CZ9902807A3 - Process for preparing organic compounds, particularly carba analogs of peptides - Google Patents

Abstract

The invention relates to a process for the production of organic substances, in particular carba analogs of peptides using new derivatives cysteine, which allows the compounds to be produced creating a "karba bridge". In particular, the solution has proved its worth producing 1-desamino-1-monocarb-2-O-methyl-tyrosinoxytocin, whose net net worth was less than 2% total area of all detectable peaks. This substance does used in veterinary medicine and its new simpler way production, but much more effective than current methods production, will allow its use in human medicine.

Description

Process for the production of organic substances, in particular carba analogues of peptides

Technical field

BACKGROUND OF THE INVENTION The present invention relates to a process for the production of organic compounds, in particular carbo analogues of peptides. In their preparation, the novel compounds described below are used as intermediates for simplification.

BACKGROUND OF THE INVENTION

A key problem in the current synthesis of monocarbas compounds is the formation of so-called "carba bridges" in their molecule. This step generally has a major effect on the purity and yield of the compounds to be prepared, such as the production of the pharmaceutically used 1-desamino-1-monocarb-2-O-methyl-tyrosinoxytocin (carbetocin). Its synthesis was described in 1971 [1] and since then the original procedure has been improved several times [2], [3] both by solution and solid phase synthesis. Its syntheses have been patented. Said peptide is used in veterinary medicine.

The original syntheses are based on the use of S-benzylcysteine derivatives. The protected peptide, obtained by ammonolytic cleavage of the carrier, is subjected to sodium reduction in liquid ammonia, removing the benzyl protecting groups and replacing the 4-bromobutyric acid ethyl or t-butyl ester. After alkaline hydrolysis of the ethyl ester or acid cleavage of the t-butyl ester and cleavage of the benzyloxycarbonyl- or t-butyloxycarbonyl group from the N-terminal methyl ether of tyrosine, cyclization is carried out. In the current synthesis processes, problems arise with the yield and quality of the end product, since both of these parameters are negatively influenced by the presence of a number of impurities of different composition and amount. These serious problems are mainly due to the reduction in sodium in liquid ammonia, which is accompanied by a number of side-reactions and the composition of the by-products is unpredictable and almost uninvitable. This is not always detrimental to the purification and, last but not least, to the standard quality of the end product, which, among other things, has caused the delayed use of e.g.

SUMMARY OF THE INVENTION

The above-mentioned drawbacks are completely eliminated by the process according to the invention for the production of organic compounds, in particular carbo analogues of peptides, which comprises using a cysteine derivative of the general formula A in the construction of a linear chain in their molecule. · ··· · · ··· ·

(And where

R ¹ represents H, saturated or mono-unsaturated alkyl having a number of C 1 -C 6 carbon atoms including branched derivatives and cycloalkyl, optionally substituted benzyl with a substituent selected from H, 2- or 4-methoxy, 2- or 4-chloro and 2- or 4-nitro or 9-fluorenylmethyl,

R ² represents a saturated or mono-unsaturated alkyl having a carbon number of C 1 to C 8 including branched derivatives and cycloalkyl, optionally substituted benzyl with a substituent selected from H, 2- or 4-methoxy, 2- or 4-chloro and 2- or 4- nitro or 9-fluorenylmethyl, wherein R ¹ and R ² are different, which during the production of organic substances, in particular carbonic analogs of peptides, is altered by removal of the protecting group R ¹ and subsequent condensation to form an amidic bond organic substances, especially peptides.

The process according to the invention is further carried out by using cysteine derivatives of the general formula I for the construction of 1-carb modified disulfide bonds.

X-NH-CH-CO-Y (I). · Kde kde kde kde kde kde kde kde kde kde kde kde kde kde kde kde kde kde where

X denotes H, Boc-, Fmoc-, Boc-Tyr (Me) -Ile-Gln-Asn-, H-Tyr (Me) -Ile-Gln-Asn-, ¥ denotes -OH, -Pro-Leu-Gly -NH ₂ , -Pro-Leu-Gly-P,

Z is allyloxycarbonylpropyl, carboxypropyl, p-nitrophenyloxycarbonylpropyl, where Z is carboxypropyl or p-nitrophenyloxycarbonylpropyl, optionally forming an amide bond with one of X.

Furthermore, the process according to the invention is characterized in that the deprotection is carried out by cleaving the R ¹ in the presence of homogeneous palladium catalyst is used in an amount of 0.01 to 0.1 equivalent of the digested substance in a solvent mixture consisting of dimethylformamide, acetic acid and morpholine in volume ratio of 10: 2: 1.

According to the invention, mainly cysteine derivatives of the general formula (A) are produced, wherein R is allyl and R is t-butyl or 9-fluorenylmethyl.

The process according to the invention further comprises using cysteine derivatives of the general formula A in the preparation of 1-desamino-1-monocarb-2-O-methyltyrosine-oxytocin, which always contains the same definable amount of biologically harmless by-products less than 2% of the total area of all peaks detectable in its analysis.

The cysteine derivatives according to the invention are novel substances useful for the simple and efficient production of not only analogues of various peptides containing a disulfide bridge in their natural structure. Said derivatives allow isostemic substitution of the sulfur atom by a methylene group and are used for the relatively easy preparation of the so-called desamino-1-monocarbone analogues of peptides, which has been verified by the inventors. Carba analogs are usually more stable to enzymatic degradation and for this reason the novel cysteine derivatives of the invention may have biological properties enabling them to be used in pharmacy [1]. Karba modifications in the molecule of various natural peptide analogues can interestingly influence the profile of their biological activities.

The cysteine derivatives of the general formula (I) according to the invention are now used mainly for the preparation of 1-desamino-1-monocarba-2-O-methyl-tyrosinoxytocin. This oxytocin analog is used in veterinary medicine to treat agalactia and to influence the course of labor. It is also intended to be used for the treatment of similar indications in human medicine, in particular because it has been prepared in a very high and stable purity by the method of manufacture of the invention. In general, the cysteine derivatives of formula (I) can be used in all cases where desamino-1-carb substitution is required in the molecule.

• · · · · · · · · · · · · · · · · ·

The process for preparing the desamino-1-monocarb analogues of the invention is carried out in several stages, as shown schematically in Annex 1.

It is first necessary to prepare N-protected S- (3-allyloxycarbonylpropyl) cysteines. Reaction of L-cysteine monohydrate hydrochloride with 4-bromobutyric acid allyl ester (III) results in CysC ₃ All (IV). The amino group of this compound is protected in the next step with either a t-butyloxycarbonyl or fluorenylmethyloxycarbonyl group. The Boc-group is introduced by reaction with di-t-butyldicarbonate to form BocCysC ₃ All (V). The Fmoc-group is introduced by reaction with 9-fluorenylmethylsuccinimidyl carbonate to form FmocCysC ₃ All (Va). The cysteine derivatives of the present invention have been used primarily to produce 1-desamino-1-monocarb-2-O-methyl-tyrosine-oxytocin, as shown schematically in Appendix 2 for solid carbide bridging and in Appendix 3, where A "karba bridge" formed in reaction in solution.

As shown in Annex 2, the immobilized open chain derivative (VII) was prepared by solid-phase synthesis [4], [5] using either a cysteine derivative (V) or (Va) by the method of the invention. Both methods resulted in the same compound. The allyl protecting group was removed from the side chain with tetrakis (triphenylphosphine) palladium as a homogeneous catalyst. The result was derivative (VIII). The Boc group was removed from the N-terminal amino group with 50% trifluoroacetic acid in dichloromethane with 5% anisole. The deprotected peptidyl resin, treated with a mixture of diisopropylcarbodiimide and N-hydroxybenzotriazole in N-methylpyrrolidone, gave the immobilized cyclic product (X). The desired 1-desamino-1-monocarb-2-O-methyltyrosine-oxytocin (XI) was released from the polymer by ammonolytic ammonia gas [7] and purified by IEC and HPLC.

The method of the invention, shown in Annex 3, yielded an open chain derivative (XII) by ammonolysis of compound (VIII). The active ester (XIII) was prepared from this by reaction with p-nitrophenyltrifluoroacetate. The Boc-group was removed from the N-terminal amino group with trifluoroacetic acid with 5% anisole. The final 1-desamino-1-monocarb-2-O-methyl-tyrosine-oxytocin (XI) was prepared from the deprotected active ester (XIV) by intramolecular reaction in triethylamine / DMF solution. The crude material was purified again by IEC and HPLC.

The obtained 1-desamino-1-monocarba-2-O-methyl-tyrosine-oxytocin, prepared according to the methods of the invention described above, contained very few impurities, the composition of which, however, was validated. The quality of the final product fully complies with pharmacology requirements and articles published in PhEu.

• 9999 9 9 9 9 9 9 9 9 99 99 99 999 99 999 9999 9999

Said amount and composition of impurities provides the desired biological activity profile known for 1-desamino-1-monocarb-2-O-methyl-tyrosine-oxytocin [6].

The process of the invention can easily be used to produce industrial quantities of the desired substance with yields exceeding several times the yields of the known and used synthetic methods. All economic parameters are better than known methods.

The process of the invention may also be used to prepare other organic compounds, particularly peptides, having the same structural modification in the molecule as the compounds of the invention. This has been verified by the inventors by the preparation of a desamino-1-fragment for D-phenylalanyl-cysteinyl-phenylalanyl-D-tryptophyll-lysyl-threonyl-cysteinyl-threoninol and analogs of 1-desamino-1-monocarb-2-O-methyl-tyrosine-oxytocin. 1-desamino-1-monocarbba-2-O-methyl-tyrosine-5-D-asparagine-oxytocin and others. Details are described later in the examples.

The following examples illustrate, but are not limited to, the process for the production of organic compounds, in particular carbonic analogues of peptides by means of the novel compounds.

Example 1

Production of S- (3-allyloxycarbonylpropyl) cysteine

1777.5 g of 4-bromobutyric acid allyl ester was dissolved in 42 liters of 50% (v / v) aqueous i-butanol. The solution was vented with helium and argon. Nitrogen was then bubbled through the solution until the reaction was complete. To the solution was added 1005 g of _L-2 O.HCl Cys.H and 3,500 ml of N, Ndiisopropyletylaminu. The mixture was stirred for an additional 44 hours. The pH of the reaction mixture was maintained between 9.5 and 10 with N, N-diisopropylethylamine. The reaction was quenched by acidification to pH 6 with 2M aqueous HCl. The expected crude compound precipitated at +4 ° C overnight and was filtered off. A second fraction precipitated from the mother liquors at + 4 ° C overnight. In the same way, a third crop was obtained from the mother liquors after filtration.

The combined solids were mixed with 36 liters of diethyl ether to a homogeneous suspension after drying in air and the final material obtained after filtration and drying.

The yield of S- (3-allyloxycarbonylpropyl) cysteine was 1283 g (90.6%). Analytical properties were confirmed by H, C NMR and MS spectra. Optical rotation [α] _D ²⁰ = 15.8 ± 0.8, c = 1 (AcOH), TLC, HPLC.

• · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

Example 2

Preparation of N-t-butyloxycarbonyl-S- (3-allyloxycarbonylpropyl) cysteine

900 g All _three CYSC prepared according to Example 1 were suspended in a mixture containing 4000 ml of dioxane and 2400 ml of water. To the suspension cooled to +2 ° C, 830 ml of N, N -diisopropylethylamine and a freshly prepared solution of 1191 g of di-t-butyldicarbonate in 1470 ml of dioxane were added with stirring. The reaction mixture was rapidly warmed to 18-22 ° C, where the reaction was continued for an additional 2 hours (TLC control). The pH of the reaction mixture was maintained between 8 and 10 with N, N -diisopropylethylamine.

The solvents were evaporated under reduced pressure at + 45 ° C ± 5 ° C. The oily residue was dissolved in 6500 mL of 10% (w / v) aqueous sodium carbonate. The resulting solution was shaken 3 times with 3700 mL of diethyl ether and then acidified with saturated aqueous potassium bicarbonate solution to pH = 3. The compound was extracted 4 times with 2800 mL of ethyl acetate. The combined ethyl acetate solutions were shaken with water (2 x 4600 mL), saturated aqueous sodium chloride solution (2 x 4600 mL), dried with magnesium sulfate and, after filtration, concentrated by evaporation under reduced pressure at + 45 ° C ± 5 ° C to an oil. The crude compound was dissolved in 3700 mL of methanol and the solution was evaporated. The oil obtained was dissolved in 3700 ml of dichloromethane. The final material was obtained by evaporating the dichloromethane solution.

The yield of N 2 -butyloxycarbonyl-S- (3-allyloxycarbonylpropyl) cysteine was 1044 g (82.6%). Analytical properties were confirmed by MS spectroscopy. Optical rotation [α] _D ²⁰ = 13.0 ± 0.4, c = 1 (methanol), TLC, HPLC.

Example 3

Preparation of N-fluorenylmethyloxycarbonyl-S- (3-allyloxycarbonylpropyl) cysteine

1.98 g CYSC All ₃ prepared according to Example 1 were suspended in 80 ml of 50% aqueous acetonitrile. All _three CYSC All dissolved after the slurry was pH adjusted VV-diisopropylethylamine at 10. The solution was added 2.97 g of 9-fluorenylmetylsukcinimidyluhličitanu and reaction was performed for further 4 hours. The pH of the reaction mixture was maintained between 7.5 and 8 by N, V-diisopropy lamination years. The reaction was terminated by evaporating the solvents under reduced pressure at + 45 ° C ± 5 ° C. The oily residue was dissolved in 30 ml of water containing N, V-diisopropy years lamin (pH = 9). The resulting solution was shaken 3 times with 50 ml diethyl ether and then acidified with 1M aqueous HCl solution to pH = 3 and pH 9 9. 9 9 9 99 999 999

9 9 9 9 9 9 9 9 φ The obtained substance was extracted 3 times into 50 ml of ethyl acetate. The combined ethyl acetate solutions were shaken with water (2 x 50 mL), saturated aqueous sodium chloride solution (1 x 50 mL), dried with magnesium sulfate and, after filtration, concentrated by evaporation under reduced pressure at + 45 ° C ± 5 ° C to an oil.

The yield of N-fluorenylmethyloxycarbonyl-S- (3-allyloxycarbonylpropyl) cysteine was 3 g (79.8%). Analytical properties were confirmed by MS spectroscopy, TLC, HPLC.

Example 4

Preparation of N-t-butyloxycarbonyl-O-methyltyrosyl-isoleucyl-glutaminylasparaginyl-S- (3-allyloxycarbonylpropyl) cysteinyl-prolyl-leucyl-glycyl-copoly (1% divinylbenzene-styrene)

Boc-Tyr (Me) -Ile-Gln-Asn-Cys (S- (CH ₂ ) ₃ COOAll) -Pro-Leu-Gly-OP

Production was carried out on a hand-held synthesizer with 920 g of chloromethylated polymeric carrier esterified with Boc-Gly at a 1 mmol / g substitution [4].

Each amino acid residue was attached in a synthetic cycle consisting of deprotection, wash, neutralization, and the actual condensation reaction as shown in Table 1. All cycles were the same except for the condensation step, each using 4.5 liters of solvents or reagents . The deprotection was carried out with hydrogen chloride gas bubbled through a suspension of the carrier in dichloromethane [5].

Table 1

Step Agent Time (min) Number of operations Deprotection of the Boc group HCl / DCM 60 1 Wash DCM 2-4 3 Neutralization 10% DIPEA / DCM Mon 4 2 Wash DCM (+ DMF) 2-4 1 (+1) * Condensation active component ifcsk 60-120 1-2 Wash DCM 2-4 3

* It is necessary to wash the support once with dimethylformamide before condensing Boc-Tyr (Me) -OH.

** The condensation reaction lasts until the negative ninha of the dogwood test [4].

HOBt protected amino acid esters were prepared as follows:

• ·

0 ·

The amount of amino acid derivatives used is shown in Table 2.

Dicyclohexylcarbodiimide and N-hydroxybenztriazole were used in equimolar amounts.

Table 2 0 0 0 0 0 0 0 0

Cycle in C. Amino acid derivative Molar Mass Molar surplus DCM: DMF 1 B α-Leu-OH.H ₂ O 249.3 1,8 2 - 1 2 Boc-Pro-OH 215.3 1,8 2 - 1 3 Boc-Cys (S- (CH ₂ ) ₃ COO-allyl) - OH * 347.5 1,8 2 - 1 4 Boc-Asn-OH 232.2 2.2 1 - 1 5 Boc-Gln-OH 246.3 2.2 1 - 1 6 Boc-Ile-OH.7 ₂ H ₂ O 240.3 2,0 1 - 1 7 Boc Tyr (Me) - OH 295.4 2,0 only DMF

prepared according to Example 3.

The reaction was terminated by washing the fully protected peptidyl resin 3 times

4.5 liters of dichloromethane and drying under reduced pressure at + 40 ° C to constant weight.

The yield of Boc-CB (C3A11) -P was 1815 g (98.8%). The sequence was confirmed by amino acid analysis.

A sample of the fully protected peptide was ammonolytically cleaved from 250 mg of the compound of Example 7A. The analytical properties of the compound were confirmed by amino acid analysis and MS spectrometry, HPLC.

Example 5

Cleavage of the allyl group. Preparation of N-t-butyloxycarbonyl-O-methyltyrosylisoleucyl-glutaminyl-asparaginyl-S- (3-carboxypropyl) cysteinyl-prolyl-leucylglycyl-copoly (1% divinylbenzene-styrene)

Boc-Tyr (Me) -Ile-Gln-Asn-Cys (SO ₃ COOH) -Pro-Leu-Gly-OP

2.4 L of acetic acid was slowly added to a mixture of 12 L of DMF and 1.2 L of morpholine. 400 g Boc-CB _(C3 A11) -P prepared according to Example 4 was reacted in a reactor for solid phase washed 3 times with 3.2 1 of the above mixture. The washed peptidyl resin was then suspended in 4 L of the same mixture.

* ♦ <<<<<t t t t t t t t t t t t t t t t t t t · * · · · · · · · · ·

Tetrakis (triphenylphosphine) palladium (10 g) was added to the suspension stirred by bubbling argon. The reaction was continued under argon for a further 20 hours. Terminated by filtering the reaction solution. The resin was washed according to the following scheme:

1st wash, DCM, 3 x 1400 mL

2. neutralization, 10% N-diisopropylethylamine / DCM, 3 x 1400 mL

3. wash, DCM, 3 x 1400 mL; MeOH, 3 x 1400 mL

The yield of Boc-CB (C3OH) -P was 398 g (95.5%). The compound was analyzed by amino acid analysis.

A partially protected peptide sample was ammonolytically cleaved from 250 mg of Example 7A. The composition of the final compound was confirmed by amino acid analysis and MS spectrometry, HPLC.

Example 6

Production of crude 1-desamino-1-monocarb-2- (O-methyl) -tyrosine-oxytocin.

Cyclization on polymer support.

A. Cleavage of the t -butyloxycarbonyl group. Preparation of O-methyltyrosylisoleucyl-glutaminyl-asparaginyl-S- (3-carboxypropyl) cysteinyl-prolyl-leucylglycyl-copoly (1% divinylbenzene-styrene)

H -Tyr (Me) -Ile-Gln-Asn-Cys (S- (CH ₂ ) ₃ COOH) -Pro-Leu-Gly-OP

398 g of Boc-CB (C3OH) -P prepared according to Example 5 was charged to a solid phase reactor and treated according to the following scheme:

1st wash, DCM, 3 x 2000 mL

2. deprotection, 5% anisole in 50% trifluoroacetic acid / DCM, 3000 mL, 45 min

3. wash, DCM, 2 x 1800 mL; DMF, 2 x 1800 mL; DCM, 2 x 1800 mL

4. neutralization, 6% N, N-diisopropylethylamine / DCM, 2 x 1800 mL

5. wash, DCM, 3 x 1800 mL

B. Creation of "bridge bridge". Cyclization of O-methyltyrosyl-isoleucyl-glutaminylasparaginyl-S- (3-carboxypropyl) cysteinyl-prolyl-leucyl-glycyl-copoly (1% divinylbenzene-styrene)

398 g of NH ₂ -CB (C ₃ OH) -P prepared according to Example 6A was charged to a solid phase reactor and treated according to the following scheme:

1st wash, N-methylpyrrolidone, 3 x 2000 mL

99 9900 99

9 9 9 9 9 9 9 9 9

9999 9 999 9 9 9 9 • · · ·

9 9 9 9 9

9 9 9 9 9 9 9 9 9 9

2. 1st cyclization: A solution of 77.4 ml of diisopropylcarbodiimide and 75 g of Nhydroxybenzotriazole in 3200 ml of N-methylpyrrolidone, 20 hours

3. wash, N-methylpyrrolidone, 1 x 2000 mL

4. 2nd cyclization: A solution of 77.4 ml of diisopropylcarbodiimide and 75 g of Nhydroxybenzotriazole in 3200 ml of N-methylpyrrolidone, 20 hours

3. wash, DMF, 3 x 1200 mL; DCM, 2 x 1200 mL; MeOH, 2 x 1200 mL; DCM, 2x1200 mL

The final compound was dried under reduced pressure at +40 ° C. The yield of CBP was 368 g (99%). The sequence was confirmed by amino acid analysis.

C. Ammonolytic cleavage of polymer carrier and isolation of 1-desamino-1-monocarb-2- (O-methyl) -tyrosine-oxytocin by IEC.

368 g of CB-P prepared according to Example 6B was placed in a canvas bag and placed in a pressure vessel equipped with a pressure relief valve attached to an ammonia cylinder. The vessel was charged with ammonia (pressure 400 ± 50 kPa). The reaction was run at 18-22 ° C for 70 hours.

The peptide was obtained by extraction of the 5x21 methanol support. The combined methanolic solutions were filtered through 0.45 µm and 0.2 µm membranes and evaporated under reduced pressure to 1 L.

The methanolic solution was passed through a column packed with 1 kg of anion exchange resin, OH in methanol. The column was washed with 4x21 methanol and the resulting solution was evaporated under reduced pressure. The oily residue was dissolved in 1 L of water and passed through a 1 kg cation exchange column, H + in water. The column was washed with 4x21 water and the resulting solution was concentrated by evaporation under reduced pressure to 1.5 L. The final solution was, after addition of 80 ml of acetic acid, isolated by lyophilization.

The yield of crude 1-desamino-1-monocarbba-2- (O-methyl) -tyrosinoxytocin was 94.2 g (47.1%). The sequence was confirmed by amino acid analysis.

Example 7

Preparation of crude 1-desamino-1-monocarb-2- (O-methyl) -tyrosine-oxytocin

Cyclization in solution.

A. Ammonolytic cleavage from polymer carrier.

Preparation of N-t-butyloxycarbonyl-O-methyltyrosyl-isoleucyl-glutaminylasparaginyl-S- (3-carboxypropyl) cysteinyl-prolyl-leucyl-glycyl-amide • 0 00 «0 90 00 90 • 00 0909« «» ·

0 000 0 0 00 9 0 0 0 * · · 090 90 99 990 099 .

Boc-Tyr (Me) -Ile-Gln-Asn-Cys (S- (CH 2) 3 COOH) -Pro-Leu-Gly-NH ₂

100 g of Boc-CB (C3OH) -P prepared according to Example 5 was reacted with ammonia according to Example 6C.

The peptide was obtained by extraction of the support after ammonolysis 4 times with dimethylformamide. The combined DMF extracts were evaporated under reduced pressure at + 45 ° C ± 5 ° C to an oil from which the material was precipitated with 1.5 L of methanol. The precipitate was washed twice with 1.5 L of methanol, filtered and air dried at 18-22 ° C.

The yield of Boc-CB (C 3 OH) -NH 2 was 40.5 g (76.4%). Analytical properties were confirmed by MS spectroscopy, TLC, HPLC.

B. Preparation of N-t-butyloxycarbonyl 1-O-methyltyrosyl-isoleucyl-glutaminylasparaginyl-S- (3- [p-nitrophenyl] oxycarbonylpropyl) cysteinyl-prolyl-leucylglycyl-amide

Boc-Tyr (Me) 41e-GBI-Asn-Cys (S- _{(CH2) 3} COONp) -Pro-Leu-Gly-NH 2 g of Boc-CB (C3OH) -NH 2 prepared in Example 7A was dissolved in 1280 ml of dimethylformamide . To the nitrogen-bubbled solution was gradually added 1280 mL of pyridine and 51 g of p-nitro phenyl trifluoroacetate. The reaction was run under nitrogen at 18-22 ° C for 52 hours. The solvents were evaporated under reduced pressure at + 45 ° C ± 5 ° C to an oil. The substance was precipitated from the oil by

2.5 L of ethyl acetate. The precipitate was washed with 500 mL of ethyl acetate and 500 mL of diethyl ether. After filtration, air-dried at room temperature of 18 to 22 ° C.

The yield of Boc-CB (C ₃ ONp) -NH ₂ was 40.2 g (90.5%). HPLC.

C. Cleavage of t-butyloxycarbonyl group.

Preparation of O-methyltyrosyl-isoleucyl-glutaminyl-asparaginyl-S- (3- [p-nitrophenyl] oxycarbonylpropyl) cysteinyl-prolyl-leucyl-glycyl-amide H-Tyr (Me) -Ile-Gln-Asn-Cys (S- ( CH ₂ ) 3 COON (P) -Pro-Leu-Gly-NH ₂ g of Boc-CB (C ₃ ONp) -NH 2 prepared according to Example 7B was dissolved in a mixture of 34 ml of anisole and 640 ml of trifluoroacetic acid. The reaction was carried out at 18-22 ° C for 1 hour. The solvents were evaporated under reduced pressure at + 40 ° C ± 5 ° C to an oil from which the final material was precipitated with 2 L of diethyl ether. The precipitate was washed with 500 mL diethyl ether, filtered and air dried at 18-22 ° C.

The yield of NH ₂ -CB (C ₃ ONp) -NH 2 was 42.8 g. HPLC.

• 4 9 9 · 4 ·

9 4 4

4 9 • 4 4 44 4

9 4 · · 5 ·

· ·

D. Creation of "bridge bridge". Cyclization of O-methyltyrosyl-isoleucyl-glutaminylasparaginyl-S- (3R-nitrophenyl) oxycarbonylpropyl) cysteinyl-prolyl-leucylglycyl-amide. Cleaning with IEC.

42.4 g of NH ₂ -CB (C ₃ ONp) -NH ₂ prepared according to Example 7B was dissolved in 8 L of dimethylformamide. 24 ml of triethylamine were added to the solution with stirring. The reaction was carried out for 21 hours at 18-22 ° C. The solvents were evaporated under reduced pressure at + 40 ° C ± 5 ° C to an oil which was dissolved in 11 methanol. The solution was allowed to stand at 18-22 ° C for 1 hour and was then filtered through a 0.45 µm and 0.2 µm membrane. The filtrate was evaporated under reduced pressure at + 40 ° C ± 5 ° C to an oil and the desired material was precipitated from the oil with 2 L of ethyl acetate. The precipitate was washed with 500 mL of ethyl acetate, filtered and air dried at 18-22 ° C.

The precipitate (34.5 g) was dissolved in 70 mL of methanol. 630 ml of water was added to the solution to give a 10% (v / v) aqueous methanol solution. The undissolved fractions were filtered off. The clear solution was passed through a column packed with 1 kg of cation exchanger, EA in water, according to the procedure of Example 6C. The resulting solution was evaporated under reduced pressure at + 45 ° C ± 5 ° C to a final volume of 500 ml and filtered through a 0.45 µm and 0.2 µm membrane. From the final solution, after addition of 25 ml acetic acid, the final compound was isolated by lyophilization.

The yield of crude 1-desamino-1-monocarbba-2- (O-methyl) -tyrosinoxytocin was 27 g (78.3%). The sequence was confirmed by amino acid analysis, HPLC.

Example 8

Purification of crude 1-desamino-1-monocarb-2- (O-methyl) -tyrosine-oxytocin by HPLC.

A. Purification of said compound by cyclization on a polymeric support

A preparative HPLC column packed with a reverse ODS silica gel-based stationary phase was equilibrated with a mobile phase consisting of 85% (v) component A = 2% acetic acid in water and 15% (v) component B = 2% acetic acid in methanol. Equilibration was performed for 30 minutes at a flow rate of 50 liters per hour.

g of crude 1-desamino-1-monocarb-2- (O-methyl) -tyrosine-oxytocin prepared according to Example 6C was dissolved in 500 ml of 6% acetic acid in water and the solution was applied to the column at a flow rate of 3.5 liters per hour. Elution was performed with a mobile phase consisting of 54% (v) of component A and 46% (v) of component B at a flow rate of 50 liters per hour.

• 99 · · · · · · · · · ·

9999 9,999 · «9 9 • · 9 9 * 9,99 9» 999,999

9999 999 · · ·

The composition of the material in each fraction was determined using HPLC. Fractions containing 1-desamino-1-monocarb-2- (O-methyl) -tyrosine-oxytocin of > 96% purity were pooled, concentrated by evaporation of the solvents under reduced pressure at + 45 ° C ± 5 ° C and lyophilized.

The yield of purified 1-desamino-1-monocarbba-2- (O-methyl) -tyrosine-oxytocin was 53 g (66%). The following analytical data were obtained by analysis:

Amino Acid Analysis: Asx 0.95 - 1.05 Gly 0.95-1.05 For 0.95-1.05 Glx 0.95-1.05 Ile 0.95-1.05 Leu 0.95-1.05 Tyr 0.95-1.05 CysC ₃ 0.95-1.05

2. Optical rotation, [ot], 598 nm, c = 5 mg / ml (1% acetic acid in water)

- 83 ° to - 73 °

3. Water content: Karl Fischer, coulometric detection <6%

4. Peptide impurities: HPLC, sum of impurities <4%

5. Residual solvents: Isotachophoresis, acetic acid <5%

6. Content of 1-desamino-1-monocarb-2- (O-methyl) -tyrosine-oxytocin (free base) in% of the total weight of the substance: HPLC,> 85%

7. Content of 1-desamino-1-monocarb-2- (O-methyl) -tyrosine-oxytocin (free base): HPLC, 95-105%

B. Purification of the compound by cyclization in solution g of crude 1-desamino-1-monocarb-2- (O-methyl) -tyrosine-oxytocin prepared according to Example 7D was purified according to Example 8A.

The yield of purified 1-desamino-1-monocarb-2- (O-methyl) -tyrosine-oxytocin was 58 g (72.2%). The analytical data of the purified material was identical to that shown in Example 8.

Example 9

Preparation of 1-desamino-1-monocarb-2- (O-methyl) -tyrosine-5-D-asparagine oxytocin • 1111

Φ 11 1 1 1 11 111 111

11111111 1 ·

A. Preparation of N-t-butyloxycarbonyl-O-methyltyrosyl-isoleucyl-glutaminyl-Dasparaginyl-S- (3-allyloxycarbonylpropyl) cysteinyl-prolyl-leucyl-glycyl-copoly (1% divinylbenzene-styrene)

Boc-Tyr (Me) -Ile-Gln-D-Asn-Cys (S- (CH ₂ ) ₃ COOAll) -Pro-Leu-Gly-OP

The reaction was carried out with 2.8 g of a chloromethylated polymeric carrier esterified with Boc-Gly to 1 mmol / g substitution [4] according to the procedure of Example

4. Boc-D-Asn was used in the sequence instead of Boc-L-Asn.

The yield of Boc- [D-Asn ⁵ ] CB (C ₃ All) -P was 5.5 g (92.3%). The sequence was confirmed by amino acid analysis.

B. Cleavage of the allyl group. Preparation of N-t-butyloxycarbonyl O-methyltyrosylisoleucyl-glutaminyl-D-asparaginyl-S- (3-carboxypropyl) cysteinyl-prolyl-leucylglycyl-copoly (1% divinylbenzene-styrene)

Boc-Tyr (Me) -Ile-Gln-D-Asn-Cys (S- (CH ₂ ) ₃ COOH) -Pro-Leu-Gly-OP

4.6 g of Boc- [D-Asn ^5] CB (C ₃ ALL) -P prepared in Example 9A was deprotected according to Example fifth

The yield of Boc- [D-Asn ⁵ ] CB (C ₃ OH) -P was 4.5 g (93.8%). The sequence was confirmed by amino acid analysis.

C. Cleavage of the t -butyloxycarbonyl group. Preparation of O-methyltyrosylisoleucyl-glutaminyl-D-asparaginyl-S- (3-carboxypropyl) cysteinyl-prolylleucyl-glycyl-copoly (1% divinylbenzene-styrene)

H -Tyr (Me) -Ile-Gln-D-Asn-Cys (S- (CH ₂ ) ₃ COOH) -Pro-Leu-Gly-OP

4.5 g of Boc- [D-Asn ⁵ ] CB (C ₃ OH) -P prepared according to Example 9B was deprotected according to Example 6A.

The obtained material was used without isolation directly in the next step - Example 9D.

D. Creation of "bridge bridge". Preparation of 1-desamino-1-monocarb-2- (O-methyl) tyrosine-5-D-asparagine-oxytocinyl-copoly (1% divinylbenzene-styrene)

NH ₂ - [D-Asn ⁵ ] CB (C ₃ OH) -P prepared according to Example 9C was cyclized according to Example 6B.

The yield of [D-Asn ⁵ ] CB-P was 3.8 g (89.6%). The sequence was confirmed by amino acid analysis.

• · · · * * * * * * * * * * * * * * * * * * * *

E. Ammonolytic cleavage of polymer carrier and isolation of 1-desamino-1-monocarb-2- (O-methyl) -tyrosine-5-D-asparagine-oxytocin by IEC.

[D-Asn ⁵ ] -CB-P prepared according to Example 9D was ammonolyzed according to Example 6C.

The yield of crude [D-Asn ⁵ ] CB was 490 mg (23.7%). Analytical properties were confirmed by HPLC.

in

F. Purification of crude 1-desamino-1-monocarb-2- (O-methyl) -tyrosine-5-Dasparagine-oxytocin by HPLC.

A semi-preparative HPLC system column packed with a reverse ODS stationary phase based on silica gel was equilibrated for 7 minutes at a flow rate of 20 ml / min with a mobile phase consisting of 90% (v) component A = 0.04% trifluoroacetic acid in water and 10% (v) component B = acetonitrile.

400 mg of crude 1-desamino-1-monocarb-2- (O-methyl) -tyrosine-5-D-asparaginoxytocin prepared according to Example 9E was dissolved in 8 ml of water and applied to the column at a flow rate of 4 ml / min. Elution was performed with a mobile phase consisting of components A and B mixed according to the gradient program shown in Table 3 at a flow rate of 20 ml / min.

Table 3

Gradient program:

IN Time (min) % A % B 0 90 10 7 90 10 25 80 20 May 110 60 40 130 50 50 140 15 Dec 85

Fractions containing the desired compound were evaluated by HPLC, pooled, concentrated by evaporation of the solvents under reduced pressure at + 45 ° C ± 5 ° C and lyophilized.

The yield of purified 1-desamino-1-monocarbba-2- (O-methyl) -tyrosine-5-Dasparagine-oxytocin was 45 mg (11.2%). Analytical properties were confirmed by MS spectroscopy, chiral amino acid analysis and HPLC.

Example 10

Preparation of 1-desamino-1-monocarbba-phenylalaninyl-D-tryptophyll-lysyl-threonyl-S (3-carboxypropyl) cysteinyl-threonyl-amide

CO-NH-Phe-D-Trp-Lys-Thr-Cys-Thr-NH ₂ 1 CH ₂ -CH ₂ -CH ₂ - S-I

A. Preparation of N-fluorenylmethyloxycarbonyl-phenylalaninyl-D-tryptophyl-N ^£ (řbutyloxykarbonyl) lysyl-O-R-butylthreonyl-S- (3-allyloxykarbonylpropyl) cy steinyl-threonyl-copoly (1% di-vinyl benzene, styrene)

Fmoc-Phe-D-Trp-Lys (Boc) -Thr (tBu) -Cys (S- (CH ₂ ) ₃ COOAll) -Thr -OP

The reaction was carried out with 1.8 g of a chloromethylated polymeric carrier esterified with Boc-Thr at a substitution of 0.39 mmol / g [4].

An overview of the amino acid derivatives used for coupling is shown in Table 4. The washing, deprotection and condensation of each amino acid residue were performed in accordance with the methodology routinely used for Boc / Bzl or Fmoc / tBu solid phase peptide synthesis tactics [4] on an automatic peptide synthesizer. Condensation reactions were performed by the DIC / HOBt method. In each step, 3 equivalents of the active component in DMF were used.

Table 4

Cycle \of C. Amino acid derivative Molar mass 1 Boc-Cys (S- (CH ₂ ) ₃ All) -OH * 347.5 2 Fmoc-Thr (t Bu) -OH 397.5 3 Fmoc-Lys (Boc) -OH 468.5 4 Fmoc-D-Trp-OH 426.5 5 Fmoc-Phe-OH 387.4

prepared according to Example 3

The yield was 2.35 g (89.4%). The sequence was confirmed by amino acid analysis.

B. Cleavage of the allyl group. Preparation of N-fluorenylmethyloxycarbonylphenylalaninyl-D-tryptophyl-N ^e (t-butyloxycarbonyl) lysyl-O-t-butylthreonyl-S- (3-carboxypropyl) cysteinyl-threonyl-copoly (1% divinylbenzene-styrene) • Φ · Φ · ο · φ φ φ · · φ φ φ • • • φ φ φ φ φ φ φ φ

Fmoc-Phe-D-Trp-Lys (Boc) -Thr (tBu) -Cys (S- (CH ₂ ) ₃ COOH) -Thr -OP

2.0 g of fully protected peptide 1 resin prepared according to Example 10A was deprotected according to Example 5.

The yield was 1.9 g (95.8%).

C. Cleavage of the N-fluorenylmethyloxycarbonyl group. Preparation of phenylalaninylD-tryptophyll-N ^E (t-butyloxycarbonyl) lysyl-O-t-butylthreonyl-S- (3-carboxypropyl) cysteinyl-threonyl-copoly (1% divinylbenzene-styrene) H-Phe-D-Trp-Lys (Boc) - Thr (tBu) -Cys (S- (CH ₂ ) 3 COOH) -Thr -OP

1.9 g of N-fluorenylmethyloxycarbonyl-phenylalaninyl-D-tryptophyll-N ^E (Zbutyloxycarbonyl) lysyl-O2-butylthreonyl-S- (3-carboxypropyl) cysteinylthreonyl copoly (1% divinylbenzene-styrene) prepared according to Example 10B was deprotected in the following cycle:

1st wash, DMF, 3x20 ml

2. wash, 20% piperidine (v / v) in DMF

3. deprotection, 20% piperidine (v / v) in DMF, 20 minutes

4. wash, DMF, 3x20 ml

The obtained compound was used without isolation directly in the next step - Example 1 OD.

D. Creation of "bridge bridge". Preparation of 1-desamino-1-monocarbonyphenylalaninyl-Dtryptophyll-N ^e (t-butyloxycarbonyl) lysyl-O-t-butylthreonyl-S- (3-carboxypropyl) cysteinyl-threonyl-copoly (1% divinylbenzene-styrene)

CO-NH-Phe-DTr-Lys (Boc) -Thr (tBu) -Cys-Thr-OP I-ch ₂ -ch ₂ -ch ₂ -S-I

Phenylalaninyl-D-tryptophyll-N ^with (t-butyloxycarbonyl) lysyl-O-t-butylthreonyl-S- (3-carboxypropyl) cysteinyl-threonyl copoly (1% divinylbenzene-styrene) prepared according to Example 10C was cyclized according to Example 6B.

The obtained material was used without isolation directly in the next step - Example 10E.

E. Cleavage of t-butyloxycarbonyl and t-butyl groups. Preparation of 1-desaminol-monocarbonyphenylalaninyl-D-tryptophyll-lysyl-threonyl-S- (3-carboxypropyl) cysteinyl-threonyl-copoly (1% divinylbenzene-styrene)

CO-NH-Phe-D-Trp-Lys-Thr-Cys-Thr-OP-CH ₂ -CH ₂ -CH ₂ -S-I • · · · · · · ··· «· · · · · · · · · · · · · · · 999 · · · · · · · · · · ·

-desamino ^{-monokarba-1-phenylalaninyl-D-tryptophan-Ν} ε 1 (ίbutyloxykarbonyl) lysyl-O-R-butylthreonyl-S- (3-carboxypropyl) cysteinylthreonyl-copoly (l% divinylbenzene-styrene) prepared as described in Example 10D was stripped protecting groups in the following cycle:

1st wash, DCM, 3x20 mL

2. wash, TFA: phenol: water: EDT: TIS, 80: 5: 5: 2.5: 1 (v / v), 20 mL

3. deprotection, TFA: phenol: water: EDT: TIS, 80: 5: 5: 2.5: 1 (v / v), 20 ml, 45 minutes

4. wash, DCM, 4x20 mL; DMF, 1x20 ml; DCM, 2x20 mL

5. neutralization, 10% N, N-diisopropylethylamine in DCM (v / v), 3 x 20 mL, 5 minutes

3. wash, DCM, 3 x 20 mL; MeOH, 3 x 20 mL; DCM, 3 x 20 mL

The material was dried at 18-22 ° C. The yield was 1.56 g. The sequence was confirmed by amino acid analysis.

F. Ammonolytic cleavage from a polymeric carrier and isolation of crude 1-desamino-1-monocarbonyl-phenylalanine 1-D-tryptophylsilyl-threonyl-S- (3-carboxypropyl) cysteinyl-threonyl amide

1.5 g of 1-desamino-1-monocarbonyphenylalaninyl-D-tryptophyll-lysyl-threonyl-S- (3-carboxypropyl) cysteinyl-threonyl-copoly (1% divinylbenzene-styrene) prepared according to Example 10E was ammonolyzed according to Example 7A. The compound was precipitated from the oil obtained by evaporating the combined DMF extracts with diethyl ether. The precipitate was air dried at 18-22 ° C after filtration.

The yield of the crude material was 305 mg (75.2%).

G. Purification of crude 1-desamino-1-monocarbonyl-phenylalaninyl-D-tryptophyll-lysylthreonyl-S- (3-carboxypropyl) cysteinyl-threonyl amide by HPLC.

A semi-preparative HPLC system column packed with a reverse ODS stationary phase based on silica gel was equilibrated for 7 minutes at a flow rate of 20 ml / min with a mobile phase consisting of 90% (v) component A = 0.04% trifluoroacetic acid in water and 10% (v) component B = acetonitrile.

300 mg of crude 1-desamino-1-monocarbamphenylalaninyl-D-tryptophyllysyl-threonyl-S- (3-carboxypropyl) cysteinyl-threonyl amide prepared according to Example 10F was dissolved in 10 ml of 10% aqueous acetonitrile and the solution was applied to a column flow rate 5 ml / min. Elution was performed with a mobile phase consisting of components A and B mixed according to the gradient program shown in Table 5 at a flow rate of 20 ml / min.

··· ί · · · · · · · · ♦ · ♦ ♦ · · · · · · · · · · · · ·

Table 5

Gradient program:

Time (min) % A % B 0 90 10 30 80 20 May 200 60 40

Fractions containing the desired compound were evaluated by HPLC, pooled, concentrated by evaporation of the solvents under reduced pressure at + 45 ° C ± 5 ° C and lyophilized.

The yield of purified 1-desamino-1-monocarbonyphenylalaninyl-D-tryptophyll-lysylthreonyl-S- (3-carboxypropyl) cysteinyl-threonyl amide was 55 mg (18.3%). Analytical properties were confirmed by MS spectroscopy, chiral amino acid analysis and HPLC.

Industrial applicability

The novel cysteine derivatives according to the invention prepared by the process according to the invention can be used in particular for the preparation and production of 1-desamino-1-monocarbba- (O-methyl) -tyrosine-oxytocin, which is used to treat various gynecological disorders in veterinary medicine and now even in human medicine, since the method of the invention has greatly simplified its production and improved the quality of the final compound. Said derivatives may also be used to prepare other peptides having a typical structural carbide motif in the molecule.

• · • • •

List of abbreviations

Asn

Asx

Cys

D-Asn

Gin

Glx

Gly

Ile

Leu

Lys

Phe

For

Thr

D-Trp

Tyr

CysC ₃ All

BocCysC ₃ All

FmocCysC ₃ All

P

Boc

Fmoc

All řBu

ACN

AcOH

Asparagine

Asparagic acid, asparagine - general abbreviation

Cysteine

D-asparagine

Glutamine

Glutamic acid, glutamine - general abbreviation

Glycine

Isoleucine

Leucin

Lysine

Phenylalanine

Proline

Threonine

D-tryptophan

O-methyltyrosine

S- (3-allyloxycarbonylpropyl) cysteine

N - t -butyloxycarbonyl-S- (3-allyloxycarbonylpropyl) cysteine N-fluorenylmethyloxycarbonyl-S- (3allyloxycarbonylpropyl) cysteine Polymeric carrier

N- t -butyloxycarbonyl

N-fluorenylmethyloxycarbonyl

Allyl / butyl

Acetonitrile

Acetic acid

Boc ₂ O

DCM

DIC

DIPEA

DMF

EDT

FmocOSu

HOBt

MeOH

NMP

TEA

TFA

TFAONp

TIS

ODS

NH ₃ (g)

NMR

TLC

MS

HPLC

IEC

Boc-CB _(C3 A11) -P

Boc-CB (C ₃ OH) -P

NH ₂ -CB (C ₃ OH) -P di-Z-butyl dicarbonate

Dichlormetan

N, N-diisopropylcarbodiimide

N, N-diisopropylethylamine

Dimethylformamide

Etandithiol

9-fluorenylmethylsuccinimidyl carbonate

N-hydroxybenztriazole

Methanol

N-methylpyrrolidone

Triethylamine

Trifluoroacetic acid p-nitrophenyl trifluoroacetate

Triisopropylsilane

Octadecylsilane

Gaseous ammonia

Nuclear magnetic resonance

Thin layer chromatography

Mass spectrometry

High performance liquid chromatography

Ion exchange chromatography

Nt-butyloxycarbonyl-O-methyltyrosyl-isoleucyl-glutaminylasparaginyl-S- (3-allyloxycarbonylpropyl) cysteinyl-prolylleucyl-glycyl-copoly (1% divinylbenzene-styrene) carboxypropyl) cysteinyl-prolyl-leucylglycyl-copoly (1% divinylbenzene-styrene)

O-methyltyrosyl-isoleucyl-glutaminyl-asparaginyl-S- (322

Boc-CB (C ₃ OH) n ₂

Boc-CB (C ₃ ONp) NH ₂

NH ₂ -CB (C ₃ ONp) NH ₂

Boc- [gingival ^5] CB (C ₃ ALL) -P

Boc- [DAsn ⁵ ] CB (C ₃ OH) -P

NH ₂ - [DA 5n ⁵ ] CB (C ₃ OH) -P

CB-P [D-Asn- ⁵ ] CB-P [D-Asn- ⁵ ] -CB

PhEu carboxypropyl) cysteinyl-prolyl-leucyl-glycyl-copoly (1% di-benzene-styrene)

N-t-butyloxycarbonyl-O-methyltyrosyl-isoleucyl-glutaminylasparaginyl-S- (3-carboxypropyl) cysteinyl-prolyl-leucylglycyl-amide

N-t-butyloxycarbonyl O-methyltyrosyl-isoleucyl-glutaminylasparagines 1- S- (3- [p-nitrophenyl] oxycarbonylpropyl) cysteinylprolyl-leucyl-glycyl-amide

O-methyltyrosyl-isoleucyl-glutaminyl-asparaginyl-S- (3- [pnitrophenyl] oxycarbonylpropyl) cysteinyl-prolyl-leucyl-glycylamide

Nt-butyloxycarbonyl-O-methyltyrosyl-isoleucyl-glutaminylD-asparaginyl-S- (3-allyloxycarbonylpropyl) cysteinyl-prolylleucyl-glycyl-copoly (1% divinylbenzene-styrene) - (3-carboxypropyl) cysteinyl-prolyl-leucylglycyl-copoly (1% divinylbenzene-styrene)

O-methyltyrosyl-isoleucyl-glutaminyl-D-asparaginyl-S- (3-carboxypropyl) cysteinyl-prolyl-leucyl-glycyl-copoly (1% divinylbenzene-styrene)

-desamino-1-monocarb-2- (O-methyl) -tyrosine-oxytocinkopoly (1% divinylbenzene-styrene)

-desamino-1-monocarb-2- (O-methyl) -tyrosine-5-Dasparagine-oxytocin-copoly (1% divinylbenzene-styrene)

-desamino-1-monocarb-2- (O-methyl) -tyrosine-5-Dasparagine-oxytocin

European Pharmacopoeia Pharm · Pharm Pharm Pharm Pharm Pharm Pharm »» »» »» »» »» »» »» 4 9

References [1] Handbook of neurohypophyseal hormone analogs vol. 144, CRC Press

Boca Raton, Florida [2] CZ patent 237944; Krchnak V., Flegel M., Krojidlo M., Lebl M., Kolinsky J.

[3] CZ patent 230542; Krojidlo M., Flegel M., Lebl M., Kolinsky J.

[4] Stewart JM, Young JD: Solid Phase Peptide Synthesis, Freeman, San Francisco,

Califomia, 1969 [5] CZ patent 255500; Krchňák V., Krojidlo M., Lepša L.

[6] Handbook of Neurohypophyseal Hormone Analogs Vol. 91, CRC Press

Boča Raton, Florida [7] Flegel M., Rinnová M., Pánek Z., Lepša L., Bláha I .: Proc 14 ^th Amer. Peptide Symp.

(Mayflower Scientific Ltd., Columbus 1996)

Claims (5)

PATENT CLAIMS Process for the production of organic substances, in particular carba analogues of peptides, characterized in that a cysteine derivative of the general formula A is used in the construction of a linear chain in their molecule: R ¹ denotes H, saturated or mono-unsaturated alkyl having a number of C 1 -C ₆ carbon atoms including branched derivatives and cycloalkyl, optionally substituted benzyl with a substituent selected from H, 2- or 4-methoxy, 2- or 4-chloro and 2- or 4-nitro, or 9-fluorenylmethyl, R ² represents a saturated or mono-unsaturated alkyl having a carbon number of C 1 to C 6 including branched derivatives and cycloalkyl, optionally substituted benzyl with a substituent selected from H, 2- or 4-methoxy, 2- or 4-chloro and 2- or 4- nitro, or 9-fluorenylmethyl, wherein R and R are different, which during the manufacture of organic materials, especially carba analogues of peptides alter removing the protecting group R ¹ and the subsequent condensation of the formed amide bond closing carba bridge cycle molecule to organic substances . Process according to claim 1, characterized in that a cysteine derivative of the formula I is used in the construction of the linear chain X - NH - CH - CO - Y Oh ₂ i i (I), ··· · · 9 9 9 9 • 9 999 9 · 99 999 999 9,999,999 9 9 9 9 9 9 where X denotes Η, Boc-, Fmoc-, Boc-Tyr (Me) -Ile-Gln-Asn, H-Tyr (Me) -Ile-Gln-Asn-, Y is -OH, -Pro-Leu-Gly-NH _2, Pro-Leu-Gly-P Z is allyloxycarbonylpropyl, carboxypropyl, p-nitrophenyloxycarbonylpropyl, and when Z is carboxypropyl or p-nitrophenyloxycarbonylpropyl optionally forms an amide bond with one of X. The process according to claim 1, characterized in that the removal of the protecting group R ^{1 is} carried out by cleavage in the presence of a homogeneous palladium catalyst which is used in the reaction in an amount of 0.01 to 0.1 equivalents of the cleaved substance in the solvent mixture of dimethylformamide. acetic acid and morpholine in a 10: 2: 1 by volume ratio. The cysteine derivatives of the general formula A according to claim 1, wherein R ¹ denotes allyl and R ² denotes t-butyl or 9-fluorenylmethyl. Process according to claim 1, characterized in that the cysteine derivatives of the general formula (A) are used in the preparation of 1-desamino-1-monocarb-2-O-methyltyrosine oxytocin, which always contains the same defined amount of harmless by-products in an amount of less than 2% of the total area of all peaks detectable by analysis.