DE1804022C3 - Polypeptides and processes for their production - Google Patents

Description

'J

The most interesting effect of the product according to the invention can then be implemented with a third

'olypeppde is the antithromboplastic effect, amino acid with a similar one of two or

because they have an influence on the course of coagulation several other amino acids obtained intermediate

: u allowed earlier. How the product is already chained to a new Pepüd that

; r mentioned, however, an antithrombin effect 5 is then possibly further avoided in a similar way.

jnd a fibrinolytic effect, as is set in, until the desired chain is built up.

know cases have been found to have a vascular effect. The order in which the amino acids are assembled

The latter effect is not related to confi-is thus to a large extent optional as long as one

guration of the natural peptide A in a clear manner - just note that the amino acids in such a sequence

context. In its way, the vascular effect 10 can be coupled to one another that the desired

viewed as having a bradychinin-like activity, a sequence of amino acids is obtained in the end product,

will. Care should be taken to ensure that free

The above-mentioned effects are due to large amino groups and carboxyl groups that do not react

Test animals are greedy in vivo and in human blood should be protected in an appropriate manner,

has been checked, as explained in more detail below. 15 so that the coupling takes place in the desired manner.

The compounds in question contain op- A suitable way to protect the amino groups,

table active amino acids of L-configuration, which consists in converting the amino acid into the carbobenzyl oxy-

che are united with one another by an amide bond. to convert derivative.

In the following, both in general When implementing products containing guanidine

Description as well as in the examples contain the mentioned 20 groups (such as arginine), the guanidinine

Amino acids everywhere have the L-configuration, although the group is transformed into the ω-nitro derivative

this is not particularly emphasized. protects. In the final product obtained, residual

The table below shows the meaning of the protective groups to be removed in a suitable manner

Abbreviations used are explained. z. B. by hydrogenation, as detailed below

25 is written.

Table 1 thus means the method according to the invention

,., .. TJ '. "".'.'. ~~ Z a series of implementations of the type

Abbreviation Name of the amino acid in L-form ^{e Jr}

A - COZ -f NH ₂ B -> ACONHB,

30 where ACOZ denotes a simple amino acid or a lower peptide built up by a similar reaction, in which amino groups and carboxyl groups which should not react are protected in a suitable manner, or a derivative 35 of such an amino acid or a peptide which tends to react, and NH ₂ B denotes an amino acid or a peptide in which carboxyl groups can be protected, and amino groups that should not react to suitable

The compounds according to the invention can be protected in the nete manner, with those used

methods known per se for the synthesis of poly-amino acids and the sequence chosen in this way

peptides are produced. be that a polypeptide according to the above

In general, it means that the formula in question is created, whereupon the undesired protection

Polypeptides, starting from the simple amino groups, can be removed.

acids, step-wise by coupling them into the production of the polypeptides according to the invention

The following examples illustrate the desired shape with the formation of amide bonds builds up. light. Examples 1 to 11 together explain

So two amino acids can be taken into an intermediate the complete synthesis of

Phe - Leu - AIa - GIu (y-OMe) - GIy - GIy - GIy - VaI - Arg - OMe.

The remaining examples explain the syntheses of After cooling, 5.6 ml of triethylamine (0.04 mol)

Intermediate products for others in the context of the Er- added and the triethylamine hydrochloride formed

Finding lying connections, with the intermediate being filtered off. The filtered solution is to -1O ⁰ C

products in the form of 55 containing protective groups are cooled, and with stirring 14.92 g of carbon

Derivatives are obtained. The removal of the protective benzyloxy-valine-p-nitrophenyl ester (0.04 mol)

groups will be given in a summarized form. After 2 hours at - 10 ⁰ C, the temperature may

ßend the examples described, and for some of the door in the reaction mixture to room temperature

The derivatives produced will rise experimentally and will react at this temperature for 24 hours,

cure given in tabular form. 6 »The dimetuvlformamide is in a vacuum at 30 to

40 C evaporates and the residue in a mini

For example 1 mixture of \ thylactate, n-butyl alcohol (2: 1) dissolved.

Carbobcnzyloxy-Valyl-o-Nitroarginine-Mcthvlcsicr, P ^{ie lisu} ^ * ^ird ' ⁵ ™! ^with 5? '., igcr sodium bicarbonate

made by methods A and Ii ' ^loslln ^ ²ⁿ ? ^al T "^ u T ', ^3m . ^al T * ,, ^{1 N} ' ^Sal "f ^ure "

6 ₅ solution and finally 3 times with distilled water

A. 10.8 g finely powdered and dried oNitro extracted.

arginine methyl ester hydrochloride (0.04 mol) in The solution is dried over magnesium sulfate,

1SOmI redistilled dimethylformamide dissolved. evaporated to dryness and the residue in

VaI Valine Arg Arginine Phe Phenylalanine GIy Glycine Leu Leucine AIa Alanine GIu Glutamic acid

18th

dissolved in dry methanol. The substance crystallizes from the methanol solution through the addition of absolute Ether off. Yield 14.1 g with a melting point of 163 to 164 ° C.

By adding petroleum ether to the mother liquor, 1.1 g of substance with a melting point is obtained from 155 to 156 ° C.

Sum of the yield 81.4%;

Analysis C _i0 H _M N _e O ₇ (molecular weight 466.5), (drying 10 hours at 70 "C over Ρ, Ο ₅ );

calculated C 51.49, H 6.48, N 18.02%;

obtained C 52.02, H 6.61, N 17.99% ,.

B. 5.4 g of f'j-nitroarginine methyl ester hydrochloride »5 (0.02 mol) are dissolved in 75 ml of dimethylformamide and, after cooling, 4.84 ml of tributylamine (0.02 mol) are added. Once the temperature of the solution - is 10 ⁰ C, 7.0 g of carbobenzyloxy-VaIin be (0.028 Mo!), Which readily dissolves, and "5.77 g dicyclohexylcarbodiimide (0.028 mol) was added thereto. The reaction mixture is stirred for 2 hours at -1O ⁰ C, then for about 10 hours at 0 "C and finally for 8 hours at room temperature. It is added to 1 ml of acetic acid, and after ¹ I ₂ hour * 5, the dicyclohexylurea formed is filtered off (weight 5 , 9 g). The filtrate is evaporated and the residue is dissolved in ethyl acetate-butyl alcohol (2: 1). Traces of undissolved dicyclohexyl carbamide are filtered off. Otherwise, proceed as under A.

Yield: 7.35 g with a melting point of 160 to 161 ° C., which corresponds to 78.9% of theory. To obtain a substance that melts at about 131 ⁰ C and is probably N-carbobenzyloxy-valine-N, N'-dicyclohexylurea from the mother liquor.

Example 2

Manufacture of Carbobenzyloxy-Glycyl-Glycyl-Valyl-

(') -Nitroarginine methyl ester

40

9.33 g of carbobenzyloxy-valylco-nitroarginine methyl ester (0.020 mol) are dissolved in 120 ml of dry acetic acid and 10 g of dry, bromine-free hydrogen bromide are passed into the solution at 20 ^° C. for 45 minutes. 80 ml of acetic acid and the excess of hydrogen bromide are distilled off in vacuo at 20 ° C. and the residue is vigorously digested in 1 l of dry ether. The ether phase is decanted off and the residue is digested 3 times with dry ether. The granular substance is dried over phosphorus pentoxide in a vacuum desiccator. It turns out to be very hygroscopic. Analysis for hydrogen bromide showed about 1.5 equivalents of hydrogen bromide per mole versus the calculated one equivalent of hydrogen bromide.

Yield 9.2 g of valyl-o-nitroarginine methyl ester-1.5 HBr. The hydrobromide is dissolved in 80 ml neudestilliertem dimethylformamide and cooled to 0 ⁰ C. 4.30 ml (0.031 mol) of triethylamine are added and, after stirring for 10 minutes, the triethylamine hydrobromide formed (about 3.55 g) is filtered off. The filtrate which was cooled to -10 ⁰ C, 7.75 g of carbobenzyloxy-glycyl-glycine-p-Nitrophenylcsler added with a melting point '163-164 C with (0.020 mole). The mixture is stirred for 1 hour at -10'C, after which it is allowed to rise to room temperature. After standing for 4 days at room temperature, the solution is concentrated in vacuo at 40 ° C.

022

evaporates and the RückEiand treated with ethyl acetate, whereupon it crystallizes.

Yield 12.45 g with a melting point of 152 to 154'C. The substance is dissolved in dimethylformamide and precipitated with 5% sodium bicarbonate solution. The precipitate is washed with distilled water and ethanol. The substance is again dissolved in dimethylformamide and precipitated with 5% citric acid solution, washed with distilled water and cooled ethanol and dried in a vacuum desiccator over P ₂ O; dried.

Yield 7.6 g with a melting point of 176 to 181 ⁰ C;

Analysis C ₂₄ H _{: 1} , N _S O ₉ (molecular weight 579.6),
(Drying for 10 hours at 70 ° C. over P ₂ O- below 0.2 torr);

calculated C49.73, H 6.09, N 19.34 ",,:

obtained C 49.20, H 6.34, N 19.35 ".,.

Example 3

Manufacture of carbobenzyloxy-alanyl-γ-methyiglutamic acid

5.92 g of glutamic acid-γ-methyl ester hydrochlorici (0.03 mol) are dissolved in a mixture of pyridine water (1: 1) and ^{cooled to 5:} 'C. 10.32 g (0.03 mol) of carbobenzyloxy-alanine-p-nitrophenyl ester are added in portions to the mixture and the pH is kept constant at 8.7 with the aid of 4N NaOH. After standing at room temperature for 24 hours, the solution is diluted with water and saturated with sodium bicarbonate. The saturated solution is extracted 7 times with ethyl acetate and acidified with half-concentrated hydrochloric acid to pH 2.9 at about 0 C, then extracted with ethyl acetate and the ethyl acetate phase washed with sodium chloride-saturated water, dried over magnesium sulfate and evaporated to dryness. The residue is dissolved in absolute ether, and when petroleum ether is added the substance crystallizes out as needles.

Yield 6.3 g with a melting point of 99.5 to 100.5 ° C., which corresponds to 57.3% of theory; Analysis C ₁₇ H ₂₂ O ₇ N ₂ (molecular weight 366.4);

calculated C 55.75. H 6.05, N 7.65%;

obtained C 55.60, H 5.98, N 7.54%.

Equivalent weight:

Calculated 366.4;

obtained 367.3, 368.1.

Example 4

Manufacture of carbobenzyloxy-glycine-carbotert.-butyloxyhydrazide

20.9 g of carbobenzyloxy glycine (0.1 mol) and 13.2 g of carbo-tert-butyloxyhydrazide (0.1 mol) are in 150 ml of acetonitrile dissolved. After cooling to OC, 20.6 g of dicyclohexylcarbodiimide (0.1 mol) are dissolved in 50 ml of acetonitrile, added, whereupon the reaction mixture is left at room temperature overnight leaves standing. Dicyclohexylcarbamide formed is filtered off and the filtrate is evaporated to dryness in vacuo. The residue is dissolved in ethyl acetate and treated with 5% citric acid, water and 5% sodium bicarbonate and washed water. The ethyl acetate Phasc is dried over sodium sulfate and concentrated in vacuo to a small volume.

whereby the substance auskrislallisicrl. The substance /. is dissolved in ether and with the addition of petroleum ether 29.1 g (90.1 ") of Caiboncnzylo.xy-Cilyein-Carbo-tert-butyloxyhydrazide crystallize out. Melting point 76 to 83 ° C. Recrystallization from Ath> lacctal-Pctrolether gives 23.1 g (71.5 "") with a melting point from 81 to 84 ° C. Further recrystallization increased the melting point to 82 to 84 C.

Analysis (Γ ,, Η ,, Κ, Ο- (molecular weight 323.36):

calculated C 55.72, 116.54. N 13.00 ",,:

obtained C 55.39. 11 6.66. N 13.35 "".

Example 5

Manufacture of Glycine-Carho-Tcrt.-ButyUmhydrazid

9.7 g of carbobenzyloxy-glycine-carbo-tert-butyloxyhydrazide (0.03 mol) are dissolved in 100 ml of methanol, 0.5 g of 10% Pd / C is added and hydrogen is passed in. After 12 hours, 660 ml of hydrogen have been consumed and the hydrogenation is stopped. The catalyst is filtered off and the methanol is driven under vacuum. The residue is digested with absolute ether, a substance with a melting point of 141 to 143 ° C. crystallizing out. Yield 5.3 (93% of theory).

The melting point of the substance rises to 145 to 146 ^a C. after recrystallization from methanol - ether.

Analysis CH _n N ₁ O ₁ (molecular weight 189.22):

calculated ''. . . C 44.43, H 7.99, N 22.21 'V ₁ ;

obtained C 43.94. 117.97. N 22.35 "/.

E-weight 189.2: 191.0.

Example 6

Manufacture of Carbobcnzyloxy-Phcnvlalanyl-Leucine-Ethyleslcr

9.8 g of Lcuein ethyl ester hydrochloride (0.05 mol) will be Dissolved in 30 ml of dimethylformamide and added 5.06 g of triethylamine (0.05 mol). The educated Triethylamine hydrochloride is removed and washed with a little chilled dimethylformamide.

15.0 g Carbohenzyloxy-phenylalanine (0.05 mol) in 30 ml dimethylformamide / Azeioniiril (1: 1) and cooled to - 10 ^c C cooled, after which 10.4 g of dicyclohexylcarbodiimide (0.05 mol) and the above FII occurred, the leucine Contains ethyl ester, are added. The temperature in the reaction mixture is allowed to rise to room temperature overnight, and then ImI 50% acetic acid is added. After 30 minutes, the dicyclohexylcarbamide formed is filtered off and the filtrate is evaporated to dryness in vacuo at 40.degree. The residue is dissolved in ethyl acetate and some undissolved dicyclohexylcarbamide is filtered off. The ethyl acetate solution is washed with 1N hydrochloric acid, water, 50% strength sodium bicarbonate solution and water. The ethyl acetate phase is dried over magnesium sulfate and concentrated to a small volume in vacuo. After the addition of petroleum ether, carbobenzyloxy-phenylalanyl-lcucine-ethyl acetate crystallizes out as needles with a melting point of 116 to 117 ° C. Ausheule 19.45 gJKS.3 ⁿ "the theory). Recovered by t'mkristaHisicrunp from allyl acetate ' petroleum ether
117.5 CV

the melting point drops to 117 to

Analysis (V, 11..N. (V (molecular weight 440.55): calculated *. '..'. ".."..'. C 6SJ6. Il 7.32. N <>.} (<".,: Obtained C 67.74. II 7.1S. N (öS ",.

Example 7

Manufacture of Caibnhcnzylo \ y-Phenvl.ilan \ l-Lcucinhydrazid

X.4 g of carbbene / oxy-phylalanyl-glycylic ether (0.02 mol) are dissolved in 150 ml of methanol and 4.0 g of hydrazine hydrate (0.08 mol) are added. The mixture is stirred overnight and the carbonyl-phenylalaiyl-hydrazide formed is filtered off and washed with a little methanol. It is then recrystallized from methanol AVasscr, giving 6.6 g with a melting point of 192.5 to 193.5 ° C., which corresponds to 81.5% of theory.

Analysis C .., R _1n N ₄ O ₄ (molecular weight 426.53):

calculated C 64.77, H 7.09. N 13.14 ",,:

obtained C 64.85, H 7.12. N 13.16 ",,.

Example 8

Manufacture of Carbobenzyloxy-y-Melhylghiiamyl-Glyein-Caibo-icrt.-Butyloxyhydnizid

7.4 g of carbobenzyloxy-y-methylglutamic acid (0.025 mol) are dissolved in 50 ml of methyl chloride and cooled to -10 C, whereupon 5.4 g of dicyclohexylcarbodiimide (0.0265 mol) dissolved in 25 ml of chilled methyl chloride. can be added with stirring.

4.73 g of glycine carbo-tert-bulvloxyhydrazide (0.025 mol), dissolved in 25 ml of chilled ethyl acetate, are then added. The temperature of the mixture can rise to room temperature overnight, and 30 minutes before the dicyclohexylcarbamide is filtered off, 2 drops of 50 "strength acetic acid are added. The filtrate is evaporated to dryness in vacuo and the residue is dissolved in allyl acetate, whereupon 5", , IgCm Nalriumbicarbon.it. Water, 5 ″ citric acid solution and water and dried over magnesium sulfate. The ethyl acetate solution is concentrated to a small volume, and after the addition of petroleum ether, 2 g of substance crystallize with a melting point of 104 to 105.5 ° C. I / m crystallizes out Ethyl acetate / petroleum ether increases the melting point to 10 * to 108 C.

Example 9 Manufacture of carbobenzyloxy-alanyl-y-methylglut

amyl-glycine-carbo-tert-butyloxyhydrazide

according to methods A and B.

A. 1.83 g of carbobenzyloxy-alanyl-y-methylglutamic acid (SmMoI) are dissolved> n 10 ml of methyl chloride, and after cooling to -10 C, 1.08 of dicyclohexylcarbodiimide (5.2 mmol), dissolved in 5 n 'of cooled methyl chloride, added, and finally 0.95 g Glyciri-Carbo-tert.-Butyloxyhydrazzi (5mMol), dissolved in HImI cooled Ethylaccta 'are added. The mixture is stirred and left to stand overnight at room temperature, whereupon 2 drops of 50% acetic acid are added. After 30 minutes, the dicycloxylearhami (1.1 p) formed is filtered off and washed with a little ethyl acetate

The fi ltnit is evaporated to dryness in vacuo and the residue is dissolved in ethyl acetate, whereupon it is washed with 5 "iger sodium bicarbonate solution, water, 5" iger / Kronentäurclösung and water. The ethyl acetate phase is dried over magnesium sulfate and concentrated to a small volume, whereupon 2.3 g of substance with a melting point of 140 to 150 ° C. crystallize. After two l ^; With crystallization from allyl acetate, the melting point rises to 154 ° to 150 ° C., and the yield is 1.9 g (70.4% of theory).

Analysis C ₄ I l _Tl N-, O ₉ (molecular weight 537.58):

calculated C 53.63, H 6.56, N 13, O3 ^c \ ,:

obtained C 53.4S, H 6.52. N 12.79 ",,.

B. 4.0 g of carbobenzyloxy-y-methylglutamyl-Cilycm-Carbo-tert-butyloxyhydrazide (0.06 mol) are dissolved in 60 ml of methanol and 0.5 g of Pd C (moistened with water) is added. The carbobenzyloxy protective group is removed with hydrogen at room temperature and atmospheric pressure in the course of 2 to 3 hours. The consumption of hydrogen is 200 ml compared to the calculated 195 ml. The catalyst is filtered off and the Killrate evaporated to dryness in vacuo dissolved in absolute ether and evaporated to dryness. An oil is obtained. The oil is dried in a vacuum desiccator over phosphorus pentoxide, the oil turning into a solid hygroscopic substance that weighs 2.76 g, which corresponds to 96.5 "" theory.

The product (2.76 g) is dissolved in 40 ml of dimethylformamide and cooled to -10 ° C. and there are 3.0 g carbobenzyloxy-alanine-p-nitrophenyl ester (8, C nmoles) added. The solution can then be slow Assume room temperature, and after 24 hours it is evaporated to dryness in a vacuum. The residue is treated as under A.

Yield 3.35 g with a melting point of 151 to 153 C, which corresponds to 75.5 ° _u of theory. After three recrystallizations from ethyl acetate, the melting point increases to 160.5 to 161 ^: C (was ^{rearranged at 153;} C).

Be

10

ι s ρ i e 1

Manufacture of Carbobenzyloxy-Phcnylalanyl-Leucyl-Alanyl-7-Methylglutamyl-Glycin-Carbo-

tert-butyloxyhydrazide

1.8 g of the product obtained according to Example 9 (2 mmol) are dissolved in 50 ml of methanol , 0.2 g of 10 ° Pd / C is added and hydrogen is passed in. The hydrogenation has ended after 3 hours , during which 5 ml (theoretically 45 ml) have been consumed. The catalyst is removed and the solution is evaporated to dryness in vacuo. The residue is dissolved in 15 ml of dimethylformamide.

0.86 g of the product according to Example 7 (2 mmol) are dissolved in 3 ml of 6N hydrochloric acid -f 2 ml of 50% acetic acid and covered with 40 ml of ethyl acetate. The mixture is cooled to -10 ° C. and 2.0 ml of molar sodium nitrite is added dropwise with stirring. After 15 minutes at -10 C ^c, the ethyl acetate phase is separated from the water phase, and washed with cooled, saturated sodium bicarbonate and ice water. The ethyl acetate solution is dried over magnesium sulfate for 1 hour at -10 ° C. The ethyl acetate solution, which contains carbobenzyl oxy-phenylalanyl leucine azide, is slowly added dropwise to the dimethylformamide solution containing the free aminopeptide, with stirring and at -10 ° C. After 24 hours at 0 C and 4S hours at room temperature ablillriert the precipitation formed. yield 0:35 g having a melting point \ on 207-208 C. the mother liquor is evaporated to dryness and the residue dissolved in ethylacetate, washed with 5 _"n iüem sodium bicarbonate, water, 5" Ziironensäurelösimg and water, dried over magnesium sulfate and concentrated in vacuo. The residue is dissolved in ethyl acetate and, with the addition of petroleum pellets, 0.70 g of substance with a melting point of 180 to 200 ° C. crystallize out.

Together this gives a yield of 0.80 g, which is 50.1 "" der Theory corresponds, the smaller part is again recrystallized from ethyl acetate.

Analysis C ₁ I ₁ II ₀₁ N ₇ O ₁ , (molecular weight 797.9):

calculated C 58.7 ?, 116.95, N12.29 " _u :

obtained C 5S, 4S, H 6.77, N 12.35 ",,.

Example 11

Manufacture of carbobenzyloxy-phenylalanyl-leucyl-alanyl-v-methyl-glutamyl-glycyl-glycyl- Glycyl-valyl-ci-nitroarginine methyl ester

334 mg of the product according to Example 10 are dissolved in 1.8 ml of trifluoroacetic acid, and after 45 minutes at 22 ° C., the trifluoroacetic acid solution is added dropwise to 100 ml of absolute ether while stirring.

After 2 hours in the refrigerator, the precipitate is filtered off and several times with absolute ether washed, whereupon it is dissolved in 4 ml of dimethylformamide and 1 ml of 4N hydrochloric acid. The solution will be on

- Cooled to 10 C, and 0.42 ml of 1 M sodium nitrite solution are added dropwise over the course of 5 minutes with stirring, before the solution including the precipitate formed in 30 ml of ice-cold. 2 _"u owned Natriumbicarbo-

Natural solution is added dropwise, whereby the precipitation of the acid cazide formed is complete. The azide is washed on the filter with ice water and ice-cold 5 ° _u iger citric acid solution, then washed free of the citric acid with ice water and then

quickly dried over phosphorus pentoxide in vacuo at 5 "C and used immediately for coupling. The yield of dry carbobenzyloxy-phenylalanyl-alanyl-methylglutamyl-glycine azide is 291 mg, which corresponds to 98" _o of theory .

243 mg of the product according to Example 2 (0.42 mmol) are dissolved in 2 ml of absolute acetic acid and 1 ml

4 N-hydrogen bromide in acetic acid added, whereupon the mixture is left to stand for 45 minutes at room temperature, protected from atmospheric moisture. Then it will be

the mixture was added dropwise to 100 ml of absolute ether with vigorous stirring. The precipitate is washed and digested with absolute ether until it is becomes granular (hygroscopic). The tetra-peptide methyl ester hydrobromide is dissolved in 4 ml of dimethylformamide

dissolved and 0.09 ml of triethylamine (0.64 mmol) are added to the solution, which is cooled to -1OX, and the above azide is dissolved in 10 ml of aui

- 10 ^c C precooled dimethylformamide, added dropwise with stirring at -1OX. After 24 hours at OC

the solution can stand for 48 hours at room temperature, after which it divides to 5 ml at 20 ° C Vacuum concentrate. Then ir

5 ml of dimethylformamide dissolved and with a stirrer

j, _v

7u 40 ml 5 "" IGCR citric acid solution was added. The precipitate is washed free with water of citric acid and dried in vacuo over Phosphorpenloxyd dried. Yield 265 mg with a melting point 203-213 C, 66.7 "corresponds to _u of theory. Two unprecipitations from dimethylformamide / water raise the melting point to 211 to 215 C.

Analysis C- ₀ H-, N ₁₃ O ₁₁₁ (molecular weight 11th
calculated '....'.... C 53.99, H 6.62,
obtained C 53.51, H 6.56,

12.2);

N 16.37 "";

N 16.72 ',' _u .

Example 12

Production of carbobenzyloxy-phenylalanyloj-nitro-arginine methyl ester

10.8 g ffj-nitroarginine methyl ester hydrochloride (40 mmol) are dissolved in dimethylformamide and 4.05 g triethylamine (40 mmol) are added while cooling. Triethylamine-HCl formed is filtered off, the filtrate lind a cooled solution (- 10 C) of 12.0 g Carbobenzyloxy-phenylalanine (4OmMoI) in acetonitrile is added, at the - 10 9.1 g Dieyclohexylcarbodiimid (40 mmol) were added ^c C. It is stirred for 2 hours at -10.degree. ^C. and brought to room temperature overnight.

The dicyclohexylcarbamide formed is filtered off and the filtrate is evaporated to dryness. The residue is dissolved in ethyl acetate and washed as usual. From the ethyl acetate solution are at Addition of absolute ether crystals with a melting point of 87 to 89; C obtained. After recrystallization from 60% ethanol, crystals of a different modification with a melting point become from 153.5 to 155 ° C. Yield 17.9 g (87.1% of theory).

Analysis of Cj ₄ H ₃₀ N ₆ O ₇ (molecular weight 514.55):

calculated C 56.02, H 5.88, N 16.33%:

obtained C 55.94, H 6.09, N 16.42%.

Example 13

Preparation of carbobenzyloxy-phenyialanylvalyl- (i) -nitroarginine methyl ester

9.33 g of the product according to Example 1 (20 mmol) are treated as according to Example 2. The yield of hygroscopic valyl-ro-nitroarginine methyl ester-1.5 HBr is _{9.15 g after drying in vacuo over KOH and P 2} O ₅ , which is quantitative.

The Hydrobromiddipeptidester is dissolved in 50 ml of dimethylformamide and cooled to -5 ⁰ C, herds added "preceded 4.2 ml of triethylamine (3OmMoI). Triethylamine HBr formed is filtered off and 8.4 g of carbobenzyloxy-phenylalamin-p-nitrophenyl ester (20 mmol) are added to the filtrate. After 24 hours at room temperature, the solution is cooled to -1O ⁰ C and a further 1.4 ml of triethylamine added. The mixture can then stand for 24 hours. After adding 1 ml of acetic acid, the solution is evaporated to dryness in vacuo and the residue is dissolved in a mixture of ethyl acetate / butyl alcohol (3: 1) and, as usual, with water, 5? _o iger citric acid and water washed. The solution is ingedunstet liiber MgSO ₄ and to a small volume, "which after the addition of absolute ether g 10.1 substance obtained with a melting point 108-111 ⁰ C, which corresponds to 82.3% of theory.

Recrystallization from ethyl acetate / methanol after addition of absolute ether increases the melting point to 112 to 114 ¹ C.

Analysis C ₂₈ H ₁₉ N ₇ O ₈ (molecular weight 613.69);

calculated C 56.76, H 6.41, N 15.98%;

obtained C 56.32, H 6.48, N 16.12% '.

Example 14

Production of carbobenzyloxy-phenylalanyl-glycyl-valyl-cy-nitroarginine methyl ester

2.33 g of the product according to Example 1 (5 mmol) are treated as according to Example 2, and the one obtained Hydrobromide dipeptide methyl ester is dissolved in 15 ml of dimethylformamide, cooled to -5 C and 1.05 ml of triethylamine (7.5 mmol) were added. Triethylamine HBr is filtered off and the filtrate is as is applied as follows:

1.85 g of carbobenzyloxy-phenylalamyl-glycine hydrazide (5 mmol) are dissolved in 7 ml of dimethylformamide and treated with 1.5 ml of 6N hydrochloric acid, cooled to -10 ° C, and 5.1 ml of 1 M sodium nitrite solution (5.1 mmol) was added dropwise. After 10 minutes at -1O ⁰ C 50 ml 5% sodium bicarbonate solution is added and ^c at 0 C begins to precipitate the azide. Precipitation is complete after adding 30 ml of ice-cold water, stirring for 10 minutes, filtering and carefully washing with ice water. The precipitated material is slurried in ice water with stirring, filtered and washed with 0 ° C water. The process is repeated 3 times and dried, the substance at 5 ⁰ C over P ₂ O ₅ in vacuo, whereupon it is directly applied. Yield 1.77 g (93% of theory).

In the cooled to -10 ° C dimethylformamide solution of Aminodipeptids dissolved in 8 ml of pre-cooled to -10 ⁰ C dimethylformamide azide is added dropwise with stirring. After 10 minutes at -10 ° C., the mixture can stand for 48 hours at room temperature and is then concentrated in vacuo to 7 ml and treated as in Example 11. Yield 2.44 g (78.2% of theory). Recrystallization from 60% Ethanol gives 1.95 g of substance with a melting point of 147 to 149 ^D C.

Analysis C ₁₁ H ₄₀ N ₈ O, (molecular weight 670.74);

calculated C 55.51, H 6.31, N 16.71%;

obtained C 55.38, H 6.58, N 16.90%.

Example 15

Production of carbobenzyloxy-phenylalanyl-glycyl-glycyl-valyl-w-nitroarginine methyl ester

1.77 g of the product obtained according to Example 1 (3.8 mmol) are treated as in Example 2 and the hydrobromide dipeptide methyl ester obtained is dissolved in 10 ml of dimethylformamide, cooled to -5 ° C., and 0.79 ml of triethylamine are added (5.7 mmol) added. The triethylamine-win HBr filtered off and the filtrate is used below 1.62 g carbobenzyloxy-glycyl-phenylalanyl glycine hydrazide (3.8 mmol) are dissolved in 10 ml Dimethylform amide, 1 ml of 6N-hydrochloric acid, and au -10 ⁰ C. chilled. 3.85 m 1 M sodium nitrite solution (3.85 mmol) are added dropwise with stirring and the rest of the treatment is as in Example 14. Yield 1.55 g (0.5% of theory).

In the on - 10 ⁰ C dissolved precooled dimethylformamide carboxylic bobenzyloxy-Tripeptidazid added dropwise with stirring - 10 ° C cooled dimethylformamide solution of the Aminodipeptidcsters in 9 ml at will. Otherwise, the procedure according to Example 11 is followed. Yield: 1.93 g with a melting point of 185 to 190 ^° C., which corresponds to 73.1 % of theory. Environmental crystallization from 90% ethanol raised the melting point to 188-191 ⁰ C.

Analysis C _M H ₄₅ N _e O, "(molecular weight 727.8):

calculated C 54.45, H 6.23, H 17.32%:

obtained C 55.01, H 6.18. N 17.52%.

Example 16 Manufacture of carbobenzyloxy-phenylalanyl- Glycyl-Glycyl-Glycyl-Valyl-

w-nitroarginine methyl ester

1.74 g of the product obtained according to Example 2 (3 mmol) are dissolved in 7 ml of dry acetic acid, and 7 ml of 4 N-HBr in acetic acid are added under anhydrous conditions. After 45 minutes at room temperature, the evolution of carbon dioxide has ended and the reaction is complete. The excess of hydrogen bromide and 7 ml of acetic acid is distilled off in vacuo and the residue is added dropwise to 150 ml of absolute dry ether with vigorous stirring. The ether is decanted and worked up. The yield of 2.26 g of carbobenzyloxy-phenylalanyl-phcnylalanine azide, corresponding to 96% of theory, is dissolved in 8 ml of dimethylformamide, cooled to -10 ° C. and added dropwise, with stirring, to a precooled solution of the aminodipeptide ester . Yield 2.14 g with a melting point of 218 to 220 ¹ C. corresponding to 58.7 ",; of theory.

ίο Analysis Γ _1Κ Η ^ Ν _Λ Ο ₈ (Moleewidite 760.87);

calculated C 59.99, H 6.36, N 14.73%:

obtained C 59.49, H 6.33, N 14.81 "".

Example 18

Manufacture of carbobenzyloxy-phenylalanyl-leucyl-valyl-o-nitroarginine methyl ester

2.35 g of the product from Example 1 (5 mmol) are treated as in Examples 2 and 14 bc-

»O is written. The obtained dimethylformamide solution of Valyl-f) -nitroarginine methyl ester is applied as follows:

2.13 g of the product according to Example 7 (5 mmol) are in a mixture of 5 m! 6 N hydrochloric acid

a5 and 5 ml of 50% acetic acid dissolved with 80 ml of ethyl acetate covered and the process as described in Example 10 continued. The ethyl acetate solution with the carbobenzyloxy-Dipcptidazid is cooled to -10 "C and with stirring in the above-

The residue is _{dried over P 2} O and KOH in vacuo

gp

the residue, which assumed a granular consistency, 3 ° mentioned D ^; methylformamide solution is added dropwise, which is digested a further 3 times with dry ether . Which was pre-cooled to -10 ^{r C.} The further Aiisführerimr

corresponds to example 10. and the ethyl acetate solution obtained is evaporated to dryness. The residue is dissolved in 7 ml of dimethylformamide and cleaned according to Example 11. Yield 2.65 g with a melting point. 171 to 181 C, accordingly

₂ ,

dried. The yield of Tetrapcptidestcrhydrobromid is 1.62 g, which corresponds to 95% of theory. calculated on 1.5 equivalents of HBr per mole of peptide. The hydrobromide is dissolved in 20 ml of dimethylformamide and cooled to -10 ⁰ C, (4.28 mmol) are added followed by 0.60 ml of triethylamine and the triethylamine HBr formed is filtered off. With stirring at -10 ⁰ C to the filtrate 1.44 g Carbo-benzyloxy-phenylalanyl-p-GIycin Nilrophenylester (3 mmol) is added, and after 24 hours at room temperature the solution is again - cooled 1O ⁰ C, and there are a further 0.20 ml of triethylamine are added, whereupon the mixture is left to stand for 24 hours. The work-up is carried out as in Example 2. Yield 1.59 g with a melting point of 164 to 167 "C, which corresponds to 71.2% of theory. Recrystallization from 60% methanol increases the melting point to 169 to 172 ° C.

72.9% of theory. The product is recrystallized a further 2 times from 60% ethanol. where mcI is the melting point at 179 to 182 ^! C increased.

Analysis Q ₂ H ₅₀ N _K O "(molecular weight 726,851:

calculated C 57.84, H 6.94. N 15.42 ° ,,

obtained C 58.05. H 7.01. N 15.35%

Example 19

Preparation of carbobenzyloxy-valyl - ('(- nitroargininamide

45.8 ■. Carbobenzyloxy - o> - Nitroargininamu 50 (0.13MoI) with a melting point of 217 to 218 "C are dissolved in 300 ml of anhydrous acetic acid , and under anhydrous conditions 300 ml of 2 N-HBr in dry acetic acid are added with stirring 45 minutes at Raumtemperatu 55 is completed, the evolution of carbon dioxide, followed by di reaction mixture in vacuo at 2O ⁰ C unte anhydrous conditions is evaporated until de residue a volume of 250 has mL. the back stand is, under vigorous stirring in 2.5 1 of dry 2 35 g of the product according to Example 1 (5 mmol) of 60 ether are added dropwise, whereby a plastic mass is treated as in Examples 2 and 14. The ether phase is decanted and the return and the resulting solution of Valyl-w-nitroarginine stood another 3 times with dry ether digesteri methyl ester is applied as follows: the substance becomes granular and solid

2.3 g of carbobenzyloxy-Phcnylalanyl-phenyl-alanine is dried in vacuo over P ₁ O ₅ and KOH, i hydrazide (5 mmol) are dissolved in 7 ml dimethylformamide 65 350 ml of dimethylformamide, and it was solved and 3 ml of 4 N Hydrochloric acid added, cooled to 18.5 ml of 1 riethylamine (0.134 mol) added. After -10'C and 5.1 ml of 1 M-NaNO ₂ solution with cooling to -10'C, the triethy rcn formed is added dropwise and the mixture is filtered off as in Example 14 amine-HBr, and then with stirring

Analysis C ₃ SH ₄₈ N ₁₀ O _n (molecular weight 784.85);

calculated C 53.56, H 6.17, N 17.85%;

obtained C 54.01, H 6.08, N 17.99%.

Example 17

Manufacture of carbobenzyloxy-phenyialanyl-phenylalanyl-valyl-w-nitroarginine methyl ester

is

48.4 g of carbobenzyloxy-valine-p-nitrophenyl ester were added to the feed (0.13 mol) added. The reaction mixture is left at room temperature for 24 hours stand, concentrate it to 100 ml and shake it with stirring in 1 15% sodium bicarbonate solution. The precipitating substance is abfikrieri. With Washed water and redissolved in 75 ml of dimethylformamide and then poured into 1 1 of 1 N hydrochloric acid, filtered and washed with water. Yield 47.1g with a melting point of 5 to 63 ° C, corresponding to 80.2 ° ,, the theory. Recrystallization from 60 ° methanol increases the melting point of the Product a_uf 66 to 67 C, but after recrystallization from ethanol with the addition of water, the Melting point raised to 218-219C.

Analysis C ,,, H ₂₁₁ NjO ₆ (molecular weight 451.5, substance with melting point 218 to 219 ° C):

calculated C 50.55, If 6.47, N 21.72 ", -

obtained C 50.57. H 6.62, N 21.89 V

Example 20

Preparation of carbobenzyloxy-phenylalanyl-valyl-f) -nitroarginine amide

1.0 g of the product according to Example 19 (2.22 mmol) is dissolved in 5 ml of dry acetic acid, and 5 ml of 2N-HBr in dry acetic acid are added under anhydrous conditions. Working up is effected in accordance with Example 19. The resultant dimethylformamide solution (10 ml) of the valyl-f-nitro arginine amide is on - ^r cooled 10 C and there are 0.93 g of carbobenzyloxy-phenylalanine-p-nitrophen \ ester (22.2 mmol! ) added with stirring. The protected tri-eptidamide is purified according to the guidelines of Example 19. Yield 1.15 g with a melting point of 213 to 220 ° C., corresponding to 86.5% of theory. Two recrystallizations from 80% ethanol increase the melting point of the Product to 218 to 220 ° C.

Analysis C _K H. _{) fi} N _K O ₇ (molecular weight 598.68):

calcd C 56.18, H 6.40, N 18.72 °;

obtained C 56.29, H 6.52, N 18.87 °.

Example 21

Preparation of carbobenzyloxy-f) -nitroarginylfj-nitroarginine methyl ester

35.3 g of carbobenzyloxy-r'j-nitroarginine (0.1 mol) with a melting point of 134.5 to 136 "'C are dissolved in 200 ml of dimethylformamide / tetrahydrofuran (1: 1), and 13.9 ml of trimelhylamine (0.1 mol) are added dropwise. After cooling to -10 "C, 13.66 g of isobutyl chloroformate (0.1 mol) are added over a period of 20 minutes, the temperature in the solution being between -10 ° C and -5 ⁰ C holds. The solution is ω-nitroarginine methyl ester dissolved in dimethylformamide and freed from 27.0 g ω-nitroarginine methyl ester HCl (0.1 mol) by means of 13.9 ml triethylamine (0.1 mol) and removed

- ^{Pre-cooled 10 c} C, added, the temperature in the reaction mixture during the dropping

- 8 ^C C. The reaction mixture is allowed to slowly reach room temperature and after a few hours the reaction has ended. Triethylamine HCl, which was formed during the reaction, is filtered off and the I iliral 7ur evaporated to dryness.

The residue is dissolved in 900 ml of butyl alcohol / ethyl acetate (2: 1) dissolved with 1N hydrochloric acid, 5 "strength sodium bicarbonate solution and water, and the solvent phase is dried over magnesium sulfate. After the solvent has evaporated obtained a substance with a melting point of 136 to 14K C. Yield 34.6 g (60.8 °., Theory). The product is recrystallized a few times from 50 Ω / pounds of methanol. The melting point drops to 147 ίο increased to 150 C.

Analysis CiH ₁₁ N ₁₀ O ₉ (Molccwiehl 568.57);
calculated "...." .... C 44.36. H 5.67. N 24.64 ".; obtained C 44.38. H 5.64, N 24.72".

Example 22

Manufacture of carbobenzyloxy-phenylalanyl-valine methylsier

so 29.9 g carboben? \ loxy-phen \ lalanine (0.1 mol) are dissolved in 75 ml of dimethylformamide, cooled to -10 ° C. and then 20.6 g of dicyclohexylcarbodiimide. dissolved in 25; nl dimethylformamide, added dropwise. Valine methyl ester, dissolved in 30 ml of dimethylformamide and by adding 13.85 ml of triethylamine to 16.8 g valine methyl ester HCl (0.1 mol) in dimethylformamide freed from its hydrochloride, is added dropwise so that the temperature of the reaction solution at -10 ° C. The reaction is complete after 24 hours at room temperature, and after cooling, the dicyclohexylcarbamide formed is filtered off. The filtrate becomes dry evaporated, the residue dissolved in atlnlacclate and shaken as usual. After the

Ethyl acetate, the residue is dissolved in 80 "JgCm methanol dissolved, whereby 33.5 g of substance with a melting point of 113 to 115 C crystallize out. yield 33.5 g (81.2 "" of theory).

Analysis C “H .. _V N ₂ O _S (molecular weight 412.49);

calculated C 66.97, H 6.S4. \ 6.79 ° ,,:

received C 67.09. H 6.85. N 6.91 "".

Example 23

Manufacture of carbobenzyloxy-phenylalanyl-valine hydrazide

8.25 g of the product obtained according to Example 23 (2OmMoI) are dissolved in 150 ml of methanol and 3.94g hydrazine indral (8OmMoI) added. Is will Refluxed with stirring for 48 hours. concentrated, and after adding a little water a product crystallizes out and is recrystallized from methanol / water. Yield 7.35 g with a melting point of 210 to 211.5'C, accordingly 89 "" of the theory

Analysis C ₂ , H ₂ .N ₄ O ₄ (molecular weight 412.5):

calculated. "C 64.06. 1! 6.84. N 13.58" ^

obtained C 64.64, 116.5S, N 13.63 ";

Example

Making Ca 1 ho)

PhcriN lalanine-M

24

m-Phenyl.ihn vlv lcster

16.2 g of pheinlalanine methyl ester IICI (75 mmol) mi a melting point 15S.5 to 1 59 C are ii Dimcilnlfnrniamid dissolved, cooled to 0 ° C.

04

10.5 ml of triethylamine (75 mmol) were added. Triethylamine-HCl formed is filtered through, and the filtrate is cooled to -10 ° C., whereupon 31.5 g of carbobenzyloxy-phenylilanine-p-nitrophenyl ester (75 mmol) are added. After 24 hours at room temperature, the solution is cooled again to -10 ° C. and 3.5 ml of triethylamine (25 mmol) are added, whereupon the solution is left to stand for a further 24 hours. Then the residue is eingedunstet in vacuo to dryness, dissolved in ethyl acetate and washed with 5% sodium Natriumbicarbonatlösurig, water, 3 _cent strength hydrochloric acid and water. The ethyl acetate solution is dried over magnesium sulfate, concentrated to a smaller volume. After the addition of petroleum ether, the product crystallizes out as needles, which are recrystallized twice from ethyl acetate / petroleum ether. Yield 27.85 g with a melting point of 148.5 to 149 "C, corresponding to 80.7% of theory.

Analysis C, ₇ H, _S N., O, (molecular weight 460.54);

calcd C 70 ^ 42, H 6.13, N 6.08%;

obtained C 70.69, H 6.15, N 6.12%.

Example 25

Manufacture of carbobenzyloxy-phenylalanyl-phenylalanine hj drazid

23.0 g of the product obtained according to Example 25 (50 nmoles) are dissolved in 500 ml of dry methanol. 7.5 g of hydrazine hydrate (0.15 mol) are added and the mixture is refluxed for 4 hours, after which it is left to stand at room temperature overnight. The precipitation of 18.1 g formed with * INEM melting point of 198 to 2OO, ^C 5 C is abgetaugt and eingedunstet the Fillrat to about 100 ml. After adding a little water, 4.15 g of substance crystallize with a melting point of 196.5 to J97.5 ^; C off. Both fractions are limcrystallized from methanol, the melting point increasing to 201 to 203.5.degree. Bulge 19.5 g (84.8% of theory).

Analysis C ₆ H., _S N ₄ O ₄ (molecular weight 460.54);

calcd C 67.81, H 6.13, N 12.17 ° _O ;

obtained C 67.57, H 6.08, N 12.37%.

Example 26

Manufacture of carbobenzyloxy-phcnylalanyl-glycine-ethyl ether

2.8 g of glycine ethyl ester HCl (20 mmol) are dissolved in 20 ml of dimethylformamide and 2.05 g of triethylamine are added. The mixture is cooled to OC and the triethylamine-HCl formed is filtered off. A mixture of 6.0 g of carbobenzyioxy-phenylalanine (20 mmol) and 4.2 g of dicyclohexylcarbodiimide (20 mmol) in 20 ml of dimethylformamide is added to the filtrate at -10.degree. After 24 hours at room temperature, the mixture is cooled to OC and dicyclohexylcarbaniide which has formed is filtered off. The t iltrai *> ird evaporated to dryness, the residue dissolved in Athylacclul and as usual with 4% SaIztäure, 5 _"u IGCR sodium untl water shaken. After drying over magnesium sulfate and concentrating, the product crystallizes upon addition of petroleum ether from. Yield 6 , 45 g with a melting point of 109 to 111,5'C, corresponding to t! 3.9% of theory. the melting point is increased by recrystallization from Äthylacetai-Petroiiither to H2 to 112.5 ⁼ C.

Analysis C ₁ H ₄₁ NO ₃ (molecular weight 384.44):

calculated "...". C 65.64, H 6.29, N 7.29%;

emahen C 65.35, H 6.35, N 7.40-.

Example 27

Manufacture of carbobenzyloxy-phenylalanyl-glycyl-glycine ethyl ester

1.97 2 Glycyl-Glycine-Ethyiester-HCl (10 mmol) with a melting point of 182 to 184 C are dissolved in 30 ml of dimethylformamide, 1.4 ml of triethylamine (10 mmol) added and triethylamine-KCI formed filtered off. 4.2 g (10 mmol) of carbobenzyloxy-phenylalanine-p-nitrophenylsier are added to the filtrate admitted. It is worked up as described in previous examples (13). The product is made from dimethylformamide / water recrystallized. Yield 3.85 g with melting point 93 to 95C, which is 87.2% of theory is equivalent to.

Analysis C ₃ H _n N ₁ O ₆ (molecular weight 441.49); calcd "C 62.57, H 6.16, N 9.52"..;

obtained C 62.20, H 6.05, N 9.72 V

Example 28

Manufacture of carbobenzyloxy-phenylalanyl-glycine hydrazide

6.35 g of the product obtained according to Example 27 (16.5 mmol) are dissolved in 50 ml of methanol and 3.2 g of hydrazine hydrate (64 mmol) were added. To

The solution is evaporated to dryness with stirring at room temperature for 24 hours. The residue is dissolved in a little warm methanol, whereupon the product crystallizes out. Yield 4.95 g with a melting point of 151.5 to 152.5 ⁰ C, corresponding to 81.0% of theory.

Analysis C ₁₈ Hj ₂ N ₄ O ₁ (molecular weight 370.42):

calcd C 61.61, H 5.99, N 15.13 ^C _O ;

obtained C 61.80, H 5.87, N 15.22 ";.

45

Example 29

Manufacture of carbobenzyloxy-phenylalanylglycyl-glycine hydrazide

2.1 g of the product obtained according to Example 28 (4.75 mmol) are dissolved in 25 ml of methanol and 0.9 g of hydrazine hydrate (18 mmol) was added. After 48 hours of standing at room temperature, the The precipitate formed is filtered off and washed with water. The product is recrystallized from methanol. Yield 1.75 g with a melting point of 190 to 191C, corresponding to 86.6% of theory.

Analysis C ₂₁ H ₀₅ N ₅ O ₅ (molecular weight 427.47);

calculated C 59.01. H 5.90, N 16.38%;

obtained C 59.49, H 5.95, N J 6.50%.

Example 30

Preparation of carbobenzyloxy-valylc) nitroarginine isopropyl ester

2.5 g of n-nitroarginine isopropyl ester hydrochloride (8.4 mmol) are dissolved in 30 ml of dimethylformamide.

After cooling, 1.2 ml of triethylamine are added and the triethylamine hydrochloride which precipitates is removed by filtration. The filtrate is cooled to -1O ⁰ C and 3.2 g of carbobenzyloxy-Valinp-nitrophenyl ester (8.4 mmol) are tugesetzt with stirring. After stirring for 2 hours at -10 ^J C is allowed to take the substance room temperature and react for one day at this temperature. The reaction mixture is cooled again to -10 ° C., after which a further 1.2 ml of triethylamine are added. The reaction is practically over after a few hours. Dimethylformamide is evaporated off in vacuo at 30 to 40 ° C. and the residue is dissolved in about 500 ml of ethyl acetate saturated with water. The solution is extracted as usual with 5% aqueous sodium bicarbonate solution, water, 1 N aqueous hydrochloric acid and water The phase is dried over magnesium sulphate and evaporated to dryness in vacuo, the residue is dissolved in hot ethyl acetate, and after the addition of dry isopropyl ether the substance crystallizes out when it is left to stand in a cooling chamber a melting point of 162 to 166 ° C., which is 88% of theory. After a further recrystallization from ethyl acetate / isopropyl acetate, the melting point increases to 165 to 168 ° C. When recrystallized from methanol / ethyl acetate, the substance has a melting point of 115 to 116 ° C. The substance is uniform in thin-layer chromatography on silica gel in three different solution systems and has e an optical rotation of [λ]? = -24.6 ^C (C = 1st methanol).

Analysis C ₂₂ H ₃₄ N ₈ O ₇ (molecular weight 494.56);

calculated C 53.43, H 6.93, N 16.99%;

obtained C 53.20, H 6.99, N 17.11%.

Example 31

Manufacture of carbobenzyloxy-phenylalanyl-

Valyl-w-nitroarginine isopropyl ester

2.0 g of carbobenzyloxy-valyl-w-nitroarginine isopropyl ester (4.05 mmol) are dissolved in 10 ml of dry glacial acetic acid, then 5 ml of 4N hydrogen bromide in glacial acetic acid are added and the mixture is stirred for 1 hour at room temperature, the reaction is protected from moisture in the air. During the reaction time a quantity of tripeptide window hydrobromide crystallizes out, and indeed completely after adding 2.50 ml of dry ether. The ether phase is decanted and the precipitate is treated 3 to 4 times with dry ether. The precipitate is dried in vacuo over potassium hydroxide and P ₂ O ₅ and is very hygroscopic. The tripeptide ester hydrobromide obtained is dissolved in 15 ml of dimethylformamide and cooled to 0 ^c C and treated with 1 ml triethylamine (7.3 mmol). The triethylamine hydrobromide obtained is filtered off and 1.7 g of carbobenzyloxy-phenyl-alanine-p-nitrophenyl ester (4 mmol) are added to the filtrate. After a few hours at SSO - 10 ^c C was allowed to stand, the temperature is allowed in the Reakticnslösung to 20 C increase. The reaction solution is then cooled to 10 ° C., whereupon 1 ml of triethylamine is added once more and the reaction mixture is left to stand overnight. The solution is evaporated to dryness in vacuo and the residue is dissolved in a mixture of ethyl acetate / n-butanol (2: 1).

The organic phase is extracted as usual with 5% aqueous sodium bicarbonate solution, water, normal aqueous hydrochloric acid and water and dried over magnesium sulfate. The dried solution is evaporated to dryness and the residue is dissolved in a hot methanol-ethyl acetate mixture (1: 1), whereupon the solution remains in the cooling chamber for a day. 1.93 g of a crystallized substance with a melting point of 175 to 177 ° C. are obtained. Recrystallization increases the melting point to 178 to 179 ^° C. The yield is 1.7 g, which corresponds to 68% of theory. The substance is uniform in thin layer chromatography on silica gel and has an optical rotation of [χ} Ζ = 25.7 ° (C = 1, methanol).

Analysis C ₃₁ H ₁ -N ₇ O ₈ (molecular weight 641.74);
calcd .... * .... C 58.01, H 6.75, N 15.28%; obtained C 58.32, H 6.92, N 15.19%.

Example 32

Preparation of carbobenzyloxy-valylw-nitroarginyl-w-nitroarginine methyl ester

5.7 g of carbobenzyloxy-cu-nitroarginyl-io-nitroarginine methyl ester (10 mmol) and 30 ml of dry glacial acetic acid are placed in a flask protected from atmospheric moisture, whereupon 10 ml of 4N hydrogen bromide in dry glacial acetic acid are quickly added. After stirring for 1 hour at room temperature, the carbobenzyloxylation is complete and a plastic substance has formed. The reaction mixture is poured into 700 ml of dry ether while stirring vigorously, as a result of which a plastic hard mass is formed, the surface of which is partially crystalline. The ether phase is decanted off and the residue digested with dry ether, which is then decanted off again. The process is repeated until the product has become crystalline, whereupon the product _{is dried over P 2} O ₅ and potassium hydroxide in vacuo. The yield of dipeptide ester hydrobromide is 6.5 g, and the determination of the bromine content shows that the substance contains 2.3 equivalents of hydrogen bromide. The substance is dissolved in 40 ml of dimethylformamide, ^{cooled to 0} ° C. and mixed with 3.5 ml of triethylamine (25 mmol). The resulting triethylamine hydrobromide is removed by nitration, and 3.75 g of carbobenzyloxy-valine-p-nitrophenyl ester are added to the filtrate, whereupon the reaction mixture is allowed to slowly come to room temperature overnight. Another 1.4 m of triethylamine (10 mmol) to the mixture, after cooling to -10 ⁰ C were added, followed by allowing the reaction to continue fell · overnight. The solution is evaporated to dryness and the residue treated with ether, a small amount of ethyl acetate, a 5% aqueous sodium bicarbonate solution, a 10% aqueous citric acid solution and water and dissolved in hot methanol. On subsequent addition of ethyl acetate, 5.1 g of a yellow crystalline product are obtained. Two recrystallization ions from methane / ethyl acetate (1: 1) finally give a product with a melting point of 119 to 122 C and an optical rotation of [α] "= -12.3 ° (C - = 1, Dimethylformamide).

Thin layer chromatography on silica gel revealed the product in three different solutions systems only have a colored zone, which indicates that

the substance is uniform. Yield 4.35 g (65.2% theory).

Analysis CmH ₄₁ N ₁₁ O ₁₀ (molecular weight 643.68);

calculated C 44.78 H 6.42, N 23.94%:

obtained C 44.52, H 6.38, N 24.11%.

Example 33

Preparation of carbobenzyloxy-phecylalanyl-valyl-w-nitroarginyl-tu-nitroarginine methyl ester

1.30 g carbobenzyloxy-valyl-oj -nitroarginyl-i'j-nitroarginine methyl ester are deearbobenzyloxylated as described in the previous examples. During the subsequent work-up, 1.55 g of a highly hygroscopic tripeptide ester hydrobromide are obtained obtained, based on a determination of the bromine content 2.4 equivalents of hydrogen bromide contains. The tripeptide ester hydrobromide is dissolved in 15 ml of dimethylformamide, cooled to -10 "C and 0.67 ml of triethylamine (4.8 mmol) were added. The resulting Triethylamine hydrobromide is removed by filtering, and 0.84 g of carbobenzyloxy-phenylalanine p-nitrophenyl ester (2 mmol) are added to the filtrate, whereupon the reaction solution is left overnight Can accept room temperature. 0.28 ml of triethylamine are added to the solution, which takes several hours is stirred for a long time. The reaction mixture is evaporated to dryness and the residue with Treated with ether, which contains a small amount of ethyl acetate, and then with aqueous, 5% sodium bicarbonate solution, Water, a 10% aqueous citric acid solution and water.

The residue is dried and then in methanol / dimethylformamide (4: 1) dissolved, treated with activated charcoal and the clear-filtered solution in the Kühlkar jner used, whereby 1.48 g of a product are obtained that has a melting point from 234 to 240 ° C. Further recrystallization of the substance increases the melting point to 239 to 241 ° C. The yield from chromatographic uniform substance is 1.15 g with an optical rotation of [α]? = -9.6 ° (C = 0.5 Dimethylformamide), which corresponds to 70.6% of theory.

Analysis CjJHs ₀ N ₁₁ O _n (molecular weight 814.88);

beredinet C 51.59, H 6.19. N 20.62%;

obtained C 51.81, H 6.09, N 20.95%.

Example 34

Preparation of carbobenzyloxy-phenylalanyl-valyl-w-nitroarginyl-w-nitroarginine methyl ester

408 mg of carbobenzyloxy-phenylalanyJ-valyl-w-nitroarginyl-cu-nitroarginine methyl ester (0.5 mmol) are decarbobenzyloxylated as described in the previous examples. 538 mg of the resulting tetrapeptide ester hydrobromide, which contains 2.4 chemical equivalents of hydrogen bromide, are dissolved in 10 ml of dimethylformamide, cooled to -10 ° C and treated with 0.17 ml of triethylamine, whereupon the resulting triethylamine hydrobromide is removed by filtration. 0.21 g of carbobenzyloxy-phenylalanine p-nitrophenyl ester (0.5 mmol) are added to the filtrate. The reaction sequence is the same as described in the previous examples. The solution is evaporated to dryness in vacuo and the residue is digested with ether, a 5% strength aqueous sodium bicarbonate solution, water, a 10% strength aqueous citric acid solution and water. The product treated in this way is dissolved in hot methanol, and the solution, after being left to stand in the cooling chamber for one day, yields 4.18 mg of a product with a melting point of 230 to 238 ° C. The substance is then recrystallized twice from methanol, which is a chromatographically uniform substance with a melting point of 236 to 240 ⁰ C and an optical rotation

ίο of [*]% = -20.0 ° (C = 3.8, dimethylformamide) results. The yield is 338 mg, which corresponds to 703% of theory.

Analysis C ₄₄ H ₅₁₁ N ₁₃ O ₁₂ (molecular weight 962.06);
calculated C 54.93, H 6.18, N 18.93%:

> 5 obtained C 54.62, H 6.25, N 19.10%.

The terminal α-amino groups as well as the other amino groups protected by carbobenzyloxy groups the peptides are protected by these groups

ίο by hydrogenation of the carbobenzyloxy group in Form of toluene and carbonic acid released. The nitro group in arginine is used simultaneously as ammonia reduced, with the help of hydrogen as a reducing agent and palladium on carbon

as a catalyst. After all the reductions on can be carried out in the same way, with the exception of minor variations which are shown in a table only a general description of the experiments follows (experimental details see Table 2) The protected peptide ester is dissolved in an alcohol - methanol or ethanol - and the required Number of equivalents of the salt-forming acid added in the form of a water or alcohol solution. The catalyst moistened with water (10% palladium on carbon) is added in an amount corresponding to 100 mg per millimole of peptide. the The amount of catalyst is increased somewhat as the length of the peptide increases. As soon as the catalyst is removed from the Walls of the reaction vessel is rinsed. gets all air out of the reactor with nitrogen repressed. The nitrogen is in turn expelled with hydrogen. The reduction system is closed, and the hydrogen for the reduction is introduced via a measuring vessel as a hydrogen reservoir. The reaction begins with vigorous stirring in the reduction vessel, and the carbon dioxide formed is absorbed with stirring in a flask connected to the reaction vessel, the 50% Kalilau · * - contains. With the help of a

so reservoir connected level bottle, the water contains as sealing liquid, the hydrogen consumption for the complete reaction is read off (corrected to normal pressure and normal temperature as well as reduced with the partial pressure of water at the relevant temperature). The reaction time varies depending on the length of the peptides and presence of nitro groups in arginine between 2 and 12 hours. Once the reaction has ended nitrogen is passed through the reaction vessel to displace the hydrogen. The catalyst is filtered off, the filtrate evaporated to dryness, and the residue a few times with dry ether digests, whereupon the product becomes firm and grainy. All peptide esters are very much called hydrochlorides hygroscopic and must therefore be processed and isolated in a drying chamber and also cool and stored under anhydrous conditions.

23

Table 2

Peptide derivative
for reduction
of the example

Amount of the peptide derivative by weight in M oil

HCL ■

H - Art- (NO ₂ ) - OMc

1
11th
12th
13th
14th
15th
16
17th
18th
20th

5.39 g

4.66 g 253 mg 515.6 mg

5.0 g 826 mg 773 mg 450 mg 352 mg 1 374 mg 675 mg

20th

10

0.227 1

8.15 1.23 1.06 0.57 0.46 1.89 1.13

Meniie

10 ",. '

PO ₁ C

75 mg 100mg

150 mg 125 mg 100mg

50 mg 200 mg 100 mg

24

Water loafeiMiieniic in ml Calculate Veihi.

1793

1120

25.5
112
913
138
119

64

52
212
126

1 5S0

1025

22.5 112 8S0 135 112

200 141

Response time in hours

Pcptiddcrivat Yield of reduced chloride content of the amino acid analysis

for the reduction of peptide esters - NH ₁ CI product

of example _C ; _{ewii: hl} percent Bcrcchn. F.rlialten

HCL
H - Are
(NO ₂ ) - OMe 5.3 g 87.6 31.15 33.90 Va! : Arg : NH,
0.99 : 1.0 ~ l : 0.92 Arg: NH ₃
0.96: 0.72: 1 3.25 g 78.6 25.02 24.12 Phe : Leu : AIa : GIu : GIy : Va! : Are
0.95 : 1.03 : 1 .09 : 1.04 : 3.00 : 1.03 : 0.9 ( NH ₁
0.73 11th 195 mg 80.9 04/10 9.76 Phe: Arg : NH,
1.00 : 1.00 : 0.85 Arg: NH ₁
0.97: 0.76 12th 375 mg 81.2 23.03 21.92 Phe: VaI : Arg : NH,
1.01 : 1.02 : 0.97 : 0.83 NH ₁
1.94 13th 4.45 g 97.4 18.96 17.90 Phe: GIv : VaI : Arg : NH ₃
1.02 : 1.00 : 1.02 : 0.96 : 0.97 14th 695 mg 91.3 17.21 05/17 Phe: GIy : VaI: Arg : NH,
1.02 : 2.03 : 0.98 : 0.97 : 0.84 15th 650 mg 90.7 15.76 14.98 Phe: Uly: VaI
1.00: 2.98: 0.98 16 390 mg 92.9 14.53 14.13 Phe: VaI: Arg
2.01: 1.02: 0.97 17th 283 mg 86.4 15.02 14.27 Phe: Leu: VaI
1.01: 0.99: 1.02 18th 1050 mg 82.4 15.78 14.54 Phe: VaI: Arg
1.00: 1.02: 0.97 20th 515 mg 83.7 19.49 18.64

The antithromboplastin activity for some of the In Table 3 gives the values in column 3

The most interesting compounds are shown in Table 3 with the following 35 50 "ige inhibition of thrombopin instinct

calculated as an effect equivalent to one millimole of peptide. and / was expressed in

certain amount of trypsin hcmcr from the speaking amount of trypsin hcmcr (mg). O >>

Lungs in standardized form. in the tabular formulas means a methyl ester

/H

Table 3

Peptides

Number Relative amino-tartaric acid mung

Phe --VaI -Arg -OMe 3 0.18

Phe GIy - Val - Arg OMe 4 0.10

Phe -Phe -Val -Arg -OMe 4 0.19

Phe - Leu - Val - Arg - OMe 4 0.11

Phe - (GIy), - Val - Arg - OMe 5 0.076

Phe- (Clv);, - Val -Arg -OMe 6 0.15 Phe-Leu-Ala -GIu- (GIy) ₃

Val -Arg-OMe 9 0.39

The vascular active bradychinin-like effect of two of the most potent peptides according to the invention was expressed as the percentage increase in blood flow in the a. femoralia for intra-arterial injection Dogs with a solution at a concentration of 1 mg peptide / ml and an injection rate measured between 1 to 3 ml / min

Table 4

peptide

corresponding activity of heparin, a commercially available one Amicoapulant. compared.

Different amounts of the heparin or the peptide were incubated with thrombin for 30 seconds. The remaining thrombin activity was then tested for fibrinogen as a substrate. The time until to polymerize fibrin - the final stages of blood clotting consist of the conversion of Fibrinogen to fibrin and a subsequent polymerisation of the fibrin - was measured and with compared to a blank sample.

Buffer:
Distilled
Water:
Heparin:

Reagents
Tris buffer, 0.15 M, pH 7.35

Amount of peptide per ml of blood

Flow rate increase in%

Phe - VaI - Arg - OMe 0.017 130 0.031 270 0.033 260 0.05 480 0.1 520 Phe -Leu -Ala -GIu (GIy) ₃ - Val - Arg - OMe 0.033 140 0.05 360 0.1 310

35

The an'icoagulation activity of the polypeptide H - Phe - Vaf - Arg - OMe • HCl was determined with the Vitrum-Heparin 5000 1 µmI, diluted with water

Peptide: H - Phe - VaI - Arg - OMe ■ HCl thrombin: bovine thrombin 340 IU; mg, dissolved
to 100 IU / ml physiological saline solution, further diluted in Tris buffer 1/15
Fibrinogen: 0.4 ^, in Tris buffer, pH 7.2

Carrying out the experiment

0.5 ml thrombin solution (dilution 1/15; 37 "C] were 30 seconds with 0.5 ml HeparinlösunE (various degrees of dilution: 37 ⁰ C) or with 0.5 ml of peptide solution (various degrees of dilution: ^37: C) or with 0.5 ml of distilled water (37 ⁰ C; incubated.

0.2 ml of each incubation preparation were zi 0.2 ml of fibrinogen heated to 37 ° C. is added. The time taken for the fibrin to polymerize was determined. The increase in the coagulation time (.1 /) was compared to the peptide concentrations (mg / m!) Or di ( Heparin concentration (IU / ml) is applied. The results are shown in Table 5 below

Table 5 Phe - VaI - Arg - OMe HCl 1.0
46.5 2.0
69 3.0
72 10.0
12.5 0.028
5.5 0.125
16 0.25
19.5 0.5
34 mg / ml
Increase in
Coagulation
time (see) Heparin 0.5
17.5 1.0
17.5 5.0
17.5 0.02
5.0 0.1
10.0 0.2
15.0 0.3
16.5 IU / ml
Increase in
Coagulation
time (see)

As can be seen from Table 5, heparin shows a good effect in relation to in small concentrations e on the coagulation time. At higher concentrations, however, the coagulation time does not increase Increasing concentrations prolonged, rather one receives a constant value, which at still high higher concentrations even drops again. This is explained by the fact that for heparin the An The essence of its coftctor is required if an anticoagulant effect is to be achieved. This

λίν

8 04 022

Cofactor is therefore for the one in a heparin treatment achievable maximum effect is decisive.

Patients who are a result of prolonged heparin therapy or have low levels of the heparin cofactor for other reasons therefore respond poorly to the heparin treatment.

On the other hand, with the tested peptide, a favorable therapeutic response will be given Dose achieved over the entire measured area. Treatment with the tested peptide is therefore preferable, especially in the presence of a cofactor deficiency.

Claims (1)

1 2 lare sulfur bridges in insoluble coagulates patent claims: be converted. The preliminary formation of thromboplastin will be 1. Phenylalanine and arginine in terminal posi- tion in the blood vessels activated in places where a polypeptides of the formulas 5 which have been violated. This thromboplastin, which is "intrinsic" thrombus Phe - VaI - Arg - OMe is called boplastin or plasma thromboplastin, However, Phe - GIy - VaI - Arg - OMe can be replaced by an active product, pj, _e Qiy QJy y _a i A _rg OMe which is called "extrinsic" thromboplastin and p _he _GIy_GIy_GIy_VaI-Arg-OMe ^{10 the u} _K ^nter ? ° ^{EFFECT OF THE FACTOR IN THE VESSEL} - . _v , weaving is formed. "" ^e ™ " ^e ^ aI ^ rg OMe Q _er g _an2e coagulation process is a series of Phe - Leu - VaI - Arg - OMe enzymatic reactions in which different Phe - VaI - Arg - NH ₂ or factors activate each other successively. Phe - Leu - AIa - Glu "(y-OMe) - GIy - ¹ S ^{There are} several substance groups with a different known to have a reluctant effect on blood coagulation. - GIy - GIy - VaI - Arg - OMe. D ^ ₆ substances can have their effect on various processes in the complicated coagulation 2. Process for the production of the polypeptides exercise, namely by influencing the according to claim 1, characterized in that ₂₀ blood vessels to strong fibrinolysis. Influencing them in a manner known _{per se from the solution of the} coagulation mechanism can thus build up an amino acids VaI, Arg, Phe, GIy, Leu, AIa and vascular effect, an antithrombin effect, a fibri-GIu. nolytic effect or an antithromboplastin be effect. In anticoagulation therapy, e.g. B. 25 in connection with surgical interventions applies it means at as early a stage as possible to use that prevent the coagulation process, The Ernndung relates phenylalanine and arginine in _when pathological changes of presuming termialen positions having polypeptides of the danger of a high torque for thrombosis formulas ₃₀ ^ _n j _a ß have given. Such a moment of danger p, _yi δ OM is often found in younger patients after a surgical _n , ^C "" . * ^S. . ^e _ .. previous surgery. Phe - GIy - VaI - Arg - OMe Substances that affect the coagulation system Phe - GIy - GIy - VaI - Arg - OMe flows, are z. B. heparin, coumarin derivatives, strepto- Phe - GIy - GIy - GIy - VaI - Arg - OMe 35 chinase, proteinase inhibitors, etc., and these sub- Ph _e phe VaI Arg OMe punches were previously used in anticoagulation p, _{T used} via n \ yf therapy. When using this substance 1 ne ueu vai arg uMe _{zen lfeten abej} . _certain difficulties, in particular Phe VaI Arg NH ₂ or ^ _{£ Γε} j _{st es e} j _{n mc} h _t insignificant problem that Phe - Leu - AIa - GIu (y-OMe) - GIy _{40 to determine an} individually suitable dose. Improved GIy - GIv --VaI - Are OMe drugs were used to improve the therapy situation. The present invention relates to new sub- and processes for their manufacture. punch that is primarily an antithromboplastic The polypeptides of the invention exhibit interesting effects and thus thrombin formation as well pharmacological properties, especially blood-45 affect the antithrombin effect, so that they the anti-coagulation effects. Inhibit conversion of fibrinogen into fibrin. The coagulation of the blood is known to be an In addition, certain complicated process produced in accordance with the invention. Schematically, other interesting effects can be shown according to all polypeptides, commonly accepted hypotheses of normal coagulation. For example, some of them have a strong influence on the blood lation process. They are divided into three phases, viz. Vessels, which in the treatment of various lent a first phase, in which circulatory disorders are of great interest due to interaction.
between certain factors in the blood thromboplastin. The polypeptides according to the invention have a is formed, a second phase, during which the chain of amino acids is formed, which is in close correspondence with the peptide A prothrombin of the blood under enzymatic contact with humans. The action of a factor that has been activated by the natural Fibnnopeplide concerned by thromboplastin 55 is converted into thrombin, 14 to 16 amino acids, but it has according to the invention as well as a third phase in which the thrombin, which measured, has been shown to have good effects a protheolytic enzyme is already in use , the fibrinogen can be obtained in the formation of polypeptides which convert to fibrin, which forms a solid koalate. 3 to 9 amino acids are built up, although polypeptides are built up from fibrinogen into fibrin. This conversion of fibrinogen into fibrin is likely to be 60 de with longer chains within the scope of the invention to ensure that the fibrinogen is produced by impairing the thrombin Helix in the polypeptide chains contain short chains which cleave peptide Λ and Pep- d. Iv. when the number of amino acids is a multiple before tid B. 3 is. Specifically two amino acids, namely phenyl Through aggregation via the active Abspalumg- 65 alanine and arginine, a the i ibi inmoleküic end up in a long fiber-good anüihromboplastic effect as essential Similar complexes in the form of soft aggregates were found when "these amino acids in endposi which are located on it through intermolecu- tionci ".