Pentapeptide, the methods of its production and peptide compounds which forproducts of pentapeptide

 

(57) Abstract:

Describes hydrochloride Pentapeptide formula (I) Me2Val-Val-MeVal-Pro-Pro-NHBzl HCl (I), where Me is methyl, Bzl is benzyl. The connection has antitumor activity. Also describes the means of obtaining it. 4 C. and 1 C.p. f-crystals.

The invention relates to new peptide exhibiting antitumor activity, more specifically to Pentapeptide, the methods of its production and peptide compounds exhibiting antitumor activity.

In the international application 93/23424 describes the active substance is a peptide-based, which have interest antitumor activities. Especially good action has Pentapeptide example 234 said application that meets the following formula:

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in which Me2Val means N,N-dimethyl-L-valine; MeVal means N-methyl-L-valine; Bzl means benzyl residue.

The peptide according to the abovementioned international application can be obtained by a solid phase method, on the basis of Proline. This forms the active substance with a small output and contaminated form. Required chromatographic purification. Solid-phase method, in addition, suitable only the application 93/23424 in crystalline form. The active substance is in the form of a resin. As a consequence difficult to complete separation of residual solvent. Require costly cleanup phase (spray drying, spray drying, freezing). Further herbal processing of substances difficult. For test and introduction requires a large number of substances.

The task of the invention to provide the above Pentapeptide in the form of a crystalline salt, hydrochloride.

The problem is solved in two ways to obtain hydrochloride of Pentapeptide formula (I)

Me2Val-Val-MeVal-Pro-Pro-NHBzl HCl, (I)

where Me denotes methyl, a Bzl is benzyl,

the first of which is that the compound of General formula (II)

Z-Val-Val-MeVal-Pro-OR1(II)

R1means alkyl with 1-5 carbon atoms, and Z means benzyloxycarbonyl protective group which can be substituted in the phenyl ring, is subjected to removal of the protective group Z on the terminal amino group, the resulting compound is subjected to demethylation at the free terminal amino group and subsequent saponification CNS group OR1on the terminal carboxyl group and the resulting compound of formula (V)

Me2Val-Val-MeVal the chloride and polybenzimidazole with subsequent transfer to the hydrochloride, and the second that the compound of General formula (II)

Z-Val-Val-MeVal-Pro-OR1(II)

where R1means alkyl with 1-5 carbon atoms, and Z means benzyloxycarbonyl protective group which can be substituted in the phenyl ring, is subjected to the saponification CNS group OR1at the end carboxyl group of the resulting compound of formula (IX)

Z-Val-Val-MeVal-Pro-OH, (IX)

where Me is the above value,

sequentially subjected to interaction with revalorisation and polybenzimidazole and the obtained compound of the formula (XI)

Z-Val-Val-MeVal-Pro-Pro-NHBzl, (XI)

where Me is the above value,

subjected to removal of the protective group Z on the terminal amino group and subsequent demethylation of free terminal amino group and saponification CNS group or SIG1on the terminal carboxyl group and the resulting compound is transferred into the hydrochloride.

The first way, which is hereinafter referred to as "method A", proceeds according to the following reaction scheme A:

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Shown in scheme A, the radicals R1, R2, Z, Me and Bzl have the above values, Mmeans the potassium ion, sodium ion, lithium ion or ammonium ion, as triethylamine.

Complex tetrapeptides ether of the formula (II) is dissolved in a suitable solvent, such as alcohol, like methanol, ethanol, isopropanol, butanol; in simple ether like tetrahydrofuran, dioxane, methyl-tert.-butyl ether; ether complex as ethyl acetate; or in glacial acetic acid. After the addition of suitable catalyst, such as palladium-on-coal-or platinum-on-charcoal, at temperatures in the range of 0oC to 50oC, preferably in the range from 10oC to 30oC, miss hydrogen. The introduction of hydrogen can be carried out at normal pressure or at elevated pressure up to 10 bar. The reaction can be accelerated by assuming a known current to the exhaust gas. After the hydrogen absorption add 2-5 equivalents of formaldehyde in aqueous solution or in gaseous form or in the form of paraformaldehyde. Then enter the hydrogen under the above described conditions. Then the catalyst is filtered off. Rastvoritelei. This is a useful mixture of isopropanol with methyl-tert.-butyl ether. Trace amounts of complex Z-tetrapeptide ether of the formula (II) present in the compound of formula (IV) can be removed also by using extractive separation processes.

Saponification of ester of the formula (IV) is carried out in a suitable solvent, such as alcohol, like methanol, ethanol, isopropanol; in simple ester as methyl-tert.-butyl ether, tetrahydrofuran, dioxane; hydrocarbons like toluene, xylene; or a chlorinated hydrocarbon, as 1,2-dichloroethane, methylene chloride, chloroform, with the addition of water or without the addition of water and using a suitable base, like sodium hydroxide, potassium hydroxide, lithium hydroxide. Cleavage of ester can be carried out using acids. In the case where R1means tert.-butyl, for this purpose, especially suitable triperoxonane acid and a solution of hydrogen chloride in dioxane.

Received tetrapeptide acid of formula (V) next you need to associate with polybenzimidazole formula (VIII) to obtain Pentapeptide formula (I). When such coupling reaction easily proceeds racemization. So G. Pettit and others (J. Am. Chem. Soc. 113. 6692-6693 (1991) for similar binding of storecanada in large enough quantities in stock. Because this method requires additional stages of the method using poisonous phosphorus and cyanide reagents. Landstrasse waste cause problems in relation to environmental pollution. Therefore, the method is unsuitable for implementation. Method binding peptide, which can be particularly easy to implement on an industrial scale, is the method of the mixed anhydride (see, for example, J. Meienhofer in "The Peptides, Analysis, Synthesis, Biology", vol. 1, Academic Press, Orlando, 1979, S. 264 -314). The acid of formula (V) deprotonated using a suitable base, e.g. a tertiary amine like triethylamine, N-methylmorpholine, dicyclohexylamine, aminobutiramida ethylamine with obtaining the compounds of formula (VI). Esters of the formula (IV) by using the bases as sodium hydroxide, potassium hydroxide, lithium hydroxide, can be directly converted into salts of the formula (VI). The compounds of formula (VI) by entering into interaction with the acid chloride of the acid ClCOR1turn into a mixed anhydride of the formula (VII). Along with revalorisation it is also possible to use other acid anhydrides, such as, for example, the acid chloride of 2-ethylhexanoic acid, ethyl ester Harborview acid, methyl ester chloraniline (see, for example, J. Meienhofer in "The Peptides", volume 1, Academic Press, Orlando, 1979, S. 276 and later).

Currently, it was unexpectedly found that tetrapeptide acid of formula (V) can be transformed according to the method of the mixed anhydride is completely without racemization. Especially good results are achieved with the mixed anhydride, which is obtained from the acid of formula (V) and pivaloyloxy. In contrast, more recent published results (N. L. Benoiton, and others, Can. J. Chem., 65, 619-625 (1987)) interaction with revalorisation, in terms of selectivity and yield, gives better results than the interaction with the esters of Harborview acid. Getting mixed anhydride of the formula (VII) and subsequent binding to polybenzimidazole carried out at temperatures from -20oC to +5oC in a suitable solvent, like dioxane, N-organic, tetrahydrofuran, toluene, methylene chloride, dimethylformamide. Instead of polybezierto formula (VIII) can also be used suitable salt of this compound, as, for example, hydrosulfate, methylsulfonate, hydrochloride or hydrobromide. When you do this then you need to add a further equivalent of a base, such as triethylamine. After made binding peptide and Obi toluene, xylene; or a simple ester as diethyl ether, tetrahydrofuran, dioxane, methyl-tert.- butyl ether; a ketone, such as acetone, methyl ethyl ketone, diethylketone, cyclohexanone; or in chlorinated solvents, such as methylene chloride, chloroform, 1,2-dichloroethane. By introducing gaseous hydrogen chloride or adding a solution of hydrogen chloride in a suitable solvent, such as tetrahydrofuran, methanol, isopropanol, n-pentanol, diisopropyl ether, precipitated hydrochloride of the formula (I). Particularly suitable is a technique in which the Pentapeptide in the form of a free base is first dissolved in methyl ethyl ketone and then add a solution of hydrogen chloride and isopropanol.

The second method, which is hereinafter referred to as method B, proceeds according to the following reaction scheme B:

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Saponification of ester of the formula (II), obtaining a mixed anhydride of the formula (X) and the binding of the peptide to obtain the compounds of formula (XI) is carried out by analogy with the sequence of the synthesis of IV---> V---> VI---> VII---> I. Cleavage of the protective group Z and demethylation to obtain the compounds of formula (I) is carried out by analogy with the transformation of II---> III---> IV.

Also in the case of the variant of method B method smexeh outputs reach a mixed anhydride, which is derived from the acid of formula (IX) and pivaloyloxy.

Necessary to obtain a peptide of formula (I) parent compound of formula (II) can be obtained from Z-Val-O-CO-R2(XII) and Val-MeVal-Pro-OR1(XIII).

On the proposed methods A and B active substance of the formula (I) are obtained in crystalline form. The peptide can be cleaned just by recrystallization. Mandatory chromatographic purification stages is not required.

The invention relates also to the following forproducts to obtain the compounds of formula (I):

Z-Val-Val-MeVal-Pro-OR1; (II)

Val-Val-MeVal-Pro-OR1; (III)

Me2Val - Val-MeVal-Pro-O-R1; (IV)

Z-Val-Val-MeVal-Pro-OH; (IX)

Z-Val-Val-MeVal-Pro-Pro-NHBzl, (XI)

in which R1, R2and Z have the above values.

The compound of formula (I) is effective against solid tumors (tumors of the lung, chest, intestines, bladder and gallbladder, rectum, uterus, prostate) against leukemia, lymphoma and other neoplastic diseases.

Antitumor activity hydrochloride Pentapeptide formula (I) is illustrated by the following experiments.

Experience 1 (in vitro)

Cytotoxicity was determined using the standard methodology, iseem tetrazolium connection test (MTT). The details of this experience was published (see Alley, MS and others, Cancer Research 48, pp. 589-601, 1988). Exponentially growing cultures of cancer cells, such as cells HT-29 colon cancer or LX-1 lung cancer used to prepare cultures grown in microtiter plates. Cells were inoculated in an amount of 5000-20,000 cells per cell in 96 - cellular plate (150 μl medium) and grown over night at 37 ° oC. was Added investigated the connection in a 10-fold dilution, and concentration was varied in the range from 10-4M to 10-10M. the cells are Then incubated for 48-72 hours. To determine the number of viable cells in each cell, was added 50 μl of a solution with a concentration of 3 mg/ml bromide 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium in saline solution. The resulting mixture is incubated at 37oC for 5 hours and then in each cell was added 50 μl of 25% sodium dodecyl sulfate, pH 2. After incubation overnight, the absorption at 550 nm of each cell was measured in the standard unit is the meter used in the case of enzyme linked immunosorbent assay. The average percentage of activity of the compounds according to the four cells (+/-standard OTC what

The concentration of tested compound providing 50% inhibition of cell growth was 3 x 10-10.

Experience II (in vivo)

The compound of the present invention was tested in pre-clinical experience on activity in vivo, which is very revealing about the usefulness of the substance in clinical practice. This experiment was carried out on shaved mice, in which are embedded tumor tissue, preferably from the human body, by transplantation of well-known techniques. Antitumor activity of the compounds was determined on the treated mentioned manner mice. The human breast tumor (MX-1) grown on shaved mice deprived of thymus, transplanted to a new recipient animals, using fragments of the tumor, the size of which about 50 mg. a Day of transplantation was defined as day 0. After 6-10 days to mice, divided into groups of 5-10 animals were given an investigational compound, is introduced intravenous or intraperitoneal way through the shot. The connection was given in doses of 25 mg/kg of body weight three times a week for 3 weeks. The diameters of the tumors and the weight of the bodies of the animals were measured twice a week. The volume of tumor was calculated using the values of the diameters, Izmir CLASS="ptx2">

The average volume of the tumors was calculated for each treated group and the determined values of A, defined above. Activity assessed as follows. The value of A equal to 1.0 or greater indicates that the compound did not have an impact on tumor growth, whereas the value of < 1.0 indicates a slight reduction in the tumor mass. Values of 0.15-0.49 can be seen as a reflection of the moderate slowdown in activity, < 0.01-0.14 - good to excellent activity.

Under the above conditions the investigated connection provides complete degeneration of the tumor disappeared cancerous mass after therapy).

Hydrochloride Pentapeptide formula (I) belongs to the category of low-toxic substances.

The following examples illustrate how the proposed methods.

Example 1 (method A)

A. Receipt of parent compounds

a. Z-Val-Val-MeVal-Pro-OMe (II; R1= Me)

In a reactor for the hydrogenation capacity of 400 liters contribute to 39.6 kg (83,3 mole) of Z-Val-MeVal-Pro-OMe (VIII) 320 l of methanol together with 4 kg of a 5% palladium-on-charcoal grill. Then when cooled at 20-30oC miss hydrogen up until in the reaction solution is no longer possible to detect any of the educt. With the release of sleep reactor with a capacity of 400 l in water-jet vacuum pump up to a volume of 50 L. Then poured 50 l of toluene and extracted with 40 l of 2n. of hydrochloric acid. Toluene phase again additionally extracted with 40 l of 1N. hydrochloric acid and then drained. The combined acidic aqueous phase contribute back to the reactor, add 40 l of methylene chloride in the form of lower layer, and then by prilipanie 50%- aqueous sodium hydroxide solution, under vigorous stirring and cooling, set pH 9. After separation of the phases methylenchloride phase is drained, and the aqueous phase is additionally extracted twice more in 40 l of methylene chloride. United methylenchloride the solution is washed with water until neutral. After that methylenchloride phase is concentrated to a volume of 90 liters Receive Val-MeVal-Pro-OMe (XIV; R1= methyl).

The output is 24.2 kg, respectively, of 85.2%.

In the reactor with a capacity of 400 l 17,84 kg (to $ 70.88 mol) of Z-valine and 4.59 kg (74,42 mol) of triethylamine are dissolved in 170 l of methylene chloride. To this solution at a temperature of from -5oC to -10oC add 8,58 kg (to $ 70.88 mol) of acid chloride of pavlinovoi acid. After 2 hours reaction time at -5oC, at a temperature of -5oC poured the solution 24,2 kg Val-MeVal-Pro-OMe 86 l of methylene chloride. Later, following 2 hours of incubation at s 50 l of water. After separation of the aqueous phase the organic phase is extracted once with 40 l of 2n. hydrochloric acid and twice with 40 l each time 2n. the sodium hydroxide solution. After washing the organic phase with water to neutral reaction, the methylene chloride is distilled off and replaced with 300 l of diisopropyl ether. For crystallization of the product emulsion in the form of oil product heated to 60oC, is mixed with the seed crystals and incubated for 7 hours at 60oC. To complete the crystallization continue to stir consistently for 5 hours at 50oC and for 5 hours at 40oC and then cooled to 20oC. Suspension of crystals is filtered through a pressure filter capacity 120 l and dried in a stream of nitrogen.

The output is 32.2 kg, respectively, 79%.

Melting point is 134-135oC.

B. Hydrochloride polybezierto (XII HCl)

To a solution of 99.7 g of Z-Proline and 58 ml of triethylamine in 1 l of methylene chloride at a temperature of from -10oC to -15oC was added dropwise 48,2 g of acid chloride of pavlinovoi acid. Stirred additionally for 45 minutes at -10oC and then within half an hour at a temperature of -10oC we use the re. Methylenchloride the solution is then washed with 500 ml of water, twice with 500 ml of 10% aqueous solution of sodium bicarbonate, twice with 500 ml of water, twice with 500 ml of 5% aqueous citric acid solution and twice with 500 ml of water, dried over sodium sulfate and evaporated. Remain 120 g of residue which is dissolved in 200 ml of ethyl acetate. To an ethyl acetate solution was added to 1.2 l of n-heptane, stirred for one hour, the solid is sucked off and dried at -50oC in vacuum.

The output is 110 g, respectively, 81,3%.

Melting point is 93-94oC.

110 g of the thus obtained Z-polybezierto dissolved in 1.5 l of methanol. After adding 0.5 g of 10% palladium-on-coal-miss hydrogen. At room temperature for one and a half hours, the solution absorbs 0.5 liters of hydrogen. After filtering off the catalyst and evaporating remain 4.6 g of yellow oil.

413 g thus obtained polybezierto dissolved in 400 ml of isopropanol. Add 630 ml of a saturated solution of hydrogen chloride in isopropanol, mix the resulting suspension for two hours at a temperature of from 0oC to 5oC, and acidified with DV the reed of polybezierto;2D0is - 45o.

B. Obtaining the target product

A. 1 Me2Val-Val-MeVal-Pro-OMe HCl (HCl IV; R1= Me)

In a reactor for the hydrogenation capacity of 400 liters made of 20 kg (34.8 mol) of Z-Val-Val-MeVal-Pro-OMe (II; R1= methyl) together with 2 kg of a 5% palladium-on-coal in 200 l of methanol. Then when cooled at a temperature of 20oC miss hydrogen up until in the reaction solution is no longer possible to detect any of the educt. Then add 8,46 kg (104 mol) of 37% aqueous formaldehyde solution and continue to gidrirovanii at 20oC until the cessation of hydrogen absorption. With the release of the contents of the reactor the catalyst is filtered off. To perform processing concentration in enameled reactor with a capacity of 400 l in water-jet vacuum pump up to a volume of 50 L. Then add 200 l of isopropanol and again concentrated to a volume of 50 L. then dissolved in 135 l of methyl-tert.-butyl ether and cooled at 20oC add one equivalent isopropanolic solution of hydrogen chloride. The resulting suspension is stirred further for another 3-4 hours at 20oC and 2 hours at a temperature of from 0oC to 5oC and then filtered through a pressure filter capacity 120 HP On the place of 16.2 kg accordingly, 92,3%.

The melting temperature is 224oC (decomposition).

A. 2 there are Also intermediate connection Val-Val-MeVal - Pro-OMe (III; R1= methyl), when after the first stage hydrogenation treatment is carried out as follows:

The reaction solution is separated from the catalyst and concentrated. The residue is dissolved in ethyl acetate. An ethyl acetate solution is extracted twice with 2n. of hydrochloric acid. In the acidic aqueous phase establish a pH-value of 9 using sodium hydroxide solution and extracted twice with methylene chloride. Methylenchloride phase is then washed until neutral and evaporated.

High-performance liquid chromatography (HPLC):

the content of the compound is 96.8%,

1H-NMR (400 MHz, deuterochloroform, tetramethylsilane was as internal standard):

(M. D.); 0,84-1,08 (m, 18 H); 1,45-1,6 (C., widen, ); 1.85 to to 2.15 (m , 4H); 2,18-of 2.38 (m, 3H); 3.15 in (C., ); 3,25 (D., 1 H); 3,65 of 3.75 (m , 1H); to 3.73 (C., ); 3,9-of 4.05 (m, 1H); of 4.38 is 4.45 (m, 1 H); 4,73 of 4.83 (m, 1H); 5,12 (D., 1H): 7,9 (D., ).

A. 3 Me2Val-Val-MeVal-Pro-OMe HCl (HCl IV; R1= Me) can also be obtained by the following method, which is not necessary to isolate and purify the intermediate compound Z-Val-Val-MeVal-Pro-OMe (II; R1= Me).

The output is 182,8 g, respectively, 71%.

The melting temperature is 224oC (decomposition).

B. Me2Val-Val-MeVal-Pro-Pro-NHBzl HCl (I)

In a reactor with a capacity of 400 l pre contribute 15.9 kg (31,5 mol) Me2Val-Val-MeVal-Pro-OMe HCl (HCl IV; R1= methyl) with 140 l of toluene and 15 l of methanol. To the resulting mixture are added 3.15 kg (76,38 mol) of sodium hydroxide tablets. After complete saponification, i.e. after 3 hours at 20oC, are neutralized by adding isopropanolic solution of hydrogen chloride. Then carry out azeotropic distillation with toluene at a pressure of 100 mbar until the liberation of alcohol and water. Distilled solvent is gradually replaced by toluene. Then add 80 l of methylene chloride and 6,44 kg (63.0 mol) of triethylamine (content: 99%), cooled to -5oC and at this temperature, add 3.84 kg (31,5 mol) of acid chloride of pavlinovoi acid. After reaction for two hours at a temperature in the range from -5oC and 0oC portions add (7.6 kg 31,5 mol) Pro-NHBzl HCI. After standing for two hours at -5oC is heated to 20oC and left to react for six hours. Then at a pressure of 500 mbar is enableval pH-value, equal to 9. After intensive mixing of the aqueous phase is separated and the organic phase is optionally washed once with 25 l of water. Then the organic phase is extracted with twice 50 l 2n. of hydrochloric acid. The product is again extracted from the acidic aqueous phase, after the establishment of the pH-value of 9 by triple extraction with 50 l of methylene chloride each time. After washing methylenchloride phase with water to neutral reaction, the methylene chloride is distilled off and replaced with 180 l of methyl ethyl ketone. The solution is heated to 40oC and mixed with one equivalent of 31.5 mol) isopropanolic solution of hydrogen chloride. The resulting suspension is heated to 60oC and then without further heating is stirred for 12 hours. Then cooled to 20oC and stirred for another 5 hours. Then cooled to 5oC and filtered through a pressure filter capacity 120 liters of the Precipitate on the filter is washed with 60 l of fresh, chilled to 5oC, methyl ethyl ketone. After pre-drying on the filter, the product is dried in a vacuum drying Cabinet at 40oC to constant weight.

The output is 14,36 kg, respectively, 67%.

The melting point of the state-OH (IX)

In the flask with a capacity of 2 l 117 g (0.2 mole) of Z-Val-Val-MeVal-Pro - OMe (II; R1= Me, example 1Aa) is dissolved in 900 ml of methanol and of 47.5 ml of water. Then add 18 g (0.45 mol) of sodium hydroxide in pellets and stirred for 12 hours at 20oC. For processing add 250 ml of distilled water and methanol. Then add enough ethyl acetate to happen a clear separation of the phases (approximately 500 ml). An ethyl acetate phase is separated. The aqueous phase is acidified to pH-values equal to 1, and extracted twice with 500 ml of methylene chloride. The organic phase is then evaporated to dryness.

The output is 105 grams, respectively, 96,4%.

1H-NMR (200 MHz, deuterochloroform, tetramethylsilane was as internal standard):

(M. D.): 0,6-1,2 (m, 18H); 1,7-of 2.45 (m,7H); 3,2 (C., ); 3,55-3,95 (m , 2H); 4,05-4,2 (m, 1H); 4,35 to 4.5 (m, 1H); 4,68-is 4.85 (m, 1H); 4,98 with 5.2 (m , 3H); 5,93 (doctor, Val-1 ); to 7.2 to 7.4 (m, 5H); 7,53-7,68 (Val-2 ); from 9.6 to 10.2 (C., widen, ).

B. Z-Val-Val-MeVal-Pro-Pro-NHBzl (XI)

5 g of Z-Val-Val-MeVal-Pro-OH (8,75 mmol) (IX) is dissolved in 50 ml of methylene chloride, was added dropwise to 1.79 g (17.5 mmol) of triethylamine, cooled to 10oC and at this temperature was added dropwise 1,08 g (is 8.75 mmol) of the acid chloride pavlinovoi acid. After stirring for two hours at 10oC at this temperature was added dropwise a solution 2,11 g (is 8.75 mmol) is room temperature.

The reaction mixture is washed three times with 50 ml water, once with 50 ml of water at a pH-value of 9, and again twice with 50 ml of water. Methylenechloride solution processed on a rotary evaporator. As the residual gain of 5.3 g (81,4%) of white crystalline product (purity: 88,7%).

Melting point is 118-122oC.

C. Me2Val-Val-MeVal-Pro-Pro-NHBzlHCl (I)

12 g of Z-Val-Val-MeVal-Pro-Pro-NHBzl (XI) is dissolved in 200 ml of methanol. To the resulting solution was added 2 g of a 5% palladium-on-coal (suspended in 20 ml of water) and hydronaut at 20oC until the cessation of hydrogen absorption. Then add 6.5 g of 37% aqueous formaldehyde solution and hydronaut forth until the cessation of hydrogen absorption. Then the catalyst is separated and the reaction solution is evaporated. The residue is treated with toluene and again concentrated, again mixed with 200 ml of toluene and filtered. Toluene solution is then extracted twice with 50 ml of 2n. of hydrochloric acid. An acidic aqueous phase is brought to pH 9 using sodium hydroxide solution and extracted with three times 50 ml of methylene chloride. Methylenchloride phase is washed with water until neutral and concentrated. The crude base is dissolved in a mixture of 150 ml matile the Le at 40oC precipitated the product as a salt. The suspension is additionally stirred for three hours at 20oC and for one hour at a temperature of from 0oC to 5oC and then sucked off.

The output is 7.1,

The content of the compound is 99.1% (area percentage according to HPLC).

Melting point is 214oC (decomposition). []2D0- 180,3o.

1. Hydrochloride Pentapeptide formula I:

Me2Val-Val-MeVal-Pro-Pro-NHBzlHCl,

where Me is methyl;

Bzl is benzyl.

2. Pentapeptide under item 1, available in crystalline form.

3. The method of producing Pentapeptide formula I:

Me2Val-Val-MeVal-Pro-Pro-NHBzlHCl,

where Me is methyl;

Bzl is benzyl,

characterized in that the compound of General formula II

Z-Val-Val-MeVal-Pro-OR',

where R' is alkyl with 1-5 carbon atoms;

Z - benzyloxycarbonyl protective group which can be substituted in the phenyl ring,

subjected to removal of the protective group Z on the terminal amino group, the resulting compound is subjected to demethylation at the free terminal amino group and subsequent saponification CNS group - OR' on the terminal carboxyl group and the resulting connection is up interaction with revalorisation and polybenzimidazole with subsequent conversion of the resulting compound in hydrochloride.

4. The method of producing Pentapeptide formula (I)

Me2Val-Val-MeVal-Pro-Pro-NHBzlHCl,

where Me is methyl;

Bzl is benzyl,

characterized in that the compound of General formula (II)

Z-Val-Val-MeVal-Pro-OR' (II)

where R' is alkyl with 1-5 carbon atoms;

Z - benzyloxycarbonyl protective group which can be substituted in the phenyl ring,

subjected to saponification CNS group - OR' at the end carboxyl group of the resulting compound of formula (IX)

Z-Val-Val-MeVal-ProOH (IX)

where Me is the specified value,

sequentially subjected to interaction with revalorisation and polybenzimidazole and the obtained compound of the formula (XI)

Z-Val-Val-MeVal-Pro-Pro-NHBzl (XI)

where Me is the specified value,

subjected to removal of the protective group Z on the terminal amino group and subsequent demethylation of free terminal amino group and saponification CNS group - OR' at the end of the carboxyl group and the resulting compound is transferred into the hydrochloride.

5. Peptide compounds selected from the group comprising Z-Val-Val-MeVal-Pro-OR'; Val-Val-MeVal-Pro-OR'; Me2Val-Val-MeVal-Pro-OR'; Z-Val-Val-MeVal-Pro-OH; Z-Val-Val-MeVal-Pro-NHBzl, where Me, Z and R' are specified in paragraph (3 values representing forproduct for receipt of the Sabbath.

 

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in which Rameans a hydrogen atom or a hydroxyl radical, CNS radical with 1-4 carbon atoms, acyloxy radical with 1-4 carbon atoms or alkoxyacetic-radical, the alkyl part of which contains 1-4 carbon atoms;

Rbmeans a hydrogen atom; or

Raand Rbtogether with the carbon atom to which they are bound, form a ketone function;

Z means a hydrogen atom or a radical of General formula (II)

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in which R1means bentely radical, possibly substituted by one or more identical or different atoms or radicals selected among halogen atoms and alkyl radicals with 1-4 carbon atoms, CNS radicals with 1-4 carbon atoms or trifloromethyl; tenor or furoyl, or the radical R2-O-CO-, in which R2means:

is an alkyl radical with 1-8 carbon atoms, alkanniny radical with 2-8 carbon atoms, alkynylaryl radical with 3-8 carbon atoms, cycloalkenyl radical of 3-6 carbon atoms, cycloalkenyl radical with 4 to 6 atoms in or more substituents, selected among halogen atoms and hydroxyl radical, CNS radical with 1-4 carbon atoms, dialkylamino radical, each alkyl portion of which contains 1-4 carbon atoms, piperidino radical, morpholino radical, piperazine-1-ilen radical (possibly substituted in position 4 alkyl radical with 1-4 carbon atoms or phenylalkyl radical, the alkyl part of which contains 1-4 carbon atoms), cycloalkyl radical of 3-6 carbon atoms, cycloalkenyl radical with 4 to 6 carbon atoms, phenyl radical (possibly substituted by one or more atoms or radicals, selected among halogen atoms and alkyl radicals with 1-4 carbon atoms or CNS radicals with 1-4 carbon atoms), a cyano radical, carboxyl or alkoxycarbonyl, the alkyl portion of which contains 1-4 carbon atoms;

is phenyl or- or-nattily radical, possibly substituted by one or more atoms or radicals selected among halogen atoms and alkyl radicals with 1-4 carbon atoms or CNS radicals with 1-4 carbon atoms; or a heterocyclic aromatic patterollers radical with 4 to 6 carbon atoms, possibly substituted by one or more alkyl radicals with 1-4 carbon atoms;

R3means a linear or branched alkyl radical with 1-8 carbon atoms, a linear or branched alkanniny radical with 2-8 carbon atoms, linear or branched alkynylaryl radical with 2-8 carbon atoms, cycloalkyl radical of 3-6 carbon atoms, phenyl or- or-nattily radical, possibly substituted by one or more atoms or radicals selected among halogen atoms and alkyl, alkenyl, etkinlik, aryl, kalkilya, CNS, alkylthio, aryloxy, aaltio-, hydroxyl, hydroxyalkyl, mercapto-, formyl, acyl, acylamino, aroylamino, alkoxycarbonyl-, amino-,

alkylamino, dialkylamino-, carboxyl, alkoxycarbonyl, karamolegos, alkylcarboxylic, dialkylanilines, cyano-, nitro - and triptorelin radicals; or an aromatic five-membered a heterocycle containing one or more, identical or different heteroatoms selected among nitrogen atoms, oxygen or sulfur, and possibly substituted by one or more of the ylamino - dialkylamino-, alkoxycarbonyl-, acyl, arylcarboxylic, carboxyl, karamolegos, alkylcarboxylic, dialkylanilines or alkoxycarbonyl radicals, provided that the substituents phenyl,- or-afternova and aromatic heterocyclic radicals, the alkyl radicals and alkyl portions of other radicals contain 1-4 carbon atoms; alkeneamine and alkyline radicals contain 2 to 8 carbon atoms, and aryl radicals are phenyl or- or-raftiline radicals; and

R4means:

- linear or branched alkyl radical with 1-8 carbon atoms, a linear or branched alkanniny radical with 2-8 carbon atoms, linear or branched alkynylaryl radical with 2-8 carbon atoms, cycloalkyl radical of 3-6 carbon atoms, cycloalkenyl radical with 4 to 6 carbon atoms or bicycloalkyl radical with 7 to 11 carbon atoms, and each of these radicals may be substituted by one or more substituents selected among halogen atoms and hydroxyl radical, alkoxyl carbon piperidino radical, morpholino radical, piperazine-1-ilen radical (possibly substituted in position 4 alkyl radical with 1-4 carbon atoms or phenylalkyl radical, the alkyl part of which contains 1-4 carbon atoms), cycloalkyl radical of 3-6 carbon atoms, cycloalkenyl radical with 4 to 6 carbon atoms, possibly substituted phenyl radical, cyano radical, carboxyl or alkoxycarbonyl, the alkyl portion of which contains 1-4 carbon atoms;

- or aryl radical, possibly substituted by one or more atoms or radicals selected among halogen atoms and alkyl, alkenyl, etkinlik, aryl, kalkilya, CNS, alkylthio, aryloxy, aaltio-, hydroxyl, hydroxyalkyl, mercapto-, formyl, acyl, acylamino, aroylamino, alkoxycarbonyl-, amino-, alkylamino, dialkylamino-, carboxyl, alkoxycarbonyl, karamolegos, alkylcarboxylic, dialkylanilines, cyano-, nitro-, azido-, triptorelin or triptoreline radicals;

or saturated or unsaturated 4-6 membered heterocyclyl radical, possibly substituted by one or more alkyl radicals with 1-4 of carbon atoms is a branched alkanniny radical with 2-8 carbon atoms, linear or branched alkynylaryl radical with 2-8 carbon atoms, cycloalkyl radical of 3-6 carbon atoms, cycloalkenyl radical with 4 to 6 carbon atoms or bicycloalkyl radical with 7 to 11 carbon atoms, these radicals can be substituted by one or more substituents selected among halogen atoms and hydroxyl radical, CNS radical with 1-4 carbon atoms, dialkylamino radical, each alkyl portion of which contains 1-4 carbon atoms, piperidino radical, morpholino radical, piperazine-1-ilen radical (possibly substituted in position 4 alkyl radical with 1-4 carbon atoms or phenylalkyl radical, the alkyl portion of which contains 1-4 carbon atoms), cyanoaniline radical of 3-6 carbon atoms, cycloalkenyl radical with 4 to 6 carbon atoms, possibly substituted phenyl radical, cyano radical, carboxyl or alkoxycarbonyl, the alkyl portion of which contains 1-4 carbon atoms;

provided that cycloalkyl, cycloalkenyl or bicycloalkyl radicals can be substituted by one or more alkyl radicals with 1-4 carbon atoms
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