, then step (3) must be performed, (3) resolving the compound of Step (2) in a known manner to provide a product wherein the -CH ₂ CH (CH 3) group is in the D configuration; obsession f) (1) an amino acid as defined in A with a reagent suitable for introducing a R group 35, whereby R and A are reacted with the above (2) thioacetic acid with acrylic acid or methacrylic acid, then the product is cleaved off with the acetyl group, (3) reacting the compound of formula RA obtained in step (1) with the compound obtained in step (2) to give RAS-CH ₂ -CH (R 1) -COOH, wherein R 1 is hydrogen or methyl; with the proviso that If Rt is methyl, then step (4) is required, (4) resolving the product of step (3) in a known manner to give the compound in which the -CH ₂ CH (CH 3) group is in the D-configuration, and finally 50 (5) the compound obtained in steps (3) or (4) R ₂ is condensed with an amino acid as defined above, or g) (1) reacting a compound of formula R-A-OH, wherein R 3 is as defined above, with an aryl mer 55 captan or alkyl mercaptan in the presence of a condensing agent, and (2) reacting the compound obtained in step (1) with an alkali metal 60 (3) obtained in step (2) R3 -A-SH compound is reacted with acrylic acid or methacrylic acid to give R3- AS-CH ₂ -CH (R i) -COOH, a compound of formula wherein R is hydrogen or methyl, with the proviso that if Ri 65 is methyl, then we have to do 35; -351 Β EN 199 786 (4), step (4) of step (3) product may be resolved in known manner to give a product in which the CH ₂ CH (CH 3) - D-configuration; (5) cleaving the R3 protecting radical, and the resulting compound is reacted with a reagent capable of introducing benzoyl reagent to give benzoyl-AS-CH ₂ CH (R) -COOH, a compound of formula and (6) to (4) or (5) - condensing the compound prepared in Step ₁ with an amino acid corresponding to R ₂ above, or h) (D) reacting an amino acid as defined above with a reagent suitable for introducing one of the above R groups, and (2) reacting a compound of formula RA prepared in step (1) with a compound of formula HS- (CH ₂ ) _n -CH (R 1) -COOH wherein n and R1 are as defined above, with the proviso that when n is 1 and R1 is methyl, then step (3) must be carried out, (3) resolving the compound obtained in step (2) in a manner known per se wherein the CH ₂ -CH (CH 3) - group is in the D configuration and (4) condensing the compound obtained in steps (2) or (3) with an amino acid as defined above for R ₂ , or i) (1) a-R4-OH compound of formula - wherein R4 is known in peptide chemistry, light-sensitive amino protecting group and A is as hereinbefore defined - of a HS- _{(CH2) n} CH (R) -COOH, compound wherein n and R 1 are as defined above, provided that when n is 1 and R 1 is methyl, then step (2) must be carried out, (2) by resolution of the compound obtained in step (1) in a known manner to give wherein the -CH ₂ CH (CH 3) - group is in the D configuration and then (3) condensing the compound obtained in steps (1) or (2) with an amino acid corresponding to R ₂ , followed by (4) reacting the compound obtained in step (3) with removing the protecting group by irradiation and finally (5) reacting the compound obtained in Step (4) with a reagent for introducing the above R group; obsession j) (1) a R 3-OH compound of formula - wherein R 3 is the above protecting group - is reacted with CH (R) -COOH, a compound of formula wherein n and R as defined above, wherein the - a HS- _{(CH2) n} provided that when n is 1 and R is methyl, it must be done in the step (2) may be resolved (2) the compound obtained in step (1) in a known manner to give a product in which the CH ₂ CH (CH 3) - The compound of configuration D, (3) is condensed with the amino acid of R _{2 as} defined above, (4) and (4) is deprotected of (3), and (5) is (4). reacting the compound obtained in Step 1 with a reagent suitable for introducing the above R group; obsession k) (1) an H3 C-CO-S- (CH _{2) n} -CH (R) - compound of formula COOH - where n and R as defined above - is reacted with 2-methyl-propene, followed by cleavage of the acetyl group, then (2 ) the compound prepared in Step (1) with a compound of formula RA-OH wherein R and A are as defined above - reacting and (3) cleaving the tert-butyl ester group from the compound RAS- (CH ₂ ) _n -CH (R 1) COOC = (CH 3) 3 obtained in step ( ₂ ), provided that if n is 1 and R 1 is methyl, then step (4) is required, (4) resolving the compound prepared in step (3) in a known manner to give the product in which the -CH ₂ -CH (CH 3) - group is in the D configuration and (5) condensing the compound obtained in steps (3) or (4) with an amino acid as defined above for R2, or l) (1) an H3 C-CO-S- (CH _{2) n} -CH (R) - COOH, a compound of formula - wherein n is as defined above and R - reacting with 2-methylpropene and cleaving the acetyl group, (2) reacting the compound prepared in Step (1) with a compound of the formula R 3 -A-OH or R 4 -A-OH, wherein R 3 and R 4 are as defined above; ) R3 or R4 deprotected RAS (CH _{2) n} -CH prepared in step (2) (R) - compound of formula 3 COOC (CH ₃₎ and (4) the compound obtained in step (3) an the Reaction with a reagent suitable for introducing R, then (5) cleaving the tert-butyl ester from the compound obtained in Step (4), provided that when n is 1 and R 1 is methyl, then (6) must be carried out. Step (6) resolving the compound prepared in Step (5) in a known manner to give the product in which the -CH ₂ - CH (CH 3) - group is in the D configuration and finally (7) in step (5) or (6). compound is condensed with an amino acid as defined above for R ₂ , obsession m) (1) a compound of the formula R 3 -A-OH or R 4 -A-OH, wherein A, R 3 and R 4 are as defined above, with N-hydroxysuccinimide, p-nitrophenol, o-nitrophenol, N-hydroxybenzotriazole, pentachloro; with phenol, pentafluorophenol, or N-hydroxy-5-norbomene-2,3-dicarboximide, the product is reacted with an alkali metal hydrogen sulfide or hydrogen sulfide, and (2) R3-A- prepared in step (1). SH or R ₄ A-SH compound is reacted with acrylic acid or methacrylic acid, to yield a compound of formula RAS-CH ₂ CH (R) -COOH, wherein A is as defined above R @ 3 or R 4 group, and R is hydrogen or methyl, with the proviso that when R 1 is methyl, step (3) must be performed, (3) resolving the compound obtained in step (2) in a known manner to give a compound wherein -CH ₂ -CH (CH 3) - group D configuration, (4) step (2) or (3) The compound obtained with condensed in _R2 above meaning amino acids, followed by cleavage (5) the compound obtained in step (4) the R3 or R4 protecting group, and finally, (6) the compound obtained in step (5) with one of the above R groups are capable of introducing a reagent; obsession -361 HU 199786 Β η) (1) reacting an amino acid as defined above with R ₂ with acrylic acid or methacrylic acid to form a compound of the formula H ₂ C = C (R 1) -CO-R ₂ , wherein R 1 is above, (2) -OH or R 4 -A-OH R 4 -A wherein A, R 3 and R 4 are as defined above, with N-hydroxysuccinimide, p-nitrophenol, o-nitrophenol, N-hydroxybenzotriazole, N-hydroxy-5- by reacting with noibornene-2,3-dicarboximide, pentachlorophenol or pentafluorophenol, the resulting product is reacted with an alkali metal hydrogen sulfide or hydrogen sulfide to give a compound of formula R3-A-SH or R4-A-SH and (3) reacting the compound of step (1) with the compound of step (2), then (4) removing the protecting group R 3 or R 4 from the compound of step (3) in a known manner, and (5) compound with a reagent suitable for introducing the above R group Alternatively, RAS-CH ₂ -CH (R 1) -CO-R ₂ is prepared wherein R 1 is hydrogen or methyl, provided that when R 1 is methyl, step (6) and (6) are required. ) resolving the product of step (5) in a known manner to give a product in which the -CH ₂ -CH (CH 3) group is in the D-configuration, or ο) (1) an H3 C-CO-S- _(CH2) "- CH (Ri) - COOH, a compound of formula - wherein n and R as defined above - is condensed with the appropriate R ₂ above report amino acid of, and (2) (1 ), the acetyl group is cleaved to give HS- (CH ₂ ) _n -CH (R 1) -CO-R ₂ , wherein R 1 is hydrogen or methyl, provided that when n is 1 and R is methyl, then step (3) must be carried out, (3) resolving the compound obtained in step (2) in a known manner to give a compound in which the -CH ₂ CH (CH 3) - group is in the D-configuration, and finally (4) reacting the compound of steps (2) or (3) with a compound of formula RA wherein R and A are as defined above; obsession p) (1) reacting a protected amino acid of formula R ₃ -R ₂ wherein R ₂ and R 3 are as defined above with a solid phase resin capable of reacting with R ₂ ; then deprotecting the resulting product R 3, then reacting (2) with R ₂ bonded to the resin in step (1) a compound of formula RA-S- (CH ₂ ) n -CH (R 1) -COOH, wherein R , n and R 1, the complex obtained above and then (3) is cleaved in a manner known per se in peptide chemistry to give R-A-S- (CH ₂ ) _n CH (R 1) -CO-R ₂ wherein R, n, R 1 and R ₂ are as defined above, with the proviso that when n is 1 and R 1 is methyl, then step (4) is required and (4) the compound obtained in step (3) is known. resolving the compound to give a compound wherein the -CH ₂ CH (CH 3) - group is in the D configuration; obsession r) (1) coupling a protected amino acid of the formula R 3 -R ₂ , wherein R ₂ and R 3 are as defined above, to a solid phase resin capable of reacting with R ₂ , then deprotecting the R 3 protecting group; ) coupling the compound obtained in Step (1) with a compound of the formula Cbo-S (CH ₂ ) _n -CH- (Rt) -COOH, where n and R 1 are as defined above, and cleaving the Cbo group from the resulting protected compound. 3) condensing the complex obtained in step (2) with a compound of the formula R3-A-OH where R3 and A are as described above, and (4) deprotecting the compound obtained in step (3), (5) (6) cleaving the complex obtained in step (5) in a manner known per se in peptide chemistry to give RAS- (CH ₂ ) _n CH (R 1) -CO-R ₂ wherein R, A, n, R 1 and R ₂ are fe nti, with the proviso that when n is 1 and R 1 is methyl, then step (7) must be performed and finally (7) by resolution of the product of step (6) in a known manner to give the product wherein -CH ₂ - The CH (CH3) group has the D-configuration; obsession s) (1) reacting the thioester of an amino acid as defined above with hydrogen sulfide to form A-SH and then (2) reacting the compound obtained in step (1) with acrylic acid or methacrylic acid to give AS-CH CH ₂ R 1 - COOH is prepared wherein R 1 is hydrogen or methyl, provided that when R 1 is methyl, then step (3), (3) of compound (2) resolving in a known manner to provide a compound in which the -CH ₂ -CH (CH 3) - group is in the D configuration and then reacting the compound obtained in steps (2) or (3) with a reagent for introducing the above R group, and finally (5) condensing the compound prepared in Step (4) with an amino acid as defined above for R ₂ ; obsession t) (l) reacting a compound of formula RA-SH wherein R and A are as defined above with a compound of formula X (CH ₂ ) _n -CH (R 1) -COOH, wherein n and R 1 are as defined above, and X is bromine, chlorine or iodine, then (2) condensing the compound obtained in Step (1) with an amino acid as defined above for R _{2 to} give RAS- (CH ₂ ) _n -CH (R 1) -CO-R ₂ wherein R, A, n, R 1 and R ₂ are hydrogen or methyl, provided that when n is 1 and R 1 is methyl, then step (3) and (3) (2) are required. ), the compound obtained in step (a) is resolved in a known manner to give a compound wherein the -CH ₂ CH (CH 3) - group is in the D-configuration; obsession u) (1) of a R3-SH compound of formula - wherein R 3 is as defined above - is reacted with acrylic acid or methacrylic acid, to give R 3 -S-CH ₂ -CH (R i) -COOH, a compound of formula wherein R is hydrogen or methyl, with the proviso that if Ri is methyl, then we need to complete step (2), -371 HU 199786 Β (2) resolving the compound obtained in step (1) in a known manner to give a compound in which the -CH ₂ -CH (CH ₃ ) - group has the D-configuration, (3) obtained in steps (1) or (2). compound is condensed with the appropriate R ₂ above meaning amino acids, and (4) removing the R ₃ protecting group of the compound obtained in step (3), then (5) the compound obtained in step (4) is reacted with an R-OH compound wherein R and A upper -; obsession v) (1) a compound of formula A-SH wherein A is reacted with acrylic acid or methacrylic acid, and (2) reacting the compound prepared in Step (1) with a reagent suitable for introducing the above R group to form a compound of formula RA-S-CH ₂ -CH (R 1) -COOH wherein R1 is hydrogen or methyl, with the proviso that when R1 is methyl, step (3) is performed, (3) resolving the compound obtained in step (2) in a known manner to give a compound wherein -CH ₂ -CH (CH ₃ ) - is in the D configuration and (4) condensing the compound obtained in steps (2) or (3) with an amino acid as defined above for R ₂ ; obsession z) (1) a compound of formula H ₃ C-CO-S- (CH ₂ ) _n -CH (R 1) - COOH - wherein n and R 1 are as defined above - reacting with o-nitrobenzyl alcohol to give H ₃ CCO-S- (CH 2) _n -CH (R 1) -COO- (o-nitrobenzyl), wherein R 1 is hydrogen or methyl, provided that if n is 1 and R 1 is methyl, then step (2) must be performed, (2) resolving the compound obtained in step (1) in a known manner to give a compound in which -CH 2 -CH (CH ₃ ) - is D- then (3) cleaving the o-nitrobenzyl group from the compound obtained in steps (1) or (2) by irradiation, then (4) condensing the compound obtained in step (3) with an amino acid as defined above for R ₂ , and (5) and cleaving the acetyl group from the compound obtained in Step (4); and (6) reacting the compound obtained in Step (5) with a compound of formula RA-OH wherein R and A are as defined above; or aa) (1) reacting a compound of the formula H 2 N- (CH 2) n -CH (R 1) -COOH, wherein Na and R 1 are hydrogen or methyl, with a compound of the formula HX, wherein X is bromine or chlorine; X- (CH ₂ ) n -CH (R 1) COOH is obtained wherein X, n and R 1 are as defined above, provided that when n is 1 and R 1 is methyl, then the starting compound is resolved in a known manner to wherein the -CH ₂ - CH (CH ₃ ) group is in the D configuration and then reacting this resolved compound with the compound of formula HX followed by (2) the compound obtained in step (1) with a compound of formula RA-SH R and A are as defined above, and (3) condensing the compound prepared in Step (2) with an amino acid as defined above for R ₂ ; or ab) (1) reacting a compound of the formula H ₂ N- (CH ₂ ) n -CH (R 1) -COOH, wherein n and R 1 are hydrogen or methyl, with a compound of the formula HX, wherein X is bromine or chlorine; to give X- (CH ₂ ) n CH (R 1) -COOH, where X, n and R 1 are as defined above, with the proviso that when n is 1 and R 1 is methyl, then the starting compound is first resolved in a known manner yielding a starting compound in which the -CH ₂ -CH (CH ₃ ) - group is in the D configuration and reacting this resolved starting compound with the compound of formula HX followed by (2) reacting the compound obtained in step (1) with thiobenzoic acid or thioacetic acid. and (3) cleaving the benzoyl or acetyl group from the compound prepared in step (2) in a known manner, and (4) reacting the compound prepared in step (3) with a reacting a needle compound wherein R and A are as defined above and (5) condensing the compound obtained in Step (4) with an amino acid corresponding to R ₂ ; or ac) reacting a compound of the formula HO- (CH ₂ ) _n -CH (R 1) -COOH, where π is the above and R 1 is hydrogen or methyl, with phosphorus tribromide to give Br- (CH ₂ ) n -CH ( R 1) -COOH is obtained, provided that when n is 1 and R 1 is methyl, the starting compound is first resolved in a known manner to give a starting compound in which - CH ₂ CH (CH ₃ ) - is D-. and then reacting this resolved compound with phosphorus tribromide, (2) reacting the compound prepared in step (1) with a compound of formula RA-SH wherein R and A are as defined above, and (3) reacting the compound obtained in step (2). reacting the compound with an amino acid as defined above for R ₂ ; or ad) (1) 3-benzoylmercapto-2-D-methylpropionic acid With 2-methylpropene, (2) cleaving the benzoyl group from the 3-benzoylmercapto-2D-methylpropionic acid tert-butyl ester obtained in Step (1), and then (3) reacting the compound obtained in Step (2) with With a compound of formula RA-OH wherein R and A are as described above, (4) thereafter from the tert-butyl ester of 3- (RA-mercapto) -2D-methylpropionic acid prepared in Step (3) - cleaving the group and (5) condensing the compound prepared in Step (4) with an amino acid as defined above for R ₂ ; or ae) reacting (1) 3-mercapto-2-D-methylpropionic acid tert-butyl ester with a compound of formula R ₃ -A-OH wherein R ₃ and A are as defined above, and (2) reacting (1) deprotecting the R ₃ protecting group from the 3- (R ₃ -A-mercapto) -2D-methylpropionic acid tert-butyl ester obtained in Step ₃ , followed by (3) reacting the compound prepared in Step (2) with a reagent for introducing the above R group. and then -381 HU 199786 Β (4) cleaving the tert-butyl ester from the 3- (RA-mecapto) -2-Dimethylpropionic acid tert-butyl ester obtained in step (3) and finally (5) the (4) condensing the compound prepared in Step 1 with an amino acid corresponding to R2 as defined above; or (f) (1) cleaving the benzoyl group from 3-benzoylmercapto-2-D-methylpropionic acid, and (2) reacting the compound prepared in Step (1) with a compound of the formula R 3 -A-OH (3) deprotecting R 3 from the 3- (R 3 -A-mercapto) -2-dimethylpropionic acid obtained in Step (2), followed by (4) reacting the compound obtained in Step (3) with one of the above R and (5) condensing the 3- (RA-mercapto) -2-D-methylpropionic acid obtained in Step (4) with an amino acid corresponding to R 2; or (b) reacting (1) 3-mercapto-2-D-methylpropionic acid with a compound of the formula R 3 -A-OH wherein R 3 and A are as defined above, followed by (2) reacting the 3- (R 3) compound of Step (1). -A-mercapto) 2-D-methylpropionic acid is condensed with an amino acid as defined above for R2, (3) deprotecting R3 from the compound obtained in step (2), and (4) reacting the compound prepared in step (3) with one of the above With a reagent suitable for introducing R; or ag2) (1) condensing a 3- (R3-A-mecapto) -2-D-methylpropionic acid, wherein R3 and A are as defined above with R2, (2) cleaving the compound obtained in step (1) to give R3; and (3) reacting the compound prepared in Step (2) with a reagent suitable for introducing the R group and optionally converting the resulting free compound into a pharmaceutically acceptable base with a salt thereof. (Priority: March 27, 1985) A process for the preparation of a compound of formula (I) and pharmaceutically acceptable base salts thereof according to claim 1, wherein L-phenylalanyl, D-phenylalanyl, D-alanyl, D-tryptophyl, D-valyl, or D-leucyl, the a-amino group of which is amide bonded to the R group , and R1, R1 and R2 and n are as defined in claim 1, characterized in that they are based on appropriately substituted materials. (Priority: March 3, 1981) A process for the preparation of a compound of formula (I) according to claim 1 wherein R, A, R 1, R 2 and n are as defined in claim 1 and when R 1 is methyl, the -CH 2 -CH (CH 3) - group is D-. configuration, characterized in that it is based on appropriately substituted materials. (Priority: March 9, 1981) A process according to claim 1 for the preparation of a compound of formula I wherein: R, A and R 2 are as defined in claim 1, n is 1 and R 1 is methyl and the -CH 2 -CH (CH 3) - group is in the D-configuration, characterized in that it is derived from appropriately substituted materials. (Priority: June 10, 1980) A process for the preparation of a salt of a compound of formula (I) with pharmaceutically acceptable bases according to claim 1, wherein R, A, R 1, R 2 and n are as defined in claim 1, characterized in that wherein R, A, R 1, R 2 and n are reacted with an organic or inorganic base as defined in claim 1. (Priority: June 5, 1980) Processes a) -z) and aa) -ac) according to claim 1 for the preparation of compounds of formula I wherein R, A, R 1, R 2 and n are as defined in claim 1, characterized in that they are suitably substituted. materials. (Priority: March 10, 1980) A process for the preparation of a compound of formula (I) according to claim 1, wherein L-phenylalanyl and R, R 1, R 2 and n are as defined in claim 1, characterized in that they are derived from appropriately substituted materials.