NO122126B - - Google Patents

Description

Analogy progress in the production of polypeptides with therapeutic effects.

The invention relates to an analogue method for the production of polypeptides with therapeutic effects, in particular blood coagulation-inhibiting effects.

Blood coagulation is, as is well known, a complicated process.

Schematically, according to relatively generally accepted hypotheses, the normal coagulation process can be divided into three phases, namely a first phase where thromboplastin is formed by interaction between certain factors in the blood, a second phase during which the blood's prothrombin changes. is formed into thrombin under the enzymatic involvement of a factor' which is activated by the thromboplastin, and a third: phase where the thrombin, which is a proteolytic enzyme, converts the fibrinogen into fibrin which forms a solid clot. This conversion of fibrinogen into fibrin presumably takes place by the fact that the fibrinogen, under the influence of thrombin, cleaves off a pair of polypeptides with short chains and. cold peptide A and peptide B. When aggregating over the active cleavage terminals, . the fibrite molecules form long fiber-like complexes in the form of soft aggregates which are then converted into insoluble clots by meHorn molecular sulfur bridges. The initial form-

see of thromboplastin is activated at the place in the blood vessels where an injury has occurred. However, this thromboplastin, called intrinsic thromboplastin or plasma thromboplastin, can be replaced by an active product called extrinsic thromboplastin, which is formed under the influence of a factor in the vascular tissues. The entire coagulation process is a series of enzymatic reactions where various factors successively activate each other.

.Several groups of substances with a delaying effect on blood coagulation are known. These substances can exert their effect on various steps in the complicated process from blood vessel influence to strong fibrinolysis. Thus, the influence on the coagulation mechanism can be a vessel effect, an antithrombin effect, an antithromboplastin effect or a fibrinolytic effect. In the case of anticoagulation therapy, e.g. in in connection with surgical interventions, it applies to

at as early a stage as possible to use agents that prevent the coagulation process if pathological changes have given reason to assume that there is a high risk factor for thrombosis formation. Such a risk element is often present even in younger patients after surgical interventions.

Substances that affect the coagulation system are e.g. heparin, coumarin derivatives, streptokinase, trypsin preparations etc., and these substances have been used in anticoagulation therapy. When using these substances, however, certain difficulties occur, and it is in particular a not insignificant problem to titrate the individually suitable dose. Improved pharmaceutical preparations should be able to improve the therapy situation.

The invention relates to the production of new substances which, firstly, show an antithromboplastic effect and thereby affect thrombin formation, and an antibromine effect whereby they inhibit the transfer of fibrinogen to fibrin. Some of the polypeptides produced according to the invention have also been shown to have other interesting effects. Thus some of them have been shown to have a strong vascular effect which is of great interest in the treatment of various circulatory disorders.

The polypeptides produced by the present analogous method have a sequence of amino acids which is in close agreement with peptide A from humans. The natural fibrinopeptides in question consist of lh - 16 amino acids, but according to the invention it has been shown that good effects can already be achieved by using polypeptides made up of 3 - 9 amino acids, although polypeptides with longer chains also fall within the scope of the invention. A maximum antithromboplastic effect is obtained with smooth spirals in the polypeptide chains, i.e. when the number of amino acids is a multiple of 3. In particular, two amino acids, f.enylalanine and arginine, have been shown to be of significant importance for achieving a good antithromboplastic effect, and especially if these amino acids are in terminal positions.

The antithromboplastic effect is the most interesting effect of the polypeptides produced according to the invention as it enables an influence on the coagulation process at an early stage.

As mentioned, however, an antithrombin effect and a fibrinolytic effect and in some cases a vascular effect could also be determined. It has not been possible to establish the latter effect in any clear connection with the configuration of the natural peptide A. The vascular effect can be regarded as a bradykinin-like activity.

The above-mentioned effects have been investigated in large experimental animals in vivo and in blood from humans, which will be described in more detail.

The compounds in question contain optically active amino acids with L-configuration and are connected to each other through amide bonds. In the following, both in the general description and in the examples everywhere in connection with the mentioned amino acids, it is intended that these have L-configuration, even if this is not specifically mentioned.

The invention therefore relates to an analogue method for the production of new peptides with antithromboplastin, antithrombin and antifibrinolytic action and vasodilating action and with the formula:

where R-^ is hydrogen, a butyl, isobutyl, secondary butyl or benzyl group, m is 0 or 1, X is -(HNCH2CO)n -, with n^ varying

■ from 0 to 3, or <1>

with n^ varying from 0 to 3> and R^. constitute a carboxyalkyl group,

where the carboxyl group can be free, esterified. with an alkanol with at most 8 carbon atoms.or amidated, or

with n-^ and R^ with the above meaning, where Ala means the remainder of alanine, and R^ is a straight or branched alkyl group with at most 8 carbon atoms, and the method is characterized by the necessary amino acids in the specified order and/or peptide fragments containing these amino acids in the specified sequence are condensed to the specified polypeptide, whereby for the peptidic link a terminal carboxyl group and/or an amino group is activated and the other reactive groups are intermediately protected.

The meaning of the abbreviations used is indicated in the table below. The present method thus involves a number of conversions of the type

where ACOZ is a simple amino acid or a lower peptide built up by a similar reaction where amino groups and carboxyl groups that are not to react are protected in a suitable way, or a reactive derivative of such an amino acid or peptide, and where NH"2B means a amino acid or peptide where carboxyl groups can be protected and where amino groups that are not to react are protected in a suitable way, whereby the amino acids used and the reaction sequence are chosen so that a polypeptide is formed according to the above-mentioned formula, after which unwanted protective groups are removed.

The method according to the invention is illustrated by the following examples. Examples 1-11 together show the complete synthesis of

Phe - Leu - Ala r Glu (gamma - 0 Me) - Gly - Gly - Gly -Val - Arg - 0 Me. The other examples show syntheses of other compounds within the scope of the invention and various intermediate products for these, whereby the products are similarly obtained in the form of derivatives containing protective groups. The removal of the protective groups is described in summary form after the examples, and for some of the derivatives produced, experimental details are given in tabular form.

Example 1

Carbobenzyloxy - valyl - omega -> nitroarginine - methyl ester produced according to two different methods (A and B).

-A. 10.8 g of finely powdered and dried omega-nitroarginine methyl ester hydrochloride (0.0*f) mol was dissolved in 150 ml of freshly distilled dimethylformamide (DMF). After cooling, 5.6 ml of triethylamine (0.0 mol) was added and the formed triethylamine hydrochloride was filtered off. The filtered solution was cooled to -10°C, and 1U-.92 g (0.04-mol) of carbobenzyloxy-valine-p-nitrophenyl ester was added with stirring. After 2 hours at 10°C, the temperature of the reaction mixture was allowed to rise to room temperature, and the reaction took place at this temperature for 1 dbgn. DMF was evaporated under vacuum at 30 - 0°C, and the residue was dissolved in a mixture of ethyl acetate/n-butanol (2:1). The solution was extracted 5 times, with 5% sodium bicarbonate solution, 2 times with water, 3 times with 1 N hydrochloric acid solution and finally 3 times with distilled water.

The solution was dried over magnesium sulfate, evaporated to dryness and the residue dissolved in dry methanol. The compound was crystallized from the methanol solution by addition of absolute ether. The yield was 1^.1 g, and the product had a melting point of 163 - 16<i>+<0>C.

By adding petroleum ether to the mother liquor, L.L g compound with a melting point of 155 - 156° C was obtained. The total yield was thus 8L.4%.

Analysis: C20H30^6°7 Cmol-equyl weight 466.5) (tbrk for 10 hours at 70° C over P2O5).

Calculated: C = 51.49 7„, H = 6.48 7,, N = L8.02 /,.

Obtained: 52.02% 6.61 7. 17.99 /„

B. 5.4 g (0.02 mol) of omega-nitroarginine methyl ester hydrogen chloride was dissolved in 75 ml of DMF, and after cooling, 4.84 ml (0.02 mol) of tributylamine was added. After the temperature of the solution had become -10° C., 7.0 g (0.028 mol) carbobenzyloxyvaline was added which dissolved easily, and then 5.77 g (0.028 mol) dicyclohexylcarbodiimide (DCCI).

The reaction mixture was stirred for 2 hours at -10° C, then for approx. 10 hours at 0° C and finally for 8 hours at room temperature. 1 ml of AcOH was added, and after 1/2 hour dicyclohexylurea was formed and filtered off, (weight 5.9 g). The filtrate was evaporated and the residue dissolved in EtOAc - BuOH (2:1). Traces of undissolved dicyclohexylurea were filtered off. The procedure was otherwise as described under A.

Yield: 7.35 g with a melting point of 160 - 161° C, i.e. a yield of 78.9% of the theoretical. From the mother liquor, a compound was obtained which melted at approx. 131° C and which was presumably N-carbobenzyloxy-valine-N,N'-dicyclohexylurea.

Example 2

Preparation of carbobenzyloxy-glycyl-rglycyl-valyl-alin-nitroarginine metayl ester.

9.33 g (0.020 mol) of carbobenzyloxy-valyl-uc-nitroarginine methyl ester was dissolved in 120 ml of dry AcOH and 10 g of dry, bromine-free hydrogen bromide was introduced into the solution at 20° C. over the course of 45 minutes. 80 ml of AcOH and the excess of hydrogen bromide were distilled off under vacuum at 20° C. and the residue was vigorously dissolved in 1 liter of dry ether. The ether phase was decanted off and the residue was washed a further 3 times with dry ether. The granulated compound was dried over phosphorus pentoxide in a vacuum desiccator. It turned out to be very hygroscopic. An analysis for hydrogen bromide resulted in approx. 1.5 equivalents of hydrogen bromide per mol, against the calculated 1 equivalent of hydrogen bromide. Yield: 9.2 8

The vinryl-cp-nitroarginine met^ HydfbgéhlirbmicleE' was dissolved in 80 ml of freshly distilled DMF and cooled to 0° C. 4.30 ml (0.031 mol) of Et^N was added, and after 10 minutes of stirring, Et^N hydrogen bromide was formed filtered by ( about 3.55 g). To the filtrate which was cooled to -10°C, 7.75 g (0.020 mol) of carbobenzyloxy-glycyl-glycine-p-nitrophenyl ester with a melting point of 163 - 164°C was added, and the mixture was stirred for 1 hour at -10 ° C, after which the temperature was allowed to rise to room temperature. After 4 days at room temperature, the solution was evaporated under vacuum at 40° C. and the residue treated with AcOEt, after which it crystallized out.

Yield: 12.45 g with melting point 154 - 152° C. The compound was dissolved in DMF and precipitated with 5% sodium bicarbonate solution. Out-! the fines were washed twice with distilled water and ethanol. i i The compound was redissolved in DMF and precipitated with 5% citric acid-i ! solution, washed with distilled water and cooled ethanol And dried in a vacuum desiccator over £>2^5*

'Yield: 7.6 g with melting point 176 - 181° C.

; Analysis: C24<H>35<N>8°9 (molecular weight 579.6). (Tbrking for 10 hours at 70° C over P205 under 0.2 Torr).

' Calculated: C = 49.73%, H = 6.89%, N = 19.34% i Obtained: 49.20% 6.34% 19.35%

In Example 3

I ' kFrloermisd tibllle in5og ,p9pa2 v lg osct a(r0bi ,o0e3 bn enmozbyllal) noxdgyilnu-g atalamanv inypslyy-rrgaeidm-mgin aam-momag et-vhmayentln hgylul(t1eas:m1tei) nrohsyg yrdraeovg-eri-

In kjbtt at 5° C. 10.32 g (0.03 mol) of carbobenzyloxy-alanine-p-nitra I phenyl ester was added to the mixture in small portions, and the pU was kept constant at 8.7 vdd with the help of 4 N NaOH. After 1 day at room temperature, the solution was diluted with water and saturated with sodium bicarbonate. The saturated solution was extracted 7 times with EtOAc and acidified with half concentrated hydrochloric acid to a pH of 2.9 at ca. 0° C, extracted with EtOAc and the EtOAc phase washed with boiling brine, dried over magnesium sulfate and evaporated to dryness. The residue was dissolved in absolute ether, and on addition of petroleum ether the compound crystallized out as needles.

Yield: 6.3 g, melting point 99.5 - 100.5 C, corresponding to a yield of 57.3% of the theoretical*. , •

•■ ■ t r-»

Analysis: CY7H22O7N2 (molecular weight 366.4).

Calculated: C = 55.75 X, H = 6.05%, N = 7.65 7, Obtained: 55.60% 5.98% 7.54 7. j

in Equivalent weight: Calculated: 366.4. Obtained: 367.3, 368.1. j Example 4 j Preparation of carbobenzyloxy-glycine-carbo-tert.butyloxyhydrazide j

20.9 g (0.1 mol) of carbobenzyloxyglycine and 13.2 g of carbo-tert.-butyloxyhydrazide (0.1 mol) were dissolved in 150 ml of acetonitrile. After cooling to 0° C, 20.6 g (0, 1 mol) of DDCCI dissolved in

50 ml of acetonitrile added and the reaction mixture allowed to stand at room temperature overnight. The DCU formed was filtered off and the filtrate evaporated to dryness under vacuum. The residue was dissolved in EtOAc and washed with citric acid, water, 5% sodium bicarbonate and water. The EtOAc phase was dried over sodium sulfate and concentrated under vacuum to a small volume, whereby the compound crystallized out. The compound was dissolved in ether, and on addition of petroleum ether, 29.1 g (90.1 g) of carbobenzyloxy-glycine and carbo-tert.butyloxyhydrazide crystallized out. The melting point was 76 - 83° C. j Recrystallization from EtOAc-petroleum ether gave 23.1 g (71.5%) with a melting point of 81 - 84° C. j

Upon further recrystallization, the melting point dropped to 82 - 84° C. I

Analysis: ci5<H>2l<N>3°5 (molecular weight 323.36) \ Calculated: C = 55.72 7., H = 6.54%, N = 13.00% Obtained: 55.39 7. 6.66 X 13.35 7. j Example 5 j Preparation of glycine-carbo-tert.butyloxyhydrazide j

9.7 g (0.03 mol) of carbobenzyloxy-glycine-carbo-tert.bntylf oxyhydrazide was dissolved in 100 ml of mebanol, and 0.5 g of 10% Fd/C was added and hydrogen gas introduced. After 12 hours, 660 ml of hydrogen gas had been consumed, dg hydeneoeringeh.'was interrupted. The catalyst was filtered off and the methanol was evaporated under vacuum. The residue was taken up with absolute ether, whereby a compound with a melting point of 141 - 143° C crystallized out.

Yield: 3.3 g (93% of theory).

The compound's melting point Looked for a recrystallizer! ig from "ethanol/ether to 145 - 146° C.

Analysis: C , :; .0-, (molecular weight 189.22)

Calculated: C = M^3 K = 7.99%, N = 22.21 Jg, E-weight = 189.2 Obtained: ^?:!9<I>+ Vs97 5? 22.35 # 191.0

Example i

Preparation of eardob-nzyloxy-phenylaianyl-leucine-ethyl ester. 9.8 g (0.0:. mol) of leucine ethyl ester hydrochloride was dissolved in 30 ml of DMF of 5.Ot ;-(0.0;- mo": ) Et,II "ilsatt. Et^N.HCl formed was filtered off and washed with er. small amount of cooled DMF. 15.0 g (0.05 mol) of benzyloxy-phenylalanine was dissolved in 30 ml of DMF-acetonitrile (1:1); and, cooled to -10°C, after which 10.4 g (0 .05) DCCI and the aforementioned :iltr.--t containing leucine ethyl ester were added. Re-.;ir ion mixture? Temperature allowed overnight to rise to room temperature, and 1 ml -.0% AcOH was added. After 30 minutes, other DCU (N,;;' -dicyclohexylurea) f Filtered off and the filtrate evaporated to dryness under vacuum at 4o° C. The residue was dissolved in EtOAc and some undissolved DCU filtered off. The EtOAc solution was washed -down in N Hydrochloric acid, water, 5% sodium bicarbonate solution and water, The ethyl acetate phase was dried over magnesium sulfate and concentrated under vacuum to a small volume. After addition of petroleum ether, carbobenzyloxy-phenylalanyl-leucine-ethyl ester crystallized out as needles with melting point 116 - 117° C. Yield: 19.5 g (88.3 of theory) After recrystallization from ethyl acetate/petroleum ether, melting point 117-117.5° C.

Analysis: C^H^.Ii^ (molecular weight M+0.55)

Calculated: C = 68.16%, H = ?.32%, N = 6.36%

Obtained: 67.74% V ,l8% 6 , 58%

Example 7

Preparation of carbobenzyloxy-phenylalanyl-leucinehydrazide »

8.4 g (0.02 mol) of carbobenzyloxy-phenylalanyl-leucine ethyl ester were dissolved in 150 ml of methanol and h.0 g (0.0§ mol) of hydrazine hydrate added. The mixture was stirred overnight to form carbobenzyloxy-phenyl-alanyl-leucine hydrazide filtered off and washed with a small amount of methanol. The product was recrystallized from methanol/water, and a yield of 6.6 g was obtained (81.5% of the theoretical), the product having a melting point of 192.5 - 193.5°C.

Analysis: C23H30'N4C'4

Calculated: C = 64.77 7=, H = 7.09 7a, N = 13.14%

Obtained: 64.85% 7.12 /, L3.16 X

Example 8

Preparation of carbobenzyloxy-eamma-methylglutamyl-glyein-carbo-tert.butyloxy-hydrazide

7.4 g (0.025 mol) carbobenzytoxy-gamma-methylglutamic acid was dissolved in 60 ml of methylene chloride and cooled to -10° C, whereupon 5.4 g (0.0265 mol) of DCCI dissolved in 25 ml of cooled methylene chloride was added with stirring, and finally 4.73 g (0.025 mol) of glycine-carbo-tert.butyloxyhydrazide dissolved in 25 ml of cooled ethyl acetate was added. The mixture temperature was allowed to rise to room temperature overnight, and 30 minutes before the DCU was filtered off, 2 drops of 50 70 AcOH were added. The filtrate was evaporated to dryness under vacuum and the residue dissolved in EtOAc which was washed with 5% sodium bicarbonate, water, 5% citric acid solution, water and dried over magnesium sulfate. The EtOAc solution was concentrated to a small volume and after addition of petroleum ether crystallized

8.2 g of compound with a melting point of 104 - 105.5° C. Upon recrystallization from EtOAc and petroleum ether, the melting point fell to 107 - 108° C.

Example 9

Preparation of carbobenzyloxy-alanyl-gamma-methylglutamyl-glycine-carbo-tert.butyloxyhydrazide by the method according to A and B

A. 1.83 g (<3 mmol) of carbobenzyloxy-alanyl-gamma-methylglutamic acid was dissolved in 10 ml of methylene chloride, and after cooling to -10° C, 1.08 g (5.2 mmol) of DCCI was dissolved in 5 ml of cooled methylene chloride was added, and finally 0.95 g (5 mmol) glycine-carbo-tert-butyloxyhydrazide dissolved in 10 ml of cooled ethyl acetate was added.

The mixture was stirred overnight at room temperature, after which 2 drops of 50% AcOH were added. After 30 min, DCU (1.1 g) formed, filtered off and washed with a small amount of EtOAc. The filtrate was evaporated to dryness under vacuum and the residue dissolved in EtOAc which was washed with 5 7D sodium bicarbonate solution, water, 5 7. citric acid solution and water. The EtOAc phase was dried over magnesium sulfate and concentrated to a small volume, whereby 2.3 g of compound with a melting point of 140 - 150° C crystallized out. After two recrystallizations from EtOAc, the melting point fell to 154 - 156° C, and a yield of 1.9 g was obtained which was 70.47 of the theoretical.

Analysis :" C24H35N5°8" (molecular weight "537.58) "j

Calculated: C = 53.62%, H = 6.56%, N = 13.03% Obtained: 53.48% 6.52% .12.79%

B. 4.0 g (0.06 mmol) carbobenzyloxy-gamma-methylglutamyl- | glycine-carbo-tert.butyloxyhydrazide was dissolved in 60 ml of methanol, ] and 0.5 g of 10 7 Pd/C (moistened with water) was added. The carbobenzyloxy-f protecting group was hydrogenated away with hydrogen gas at weather-j

* temperature and atmospheric pressure over the course of 2 - 3 hours. The consumption of hydrogen gas was 200 ml against the calculated 195 ml. The catalyst was filtered off and the filtrate evaporated to dryness under vacuum. Res-! The oil was dissolved in absolute ether and evaporated to dryness, whereby an oil was obtained. i i ■ ]

! The oil was dried in a vacuum desiccator over phosphorus pent-. oxide whereby the oil changed to a solid, hygroscopic compound weighing 2.76 g which was 96.5% of the theoretical yield.

The product was dissolved in 40 ml of DMF and cooled to -10° C., log 3.0 g (8.6 mmol) of carbobenzyloxy-alanine p-nitrophenyl ester was added. The solution was slowly allowed to rise to room temperature 1 and after 1 day evaporated to dryness under vacuum. The residue was treated as described under example A. Yield: 3.35 g or 75.5% of the theoretical with a melting point of 151 - 153° C. Three recrystallizations from EtOAc brought the melting point to 1160.5 - 161° C (restored at 153° C).

In Example 10

• Preparation of carbobenzyloxy-phenylalanyl-leucyl-alanyl-gamma-methylglutamyl-glycine-carbo-tert.butylj+oxyhydrazide

1.08 g (2 mmol) of the product from Example 9 was dissolved in 50 ml of methanol, and 0.2 g of 10% Pd/C was added and hydrogen gas introduced. After 3 hours, the hydrogenation was complete, and 55 ml (theoretically 45 ml) had been consumed. The catalyst was removed and the solution evaporated to dryness under humidity. The residue was dissolved in

5 ml of DMF.

0.86 g (2 mmol) of the product from example 7 was dissolved

in 3 ml 6 N hydrochloric acid + 2 ml 50% AcOH and 40 ml EtOAc added on top. The mixture was cooled to -10° C. and 2.0 ml of 1-molar nitrite was added dropwise with stirring. After 15 minutes at

-10°C, the EtOAc phase was separated from the aqueous phase and washed with cooled, saturated sodium bicarbonate solution and ice water. The EtOAc solution

bTe was dried over magnesium sulfate for 1 hour at -10° C. The EtOAc solution containing carbobenzyloxy-phenylalanyl-leucinazide was slowly added dropwise into the DMF solution containing the free amino peptide, with stirring and at -10° C. After 1 day at 0° C and 2 dbgn at room temperature, the formed precipitate was filtered off. Yield: 0.35 g with a melting point of 207 - 208° C. The mother liquor! was evaporated to dryness and the residue dissolved in EtOAc, washed with 5 7. sodium bicarbonate, water, 5 70 citric acid solution, water, washed over magnesium sulfate and concentrated under vacuum. The residue was dissolved in EtOAc, and upon addition of petroleum ether, 0.70 g of compound crystallized out with a melting point of 180 - 200° C. Altogether this gave a yield of 0.80 g, which was 50 7th of the theoretical. A small portion recrystallized once more from EtOAc►

Analysis: C39H55N7Ou (molecular weight 797.9) j Calculated: C = 58.71 7., H = 6.95%, N = 12.29 7. Obtained: 58.48 7. 6.77% 12.35 7.

Example 11

Preparation of carbobenzyloxy-phenylalanyl-leucyl-alanyl-gamma- \ methylglutamyl-glycyl-glycyl-glycyl-valyl-omega-nitroarginine- i methyl ester. j

334 mg (0.42 mmol) of the compound prepared according to example 10 was dissolved in 1.8 ml of trifluoroacetic acid, and after 45 minutes at 22° C, the trifluoroacetic acid solution was added dropwise to 100 ml of absolute ether with stirring.

After 2 hours in a refrigerator, the precipitate was filtered off and washed repeatedly with absolute ether, after which it was dissolved in 4 ml of DMF and 1 ml of 4 N hydrochloric acid. The solution was cooled to -10°- t, t and 0.42 ml of 1 molar sodium chloride solution was added dropwise with stirring over a period of 5 minutes, after which the solution and formed precipitate were added dropwise to 30 ml of ice-cold j 2 7, sodium bicarbonate solution, whereby the precipitation of the resulting acid azide was complete. The azide was washed on the filter with ice water and ice cold 5% citric acid solution and finally washed free of cold citric acid with ice water. Des was then quickly dried over phosphorus pentoxide under vacuum at 5° C and immediately used for coupling. The yield of tbrt carbobenzyloxy-phenylalanyl-leucyl-alanyl-gamma-methylglutamyl-glycinazide was 291 mg, which was 98% of the theoretical.

243 mg (0.42 mmol) of the compound prepared as follows

example 2 was dissolved in 2 ml absolute AcOH and 1 ml 4 itf hydrogen bromide in AcOH added. The mixture was allowed to stand for 45 minutes at room temperature and protected from humidity, after which the mixture was added dropwise to 100 ml of absolute ether under vigorous stirring. The precipitate was washed twice and taken up with absolute ether until granular (hygroscopic). The tetra-peptide methyl ester hydrobromide was dissolved in 4 mL of DMF, and 0.09 mL (0.64 mmol) of Et-jN was added to the solution which was cooled to -10°C, and the above azide was dissolved in 10 mL of DMF in advance cooled to -10° C was added dropwise with stirring at -10° C. After 1 db<g>n at 0° C, the solution was allowed to stand for 2 dbgn at room temperature, after which it was concentrated to 5 ml under vacuum at 20° C A renewed dissolution was carried out in 5 ml of DMF, and the solution was added with stirring to 40 ml of a 5% citric acid solution. The precipitate was washed free of citric acid with water and dried under vacuum over phosphorus pentoxide. Yield: 265 mg which was 66.7% of the theoretical. The melting point was 203 - 213° C. Upon two recrystallizations from DMF water, the melting point fell to 211 - 215° C.

Analysis: C50<H>73<N1>3°16 (molecular weight 1112.2)

Calculated: C - 53.99%, H = 6.62%, N = 16.37 7.

Obtained: 53.51% 6.56 7. 16.72 7.

Example 12

Preparation of carbobenzyloxy-phenylalanyl-omega-nitroarginine methyl ester.

10.8 g (40 mmol) of omega-nitroarginine methyl ester hydrochloride was dissolved in DMF, and 5.05 g (40 mmol) of Et₂N was added under cooling. Formed Et^N. The HCl was filtered off and the filtrate added to a cooled solution (-10° C) of 12.0 g (40 mmol) carbobenzyloxy-phenylalanine in acetonitrile to which at -10° C 9.1 g (40 mmol) DCCI had been added . The whole was stirred for 2 hours at -10° C and allowed to assume room temperature after standing overnight.

The DCU formed was filtered off, the filtrate evaporated to dryness and the residue dissolved in EtOAc and washed as usual. From the EtOAc solution, upon addition of absolute ether, crystals with a melting point of 87 - 89° C were obtained. After crystallization from 60% ethanol, crystals of another modification with a melting point of 153.5 - 155° C were obtained . Dividend;

17.9 g (87.1 7" of the theoretical)....

Analysis: C24H3o^6°7 (molecular weight 514.55)

Calculated: C - 56.02 '/„ H = 5.88 7o, N = 16.33 7.

Obtained: 55.94% 6.09 7„ 16.42%

Example 13

Preparation of carbobenzyloxy-phenylala.wl-vaLyl-omega-nitro-arginine ethyl ester.

9.33 g (20 mmol) of the compound prepared according to example 1 was treated as described under example 2. The yield of hygroscopic valyl-omega-nitroarginine methyl ester. 1.5 HBr was after treatment under vacuum over KOH and ?2^5 9>15 g which was quantitative.

The hydrogen bromide dipeptide ester was dissolved in 50 ml of DMF and cooled to -5° C, whereupon 4.2 ml (30 mmol) of Et-1N was added. The formed Et-jN-HBr was filtered off, and 8.4 g (20 mmol) of carbobenzyloxy-phenylalanine-p-nitroenyl ester were added to the filtrate. After 1 day at room temperature, the solution was cooled to -10° C, and a further 1.4 ml of triethylamine was added and the mixture was allowed to stand for another 1 day. After adding 1 ml AcOH, the solution was evaporated to dryness under vacuum and the residue dissolved in a mixture of EtOAc-BuOH (3:1) and washed as usual with water, 5% citric acid and water. The solution was concentrated over MgSO^ and evaporated to a small volume which, after addition of absolute ether, gave 10.1 g (£82.3 7" of the theoretical) compound with a melting point of 108 - 111° C. On recrystallization from EtOAc-methanol after addition of absolute ether, the melting point increased to 112 - 114° C.

Analysis: C29<H>3<gN>7<0g> (molecular weight 613.69)

Calculated: C = 56.76 7th, H = 6.41 7th, N = 15.98 7th.

Received: 56.32 7. 6.48 7. 16.12 7..

Example 14

Preparation of carbobenzyloxy-phenylalanyl-glycyl-valyl-omega-nitroarginine methyl ester.

2.33 g (5 mmol) of the compound prepared according to example 1 was treated as described under example 2, and the hydrogen bromide dipeptide methyl ester obtained was dissolved in 15 ml of DMF, cooled to -5° C and 1.05 ml ( 7.5 mmol) Et^N added. Et^-HBr

was filtered off and the filtrate used as described below.

1.85 g (5 mmol) of carbobenzyloxy-phenylalanyl-glycinehydrazide was dissolved in 7 ml of DMF and 1.5 ml of 6 N hydrochloric acid was added, cooled to 11-10° C and, with stirring, 5.1 ml of 15.1 mmol were added dropwise ) 1 Molar sodium nitrite solution. After 10 minutes at -10°C, 50 ml of 5% sodium bicarbonate solution was added, and at 0°C the azide began to precipitate and the precipitation was complete after the addition of 30 ml of ice-cooled water, after which the whole was stirred for 10 minutes, filtered and nbye washed with ice water. The precipitate was slurried in ice water with stirring, filtered and washed with water at a temperature of 0° C. The procedure was repeated 3 times, and the compound was dried under vacuum at 5° C over ^2^5" ;*worPa it was immediately used . Yield: 1.77 g (93 7ths of theory).

i To the DMF solution of the aminodipeptide cooled t-il

-10° C the azide was dissolved in 8 ml previously cooled to -10° C DMF was added dropwise with stirring. After 10 minutes at -10° C, the mixture was allowed to stand for two hours at room temperature, and it was then concentrated under vacuum to 7 ml and treated as described in Example 11. Yield: 2.44 g (78.2 7. of the theoretical ). Crystallization from 60% ethanol gave 1.95 g of compound with a melting point of 147-149°C.

Analysis: c^^ 2ti809 (molecular weight 670.74) j Calculated: C = 55.51 7., H = 6.31%, N = 16.71 7.

in Received: 55.38 7. 6.58% 16.90%

in t

■ Example 15

: Preparation of carbobenzyloxy-phenylalanyl-glycyl-glycyl-valyl-Iomega-nitroarginine methyl ester in 1.77 g (3.8 mmol) of the compound prepared in example 1 was treated as 1 example 2, and the hydrogen bromide dipeptide methyl ester obtained was dissolved in 10 ml DMF and cooled to 1-5° C, whereupon 0.79 ml (5.7 mmol) Bt^N was added. Et^N<*>HBr was filtered off and the filtrate used as described below.

1.62 g (3.8 mmol) of carbobenzyloxy-phenylalanyl-lp-glycyl-glycinhydrazide was dissolved in 10 ml of DMF and 1 ral of 6 H hydrochloric acid was added, cooled to -10° C. Under stirring, 3.85 ml (3.85 mmol) of 1 molar Sodium nitrite solution was added dropwise, and the whole was treated as described in Example 14. Yield: 1.55 g (95 7. of the theoretical).

To the DMF solution of the aminodipeptide ester cooled to L-10° C., under conversion, the carbobenzyloxy-trilpeptyl-adducted -opploated-in

9 ml of previously [27 7~to-10° C cooled DMF added dropwise. The treatment was otherwise carried out as described in example 11. Yield: 1.93 g (73.1% of the theoretical) with a melting point of 185 - 190° C. Recrystallization from 90% ethanol caused the melting point to rise to 188 - 191 ° C. Analysis: C33h45NqP10 (Mw 727.8) Calcd: C = 54.45%, H = 6.23%, N = 17.32% Obtained: 55.01% 6.18% 17.52%. j j ; Example 16 j Preparation of carbobenzyloxy-phenylalanyl-glycyl-glycyl-glycyl- ! valyl-omega-nitroarginine methyl ester. • 1.73 g (3 mmol) of the compound prepared in example 2; The solution was dissolved in 7 ml of tbr AcOH, and under moisture-free conditions, 7 ml of 4 N HBr in AcOH was added. After 45 minutes at room temperature, the evolution of carbon dioxide had ceased and the reaction was complete. The excess of hydrogen bromide and 7 ml of AcOH were distilled off under vacuum, and the residue, with vigorous stirring, was dropped into 150 ml of absolute tbr ether. The ether was decanted off and the residue, which assumed a granular consistency, was taken up 3 more times with tbr ether. The residue was triturated over P2O5 and KOH under vacuum, and the yield of tetrapeptide ester hydrobromide was 1.62 g, which corresponded to 95% of the theoretical value calculated on 1.5 equivalents of HBr per moles of peptide. The hydro bromide was dissolved in 20 ml of DMF and cooled to -10° C, after which 0.60 ml of (4.28 mmol) Ethyj was added and formed Et^N«HBr filtered off. With stirring at -10° C, 1.44 g (3 mmol) of carbobenzyloxy-phenylalanyl-glycine-p-nitrophenyl ester was added to the filtrate, and after 1 day at room temperature, the solution was again cooled to -10° C and a further 0.20 ml Et^N added, after which the solution was again allowed to proceed for 11 dbgn. The processing was carried out as described below; example 2. Yield: 1.59 g (corresponding to 71.2% of the theoretical^) with a melting point of 164 - 167° C. On recrystallization from 601% methanol the melting point rose to 169 - 172° C.

Analysis: C35<H>48^1<0>°11 (molecular weight 784.85)

Calculated: C » 53.56%, H = 6.17%, N = 17.85%, Obtained: 54.01% 6.08% 17.99%

Preparation of carbobenzyloxy-phenylalanyl-phenylalanyl-valyl-omega Example 17

ni troarg ininme thy1e s ter.

.._. 2.33 g (5 mmol) of the mixture prepared according to Example 1 was treated as described in Examples 2 and 14, and it obtained; solution of valyl-omega-nitroarginine methyl ester was used as ; described below.

2.3 g (5 mmol) of carbobenzyloxy-phenylalanyl-phenylalanth-hydrazide was dissolved in 7 ml of DMF and 3 ml of 4 N hydrochloric acid added, cooled to -10° C and 5.1 ml of 1 molar NaNO 2 solution was added dropwise; added while stirring. The whole thing was worked up as described in; Example 14. Yield: 2.26 g (95 7. of theory) of carbobenzy-oxy-phenylalanyl-phenylalaninazide was dissolved in 8 ml of DMF, cooled to -10° C and with stirring added dropwise to a previously cooled: solution of the aminodipeptide ester. The further procedure was; { as described in examples 11 and 14. Yield: 2.14 g (58.7 of the I theoretical) with a melting point of 218 - 220° C.

t•Analysis: C^gH^gNgOg {molecular weight 760.87) ; Calculated: C = 59.99%, H = 6.36%, N = 14.73% i i Obtained: 59.49% 6.33% 14.81%

\ Example lg j Preparation of carbobenzyloxy-phenylalanyi-leuc£l-valyi-omega-; nitroarginine methyl ester.

2.33 g (5 mmol) of the compound prepared according to Example 1 was treated as described in Examples 2 and 14. The er-j kept DMF solution of valyl-omega-nitroarginine methyl ester was '; applied as described below.

2.13 g (5 mmol) of the compound prepared according to example 7 was dissolved in a mixture of 5 ml of 6 N hydrochloric acid and 5 ml of 50% AcOH, and the solution was provided on top with a layer of

in 80 ml of EtOAc, and the further procedure was as described in section 1

j sample 10. The EtOAc solution containing the carbobenzyloxy-di-peptidazide j was cooled to -10° C and, with stirring, added dropwise to the DMF solution prepared above which had previously been cooled to

in -10° C. The further procedure was as described in example 10,

in and the obtained EtOAc solution was evaporated to dryness and res

( ten dissolved in 7 ml DMF and purified as described 1 example 11. ViM-\ exchange: 265 g (72.9% of the theoretical) with a melting point of 171 - : 181° C. By recrystallization 2 more times from 60 7 .ethaj-• noi the melting point of the product was increased to 179 - 182° C.

Analysis: C32<H>50<N>8°9 (molecular weight 726.85) Calculated: C = 57.84%, H = 6.94 7., N = 15.42 X Obtained: 58.05% 7, 01% 15.35 X-

Example 19

Preparation of carbobenzyloxy-valyl-omega-nitroargininamide

45.8 g (0.13 mol) of carbobenzyloxy-omega-nitroargininamide with a melting point of 217 - 218° C. was dissolved in 300 ml of anhydrous AcOH, and under moisture-free conditions 300 ml of 2 N HBr in dry AcOH was added with stirring. After 45 minutes at room temperature; naturally, carbon dioxide evolution had stopped, and the reaction mixture was then evaporated under vacuum at 20° C under moisture-free conditions until the residue had obtained a volume of 250 ml. The rest stayed

l

was added dropwise to 2.5 liters of ether with vigorous stirring, and a plastic mass was precipitated. The ether phase was decanted off and the residue taken up a further 3 times with tbr ether, whereby the compound became granular and solid. The compound was dried under vacuum over P 2 O 5 and KOH and dissolved in 350 mL DMF, and 18.5 mL (0.134 mol) Et 3 N was added. After cooling to -10° C, Et3N<*>HBr was formed, filtered off, and with stirring, 48.4 g (0.13 mol) of carbobenzyloxy-valine-p-nitrophenyl ester were added to the filtrate. React-; The reaction mixture was allowed to stand for 1 day at room temperature, was concentrated to 100 ml and poured with stirring into 1 liter of 5% sodium bicarbonate solution. The compound that precipitated was filtered off, washed with water and redissolved in 75 ml DMF and kept in

1 liter of 1 N hydrochloric acid, filtered and washed with water. Dividend: 47.11

g (80.2% of the theoretical) with a melting point of 61.6 - 63° C. : On recrystallization from 60 7. methanoi, the melting point of the product rose | point to 66 - 67° C, but after recrystallization from ethanol at! addition of water raised the melting point to 218 - 219°C.

Analysis: C1QH.,QN70<- (molecular weight 451.5, compound with melt-!

points 218 - 219<*> C)

Calculated: C = 50.55 7., H = 6.47 7., N = 21.72 7. j Obtained: 50.57 7. 6.62 7. 21.89 7..

Example 20

Preparation of carbobenzyloxy-phenylalanyl-omega-nitroargininamide;

1.76 g (5 mmol) of carbobenzyloxy-omega-nitroargininamide was treated as described in Example 19, and the resulting DMF solution of the omega-nitroargininamide was cooled to -10° C, after which 2.1 g (5 mmol) of carbobenzyloxy -phenylalanine-p-nitrophenyl-: ester was added and the compound worked up as described in example 19. yield: 2.05 g (82 7. of the theoretical) with a melting point of 105 - 125° c. On recrystallization from 80 7. etha--nolateg melting point to 128 - 130° C.

Analysis: <C>23<H>29N706 (molecular weight 499.54) Calculated: C = 55.30%, H = 5.85%, N = 19.63%, O = 19.22% ' Obtained: 55, 32% 6.07% 19.42 7. 19.24 7.

Example 21

Preparation of carbobenzyloxy-phenylalanyl-valyl-omega-nitroargininamide.

1.0 g (2.22 mmol) of the compound prepared according to Example 19 was dissolved in 5 ml tbr AcOH, and under moisture-free conditions 5 ml 2 N HBr in tbr AcOH was added. The work-up was carried out as described in example 19, and DMF solution was obtained

(10 mL) of the valyl-omega-nitroargininamide was cooled to -10°C, after which 0.93 µl (2.22 mmol) of carbobenzyloxy-phenylalanine-p-nitro-phenyl ester was added with stirring. The purification of the protected tripeptidamide was carried out according to the same guidelines as described; knows in example 19.

Yield: 1.15 g (86% of the theoretical) with a melting point of 213 - 220° C. On two recrystallizations from 80% ethanol, the melting point of the product rose to 218 - 220° C.

Analysis: C28H38N8°7 (molecular weight 598.68)

Calculated: C = 56.18%, H = 6.40%, N = 18.72%

Obtained: 56.29 7. 6.52% 18.87 7.

Example 22

Preparation of carbobenzyloxy-omega-nitroarginyl-omega-nitroarginine methyl ester.

35.3 (0.1 mol) of carbobenzyloxy-omega-nitroarginine with a melting point of 134.5 - 136° C was dissolved in 200 ml of DMF - TH F (1:1), and under stirring 13.9 ml ( 0.1 mol) Et-jN added dropwise. After cooling to -10° C, 13.66 g (0.1 mol) of chloroformic acid iso-butyl ester was added over a period of 20 minutes whereby the temperature of the solution during the addition time remained at -10° C and -5° C.

For the solution, omega-nitroarginine methyl ester was dissolved in DMF, liberated from 27.0 g (0.1 mol) omega-nitroarginine methyl ester-HCl with 13.9 ml (0.1 mol) Et^N, previously cooled to -10° C added, and the reaction mixture temperature was -8° C during the dropwise addition. The temperature of the reaction solution was slowly allowed to rise to room temperature, and after a few hours the reaction was complete. The EtoN.HCl which had formed during the reaction was filtered off and the filtrate evaporated to dryness. The residue was dissolved in 900 ml BuOR-EtOAc (2:1) and shaken with 1 N hydrochloric acid, 5% sodium bicarbonate-

(Solution and water and the solvent phase dried over magnesium sulfate. After evaporation of the solvent, a compound with a melting point of 136 - 148° C was obtained.

■Yield: 34.6 g (60.8 7. of the theoretical). The product was recrystallized twice from 80 7. methanol, and the melting point rose to 147 - 150°C.

Analysis: c2lH32<N>10°9 (molecular weight 568.57)

Calculated: C = 44.36%, H = 5.67 7., N = 24.64%

Earned: 44.38 7. 5.64 7. 24.72 7.

Example 23

Preparation of carbobenzyloxy-phenylanalyl-valirt methyl ester.

29.9 g (0.1 mol) of carbobenzyloxy-phenylalanine were dissolved in 75 ml of DMF and cooled to -10° C, after which 20.6 g of DCCI dissolved in 25 ml of DMF were added dropwise. Valine methyl ester dissolved in 30 ml DMF was freed from its hydrochloride by adding 13.83 ml Et^N to 16.8 g (0.1 mol) valine methyl ester.HCl in DMF and added dropwise so that the temperature of the reaction solution remained at -10° C. The reaction was complete after 1 db at room temperature, and after cooling, DCU was formed and filtered off. The filtrate was evaporated to dryness and the residue dissolved in EtOAc and shaken as usual*

corpse. After evaporation of the EtOAc, the residue was dissolved in 80% methanol, and 33.5 g of the compound with a melting point of 113 - 115 C crystallized out. Yield: 33.5 g (81.2 7. of the theoretical).

;Analysis: <C>23<H>28N2°5 (molecular weight 412.49)

Calculated: C = 66.9/ 7., H = 6.84 7., N = 6.79 7.

Received: 67.09 7. 6.85 7. 6.91 7.

Example 24

in

Preparation of carbobenzyloxy-phenylalanyl-valine hydrazide

8.25 g (20 mmol) of the compound prepared according to Example 23 was dissolved in 150 ml of methanol, and 3.94 g (80 mmol) of hydrazine hydrate was added. The whole was refluxed with stirring for 2 days and concentrated, and after adding a small amount of water the product crystallized out. It was recrystallized from methanolic water. Yield: 7.35 g (89 7. of the theoretical) with a melting point of 210 - 211.5° C.

Analysis: C^H^gNnO^ (molecular weight 412.5)

Calculated: C = 64.06 7., H = 6.84 7”, N = 13.58 7.

JfchQldt: 64.64 7. 6.58 7. 13.63 7.

.'■-. subject I 2_5

Preparation of carbobenzyloxy-phenylalanyl-phenylalanine methyl ester.

16.2 g (75 mmol) of phenylalanine aminoethyl ester "HCl with a melting point of 153.5 - 159° C, was dissolved in DMF and cooled to 0° C,

oe 10.5 ml (75 mmol) Et₂N was added. Frrcr-styled Et2.i-HCL was filtered off and the filtrate was cooled to -10° C., after which 31.5 g (75 mmol) of carbobenzyloxy-phenylalanine-p-nitrophenyl ester were added. After 1 day at room temperature, the solution was again cooled to -10°C, 3.5 ml (25 mmol) of St2' was added and the solution was allowed to stand for another 1 day. The residue was then concentrated to dryness under vacuum and the residue dissolved in EtOAc and washed with 5% sodium bicarbonate solution, water, 3% hydrochloric acid and water. The EtCAc solution was diluted over magnesium sulfate and concentrated to a smaller volume, and after the addition of petroleum ether, the product crystallized out as needles. The product was recrystallized twice from EtOAc/petroleum ether. Yield: 27.85 g (80.770 of theory) with a melting point of 148.5 - 149°C.

Analysis: c27<H>2<8i>"2°5 (molecular weight 460.54)

Calculated: C ^ 70.42 H ^ 6.13 U A = 6.08

Obtained: 70.69% 6.15 7. 6.12%

Example 26

Preparation of carbobenzyloxy-phenylalanyl-Eenylalanine hydrazide

23.0 g (50 mmol) of the compound prepared according to Example 25 was dissolved in 500 ml of methanol, and 7.5 g (0.15 mol) of hydrazine hydrate was added, and the mixture was refluxed for 4 hours and then allowed to stand overnight at room temperature. The formed precipitate of 18.1 g with a melting point of 198 - 200° C was sucked off and the filtrate evaporated to approx. 100 ml, and after the addition of a small amount of water, 4.15 g of compound crystallized out with a melting point of 196.5 - 197.5° C. Both fractions were recrystallized from methanol, and the melting point then rose to 201 - 203, 5° C. Yield: 19.5 g (84.8% of theory).

Analysis: C26<H>28<W>4°4 (molecular weight 460.54)

Calculated: C = 67.81 7., H = 6.13%, W 12.17%

Obtained: 67.57% 6.08% 12.37%

Example 27

Preparation of carbobenzyloxy-phenylalanyl-glycine ethyl ester

2.8 g (20 mmol) glycine ethyl ester-HCl was dissolved in 20 ml

DMF, 2.05 g of Et₂N was added and the mixture was cooled. 0° C os formed Et^N-HCl filtered off. A mixture of 6.5 g (20 mmol) carbobenzyloxy-phenylalanine and 4.2 g (20 mmol) DCCl in 20 ml DMF at -10°C was added to the filtrate. After 1 day at room temperature, the mixture was cooled to 0° C, formed DCU filtered off, the filtrate evaporated to dryness and the residue dissolved in EtOAc and shaken as usual with 4% hydrochloric acid, 5% sodium bicarbonate solution and water. After treatment with magnesium sulfate and concentration, the product crystallized out by adding petroleum ether1. Yield: 6.34 g (83.9 /„ of theory) with a melting point of 109-111.5° C. By recrystallization from EtOAc-petroleum ether. the melting point rose to 112 - 112.5° C.

Analysis: C21<H>24<N>2°5 (molecular weight 384.44)

Calculated: C = 65.64%, H - 6.29%, i\ = 7.29%

Obtained: 65, 35 7„ 6.35% 7.40 •/„.

Example 28

Preparation of carbobenzyloxy-phenylalanyl-<g>l<y>cvl-:lvcine ethyl ester

1.97 g (10 mmol) glycyl-glycine ethyl ester-HCl with a melting point of 182 - 184° C was dissolved in 30 ml DMF, 1.4 ml (10 mmol) Et3N was added, formed Et^HCl filtered of and 4.2 g (10 mmol) carbobenzyloxy-phenylalanine-p-nitro-phenyl ester added to the filtrate. It was all worked up as described in example 13. The product was recrystallized from DMF/water. Yield: 3.85 (87.2 /„ of the theoretical) with a melting point of 93 95° C.

Analysis: <C>23<H>27lS3°6 (molecular weight 441.49)

Calculated: C = 62.57 H = 6.16%, W 9.52 70

Obtained: 62.20% 6.05 ;Q 9.72%

Example 29

Preparation of carbobenzyloxy-phenylalanyl-glycine hydrazide

6.35 g (16.5 mmol) of the compound according to example 27 was dissolved in 50 ml of methanol, 3.2 g (64 mmol) of hydrazine hydrate was added and after stirring for 1 day at room temperature, the solution was evaporated to dryness. The residue was dissolved in a small amount of methanol, after which the product crystallized out. Yield: 4.95 g (81.0 7o of the theoretical) with a melting point of 151.5 - 152.5° C.

Analysis: '-'19^22^4^4 (molecular weight 370.42)

Calculated: C = 61.61%, H = 5.99%, N = 15.13%

Obtained: 61.80 5.87% 15.22

Example 30 i

in Preparation of carbobenzyloxy-phenylalanylglycyl-glycine hydrazide

2.1 g (4.73 mmol) of the compound prepared according to example 28 was dissolved in 25 ml of methanol, 0.9 g (18 mmol) of hydrazine hydrate was added and after 2 days at room temperature the formed; precipitate filtered off and washed with water. The product was recrystallized from methanol. Yield: 1.75 g (86.6% of theory) j with a melting point of 190 - 191° C. j Analysis: C21<H2>5N5°5 (molecular weight 427.47) j Calculated: C = 59.01 7o, H = 5.90%, N = 16.38% j Obtained: 59.49% 5.95% 16.50% \

i The peptides' gamma-amino groups at the ends and other amino groups that were protected by carbobenzyloxy groups were released from their protective groups by hydrogenating away the carbobenzyloxy groups in the form of tbluen and carbondioxyd. The nitro groups in the arginine were simultaneously reduced away in the form of ammonia with hydrogen gas as a reducing agent and palladium on carbon as a catalyst. , As all reductions were carried out in the same way, apart from minor variations that can be included in a table, only a general description of the experiments follows. (For experimental details see Table 1).

The protected peptide ester was dissolved in an alcohol, methanol or ethanol, and the necessary number of equivalents of the salt-forming acid was added in the form of an aqueous or alcoholic solution. The catalyst moistened with water (10%, palladium on carbon) was added in an amount corresponding to 100 mg per millitnol peptide. The amount of catalyst was increased somewhat by increasing length of the peptide. After the catalyst had been flushed down from the sides of the reaction vessel, all air was driven out of the reducer with nitrogen gas. This in turn was driven out with hydrogen gas. \ The reduction system was closed, and via a measuring vessel which was used as a reservoir for hydrogen gas, the hydrogen gas for the reduction was supplied. The reaction was started by vigorous stirring in the reduction vessel, and the carbon dioxide formed was absorbed during stirring in a flask connected to the reaction vessel containing 50% potassium lye. By means of a level bottle connected to the hydrogen gas reservoir i• and containing water j as a barrier liquid, the hydrogen gas consumption was ; for complete reaction (corrected to normal pressure and temperature and reduced by the water's partial pressure at the relevant temperature) read. The reaction time varied considerably from 2 to 12 hours depending on the length of the peptides and the presence of nitro groups in the arginine. After completion of the reaction, nitrogen gas was passed through the reaction vessel to expel the hydrogen gas. The catalyst was filtered off, the filtrate evaporated to dryness and the residue taken up a number of times with dry ether, after which the product became solid and granular. All peptide esters were very hygroscopic as hydrogen chlorides and therefore had to be worked up and separated in a drying chamber and stored safely under moisture-free conditions.

The antithromboplast effect for some of the interest-

antest structures calculated as an effect corresponding to a specific amount of trypsin-inhibiting agents from lung in standardized form.

In Table 2, the value<p> in column 3 indicates the 50th percentile

inhibition of thromboplastin activity expressed in the equivalent amount of trypsin inhibitor (mg) per raillimol peptide. -OMe in the table formulas means a methyl ester group, The five first compounds i

the table, which is not produced according to the invention, has been included for the sake of comparison. It is clear from the table that a marked decrease in activity occurs if three amino acids occur in the peptides, i.e. when even spirals are obtained.

The curative bradykinin-like effect of two of the most active peptides produced according to the invention has been measured as a percentage reduction of the blood flow in the femoral artery by intra-arterial injection in a dog with a solution with a concentration of 1 mg peptide/ml and an injection rate of 1 - 3 ml/min.

Example 31 ! i Preparation of carbobenzyloxy-valyl-omega-nitroarginine-iso-propyl ester 2.5 g (8.4 mmol) omega-nitroarginine-isopropyl ester hydrochloride was dissolved in 30 ml of DMF, and after cooling, 1.2 ml (8, 4 mmol) triethylamine added and formed triethylamine hydrochloride ! filtered out. The filtrate was cooled to -10° C, and with stirring 3.2 g (8.4 mmol) of carbobenzyloxyvaline p-nitrophenyl ester were added. After 2 hours at -10° C, the temperature was allowed to rise to room temperature, and the mixture was allowed to react at this temperature for | 1 dbgn. The reaction mixture was again cooled to -10° C, where a further 1.2 ml of triethylamine was added, and after a few hours the reaction was practically complete. DMF was evaporated under vacuum at 30 - 40° C, and the residue after evaporation was dissolved in approx. 500 ml ethyl acetate saturated with water. The solution was extracted as usual with 5% sodium bicarbonate solution, water, 1 N hydrochloric acid solution and water and the organic phase was dried over magnesium sulfate and evaporated to dryness under vacuum. The residue was dissolved in hot ethyl acetate, and after addition of tbr isopropyl ether, the compound crystallized out after storage in a refrigerator. The yield was 3.65 g, which corresponds to 88 7. of the theoretical, and ;

the product produced had a melting point of 162 - 166° C. After a further recrystallization from ethyl acetate-isopropyl ether, the melting point changed to 165 - 168° C. On recrystallization from methanol-ethyl acetate, the compound had a melting point of 115 - 116° cj The compound was uniform by thin-layer chromatography on silica gel ! in three different solvent systems and had an optical rotation of |>]d<*> = "24»°°» Cc=1» methanoi).

Analysis: C22<H>3<4>N6°7 (molecular weight 494.56)

Calculated: C=53.43 7th, H = 6.93 7th, N = 16.99 7th.

Achieved: 53.20 7. 6.99 7. 17.11 7.

o

Example 32

Preparation of carbobenzyloxy-phenylalanyl-valyl-omega-nitro-arginine isopropyl ester

2.0 g (4.05 mmol) of carbobenzyloxy-valyl-omega-nitro-arginine isopropyl ester was dissolved in 10 ml of sterile glacial acetic acid, 5 ml of 4 N hydrogen bromide in glacial acetic acid was added and the mixture was stirred for 1 hour at room temperature while being protected from atmospheric humidity . During the reaction time, some crystalline tripeptide ester hydrogen bromide precipitated, and the precipitation became complete after the addition of 250 ml of tbrr ether. The ether phase was decanted off and the precipitate was treated a further 3-4 times with tbrr ether. The precipitate was dried over potassium hydroxide and P2O5 under vacuum and was very hygroscopic.

i The obtained tripeptide ester hydrogen bromide was dissolved in ; 15 ml of DMF, cooled to 0° C. and 1 ml (7.3 mmol) of triethylamine were added. Triethylamine hydrogen bromide formed was filtered off, and 1.7 g (4 mmol) of carbobenzyloxy-phenylalanine-p-nitrophenyl ester was added to the filtrate. After a few hours at -10° C, the temperature of the reaction solution was allowed to rise to 20° C. The reaction solution was again cooled to -10° C, after which a further 1 ml of triethylamine was added, and the reaction mixture was allowed to proceed overnight. evaporated to dryness under vacuum and the residue dissolved in a mixture of ethyl acetate/n-butanol in a ratio of 2:1. The organic phase was extracted as usual with 5 7. sodium bicarbonate, water, 1 N hydrochloric acid, in water and dried over magnesium sulfate. The concentrated solution was evaporated to dryness and the residue was dissolved in a mixture of hot methanol and ethyl acetate in a ratio of 1:1, and after 1 day in the refrigerator 1.93 g of a compound with a melting point of 175 - 177°C crystallized Upon recrystallization, the melting point dropped to 178 - 179° C. The yield was 1.75 g, which was 68% of the theoretical. The compound [was uniform by thin layer chromatography on silica gel and had an optical rotation of £<*]n = 25.7 (C=1, methanol).

Analysis: '-'31.^4j-1 (molecular weight 641.74)

Calculated: C •- 58.0? H - 6.7 5 70, N 15.28 /„

!;:rhnldt: 58.32 7. 6.92 $ 15.19 \

Example 33

Preparation of carbobenzyloxy-valyl-orrera-nitroarginyl-omega-nitroarginine methyl ester

5.7 g (10 mmol) of carbobenz<y>loxy-ome^a-nitroarginyl-omega-nitroarginine methyl ester and 30 ml of tbr glacial acetic acid were placed in a flask that was protected from air moisture, after which 10 ml of 4N hydrogen bromide in tbr glacial acetic acid was quickly added. After stirring for 1 hour at room temperature, the decarbobenzyloxylation was complete and a plastic mass was formed. The reaction mixture was immersed under vigorous stirring in 700 ml of ether, whereby a hard, plastic, partly crystalline mass was formed on the surface. The ether phase was decanted off and the residue taken up with tbr ether from which the ice was decanted. The process was repeated until the product had become crystalline, whereupon it was treated under vacuum over ?2^5 °£ potassium hydroxide. The yield of dipeptide ester hydrogen bromide was 6.5 g, and a bromide content determination showed that the compound contains 2.3 equivalents of hydrogen bromide. The compound was dissolved in 40 ml of DMF, cooled to 0° C. and 3.5 ml

(25 mmol) of triethylamine added. The formed triethylamine hydrogen bromide was filtered off and 3.75 g of carbobenzyloxy-valine-p-nitrophenyl ester was added to the filtrate, after which the reaction mixture was slowly allowed to reach room temperature overnight. An additional 1.4 mL (10 mmol) of triethylamine was added to the mixture after it had been cooled to -10°C and allowed to react again overnight. The solution was evaporated to dryness, the residue treated with ether, a small amount of ethyl acetate, 5% sodium bicarbonate solution, 10% citric acid solution and water and dissolved in hot methanol which after addition of ethyl acetate gave 5.1 g of a yellow colored crystalline product. Two re-crystallizations from methanol-ethyl acetate (1:1) finally gave a product with a melting point of 119 - 122° C and an optical rotation of Ck]q^ ~ -12.3° (C-1, DMF) . Thin-layer chromatography of the product on silica gel in three different solvent systems gave only one spot, indicating that the compound was uniform. The yield was 4.35 g, which corresponds to 65.27, of the theoretical.

Analysis: C24<H>41^11°10 (molecular weight 643.68)

Calculated: C = 44.78 7", H = 6.42 7=, N = 23.94%

Obtained: 44.52% 6.38% 24.11%

Example 34

Preparation of carbobenzyloxy-phenylalanyl-valyl-omega-nitro-arginyl-omega-nitroarginine methyl ester

1.30 g of carbobenzyloxy-valyl-omega-nitroarginyl-omega-nitroarginine methyl ester was decarbobenzyloxylated as described in previous examples. After work-up, 1.55 g of a very hygroscopic ttipeptide ester hydrogen bromide was obtained which, upon determination of its bromide content, was found to contain 2.4 equivalents of hydrogen bromide. The tripeptide ester hydrogen bromide was dissolved in 15 ml of DMF and cooled to -10° C, after which 0.67 ml (4.8 mmol) of triethylamine was added. The formed triethylamine hydrogen bromide was filtered off, and 0.84 g (2 mmol) of carbobenzyloxy-phenylalanine-p-nitrophenyl ester was added to the filtrate, after which the reaction solution was allowed to assume room temperature overnight. 0.28 ml of triethylamine was added to the solution which was stirred for a further few hours. The reaction mixture was evaporated to dryness and the residue treated with ether containing a small amount of ethyl acetate, 5 7. sodium bicarbonate solution, water, 10 70 citric acid solution and water.

The residue was dried and then dissolved in a mixture of

methanol and DMF in a ratio of 4:1 and treated with active carbon, and the clearly filtered solution was placed in a refrigerator overnight whereupon 1.48 g of a substance with a melting point of 234 - 240° C was obtained. After one more recrystallization, the melting point I fell to 239 - 241° C. By chromatography, the yield of uniform compound was 1.15 g, corresponding to 70.6% of the theoretical yield, and ! the compound had an optical rotation of [a]j^ = -9.6° (C-0.5, j DMF) .

tAnalysis: C35H5oNl2°ll (molecular weight 814.88)

I Calculated: C = 51.59%, H = 6.19%, N = 20.62%

in Obtained: 51.81% 6.09% 20.95 7.

l

1

Oak sample 35 !

i i ! Preparation of carbobenzyloxy-phenylalanyl-phenylalanyl-valyl-omega-nitroarginyl-omega-nitroarginine methyl ester 408 mg (0.5 mmol) of carbobenzyloxy-phenylalanyl-valyl-omega!-nitroarginyl-omega-nitroarginine methyl ester was decarbobenzyloxy-: lerted as described in previous examples. The obtained tetrapep-f titester hydrogen bromide in an amount of 538 mg and containing 2.4 equivalents of hydrogen bromide was dissolved in 10 ml of DMF and cooled to -10° C, 0.17 ml of triethylamine was added and formed triethylamine-j hydrogen bromide filtered off. To the filtrate was added 0.21 g (0.5 mmol) of carbobenzyloxy-phenylalanine-p-nitrophenyl ester. The reaction profile was the same as described in previous examples.; The solution was evaporated to dryness under vacuum and the residue taken up with ether, 5% sodium bicarbonate solution, water, 10 7. Eitron? acid solution and water. The thus treated product was dissolved in hot methanol, and after 1 day in a refrigerator, 4.28 mg of product with a melting point of 230 - 238° C was obtained. After two further recrystallizations from methanol, a chromatographically uniform compound with a melting point of 236 - 240° C and an optical rotation of [a]^<5> -20.0° (C=3.8, DMF). The yield was 338 mg corresponding to 70.3 7. of the theoretical.

Analysis: c44H59lv|i3<0>i2 (molecular weight 962.06)

Calculated: C = 54.93 7., H = 6.18 7., N - 18.93%

Obtained: 54.62% 6.25% 19.10%

Example 36

Preparation of carbobenzyloxyglycylproline

7.2 g (63 mmol) of proline was dissolved in 160 ml of a mixture of pyridine and water in a ratio of 1:1 and the pH of the solution adjusted to 8.8 with 4N NaOH. While stirring at room temperature, 20.7 g (63 mmol) of carbobenzyloxyglycine-p-nitro-phenyl ester were added in small portions, the pH during the addition being maintained between 8.5 and 8.8 with 4N NaOH. After 1 hour, 25 g of sodium bicarbonate and 200 ml of water were added, and the solution was extracted with ethyl acetate (10 x 60 ml) and the aqueous phase acidified with 4N hydrochloric acid to a pH of 3.5. The acidified aqueous phase was extracted with ethyl acetate and the organic phase washed with IN hydrochloric acid solution and water and dried over magnesium sulfate. After concentration under vacuum, 14.5 g of a substance crystallized out which, after a further recrystallization from ethyl acetate, gave 12.2 g of product with a melting point of 155 - 156° C, an optical rotation of 0]j<*5> = -54 .0°

in (C=1,DMF) and an equivalent weight of 304.8 against calculated 306.3.

Example 37

Preparation of carbobenzyloxyglycylproline p-nitrophenyl ester

. 9.2 g (30 mmol) of carbobenzyloxyglycylproline were dissolved together with 4.6 g (33 mmol) of p-nitrophenyl in 75 ml of DMF. Solution-I was cooled to -5° C and 6.4 g (31 mmol) of dicyclohexylcarbodiimide dissolved in a small amount of DMF added while the temperature was not allowed to exceed 0° C. The mixture was allowed overnight to slowly assume ! room temperature and after filtering off the formed DCU (dicyclo-hexylurintoS), the filtrate was concentrated to 40 ml in vacuum and there-; after stirring, dripped into 1 liter of water. The formed half-; solid mass was separated and dissolved in ethyl acetate which was dried over magnesium sulfate. The ethyl acetate solution was evaporated to dryness and the residue dissolved in a mixture of ethyl acetate and

In ether in a ratio of 1:1, and after adding petroleum ether

<1> crystallized out 6.6 g of substance with a melting point of 104 - 105.5° C! and an optical rotation of [<x]q4 = -87.5° (C=1, DMF).

; Example 38

Preparation of carbobBnzyloxyglycylprolyl-omega-nitroarginine methyl ester

2.7 g (10 mmol) of omega-nitroarginine methyl ester hyrirogen chloride were dissolved in 30 ml of DMF and cooled to 0° C, after which <:> 1.40 ml of triethylamine was added. Triethylamine hydrogen chloride formed was filtered off, 4.3 g of carbobenzyloxyglycylproline-p-nitro-

■ phenyl ester added to the filtrate and after 1 dbgn at room temperature

j turn a further 1.4 ml of triethylamine. The reaction solution was evaporated to dryness under vacuum and the residue taken up with ether,

j 5% sodium bicarbonate solution, water, 10% citric acid solution I and water. The residue was dissolved in a small amount of warm DMF, and after addition of water 3.1 g of product crystallized out, which was recrystallized from a mixture of DMF and water. An out-j exchange of 2.8 g corresponding to 53.7 7. of the theoretical was achieved, and it is-

The product retained was chromatographically uniform and had a melting point

I of 105 - 107° C.

I Analysis: C22H31N7°8 (molecular weight 521.55)

( Calculated: C = 50.66 7th, H = 5.99 7th, N = 18.80%

! Obtained: 51.01 7. 5.84 7. 18.85%

in

in

in

Example- 39

Preparation of carbobenzyloxy-phenylalanyl-valyl-omega-nitroarginine

2.45 g (4 mmol) of carbobenzyloxy-phenylalanyl-valyl-omega-nitroarginine methyl ester was dissolved in 30 ml of 80% acetone and saponified with 4 ml of IN HaQti at a pH of 11.5-12.5. Carbon dioxide was introduced into the solution until its pH was 7.8, after which the solution was evaporated under vacuum. The residue was dissolved in water, the solution extracted with ethyl acetate and the aqueous phase "acidified" with citric acid solution. The precipitate formed was extracted with ethyl acetate and the organic phase washed hysterically with water, dried over MgSO^ and evaporated to dryness under vacuum. The residue was recrystallized from a mixture of ethanol and water. The yield of chromatographically pure product was 1.95 g corresponding to 76.7% of the theoretical, and the product had an optical rotation of [<02n 3 = - 12.2 o (c-1.1, methanol) and an equivalent weight of 625.3 against calculated 626.7 for the compound indicated in the title containing two molecules of crystal water.

Example 40

Preparation of carbobenzyloxy-phenylalanyl-valyl-omega-nitroarginyl-glycylproyl-omega-nitroarginine methyl ester

334.6 mg (0.8 mmol) of carbobenzyloxy-glycylprolyl-omega-nitroarginine methyl ester was decarbobenzyloxylated and worked up as described in the previous examples. The tripeptide ester hydrogen bromide was dissolved in 2 ml of freshly distilled DMF, 22.2 ml of triethylamine added, formed triethylamine hydrogen bromide filtered off, the filtrate cooled to -10° C and 509 mg (0.8 mmol) carbobenzyloxy-phenylalanyl-valyl-omega-nitroarginine dissolved in 2, 5 ml DMF previously cooled to -10° C added. 165 mg (0.8 mmol) DCCI was added with stirring, and the solution was allowed to stand for 3 days at room temperature. The solution was concentrated to low dryness under vacuum and the residue in an amount of 933 mg was dissolved in 3 ml DMF and cooled to -20° C, whereby 128 mg of DCU crystallized out. Preparative thin layer chromatography of the crude product on silica gel in the solvent system: n-butanol:acetic acid:water (6:2:2) gave a pure product with an Rf value of 0.64. Eh total hydrolysis of 1.2 mg of substance in 6N hydrochloric acid for 48 hours at 110° C and two-dimensional thin-layer chromatography of the hydrolyzate on silica gel yielded the amino acids included in the hexapeptide derivative. The yield of chromatographically pure substance was 185 mg corresponding to 19.1% of the theoretical. The substance had a melting point of 125 - 127° C and an optical rotation of Ca3n -32.0° (c=0.5, DMF).

Analysis: C^H^N-^O-^ (molecular weight 969.05)

Calculated: C = 52.06%, H = 6.24%, N = 20.24%

Obtained: 5l.8U-% 6.15% 20.13%

Claims (1)

Analogous procedure for the production of new peptides with antithromboplastin, antithrombin and antifibrinolytic action and vasodilating action and with the formula: where is hydrogen, a butyl, isobutyl, secondary butyl or benzyl group, m is 0 or 1, X is -(HNCH2CO)n -, with n1 varying from 0 to 3, or with n^ varying from 0 to 3* and R^ constitutes a carboxyalkyl group, where the carboxyl group can be free, esterified with an alkanol of up to 8 carbon atoms or amidated, or with n^ and Rl having the meaning given above, where Ala means the remainder of alanine, and R^ is a straight or branched alkyl group with no more than 8 carbon atoms, characterized in that the necessary amino acids in the specified sequence and/or peptide fragments containing these amino acids in the specified sequence are condensed to the specified polypeptide, since for the peptidic coupling an end- standing carboxyl group and/or an amino group are activated and the other reactive groups are intermediately protected.