DE1958383C3 - Leupeptins, processes for their preparation and pharmaceuticals containing them - Google Patents

Description

j .CH ₃ CHj - CH ₃

- CH '- CH ₂ - CH or - CH'

^N CH ₃ CH ₃ CH ₃

and R _{3 represents} a methyl or ethyl radical.

2. Process for the preparation of leupeptins and their analogs according to claim 1, characterized in that that a compound of the formula

R ₁ COOH

R ₃ _ CO - \ NH - CH - CO / "- NH - CH - CO - NH - CH - (CH ₂ ) ₃ - NH - C

in which n, R ₁ , R ₂ and R _{3 have} the meanings given above, is reduced in a manner known per se.

3. The method according to claim 2, characterized in that a compound of the formula

/ R ₂ \ R ₁ CH ₂ CH

I) ^NH

R ₃ - CO - \ NH - CH - CO / "- NH - CH - CO - NH - CH - (CH ₂ ) ₃ - NH - C '

in which n, R ₁ , R ₂ and R _{3 have} the meanings given above, is oxidized in a manner known per se.

4. Medicaments consisting of a compound according to claim 1 and customary carriers and / or Diluents and / or auxiliaries.

The invention relates to the synthesis of leupeptins inflammation in rats caused by carrageenin

and their analogs, which have been therapeutically valuable sub- caused. They are suitable for

There are and enzymatic reactions from action of pancreatitis and infiammatory

Trypsin, papain, kallikrein, plasmin and thromboki diseases, in particular, they are used to treat

able to inhibit the nose. Furthermore, these are injuries to inflammatory diseases of the skin,

bindings capable of fibrinolysis, for example burns.

Kinin formation from kininogen as well as a blood clot- The structures of leupeptins and their analogues,

inhibition. Leupeptins have a low that have been synthesized according to the invention and

Toxicity and exerting a therapeutic effect on themselves as active substances are as follows:

R ₂ \ R ₁ CHO

I, ■ ·. ' ^NH

R ₃ - CO - \ NH - CH - CO / n - NH - CH - CO - NH - CH - (CH _S ) ₃ - NH - C (I)

^X NH ₂

η = 1 or O CH ₃ - -CH ₂ - CH ₃ -CH ₂ -CH. , CH ₃ -CH , CH ₂ - -CH ₃ R ₃ = CH ₃ - - or R ₂ = -CH CH ₃ "CH ₃ CH ₃ R ₁ .

As can be seen from these formulas, according to the invention, leupeptins and theirs can exist

Leupeptides from acetyl-L-leucyl-L-leucyl-DL-argininal, analogs which are represented by the above formula

Propionyl-L-leucyl-L-leucyl-DL-argininal and their mel I are shown, (A) by oxidation of

Analogues in which one L-leucine is missing and 1 or compounds of the following general formula II,

2 L-leucine molecules with L-isoleucine and / or L-valine 5 which are produced according to various known methods

are substituted. can be synthesized:

R ₁ CH ₁ OH

. ■, ι ι

R ₃ - CO - \ NH - CH - CO / _n - NH - CH - CO - NH - CH - (CHj) ₃

NH

NH

NH.

(ID

where η stands for 1 or O and the substituents R, and R ₁ , which can be the same or different,

CH ₃

-CH.

CH,

CH ₃

- CH, - CH.

CH ₃

CK,

or CH-CH ₁ -CH ₃

and R _{3 is} CH ₃ - or CH ₃ - CH ₂ -.

The leupeptins and their analogs can also (B) by reducing chemical compounds the general formula

R ₃ -CO - \ NH

R ₁ COOH

CH - CO / _M NH - CH - CO - NH - CH - (CH ₂ ) ₃ - NH - C

NH
NH ₂

(III)

are prepared, in which, R ₂ , Rj and R _{3 have} the meanings given above.

The compound (II) can be produced by reducing the compound (HI). The connection (III), for example acyl-leucyl-leucyl-arginine can be produced with L-leucine and arginine according to a known peptide synthesis, for example according to the mixed acid anhydride method, according to the dicyclohexylcarbodiimide method, according to the nitrophenyl ester method, by the acid chloride method, by the azide method, by the carbonyldiimidazole method, by the Woodwards reagent method or by the Merrifield free-phase method. at The starting compounds, such as, for example, arginine, can be used to carry out this process Precursor (ornithine) or its derivative (NG-nitroarginine), in the l-, d- or DL-form can be used.

When carrying out the condensation of these amino acids, the amino groups are replaced by carbobenzoxy radicals, tertiary butyloxycarbonyl residues, tosyl residues, Trityl residues or phthalyl residues and the Guanide radical protected by a nitro radical, carbobenzoxy radical or tosyl radical. The carboxyl group is protected by various esters as they are generally used. Acyl residues, such as Acetyl or propionyl radicals can by known methods using the Acid anhydride are introduced. Used acetyl-L-leucine or propionyl-L-leucine as the starting material is used, then racemization can occur to a considerable extent.

According to the invention, a carboalkoxyl-L-leucine, an L-leucine ester and dicyclohexylcarbodiimide are reacted in an organic solvent to obtain a carboalkoxy-L-leucyl-L-leucine ester. This ester is saponified and gives crystals of carboalkoxy-L-leucyl-L-leucine. This compound is reacted with arginine in an organic solvent in the presence of Woodward reagent K, the guanidine ^{base of which is protected in the form of the N G} -nitro-L-arginine ester. The carboalkoxy-L-leucyl-L-leucyl-N ^G -nitro-L-arginine obtained in this way is acylated in the presence of palladium on carbon to obtain the acyl-L-leucyl-L-leucyl-L-arginine ester. In this case the

♦ o Carboxyl radical substituted with other acid derivatives being.

The acyl-L-leucyl-L-leucyl-L-arginine ester obtained in this way is in water and preferably in anhydrous organic solvents in the presence of reducing agents, such as lithium borohydride, sodium borohydride or lithium aluminum hydride, at room temperature to about 150 ⁰ C for the recovery of Acyl-L-leucyl-L-leucyl-L-argininol reduced. The reduction can also be carried out using a platinum catalyst. Alternatively, the end product of the above reaction can also be obtained in the following manner: An L-leucyl-L-leucyl-L-arginine ester or an acylated compound of this ester at the N-terminal and / or its N ^G -nitro derivative are converted by a reducing agent , such as lithium borohydride, to obtain the corresponding derivatives of L-leucyl-L-leucyl-L-argininol, whereupon this latter compound is reduced using palladium on activated carbon and then acylated. The acylated L-leucyl-L-leucyl-L-argininol obtained in this way is oxidized to obtain the corresponding leupeptins. To carry out the oxidation, the sulfoxide-carbodiimide reactor (cf. "Journal of American Chemical Society", Vol. 87 No. 24, p. 5661, 1965) is because of the smooth formation of the aldehyde radical without formation; Carboxylic acid residue of other direct oxidation

Methods to be preferred, for example an oxidation with 80 ml of ethyl acetate extracted. The extract will

Using Metallakylaten or Chrom- washed with 40 ml of water and over anhydrous

acid. Sodium sulfate and dried to approximately 40 ml

The sulfoxide-carbodiimide reaction is concentrated to the point where 200 μl petroleum ether is added suggested a reaction in which S are. After the mixture has been cooled

3'-O-acetylthymidine-5'-aldehyde is one by oxidation, 7.31 g of crude crystals are obtained and off

primary alcohol of 3'-O-acetylthymidine obtained from a mixture of 20 ml of ethyl acetate and 60 ml

will. The above Rc .k petroleum ether was found to recrystallize. 6.45 g

tion for the conversion of C-terminal pepods (85%) carbobenzoxy-L-leucyl-L-leucine in the form of

can be used to aldehydes. This io provides white crystals (mp 93 to 95 ° received Q [*]? A great advantage in the aldehyde formation in the = -29 ° (c = 2.9, methanol).

Synthesis of leupeptins. In this way, z. B. Analysis * CHON

the argininol compound described above is calculated as "^ C 63 47 H 7 99 N 7 40-

dissolved in anhydrous dimethyl sulfoxide, whereupon found ... C 63 ^! H Ί &, N 7 ^ 67. *

probably dicyclohexylcarbodumide as well as anhydrous 15

Phosphoric acid is added at room temperature 6.43 g (0.017 Mo!) Of carbobenzoxy-L-Ieucyl-L-leucine

the. The desired compound is dissolved by the reac- in 250 ml of nitromethane, whereupon the lo-

tion mixture is cooled by a customary method in an ice water bath. then

separates and by column chromatography on active 2.38 ml of triethylamine (0.017 mol) and 4.42 g

coal, the product being added in yield 20 (0.017 mol) Woodwards reagent, whereupon

of more than 80% occurs. the mixture is stirred for 1 hour. Subsequently

The presence of leupeptins in the reaction takes place the addition of 4.6 g N ^G -nitro-L-argininthyl-

mixture can be easily replaced by an anti-ester hydrochloride (0.017 mol) and 2.38 ml (0.017 mol)

Piasmin activity and / or triethylamine by a positive spot, whereupon the mixture during a

in the Rydon-Smith reaction, the Sakaguchi-keak period of 5 hours and stirred overnight

tion or, if a carbonyl reagent is used, is kept at room temperature. The reaction

when carrying out a thin-layer chromatography mixture, the mixture is evaporated to dryness in vacuo.

graphy using silica gel G and a The residue is dissolved in 400 ml of ethyl acetate.

Solvent system of butanol, butyl acetate, vinegar- The ethyl acetate solution is then successively

determine acid and water (4: 2: 1: 1). 30 with 200 ml of water, 100 ml of 0.5N HCl, 100 ml

The following examples illustrate the invention, water, 200 ml of 0.5 M NaHCO ₃ and 100 ml of water

without restricting them. washed and dried over anhydrous sodium sulfate

. -I ₁ dries. Then it goes to dryness in a vacuum

Example 1 evaporated, whereby 6.6 g (yield = 65%) Carbo- A solution of 9 g (0.034 mol) carbobenzoxy- 35 benzoxy-L-leucyl-L-leucyl-N ^G -nitro-L-arginine methyl- L-leucine, 6.7 g (0.034 mol) L-lcucine ethyl ester in the form of a white powder (m.p. 75 to 85 ° C)

chloride and 4.8 ml (0.034 mol) of triethylamine are obtained in 140 ml.

To chloroform, 7.0 g of Ν, Ν'-dicyclohexylcarbodi- 6.6 g (0.011 mol) of the ester in a mixture of imide are added with cooling and with stirring. 260 ml of methanol, 40 ml water and 20 ml of glacial acetic acid After standing overnight in a refrigerator 4 ° are by supplying hydrogen gas w ^"1" - cabinet the mixture to remove is rend a period of 14 hours in the presence of crystalline N, N'-Dicycloharnsloff nitrated. The fiI- of 10 g of a catalyst, which appeared from palladium, is evaporated to dryness in vacuo, charcoal (5% palladium) is hydrogenated. The catalyst on the residue dissolved in 150 ml of ethyl acetate is removed by filtration and added with 160 ml. Washed after removing insoluble N, N'-di-45 methanol. The filtrate and the washed cyclohexylurea by filtration, the ethyl solution are combined. The mixture is successively concentrated to acetate solution with 50 ml of 0.5 η-chlorine syrup. The syrup, namely L-hydrofluoric acid, 50 ml of water, 100 ml of 0.5 m-natricyl-L-leucyl-L-arginine methyl ester diacetate, which was then washed with bicarbonate and 50 ml of water. The solution is obtained in an amount of 6.1 g, the solution is dried over anhydrous sodium sulfate in a mixture of 80 ml of chloroform and 40 ml and evaporated to dryness in vacuo, with 12.0 g of benzene being dissolved, followed by 3 ml of acetic anhydride of a crude powder are obtained. The carbobenz should be added. After the mixture has been kept during oxy-L-leucyl-L-leucine ethyl ester for a period of 8 hours at room temperature from 30 ml of methanol and 10 ml of water, 20 ml of ethanol are crystallized in, with 8.4 g (yield = 65%) of the 55 sets, whereupon it is concentrated. 5.9 g of pure acetyl-L-leucyl-L-leucyl-L-arginine methyl ester acetate product, melting at 85.5 to 86.5 ° C., are obtained. I «]? = -50 ° (c = 2, methanol). in the form of a syrup. The colorless syrup is calculated at Analvse · C HON room temperature using 50 ml. .1 C 65.00, H 8.43, N 6.89; 1 N sodium hydroxide found in 50 ml of ethanol during a ... C 65 02, H 8.67, N 7.15. ^6o period of 4 hours hydrolyzed. Solution ⁰ is neutralized with 3 ml of 1N hydrochloric acid, 8.13 g (0.02 mol) of carbobenzoxy-L-leucyl-L-leu- and evaporated to dryness. The residue is extracted from the ethyl acetate in 22 ml with 60 ml of water at room temperature. The extract is applied to 1N sodium hydroxide in 100 ml of ethanol during a chromatography column which hydrolyzes over a period of 3 hours. The 65 wex 1X 2 (100 to 200 mesh, OH form, 210 ml, Dow solution is filled with 1N hydrochloric acid (25 ml) Chemical Co.), then deacidified with water and wound to about 50 ml under reduced pressure . The alkaline Elua.t gives a positive pressure concentrated. The concentrate is made twice and is using Sakaguchi reaction

Amberiite FRC 50 (Η-Form, Rohm and Haas Company) adjusted to a pH of 5.2. This is followed by evaporation to dryness, 250 mg of a white powder being obtained. The next neutral eluate is evaporated to dryness in vacuo. This gives 2.54 g of a white powder. The powders are combined and recrystallized from methanol / ether. This gives crystalline acetyl-L-ieucyl-L-leucyl-L-arginine in an amount of 1.67 g. The overall yield is 22% (mp. 262 to 264 ° C) (decomposition). [«] ' ₀ ⁹ - = -47 ° (c - 1, methanol).

Analysis: C ₂₀ H ₃₈ O ₆ N ₆

Calculated ... C 54.28, H 8.66, N 18.99:
Found ... C 54.50, H 8.78, N 18.75.

442 mg (0.001 mol) of acetyl-L-leucyl-L-leucyl-i.-arginine in 40 ml of dry methanol containing 0.48% hydrogen chloride are kept at room temperature for a period of 24 hours. The solution is evaporated to dryness in vacuo, 518 mg of acetyl-L-leucyl-τ -leucyl-L-arginine methyl ester hydrochloride being obtained. 400 g (18 mol) of LiHH ₄ in 40 ml of tetrahydrofuran were added to the F.sterhydrochloride, which had been dried over P ₂ O ₆ , and the mixture was then gently refluxed at 90 ° C. in an oil bath A clear solution is obtained which is then evaporated to dryness. The residue is dissolved in 20 ml of water and extracted twice in 20 ml of butanol. The butanol extracts are combined and washed with 20 ml of water and then evaporated to dryness in vacuo, giving 320 mg of a white powder, which is dissolved in 10 ml of butanol / butyl acetal / acetic acid / water (6: 6: 1: 1, based on volume) and applied to a chromatography column filled with silica (Mallinckrodt, 10 g), then developed with the same solvent, and the eluate, which gives a positive Sakaguchi reaction, is evaporated to dryness in vacuo. 150 mg (yield = 31%) of acetyl-L-leucyl-L-leucyl-i-argininol hydrochloride are obtained in the form of a white powder (melting point 60 to 75 ° C.). [>] f - -46 ° (c = 1, methanol).

Analysis: C ₂₀ H ₄₀ O ₁ N ₆ • HCl • H ₂ O

Calculated ... C 49.73, H 8.97, N 17.40, Cl 7.34;
found . C 49.92, H 8.79, N 17.44, Cl 6.98.

42.2 mg (0.09 mol) of the acetyl-l-leucy 1-l-leucyl-L-argininol hydrochloride obtained in this way are dissolved in 0.5 ml of an anhydrous dimethyl sulfoxide that is 77.9 ng (0.39 mol ) N.N'-dicyclohexylcarbodiimide and 6.9 mg (0.07 mol) of anhydrous phosphoric acid, dissolved. After standing at room temperature for 7.5 hours, the reaction mixture is diluted with 10 ml of water and adjusted to pH 7.0 using 1N sodium hydroxide. It precipitates Ν, Ν'-dicyclohexylurea, which is removed by filtration. The filtrate is applied to a chromatography column which is filled with 1 g of carbon (Wako Pure Chem. Co). The column is washed with water and then eluted with 0.02N hydrochloric acid in 80 "strength methanol. The fluid, which gives a positive Sakaguchi reaction and a red tetrazolium reaction, is evaporated to dryness in vacuo, with 38.1 mg Antiplasmin activity: ID ₆₀ 24 μg / ml. 32.6 mg of the raw powder in 2 ml of butanol are refluxed for 3 hours. 2 ml of butyl acetate and 4 ml of water are added to the solution, followed by mixing with shaking The upper layer is evaporated to dryness to give 21.1 mg of the di-n-butyl acetal hydrochloride (mp. 60 to 90 ° C.), 20.0 mg

ίο of the acetal hydrochloride in 2 ml of a 0.01 N hydrochloric acid are ^{heated to 60 0} C for a period of 3 hours. The solution is adjusted to a pH of 6.0 using Amberiite IR 45 (OH form) and evaporated to dryness in vacuo, 14.3 mg of acetyl-L-leucyl-L-leucyl-DL-argininal hydrochloride in Form of a lingering powder can be obtained. Yield = 40%. Antiplasmin activity: ID ₅₀ ^ g / ml, (F. 65 to 100 ⁰ C). [*]? - 42 ° (c - \ methanol).

Analysis: C ₂₀ H ₃₈ O ₄ N ₆ • HCl • H ₂ O

Calculated ... C 49.93, H 8.59, N 17.47, Cl 7.37;
Found ... C 50.26, H 8.46, N 16.92, Cl 7.61.

Example 2

1.8 g (0.003 mol) of carbobenzoxy-i.-leucyl-L-leucyl-N ⁿ -nitro-i.-arginine methyl ester in a mixture of 50 ml of methanol, 10 ml of water and 5 ml of propionic acid are in the presence of 2 g of a Palladium on carbon catalyst containing 5% palladium hydrogenated over a period of 18 hours to give 1.7 g of i-leucyl-L-leucyl-L-arginine methyl ester propionate (colorless syrup). The syrup is dissolved in a mixture of 20 ml of chloroform and 10 ml of benzene, whereupon 1 ml of propionic anhydride is added. After the mixture has been kept at room temperature for 18 hours, 10 ml of methanol are added and the mixture is evaporated in vacuo. This gives 1.6 g of a syrup. The syrup is dissolved in a mixture of 10 ml of methanol and 10 ml of water, whereupon 13.5 ml of a 1N sodium hydroxide solution are added. The mixture is stirred at room temperature for 4 hours. The reaction mixture is concentrated to approximately 20 ml under reduced pressure and freed from insolubles. The syrup is then applied to a column filled with Dowex 1x2 (OH form, 100 to 200 mesh, 57 ml) and eluted with water. In this case, 40 ml of an eluate collected which gives a positive Sakaguchi reaction. The eluate is brought to an iron pH of 5.2 using Amberiite IRC 50 (H-Form) and evaporated to dryness in vacuo. 412 mg of a white powder

SS vers received. Recrystallization of the powder from methanol / ether gives crystalline propionyl-L-lcucyi-L-leucyl-L-arginine in an amount of 370 mg (18% yield of carbobenzoxy-L-leucyi-L-lencin). F. 256 to 258 ⁰ C (decomposition). {<*] f = -55 ° (c = 1.5, methanol).

Analysis: C _n H ₄₀ O ₈ N, Calculated ... C 55.24, HS, 83, N 17.52; Found ... C 54.87, H 8.72, N 17.77.

254 mg (0.56 mol) of propionyl-L-leucyl-L-leucyl-L-arginine are esterified and reduced _{with LiBH 4} and purified by column chromatography (silica) in the manner described above. Included

ίο

a white powder is obtained from L-propionyl-L-leucyl- Example 6
L-leucyl-L-argininol hydrochloride in an amount of

110 mg (40%). F. 70 ° to 80 ⁰ C. [at] f = -38 ° (c = 1, by the procedure described in Example 5,

Methanol). a white powder of acetyl-L-isoleucyl-L-valyl-

Analysis: C ₁₁ H ₄₂ O ₄ N ₉ · HCl · H ₂ O ^{3 DI} - argininal hydrochloride (mp 120 to 140 ⁰ C) obtained.

Calculated ... C 50.74, H 9.13, N 16.91, Cl 7.13; Example 7

Found ... C 50.97, H 9.40, N 17.02, Cl 6.91. . ,,, " _M , .. .. _,,. . ,

12.2 mg (0.046 mol) carbobenzoxy-L-leucine

60.7 mg propionyl-L-Ieucyl-L-leucyl-L-argininoI-Hy- dissolved in 550 ml nitromethane and with 6.45 ml (0.046

Drochloride by the sulfoxyd-Carbodiimid- io mol) triethylamine and 12.0 g (0.046 mol) Woodwards

The reaction is oxidized and purified by chromatography (Koh reagent K (N-ethyl-S-phenylisoxazolium-S'-sulfonate)

lenstoff) in the manner described above with stirring and cooling during a

cleans. This gives 55.5 mg (ID ₅₀ 23 μg / ml) over a period of 1 hour. Subsequently,

of a crude powder of propionyl-L-leucyl-L-leucylden 12.4 g (0.046 mol) N ^G -nitro-i -arginine methyl-

DL-argininal hydrochloride. 52.7 mg of the crude powder 15 ester hydrochloride and 6.45 ml (0.046 mol) of triethyl

verse are added to the di-n-butyl acetal hydrochloride amine, whereupon at room temperature

(27.0 mg, m.p. 80-105 ° C). The acetal end is stirred for a period of 6 hours

hydrochloride is in an amount of 11.1 mg in Pro- and then the mixture is left overnight-

pionyl-L-leucyl-L-leucyl-L-argininal hydrochloride. The reaction mixture is

(8.8 mg) converted by hydrolysis. Evaporated to dryness total of 20 µm, whereupon the residue

loot: 40%. Antiplasmin activity: ID ₅₀ 9μg / ml, in a mixture of 300 ml of water and 600 ml

F. 75 to 90 ⁰ C [«] V - -46 ° (c = 3, methanol). Ethyl acetate is dissolved. After a shake

Analysis: C ₂₁ H ₄₀ O ₄ N ₆ · HCl · H ₂ O ^{becomes the} i ^thylaCet ₁ ^a _t: ^t ; ^Send J? _/ ^{t with l} ™ f ^ ^CYA ° ^T '

Calculated ... C 50.95, H 8.76, N 16.98, Cl 7.16; hydrogen acid, 150 ml water 150 ml 0.5 M Na-

Found ... C 50.59, H 8.91, N 16.58, Cl 7.09. ^ trium bicarbonate (twice) and 150 ml of water. The ethyl acetate washed in this way

The 13th layer is dehydrated and dried in vacuo

evaporated. It is used in an amount of 11.3 g

According to the carbobenzoxy-L-leucyl-N ^G -nitro-L-arginine methylbene described in the preceding examples, 430 mg of acetyl-L-isoleucyl-ester are obtained in a yield of 51%. 11.3 g L-valyl-L-argiiiin (decomposition point: 260 to 262 ° C) (0.024 mol) of this compound are in a 50 mg acetyl-L-isoleucyl-L-valyl-DL-argininal-hydro mixture of 390 ml Methanol, 70 ml of water and chloride (white powder, melting point 120 to 140 ^° C.). 30 ml of glacial acetic acid dissolved. This solution is 15 g

a palladium-on-carbon catalyst (5% palladium

B e i s ρ i e 1 4 35 ladium) added, whereupon with hydrogen during

a period of 15 hours at room temperature

Hydrogenation is carried out according to the procedure described in the preceding examples. The catalyst is filtered off and

In the same way, 462 mg of acetyl-L-valyl-L-leucyl- washed with 100 ml of methanol result. The methanol-L-arginine (M.p. 180 to 190 ⁰ C) 80.4 mg of a white fraction and the filtrate are combined and in the powder, namely acetyl-L-valyi-L-leucyl-DL-argininal 40 vacuum Evaporated to dryness. This gives hydrochloride (mp 85 to 100 ° C.) [, *] S ¹ = -53 "(MeOH); L-leucyl-L-arginine methyl ester diacetate in the form of a

Antiplasmin activity: ID ₈₀ = 3.2 µg / ml. colorless oil in the amount of 9.4 g. This oily

. -ic substance is in a mixture of 120 ml of chloro-

Example 5 _{{() πη resolved by} j ^ _{0 m} jg _enzo j. This solution

88 mg of acetyl-L-leucyl-L-leucyl-L-arginine are added with 45 6 ml of acetic anhydride, whereupon 1 ml of thionyl chloride are added and the whole thing is left to stand at room temperature overnight left with shaking for a period of time. After adding 40 ml of methanol 1 hour implemented. It turns an orange- the solution is evaporated in vacuo. At the same time, ergelber The precipitate is collected and afterwards 12 g of acetyl-L-leucyl-L-arginine methyl ester are kept in vacuo Adding a small amount of benzene evaporated. 50 acetate in the form of a colorless oil. The acetate is in 114 mg of the hydrochloride of acetyl in this state are obtained for a subsequent reaction ver-L-leucyl-L-leucyl-L-arginic acid chloride in the form of a reversible, to characterize this substance yellowish-red Powder. This is produced in a mixture of the however purified crystals from 4 ml of dimethylformamide and 16 ml of xylene, the hydrochloride is made up in the following manner solves. The solution is made on an oil bath at 120 to SS:

130 ° C heated and 300 mg of palladium-BaSO ₄ (5% 12 g of the compound are dissolved in 80 ml of ethanol

Palladium), whereupon by adding and adding 80 ml of 1N sodium hydroxide solution under hydrogen is hydrogenated. The catalyst will be stirred off for a period of 4 hours filtered and washed with methanol. The methanol room temperature is added. The insoluble part in fraction and the filtrate are combined and in the 60 of the reaction mixture is filtered off and with Evaporated in vacuo. The residue is neutralized in 4 ml of 1N hydrochloric acid. After a Dissolved water and after the removal of insoluble evaporation of this solution in vacuo up to borrowed ingredients twice with 4 ml of butanol extra dry, the residue in 35 ml of water at a here. The extracts are combined and dissolved in a vacuum of pH 6.4, whereupon the insoluble part is evaporated. 66 mg of a brownish 65 are removed. The soluble part is powdered onto a column obtained, the acetyl-L-Ieucyl-L-leucyl-DL-ar- brought with 380 ml Dowex 1x2 (OH type, gininal (yield = 11%, antiplasmin activity: un-100 to 200 mesh, Dow Chemical Co.) filled is approximately 15%). is developed up with water. The alkaline

11 12

Fractions as well as the neutral fractions (pH 6.6 in an amount of 913 mg (yield = 60%) with

to 5.2), which give a positive Sakaguchi reaction, an F. of 70 to 90 ⁰ C is obtained. [«] Ff = -26 °

are collected. The alkaline fraction is with (c = 1, methanol).

Amberlite CG 50 (H type, 100 to 200 mesh, Rohm 858 mg (232 mol) of this compound is added in 10 ml and Haas Co.) to adjust the pH to 5.2. S of an anhydrous dimethyl sulfoxide solution containing 2.27 g. This fraction is dissolved with the neutral fraction containing (11.0 mol) dicydohexylcarbodiimide and 119 mg (1.2 and evaporated to dryness in vacuo. Mol) anhydrous phosphoric acid. 1.85 g of the powder thus obtained are recrystallized from methanol / ether after standing for a period of time. In this way of 17 hours at room temperature, 50 ml are obtained, white crystals of acetyl-L-Ieucyl-L-ario are added to water, whereupon the pH is increased by 1N sodium ginine in an amount of 1.50 g (F. 170 to 185 ° C). hyroxyd is set to 7.0. After the unfa] f = -17 ° (c = 1, methanol); Yield - 10%. soluble components have been removed, the

1.50 g (0.005 mol) of this compound are in soluble part on a column with a diameter 150 ml of methanol containing 0.5% hydrogen chloride is applied from 17 mm, which is dissolved with 15 g of activated charcoal, whereupon the solution is filled at 15 for 24 hours. The column is washed with water and Is left to stand at room temperature. This gives an elution with 80% methanol which contains 0.02 n-chlorine and 1.76 g of a white powder of acetyl-i.-leucylhydric acid. 85 ml of the fractions that L-arginine methyl ester hydrochloride. give a positive Sakaguchi reaction

1.57 g (0.004 mol) of this hydrochloride are also collected and evaporated to dryness in vacuo.

160 ml of tetrahydrofuran and 1.7 g (0.078 mol) of lithi- 20 In this way, acetyl-L-leucyl-DL-ar-

umborhydrid added and for 6 hours at 90 ⁰ C gininal hydrochloride in an amount of 614 mg in

held at reflux. After cooling, it becomes the form of a white powder (yield = 72%). F. 90

Solution with 15.9% hydrogen chloride containing up to 100 ^° C. [»]? = -36 ° (in methanol). Methanol in an amount of 20 ml to adjust _Anal . C ₁₄ H ₈₇ N ₅ O ₃ • HCl - H, O

«Na acidic pH added. The solution is _{echnet 5 Be} i anschües- a. . _{c 45 n H 8> 2} | Ν 19.04, Cl 9.64;

send evaporated to dryness in vacuo, whereupon _ge f _and en ... C 45.81, H 8.52, N 18.94, Cl 8.22. the residue in a mixture of 50 ml each

Water and butanol is dissolved. The aqueous This product has an antiplasmin activity layer is extracted twice with 50 ml of butanol. (ID ₆₀ = 9 mcg / ml). The extracts are combined and washed with 20 ml of water for 30 examples. The butanol layer is evaporated to dryness. This gives 3.44 g of a crude powder. According to the manner described in Example 7, vers. This raw powder is in a mixture of 7 mg propionyl-L-leucyl-DL-argininal hydrochloride butanoi, butyl acetate, acetic acid and water (F. 95 to 110 ⁰ C) from 30 mg propionyl-L-leucyl-DL-ar- (4: 4: 1: 1) dissolved, whereupon the insoluble beginin is obtained, components are removed by filtration. The FiI example 9 is stepped on a column with a diameter of

32 mm, which is filled with 250 g of silica, according to the manner described in Example 7 is

brought, whereupon acetyl-L-isoleucyl-DL-argininal hydrochloride (white

(1450 ml) is developed. Another development 40 powder, F. 100 to 120 ⁰ Q from acetyl-L-isoleucyl-L-ar-

obtained by the solvent (butanol, butyl acetate, acetic ginine (30 mg),

acid and water [4: 2: 1: 1]) in an amount of _. 1 m

1500 ml provides fractions which show a positive Sakaguchi example iu

Result in a reaction. These fractions are collected. Following the manner described in Example 7 will be The combined fractions are converted into 6 mg propionyl-L-isoleucyl-DL-argininal hydrochloride in vacuo Evaporated to dryness, acetyl-L-leucyl-L-ar- (white powder, mp 100 to 120 ⁰ C) from 30 mg propionyl

gininol hydrochloride obtained in the form of a white powder L-leucyl-DL-arginm.

Claims (1)

Patent claims:
1. Leupeptine and their analogues of the general formula \ R ₁ CHO ) ι: - ^NH ι / NH-TH-CO-NlI-CH-I R ₃ - CO - \ NH - CH _{- CO / B} - NH - CH - CO - NH - CH - (CH ₂ ) ₃ - NH - C
where η is 1 or O, R ₁ and R.