DE1958383B2 - Leupeptins, processes for their preparation and pharmaceuticals containing them - Google Patents

Description

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

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

R ₃ - CO - \ nH - CH - CO / ,, - NH - CH - - CO - NH - CH - (CHJ, - NH - C

wherein η, R ₁ , R ₂ and R, which have the meanings given above, are 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

R ₃ - CO -VNH - CH - QOl _n - 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.

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

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

Oil are and enzymatic reactions from act of pancreatitis and inflammatory

Trypsin, papain, kallikrein, plasmin and thromboki diseases, in particular, they are used to treat i can inhibit nose. Furthermore, these are injuries to inflammatory diseases of the skin,

j. bindings capable of fibrinolysis, for example burns.

i- 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

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

'■: / R ₂ \ R ₁ CHO

R ₃ -CO-VNH-CH -COln - NH- CH- CO - NH- CH- (CH ₂ ) ₃ - NH- C (I)

Ϊ NH ₂

£ «= 1 or O

! ' R ₃ = CH ₃ - or CK, - CH ₂ -

CH, CH, ^ CH ₁ -CHa

R ₁ , R-2 = - CPl ^ - CH.2 - CrI ν ~~ * Crt _x

CJt ₃ Cn ₃ ^ n ₃

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

Prepionyl-L-leucyl-L-kucyl-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 U ₁

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

are substituted. can be synthesized:

R ₂

CH "OH

NH

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

'NH ₂
where η stands for 1 or O and the substituents R ₂ and R ₁ , which can be identical or different,

CH ₃

- CH:

CH ₃

CH _S

- CPL - CH:

or

- CH - CH, - CH,

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

COOH

NH

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

NH ₂

are prepared, in which, R ₂ , R ₁ and V _{1 have} the meanings given above.

The compound (II) can be produced by reducing the compound (III). The compound (III), for example acyl-leucyl-leucyl-arginine can be prepared with L-leucine and arginine by a known peptide synthesis, for example by the mixed acid anhydride method, by the DicycIohexyicarbodiimid method, by the nitrophenyl ester method, by the Acid chloride method, according to the azide method, according to the carbonyldiimidazole method, according to the Woodwards reagent method or "." Ie the Merrifield solid phase method. ^When carrying out this process, the starting compounds, such as, for example, arginine, its precursor (ornithine) or its derivative (NG-nitro- »rginine), can be used in the L-, d- or DL-form.

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 residue by a nitro residue, carbobenzoxy residue or tosyl radical protected. The carboxyl group is represented by various esters, such as those in general are used, protected. Acyl residues, as for example 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 ^{residue 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-leucy] -L-arginine ester. In this case, the carboxyl radical can be substituted with other acid derivatives.

The acyl-L-Ieucyl-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 up to about 150 ⁰ C to obtain Acyl-L-leucyl-L-leucyl-L-argininol reduced. The reduction can also be carried out by means of a platinum catalyst. Optionally, 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 N ^G -nitro derivative are reduced by a reducing agent such as lithium borohydride to obtain the corresponding derivatives of L-leucyl L-leucyl-L-argininol, whereupon this last compound is au!

Activated charcoal is reduced and then acylated. The acylated L-leucyl obtained in this way

L-leucyl-L-argininol is oxidized to obtain the corresponding leupeptins. To carry out Oxidation is the sulfoxide-carbodiimide reaction (See "Journal of American Chemical Society", Vol. 87 No. 24, p. 5661, 1965) because of the smooth running Formation of the aldehyde residue without formation of a! Carboxylic acid residue of other direct oxidation

methods such as oxidation using metal alkylates or chromic acid are preferable. .

The sulphoxide-carbodiimide reaction becomes the main event proposed a reaction in which S ^ -O-acetylthymidine-S'-aldehyde by oxidation of a primary alcohol obtained from 3'-O-acetylthymidine will. The above reaction was found to be effective for the conversion of C-terminal peptides can be used to aldehydes. This offers a great advantage in the formation of aldehydes in the Synthesis of leupeptins. In this way, z. B. the above-described argininol compound dissolved in anhydrous dimethyl sulfoxide, whereupon both Dicyclohexylcarbodiimide as well as anhydrous phosphoric acid can be added at room temperature. The desired compound is separated from the reaction mixture by a conventional method and purified by column chromatography on activated charcoal, the product: a yield of more than 80%.

The presence of leupeptins in the reaction mixture can be identified in a simple manner by means of an anti-plasmin activity and / or by a positive spot in the Rydon-Smith reaction, the Sakaguchi reaction or when using a carbonyl reagent when performing thin layer chromatography using silica gel G and a solvent system composed of butanol, butyl acetate, acetic acid and water (4: 2: 1: 1).

The following examples explain the invention without restricting it.

B e i s ρ i e 1 1

7.0 g of NjN'- Dicyclohexylcarbodiimide added with cooling and with stirring. After standing overnight in a refrigerator, the mixture is filtered to remove crystalline Ν, Ν'-dicyclourea. The filtrate is evaporated to dryness in vacuo, whereupon the residue is dissolved in 150 ml of ethyl acetate. After removing insoluble N, N'-dicycjohexylurea by filtration, the ethyl acetate solution is washed successively with 50 ml of 0.5N hydrochloric acid, 50 ml of water, 100 ml of 0.5M sodium bicarbonate and 50 ml of water. The solution is dried over anhydrous sodium sulfate and evaporated to dryness in vacuo to give 12.0 g of a crude powder. The carbobenzoxy-L-leucyl-L-leucine ethyl ester is recrystallized from a mixture of 30 ml of methanol and 10 ml of water, with 8.4 g (yield = 65%) of the pure product melting at 85.5 to 86.5 ° C can be obtained. [x] f = -50 ° (c = 2, methanol).

Analysis: ¹ C ₂₂ H ₃₄ O ₅ N ₂

Calculated ... C 65.00, H 8.43, N 6.89;
Found ... C 65.02, H 8.67, N 7.15.

8.13 g (0.02 mol) of the carbobenzoxy-L-leucyl-L-leucine ethyl ester are dissolved in 22 ml of 1N sodium hydroxide in 100 ml of ethanol at room temperature for one; Hydrolyzed for 3 hours. The solution is acidified with 1N hydrochloric acid (25 ml) and concentrated to approximately 50 ml under reduced pressure. The concentrate is extracted twice with 80 ml of ethyl acetate. The extract is washed with 40 ml of water and dried over anhydrous sodium sulfate and concentrated to approximately 40 ml, whereupon 200 ml of petroleum ether is added. After the mixture has been cooled, 7.31 g of crude crystals are obtained and recrystallized from a mixture of 20 ml of ethyl acetate and 60 ml of petroleum ether. 6.45 g (85%) of carbobenzoxy-L-leucyl-L-leucine are obtained in the form of ίο white crystals (melting point 93 to 95 ° C) [α] ² _D ^S = -29 ° (c = 2, 9, methanol).

Analysis: Co ₀ H ₃₀ O ₅ No
Calculated ... C 63.47, H 7.99, N 7.40; Found ... C 63.64, H 7.66, N 7.67.

6.43 g (0.017 mol) of carbobenzoxy-L-leucyl-L-leucine are dissolved in 250 ml of nitromethane, whereupon the solution is cooled in an ice-water bath. Then 2.38 ml of triethylamine (0.017 mol) and 4.42 g (0.017 mol) of Woodwards reagent are added and the mixture is stirred for 1 hour. Then 4.6 g of N ^G -nitro-L-argininthylesier hydrochloride (0.017 mol) and 2.38 ml (0.017 mol) of triethylamine are added, whereupon the mixture is stirred for a period of 5 hours and kept at room temperature overnight . The reaction mixture is evaporated to dryness in vacuo. The residue is dissolved in 400 ml of ethyl acetate. The ethyl acetate solution is then washed successively with 200 ml of water, 100 ml of 0.5N HCl, 100 ml of water, 200 ml of 0.5M NaHCO ₃ and 100 ml of water and dried over anhydrous sodium sulfate. It is then evaporated to dryness in vacuo, with 6.6 g (yield = 65 %) of carbobenzoxy-l-leucyl-L-leucyl -N ^G -nitro-l-arginine methyl ester in the form of a white powder (melting point 75 to 85 ° C ) can be obtained.

6.6 g (0.011 mol) of the ester in a mixture of 260 ml of methanol, 40 ml of water and 20 ml of glacial acetic acid

4P are made by supplying hydrogen gas for a period of 14 hours in the presence of 10 g of a catalyst consisting of palladium on carbon (5% palladium), hydrogenated. The catalyst is removed by filtration and with 160 ml

+5 methanol washed. The filtrate and the washed Solution are combined. The mixture is concentrated to a syrup. The syrup, namely L-leucyl-L-leucyl-L-arginine methyl ester, diacetate, thus obtained in an amount of 6.1 g

5.0 dissolved in a mixture of 80 ml of chloroform and 40 ml of benzene, followed by 3 ml of acetic anhydride can be added. After the mixture for a period of 8 hours at room temperature has been maintained, 20 ml of ethanol are added, followed by concentration. 5.9 g fall Acetyl - L-leucyl -1-leucyl-L-arginine methyl ester acetate in the form of a syrup. The colorless syrup is made at room temperature using 50 ml 1 n-sodium hydroxide in 50 ml of ethanol hydrolyzed over a period of 4 hours. The solution is neutralized with 3 ml of 1N hydrochloric acid and evaporated to dryness. The residue will extracted with 60 ml of water. The extract is applied to a chromatography column with Dowex 1x2 (100 to 200 mesh, OH shape, 210 ml, Dow Chemical Co.) is filled, whereupon developed with water will. The alkaline eluate gives a positive Sakaguchi reaction and is obtained using

1 95 ^ 383

Amberiite IRC 50 (Η-Form, Rohm and Haas Com- evaporated to dryness in vacuo, with 38.1 mg pany) adjusted to a pH of 5.2. Then a raw powder can be obtained. Antiplasmine evaporation to dryness, with 250 mg of an activity: ID ₅₀ 24 [LgImI. 32.6 mg of the crude white powder are obtained. The next neutral in 2 ml of butari oil are refluxed for 3 hours. The eluate is evaporated to dryness in vacuo. 5 held. 2 ml of butyl acetate are added to the solution and 2.54 g of a white powder are obtained. The 4 ml of water are added, whereupon the powder is combined with shaking and mixed from methanol / ether. The upper layer is single-crystallized to dryness. Crystalline acetyl-vaporized is obtained, whereby 21.1 mg of the di-n-butyl acetal-hydro-L-leucyl-L-leucyl-L-arginine in an amount of 1.67 g. chloride (F. 60 to 90 ⁰ C) can be obtained. 20.0 mg The total yield is 22% (F. 262 to 264 ° C) ιό of the acetal hydrochloride in 2 ml of a Ο, ΟΙη-chlorine (decomposition). . [*] "= -47 ° (c = 1, methanol) Hydrogen acid are heated span of 3 hours at 60 ° C during a time ^D The solution is under analysis: ,. C ₂₀ H ₃₈ O ₅ N use. from Amberiite IR 45 (OH form) to Calculated ... C 54.28, H 8.66, N lb, JJ; _{a pH VQn 6> 0 and found in} vacuo to ... C 54.50, W 8.78, N 18, IX _ig evaporated to dryness, whereby 14.3 mg of acetyl-L-leucyl-442 mg (0 001 mol) of acetyl-i.-leucyl-L-leucyl-i.-argi- L-leucyl- DL-argininal hydrochloride in the form of a nin in 40 ml 'of a dry methanol, which 0.48%, white powder is obtained. Yield = 40%. Contains hydrogen chloride, at room temperature antipbsmin activity: ID ₅₀ L ^ g / iril, (F. 65 to 100 ⁰ C). For a period of 24 hours content- [α] f -42 (c = 1, methanol),
th The solution is evaporated in vacuo to dryness einge- 20 _{Ana] ysis.} C ₀ H ₃₈ O N _{1 8} · HCI · H, 0
evaporated to give 518 mg of acetyl-L-leucyl-L-leucyl-L-ar- Calculated ... C 49.93, H 8.59, N 17.47, Cl 7.37; ginin methyl ester hydrochloride can be obtained. Found ... C 50.26, H 8.46, N 16.92, Cl 7.61. Ester hydrochloride dried over P ₂ O ₅

400 g (18 mol) of LiBH ₄ in 40 ml of tetrahydrofuran, Example 2

sets what the Mischuns easy for WC in a 25 "" ,,, "^,,

Refluxing oil bath. After 6 hours 1.8 g (0.003 mol) of carbobenzyxy-L-leucyl-L-leucyl-

the mixture is cooled, whereupon 9.5% chlorine-N ^G -niiro-L-arginine methyl ester in a mixture of

hydrogen in methanol (8 ml) may be added. DA 50 m \ methanol, 10 ml of Wa, he and 5 ml propionic acid

at a clear solution is obtained, which are then added in the presence of 2 g of a palladium

is evaporated to dryness. The residue is 30 carbon catalyst containing 5% palladium,

dissolved in 20 ml of water and added twice in 20 ml of butanol over a period of 18 hours.

extracted The butanol extracts are combined and 1.7 g of L-leuyl-L-leucyl-i.-arginine methyl-

with "OmI Wasser sevvasc ¹ - η» nd then hydrogenated in ester propionate (colorless syrup). The syrup

Evaporated to dryness in vacuo. It is in a mixture of 20 ml of chloroform »nd

32Ume ^ nes rub powder obtained. The powder 35 dissolved 10 ml of benzene, whereupon ImI Propionsaurean-

xvird in 10 ml of butanol / butyl acetate / acetic acid / water hydride is added. After the mixture has been mixed for 18 hours

(6 x 6 x 1 x 1 by volume) and stored at room temperature for a long time

applied to a 'chromatography column, which is with 10 ml of methanol are added whereupon im

Silicic acid (Mallinckrodl 10 g) is filled. Then vacuum is evaporated. This gives 1.6 g

is developed with the same solvent. The 40 of a syrup. The syrup is made in a mixture

Eluate which gives a positive Safc-vuchi reaction, 10 ml of methanol and 10 ml of water dissolved, whereupon

is "evaporated to dryness in vacuo. 13.5 ml of a 1N sodium hydroxide solution are added

150 me (yield 31%) Acctyl-i.-Ieucyl- will be. The mixture is at room temperature

ι -leucvl-i -arginW.i-1 hydrochloride in the form of emes white stirred for 4 hours. The reaction mixture will

sen powder (F 60 to 75 times. M? ~ " ⁴ ^« under reduced pressure to about 20 ml.

_M,, . centered and freed from insoluble components.

[c 1. ivietnanoii. The syrup is then poured onto a Dowex 1 2

Analysis: C _2n H ₁₁₀ O ₁ N ₁ , HCl-H ₂ O ^ ₁₇ ,, (OH form, 100 to 200 mesh, 57 ml) filled column

Calculated ... C 49.73. H 8.97, N 17.40. Cl 7.34; applied and eluted with water. Be there

found ... C 49.92. H p.79. N 17.44, Cl 6.98. ^ _{4Q mj e] nes E} , _{uats total} i _u j _{as e} j _{ne pO} sili \ e Saka-

4 "» 2 ms (0.09 mol) of the guchi reaction obtained in this way results. The eluate is mixed with

Acewl-L-feucyl-L-leucyl-i-argininol hydrochloride application of Amberiite IRC 50 (H-Form) on one

are brought to 0 s ml of an anhydrous dimethyl sulfoxide, pH 5.2 and in a vacuum to dryness

the 77 9 mg "(0 39 mol) RN'-dicyclohexylcarbodiimide evaporated. 412 mg of a white powder and 6 9 m «(0 07 Mo) of an anhydrous phosphorus verse obtained. A recrystallization of the powder from

Sure contains, 'dissolves. After leaving the methanol / ether to stand, crystalline propionyl-ueucyl-

at room temperature for a period of L-leucyl-L-arginine in an amount of 370 mg (18?

S St3en Yes the reaction mixture with 10 ml ^^^^^^^^ f ^

Diluted water and adjusted to a pH of 7.0 using 1N Na- to 258 C (decomposition), [α] "_ S5 {c - 1.5, metrium hydroxide (60 ethanol).

precipitates N N'-dicyclohexylurea, which by FiI analysis: C ₁ H ₄₀ O ₅ N ₆

tration 'is removed. The filtrate is calculated to a chromium ... C 55.24, H 8.83, N 17.52;

Matography column containing 1 g of carbon (Wako found ... C 54.87, H 8.72, N 17.77.

25 ^ SiSÄ'SfÄ ^^ «a 254 mg (0 56 mole) propionyl .leucyl- ^ ucylnn? N rhinrvunrogenic acid in S0% methanol L-arginine are esterified and reduced _{with LiBH 1 piί t} n _a Tn, _ia t da ^ If the Sakaeuchi reaction is positive and by column chromatography (silica) in Uof and a red? £ SÄ action results, it is purified in the manner described above. Included

a white powder of L-Propipnyl-L-leucyl- is obtained. For sip ie 1/6

trleucyl-LTargininol-Hydrochloride,: in an amount of

llbmg (40%). F. 70 to 80 ⁰ C. [ä] f = '- 38 ° (c = 1, ..According to the procedure described in: Example 5, methanol). ■. Becomes a white powder of acetyl-L-isoleucyl-L-valyl

Analysis: C ₂₁ H ₁₂ Op ₈ · HCl · H ₂ O ⁵ ^ -argininaU hydrochloride (F ₁ 120 to 140 ° C) obtained

- Calculated ... C 50.74, H 9.13, N 16.91, Cl 7.13; B e i s ρ i e 1 7

Found ... C 50.97, H 9.40, N 17.02, Cl 6.91. "" _M "^" r "^,,, · λ

, ''. ''. ' 12.2 mg (0.046 mol) carbobenzoxy-L-leüciii weider

60.7 mg PropionyNL-leucyl-Lrleucyl-L-argminoI-Hy- dissolved in 550 ml Nitr.omethan and with 6.45 ml (0.04i drochloride are by the Siilfoxyd-Carbodiimid- 10 mol) triethylamine and, 12.0g (0.046 mol) Woodwards reaction is oxidized and purified by chromatography (Koh reagent-K (N-EthyI-5-phe: nylisoxazolium-3'-sulfonate; fenstoff) in the manner described above while stirring and cooling during one time. This gives 55.5 mg (ID ₅₀ 23 μg / ml) over a period of 1 hour, followed by a crude powder of propionyl-L-leucyl-L-leucyldene 12.4 g (0.046 mol) N ^r -nitro -..- arginine methyl-DL-argininal hydrochloride. 52.7 mg of the crude PuI- 15 ester hydrochloride and 6.45 ml (0.046 mol) of triethylvers are added to the di-n-butyl acetal hydrochloride amine, whereupon at room temperature currency (27.0 mg, F. 80 to 105 ⁰ C) converted the acetal is stirred rend a period of 6 hours. hydrochloride is in an amount of 11.1 mg in process, and then the mixing and practice night stelienpionyl -L- leucyl -1. - leucyl - L - argininal hydrochloride is left. The reaction mixture is converted by hydrolysis in vacuo (8.8 mg). Evaporated to dryness in total 20 .mu.m, whereupon the residue yielded: 40%. Antiplasmin activity: ID ₅₀ 9μg / ml, in a mixture of 300ml water and 600ml F. 75 to 90 ° C [«] 'a --46 ° (c = 3, methanol). Ethyl acetate is dissolved. After a shake

Analysis: C ₂₁ H ₄₀ O ₄ N ₀ · HCl · H ₂ O ^{, the} ethyl acetate layer is mixed with 150 ml of 0.5 n-chlorine

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

Found ... C 50.59, H 8.91, N 16.58, Cl 7.09. ^{Washed 25} tnumbicarbonat (twice) and 150 ml of water. The Athylai-etat- washed in this way

Example 3 Layer is dehydrated and in a vacuum to dryness

evaporated. An amount of 11.3g

After dei in the above examples of carbobenzoxy-L-Ieucyl-N '^ described - nilro-L-arginine methyl surrounded way give 430 mg acetyl-L-isoleucyl ester 30 in a yield of 51 ⁿ ₀ obtained. 11.3g L-valyl-L-arginine (decomposition point: 260 to 262'Ci (0.024 mol) of this compound are dissolved in a 50 mg acetyl-i.-isoleucyl-L-valyl-DL-argininal-hydro mixture of 390 ml methanol, 70 ml of water and chloride (white powder, mp 120 to 14O ⁰ C) dissolved. 30 mL of glacial acetic acid. to this solution i5 g

of a palladium-on-carbon catalyst (5 "" PaI-

B ei game 4 35 ladium) added, whereupon with hydrogen during

a period of 15 hours

According to the manner described in the preceding examples, S ί μ '"^ ^Zimme " ™ P "^ benen manner results in 462 mg of Acety-L-valyl-L-leucvl- with 100 ml of StiS catalyst is filtered off., Or L- arginine (F. 180 to 190 ⁰ C) 80.4 mg of a white fraction "L ^ T? washed. The methanol

J ^ UDdzwarAcetyl-Lv ^ lL-leucil-SSÄ " ₄₀ ξ £ ™ zu, TrSkS ZT ^" AT ^ ^and ™ hydrochloride (F. 85 to 100 ° C) [%] f = 53 (MeOH) l ι - ,, ™ ι . I ^dry steamed. R -.an

B e i s ρ i e 1 5 substance is added to a mixture of '120 ml of chloro

88 mg of acetyl-L-le ..- yI-1-leucvI-i.-arginine are dissolved with 45 widenöml F «- ⁸ -" ²⁰¹ - ^{this ll} ^ ^{i! N} S

1 ml of thionyl chloride and stirred at room Tempe ^ Jas whole ti | added ^gSaU ^ _f ^reanh y ^d nd, whereupon

door while shaking for a period of time after H TT ' ^ ^{NaCht Slehe!} "

1 hour implemented. There will be an orance- te I & un »ίϊ Vi ^ ^V ° ^{n 4} ° ^{ml McthaIil "}

yellow precipitate and collected in a vacuum according to iah man V f Ζί ,? , "Γ T ^lgedam P ^ft · ^Dabci « "

evaporated after adding a small amount of benzene. _{so ace} tat inFomJ ^ lV ™ ^; ** ™haben ¹ ^ ¹ "-

114 mg of the hydrochloride of acetyl- "this additive ^colorless oil are obtained. The acetate is in

L-leucyl-L-leucyl-L-argminsäurechlorid in the form of a Sba ^ zur Chäraw " ⁶ · ^{anSchschlende Reakti} ^ ⁿ ^ ^er -

yellowish red powder. This is advertising in a mixture to iedoch eess ^kt "^ ^ler ™ g ^di eser substance

from 4 ml of dimethylformamide and 16 ml of xylene, Zw ₆ ^ k SrdSb ^ ^ T ^ ^her S ^estelIt - ^{zu d} iesem

solves. The solution is prepared on an oil bath at 120 to 55 ^^^ lond in the following manner

130 C and heated with 300 mg of palladium-BaSO. (5 ° 12 s der 'νrrhir, a _j

Palladium), whereupon by adding "" d Ά Z η Γ! ^ l ^{m 8} ° ^{ml Athano1 gdÖSt}

Hydrogen is hydrogenated. The catalyst is off tubes wähSnd ettr7 ° t ^ ^{hydrox> 'dlösun} S ^{under Ver} "

filtered and washed with methanol. The methanol ZimmertemS ,, ^Wedding P ^{a "ne} 4 hours at

fraction and the filtrate are combined and in the 60 ₆ Γ2Ε Γ ^ · - ^{The insoluble part in}

Evaporated in vacuo. The residue wfrd in 4 ml l £ cKSÄ ^ 5 ™ ?? ^, ^U " ^d ^

steamed. The residue is dissolved in 4 ml! -ChlomSSS

Water dissolved and after the removal of insoluble EvaporationTunf SrT "" ^eUtralized · ^{After a}

Borrowed ingredients twice with 4 ml of butanol extra dry is dS ^ ν ^^ ™ ^vacuum
hi Di Ek T ^ S ^d ^ ^{5 l W}

Evaporation SrT "

ilen twice with 4 ml of extra-dry butanol is dS ^ ν ^^ ™ ^{vacuum up to the} hiert. The extracts are combined and evaporated in a vacuum pH of T ^ St ^d ^ ^{5 ml of water with a} d 66 i bälih l Si ¥ *? ^{d &} l ^{insoluble part ent}

h rden combined and in a vacuum pH of T ^ St ^

evaporated. In doing so, 66 mg of a brownish 65 learns is D «-Si ¥ *? ^{The insoluble part of the} powder obtained, the acetyl-L-leucyl-L-leucyl-DL-ar- brought the m? t Sn ^T f ™ ^{Td to "gininal" on a column} (yield = 11%, antiplasmin activity: un- Contains 100 to 200 mesT Dow r T ^ ^{1 X 2 (ΟΗ} " ^{Τ} Φ} 'approximately 15 ⁰ Z ₀ ). Up is filled with wf Chemical Co. where-

^{is developed up with water} . The alkaline

4-69

Fractions and the neutral fractions (pH 6.6 to 5.2) that give a positive Sakaguchi reaction, are collected. The alkaline fraction is mixed with Ämberlite CG 50 (H-type, 100 to 200 mesh, Rohm and Haas Co.) to adjust the pH to 5.2. This faction is merged with the neutral faction and evaporated to dryness in vacuo. 1.85 g of the powder thus obtained recrystallized from methanol / ether. In this way, white crystals of acetyl-L-leucyl-L-ar- ; inin in an amount of 1.50 g (m.p. 170 to 185 ° C).

] f = -17 (c = 1, methanol); Yield = 10%.

1.50 g (0.005 mol) of this compound are dissolved in 150 ml of methanol containing 0.5 ° _{C. of} hydrogen chloride, and the solution is left to stand for 24 hours at room temperature. This gives • lan 1.76 g of a white powder of acetyl-L-leucyl-L-arginine methyl ester hydrochloride.

160 ml of tetrahydrofuran and 1.7 g (0.078 mol) of lithium borohydride are added to 1.57 g (0.004 mol) of this hydrochloride and the mixture is refluxed at 90 ° C. for 6 hours. After cooling, the solution is treated with methanol containing 15.9% hydrogen chloride in an amount of 20 ml to adjust it to an acidic pH. The solution is then evaporated to dryness in vacuo, whereupon the 4 residue is dissolved in a mixture of 50 ml each of water and butanol. The aqueous layer is extracted twice with 50 ml of butanol. The extracts are combined and washed with 20 ml of water. The butanol layer is evaporated to dryness. This gives 3.44 g of a crude powder. This crude powder is dissolved in a mixture of butanol, butyl acetate, acetic acid and water (4: 4: 1: 1), and the insolubles are removed by filtration. The filtrate is applied to a column 32 mm in diameter filled with 250 g of silica, followed by development with the same solvent (1450 ml). Further development by the solvent (butanol, butyl acetate, acetic acid and water [4: 2: 1: 1]) in an amount of 1500 ml gives fractions which give a positive Sakaguchi reaction. These fractions are collected. The combined fractions are evaporated to dryness in vacuo, acetyl-L-leucyl-L-ar- £ ininol hydrochloride in the form of a white powder in an amount of 913 mg (yield = 60%) with a F. of 70 to 9O ⁰ C is obtained, [oc] f = -26 ° (c = 1, methanol).

858 mg (232 mol) of this compound are dissolved in 10 ml of an anhydrous dimethyl sulfoxide solution, which is 2.27 g (11.0 moles) of dicyclohexylcarbodiimide and 119 mg (1.2 moles) of anhydrous phosphoric acid. After standing for 17 hours at room temperature, 50 ml

ίο Water added, whereupon the pH is adjusted to 7.0 with 1N sodium hydroxide. After the insoluble components have been removed, the soluble part is applied to a column with a diameter of 17 mm, which is filled with 15 g of activated carbon. The column is washed with water and eluted with 80% methanol containing 0.02 N hydrochloric acid. 85 ml of the fractions giving a positive Sakaguchi reaction are collected and evaporated to dryness in vacuo, in this way acetyl-L-leucyl-DL-argininal hydrochloride is obtained in an amount of 614 mg in the form of a white powder ( Yield = 72%). F. 90 to 100 ⁰ C. [x] S ³ = -36 ° (in methanol).

Analysis: C ₁₄ H ₂₇ N ₅ O ₃ • HCl • H ₂ O
Calculated ... C 45.71, H 8.22, N 19.04, Cl 9.64;
Found ... C 45.81, H 8.52, N 18.94, Cl 8.22.

This product has antiplasmin activity (ID ₅₀ = 9 mcg / ml).

Example

Following the procedure described in Example 7. 7 mg of propionyl L-leucyl-DL-argininal hydrochloride (F. 95 and 110 ⁰ C) of 30 mg of propionyl L-leucyl-DL-arginine is obtained.

Example 9

Following the procedure described in Example 7, acetyl-L-isoleucyl-DL-argininal hydrochloride (white powder, mp 100 to 120 ⁰ C) is obtained from acetyl-L-isoleucyl-L-arginine (30 mg).

Example 10

Following the procedure described in Example 7. 6 mg of propionyl L-isoleucyl-DL-argininal-HydrocbJorid are obtained (white powder, mp 100 to 120 ⁰ C) of 30 mg of propionyl L-leucyl-DL-arginine.

Claims (1)

1 2 Claims: 1. Leupeptine and their analogues of the general formula / ^R "- \» ι <fHO _y R ₃ - CO - \ NH - CH - COJ _n - NH - CH - CO - NH - CH - (CEy, - NH - C where η for 1 or O stands, R ₁ and R ₂ CH \ CH, / CH ₃ CH ₃ CH - \ CH ₃ -CH ₂ - , CH ₂ - CH ₃ or - CH. CH ₃