DE2014800A1 - D-analogs of secretin - Google Patents

Description

Priority: March 27, 1969, V.St.A., No. 811259

The invention relates to D-analogs of the peptide secretin, the salts and intermediates thereof _o Pig secretin have the formula His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu-Leu-Ser-Arg-Leu-Arg-Asp-Ser -

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Ala-Arg-Leu-Glu (NH ₂ ) -Arg-Leu-Leu-Glu (NH ₂ ) -GIy-LeU-VaI-NH ₂ 17 18 19 20 21 22 23 24 25 26 27

and is consequently a peptide with 27 amino acid residues of the following amino acids: L-histidine (His); L-aspartic acid (Asp); L-serine (Ser) j glycine (GIy); L-threonine (your); L-Phenylalanine (Phej; L-Glutamic acid (GIu); L-Glutamine / GIu- (NH ₂ ] /; L ^ -Leucine (Leu); L-Arginine (Arg); L-Alanine (AIa); and L- Valinamide (VaI-NH ₂ ) ₀

This peptide is the subject of the enzymatic attack, of which The effect must be short-lived because of the degradation. It was found that the D forms of histidine, serine, phenylalanine and arginine when used in the synthetic manufacture of

009841 / Ί960

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Secretin used to form analogues of secretin, the opposite are resistant to enzymatic attack and show activity of longer duration than secretin peptide.

The D-forms of secretin and their salts can be synthesized new intermediates and their association until the D-analogs of secretin are formed, synthetically produced.

According to the method of the present invention, one can produce D-secretin synthetically produced by loading ™ with L-Yalin-amide starts and adds the remaining amino acids either all at once or in groups to form the peptide. This addition is made by Activating the carboxyl groups in the amino acids to be added by protecting the amino groups in the amino acids by transferring them into its benzyloxycarbonyl derivatives and also conversion into the nitrophenyl ester derivatives and by allowing the Amino acid on the chain of a previously prepared peptide and finally removing the one originally present in the peptide Reaching protective groups.

Such groups can be included among the appropriate activating groups which have a more reactive acid function, such as mixed anhydrides (which usually involve the acylation of a Amine with the mixed anhydrides, for example one. Acyl amino acid and isovaleric acid), azides, acid chlorides, Reaction products with carbodiimides ^ reactive N ~ acyl compounds, O-acylhydroxylamine compounds, and active Esters, such as alkyl esters with electron-attracting (negative) substituents, vinyl esters, enol esters, phenyl esters, thiophenyl esters, Nitrophenyl ester, 2,4-dinitrophenyl ester, trichlorophenyl

009841/1980

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ester or nitrophenyl thiol ester. The use of nitrophenyl esters is in terms of yield. Absence of by-products and subsequent easy cleaning are particularly preferred.

In the formation of the peptide sequences according to the present invention, the amino functions can be replaced by commonly used amino protective groups be protected, such as the benzyloxycarbonyl -, Nitrobenzyloxycarbonyl, methoxybenzyloxycarbonyl, tert -.- butyloxycarbonyl, Phthalyl, o-nitrophenylsulfenyl or the tosyl group. The methyl, ethyl, tert-butyl, benzyl, nitrobenzyl or trimethylbenzyl group can protect the carboxyl groups be used. Examples of protective groups for the hydroxyl groups can be the benzyl, tert-butyl or tetrahydropyranyl groups be. Examples of protecting groups of the guanidine group can

the .'
nen / nitro ~, tosyl or p-nitrobenzyloxycarbonyl group or a

Be protonation.

The protecting groups are removed by means of known reactions, such as reduction with sodium in liquid ammonia, hydrogenation (e.g. in the presence of a palladium / activated carbon catalyst), treatment with a hydrochloric acid such as hydrobromic acid or hydrochloric acid, in acetic acid or treatment with trifluoroacetic acid «,"

To prepare the free amines after treatment with a hydrohalic acid in acetic acid, for example Hydrobromide salt either with an ion exchanger such as the Ion exchangers known under the trade name “Amberlite IR400” treated or the same with an amine, such as triethylamine, neutralized

009841/1980

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The aforementioned peptide salts include, for example, hydrochlorides, hydrobromides, Acetates, fluoroacetates, such as irifluoroacetate, or chloroacetates, like dichloroacetate.

The present invention accordingly relates to peptides of the general Formula:

R-His-R-Ser-Asp-Gly-Thr-R-Phe-Thr-Ser-Glu-Leu-Ser-R-Arg-Leu-R-1 2 3 4 5 6 7 8 9 10 11 12 13

Arg-Asp-Ser-Ala-R-Arg-Leu-Glu (NH ₂ ) -R-Arg-Leu-Leu-Glu (NH ₂ ) -Gly-

* 14 15 16 17 18 19 20 21 22 23 24 25

LeU-VaI-NH ₂

26 27

in which R is either L or D and at least one R is D.

Amino Acid Groups to Use in the Practice of the Present Invention can use are

His-R-Ser-Asp-Gly- (S-, _A );

1 2 3 4

■ Thr-R-Phe-Thr-Ser- (Sc ₈ );

* 5 6 7 8

Glu-Leu-Ser ~ R-Arg-Leu (Sq ,,); and 9 10 11 12 13

R-Arg-Asp-Ser-Ala-R-Arg-Leu-Glu (NH ₂ ) -R-Arg-Leu-Leu-Glu (NH ₂ ) -Gly-14 15 16 17 18 19 20 21 22 23 24 25

LeU-VaI-NH ₂

26 27
in which R has the meaning given above.

G G ί: δ A 1/1 9 8 0

20U800

The following equations illustrate the invention using the procedures outlined above:

S _9-13 + S ₁₄ _27 _> ^S 9-27 ^S 9-27 ^{+ S} 5-8 ^S 5-2

^S l-27

The new D-analogs of secretin made by the methods of the invention work when administered in a manner similar to secretin, say twice, longer, _{or the like}

The following examples illustrate the invention

Example 1

Benzyloxycarbonyl-D · -phenylalan.yl-L · threon.vl-L · serine meth.yl ester 2.1 g of 10% Palladiura-on activated carbon are added to a solution of 21 g (60 mmol) of benzyloxycarbonyl -L-threonyl-L-serine methyl ester in a mixture of 200 ml of absolute ethanol and 60 ml of 1 η hydrochloric acid ο The suspension is stirred for 7 hours under a water-substance atmosphere. The catalyst is filtered off and the filtrate is evaporated under reduced pressure. The oily residue is in 300 ml of dimethylformamide, the 8.4 ml

lets it contain (60 raMol) triethylamine, dissolved and / with 29 g (72 mmol)

Benzyloxycarbonyl-D-phenylalanine-p-nitrophenyl ester react. .. The mixture is left to stand for 6 hours at room temperature. The solvents are removed under reduced pressure and the residue is digested with ethyl acetate. The solid crystals are filtered off, dried and recrystallized from methanol / water ümkristallisiert (2 j 1) Yield: 16.5 g (55 $> of theory). ~

Example 2

Benzyloxycarbonyl-L-threonyl-D-phenylalanyl-L-threonyl- ■ ^ L- · serine- ^ methyl ester

To a solution of D-phenylalanyl-L-threonyl-L-serine methyl ester hydrochloride, (the by hydrogenation of 15 g (30 mmol) of benzyloxycarbonyl-D-phenylalanyl-L-threonyl-L-serine methyl ester has been prepared) in 100 ml of dimethylformamide containing 5.04 ml (36 mmol) of triethylamine, add 15 g (36 mmol) Benzyloxycarbonyl-L-threonine-2,4-dinitrophenyl ester added. the The mixture is kept at room temperature for about 15 hours. After removal of the solvent, digested under reduced pressure the residue with ethyl acetate. The solid crystals are filtered off, dried and recrystallized from 95 # ethanol. Yield: 15.4 go

Example 3

Benz _t vlox.ycarbonyl-L ^ -threonyl-D-phenylalanyl-L-threon.γl-L- ^ serine hydrazide

1.7 ml of hydrazine hydrate are added to a solution of 2.1 g (3.5 mmol) of benzyloxycarbonyl-L-threonyl-D-phenylalanyl-L-threonyl-D-serine methyl ester in 170 ml of methanol. The mixture is left to stand for 20 hours at room temperature, the precipitate formed is filtered off, dried and recrystallized from methanol. Yield! 1.56 g (74 ί> of the theory.

Example 4

t.ert.-Butylox.ycarbonyl-nitro-D-arginyl ^ If leucine-benzyloxycarboh.Yl-h.ydrazide

7-15 g (14.4 mmol) of tert-butyloxycarbonylnitro-D-arginine-2,4-dinitrophenyl ester are added to an ice-cold solution of

4 * 75 g (12 mmol) of L-leucine benzyloxycarbonylhydrazide trifluoroacetate and 1.68 ml (12 mmol) of triethylamine in 24 ml of tetrahydrofuran. Two more portions of 0.7 g of Dinitrophenylesters is added at one-hour intervals hinzuο The mixture is kept for about 15 hours at room temperature and then O _t 75 ml Dimethyläminopropylamin offset "After 1 hour, diluted the reaction mixture with 200 ml of ethyl acetate and wash the solution obtained once with 20% citric acid, once with Ttfasser, twice with 0.5 η ammonium hydrazide and four times with water. The organic layer is dried over magnesium sulfate and the solvents evaporated under reduced pressure. "The oily residue is triturated with ether until it solidifies." The crude product is recrystallized from a mixture of ethyl acetate / ether (1 sec 9). Yield 6.15 g (88% of theory)

Example 5

tert-butyloxycarbonyl-O-benzyl'-L-seryl-nitro-D-arginyl-L-leucine benzyloxycarbonyl hydrazide

Dissolve 7 g (12 mmol) of tert-ButyloxyCarbonylnitro-D-arginyl-L- \ benzyloxycärbonyihydrazid leucine in 25 ml of trifluoroacetic acid at 10 ⁰ C and the solution held lasts 15 minutes at room temperature. The trifluoroacetic acid is removed under reduced pressure at room temperature and the residue is triturated with ether. The solids are filtered off, washed with ether and dried over KOH under reduced pressure. This trifluoro acetate is dissolved in 36 ml of ice-cold dimethylformamide _{8, it is} neutralized with 1.68 ml (12 mmol) of triethylamine and allowed to react with tert-butyloxycarbon ^ 10-benzyl-L ^ serine-p-nitrophenyl ester _{9 eex}

" ⁸ 20H800

from 4 _? 4 g (15 mmol) of tert-butyloxycarbonyl-O-benzyl-L-serine has been prepared. After standing at room temperature, the reaction mixture is diluted with 300 ml of ethyl acetate and washed once with 20% citric acid and twice with water. The organic phase is dried over magnesium sulfate, the solvent is removed under reduced pressure and the residue is recrystallized from ethyl acetate. Yield: 6.3 g (70 % of theory).

Example 6

^ tert-Butyloxycarbonyl-L-leucyl-O-benzyl-L-seryl-nitro-D-arginyl-L-leucine-benzyloxycarbonylhydrazide is prepared in the same way as the above tripeptide. It crystallizes from absolute ethanol. Yield: 82 # of theory.

Example 7

tert-Butyloxycarbonyl-J ^ -benzyl-L-glutamyl-L-leucyl-O-benzyl-L-seryl-nitro-D-arginyl-L-leucine-benzyloxycarbonylhydrazide is prepared in the same manner as the above-mentioned tetrapeptide. It crystallizes from methanol. Yield: ^ 84 56 of theory.

Example 8 L-grlutamyl-L-leuxyl-L-seryl-L-arginyl-L-leucyl-nitro-D-arginyl

β-benzyl-L-aspartyl-O-benzyl-L-seryl-L-alanylnitro-D-arginyl-L- leucyl-L-gl ^ taminylnitro-D-arp: in.yl-I ^ -leucyl-L-leucyl -L · -glutaminyl -glycyl-leucyl-L-valine-amJd-trifluoroacetate A solution of 1 »35 g (1.8 mmol) tert-butyloxycarbonyl-L-glutaray1- is added to 0.9 ml of concentrated hydrochloric acid. L-leucyl-L-seryl-L-arginyl-L-leucine hydrazide, which has been prepared by hydrogenating the protected hydrazide, in 13 »5 ml of

00 98 Λ 1/1 980

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methylformamide, which has been cooled ^{to -2O 0} C in a dry ice / acetone bath. Allow the bath temperature at -15 ⁰ C Toggle rise and added 1.35 ml (2.7 mmol) of an aqueous 14- $ solution of sodium nitrite hinzuo After 5 minutes, lowering the bath temperature to 25 ° ^ C, and outputs 1 , 25 ml of N-ethylpiperidine. added. A solution of 2.8 g (1.4 mmol) of nitro-D-arginyl-β-benzyl-L-aspartyl-0-benzyl-L-seryl is added to this mixture, which contains the tert-butyloxycarbonylpentapeptide azide -L-alanyl-nitro-D-arginyl-L-leucyl-L-glutaminylnitro-D-arginyl-L-leucyl-L-leucyl-L-glutaminylglycyl-L-leucyl-L-valine-amide'-g

trifluoroacetate in 13.5 ml of dimethylformamide. The reaction mixture is left to stand at 5 ° C. and, after 48 hours, a further portion of the tert-butyloxycarbonyl pentaapeptide azide, which has been prepared from 150 mg of the corresponding hydrazide, is added. After a total of 4 days, the solvents are removed under reduced pressure and the residue is triturated with ethyl acetate. The pest body is filtered off and dried. The substance obtained in this way is dissolved in 30 ml of cold trifluoroacetic acid and the solution is kept at room temperature for 15 minutes. The trifluoroacetic acid is removed under reduced pressure, the residue is triturated with ether, filtered and dried. This trifluoroacetate is washed twice with 30 ml of water to remove salts and excess pentapeptide. The insoluble substance is separated by centrifugation and dried over KOH under reduced pressure. Yield: 2.9 g (80 io of theory),

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20U800

Example 9

L-Glutamyl-L ^^^ leucyl-L-seryl - L-arginyl-L-leucyl-D-arginyl - ^^ L-aspartyl-L-seryl-L-alanyl-D-arginyl-L-leucyl- L-glutaminyl-D - argin.vl-IJ-le ^ c.yl-L · -leucyl-L · -glutaminylglycyl-II-le ^ Gyl-l · ~ valinamide.

A solution of 1.4 g of the partially protected is hydrogenated Nonadecapeptide amides in 50 ml of aqueous 80-36% acetic acid 48 hours over 10 # palladium carbon and filtered the catalyst ab »The Piltrat is freeze-dried. Yield: 1.25 g.

Example 10

L-Threonyl-L-phenylalanyl-Ir-threonyl-L-seryl-L-glutamyl-L-leucyl-Ir-seryl-L-arginyl-L-leucyl-D-arginyl-Ir-aspartyl-L-seryl-Ii- alanyl-P-arginyl ^ -leucyl-L-glutaminyl-D-arginyl-L-leucyl-L-leuoyl-L-glutaminyiglycyl-L-leucyl-L-valine amide 0.45 ml (0.9 mmol) of a 14 56% aqueous sodium nitrite solution is added to a solution of 450 mg (0.75 mmol) of benzyloxycarbonyl-L-threonyl-Ir-phenylalanyl-L-threonyl-Ir-serine hydrazide in 5.6 ml of dimethylformamide, which is 0.37 ml contains hydrochloric acid cooled to -15 ° C in a dry ice / acetone bath. After 15 minutes the bath temperature was lowered to -25 ⁰ C and add 0.53 ml of N-ethylpiperidine "At this benzyloxycarbonyl-tetrapeptide-azide-containing mixture is added a solution of 1.2 g (0.5 mmol) of the free nonadecapeptide amide in 3.3 ml of dimethylformamide and 1 ml of water. The reaction mixture is kept at 5 ° C. After 24 hours, a large part of the benzoyloxycarbonyl-tetrapeptide-acid, which has been prepared from 90 mg of the corresponding hydrazide, is added

0 0 9 8 4 _{1/1 9 8 Q 0R1GINAL INSPECTED}

is. After a total time of 48 hours, the solvents are removed under reduced pressure and dissolve the residue in 70 ml of aqueous 90% acetic acid and hydrogenate for 7 hours

\ Activated carbon

over 500 mg of IO 5% palladium on / «, the catalyst is filtered off and the filtrate freeze-dried to this Lyophilisate is distributed for 250 transfers in the system: 1-butanol / pyridine / acetic acid / water (4: 2: 1: 7). One only observes a main peak. Yield: 740 mg.

Example 11 . pO

L-histidyl-L-seryl-L-aspartylglyeyl-L-threonyl-L-phenylalanyl-L- | threonyl-L-seryl-L-glutamyl-L-leuc.yl-LΓ ^ -seryl-L-argin.vl-L-leucyl-D-arginyl-L-aspartyl-L-seryl-L-alanyl-D-arginyl -L-leucyl-L-glutaminyl-D-arginyl-L-leucyl-L-leucyl-L-glutaminylglyeyl-L-1 eucyl-Ir-valine-amide '

0.09 ml of concentrated hydrochloric acid is added to a stirred solution of 9 »6 mg (0.18 mmol) of tert-butyloxycarbonyl-L-histidyl-L-seryl-L-aspartylglyein hydrazide, which has been obtained by hydrogenating the protected hydrazide is cooled in 1.5 ml in a dry ice / acetone bath to -20 ⁰ C dimethylformamide. The | Bath temperature is allowed to rise to -15 ^O C and added 0.15 ml (0.3 mmol) of 14 # aqueous solution of sodium nitrite hinhinzüo After 5 minutes, the bath temperature at -25 ⁰ C is lowered, and 0.13 ml H Ethyl piperidine added. A solution of 168 mg (0.06 mmol) of the free trieosapeptide amide in 2.1 ml of dimethylformamide is added to this mixture, which contains the tert-butyloxycarbonyl tetrapeptide azide. The reaction mixture is kept at 5 ° C. After 24 hours, a second portion of the tert-butyloxycarbonyl tetrapeptide azide is added, which is also 32 mg

00 98A1 / 1980

~ ¹² ~ 20H800

of the corresponding hydrazide has been prepared. After a total of 48 hours, the solvents are removed under reduced pressure and the residue is dissolved in 6 ml of cold trifluoroacetic acid. The solution is kept at room temperature for 15 minutes and the heptacosapeptide trifluoroacetate is precipitated using 100 ml of ether. The body of the plague is centrifuged off, washed with ether and dried. Yield: 330 mg. This substance is used for transmissions in the system 200: / distributed l-butanol 0, lm phosphate buffer p _H 7 (1 ί 1). The wished

-amid
Heptacosapeptide / has a separate tip and is obtained from the two-phase system by the alginic acid adsorption process: 62 mg.

Example 12 Benz.yloxycarbonyl-D-histidyl-0 ^ -benzyl - L-seryl-ß-benzyl-L-

aspartyl-gl.ycyl-L-threonyl-L-phenylalanyl-L-threonyl-O-benzyl- / • '-benzyl-L-seryl-ß-benzyl-L-glutamyl-L-leucyl-O-benzyl-L-seryl- · nitro-L-arginyl-L-leucyl-nitro-L-arginyl-ß - benzyl-L - aspartyl ~ 0- benzyl-L-seryl-L-alanyl-nitro-L-ar ^ inyl-L-leucyl-L-glutaminyl- nitro-L - arginyl-L-leuc.yl * -L-leucyl-L- glutamin.ylglycyl-L -leucyl-

L-valine amide

70 mg (1 mmol) of sodium nitrite are added to a solution of 305 mg (1 mmol) of benzyloxycarbonyl-D-histidine hydrazide in one Mixture of 4 ml of ethyl acetate and 3 ml of 1 η hydrochloric acid with stirring in an ice / salt bath. After 3 minutes, 0.8 ml of a 50% strength aqueous solution of potassium carbonate is added and the process is interrupted after a further 3 minutes, stop stirring and allow the mixture to separate into two phases. The aqueous phase is extracted one second portion of 1 ml of ethyl acetate and discarded. The Ver-

0098 4 1/1980

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some organic phases are added over magnesium sulfate O C dried. '■

3.5 ml of this ethyl acetate solution of benzyloxycarbonyl-D-histidinazids becomes an ice-cold solution of 780 mg of the hexacosapeptide trifluoroacetate in a mixture of 6 ml of dimethylforma- ' mid and 0.4 ml of triethylamine (i.e. a 14% by volume triethylamine-dimethylformamide solution) given. The reaction mixture is kept at 5 ° C. Another portion is added after 24 hours of benzyloxycarbonyl-D-histidine azide (1/3 of the initial amount) added o 24 hours after this addition, 500 ml of ethyl acetate are added dropwise to the reaction mixture. The solid substance will filtered off, washed with ethyl acetate and dried over sodium hydroxide Yield dried under reduced pressure: 717 mg.

Example 13 .

N ~ Benzyloxycarbonyl-nitro-D ~ argin.yl-L · -leuc.yl-L · ~ leucyl-L · - glutaminyl-glycyl-L-leucyl-L-valine amide To a solution of 3.53 g of N ⁰ '-Benzyloxycarbonyl-nitro-D-arginine and 2.0 g of 2,4-dinitrophehol in 60 ml of tetrahydrofuran are added 2.1 g of dicyclohexylcarbodiimide while cooling with ice. After about 1 hour at room temperature, the precipitate (dicyclohexylurea) is filtered off and washed with 40 ml of tetrahydrofuran, and washing solutions are combined and evaporated to dryness under reduced pressure. The residue is dissolved in about 10 ml of ethyl acetate and washed with about 50 ml of ether precipitated The ester is washed with about 50 ml of ether and dried under reduced pressure.

.0 09841/1980

3.9g hexapeptide amide are heated in 150 ml to about 80 ⁰ C. Dissolved dimethylformamide and mixed with a solution of the aforementioned active ester in 50 ml of the same solvent. The mixture is cooled to room temperature and, after about 4 hours, diluted with 1 liter of ether. The precipitate is filtered off, washed with 0.6 liters of ether and 0.3 liters of ethyl acetate and air-dried. Yield: 4.5 g.

Example 14

Benzyloxycarbonyl-L-glutaminyl-nitro-D-arginyl-L-leucyl-L-leucyl-L-glutaminylglycyl-L-leucyl-L-valine amide Dissolve 9.8 g of the protected heptapeptide in 50 ml of warm acetic acid, cool and treat it with about 50 ml of 4 η hydrobromic acid in acetic acid. After one hour at tree temperature, 1.2 liters of ether are added to the solution, the hydrobromide is filtered off, washed with ether and dried briefly under reduced pressure over sodium hydroxide. Then it is dissolved in 100 ml of dimethylformamide and the solution is made alkaline with 10.4 ml of triethylamine. 5.0 g of p-nitrophenyl benzyloxycarbonyl-L-glutamic acid are added to the mixture, and the mixture is left to stand at room temperature for about 15 hours. The separated crystals form a semi-solid mass. The mixture is diluted with 1 liter of ethyl acetate, the precipitate is filtered off and this is washed one after the other with 500 ml of ethyl acetate, 500 ml of ethanol, ethyl acetate, 250 ml of hot ethyl acetate and 250 ml of chloroform. The product is dried first in air and then at 50 ° under reduced pressure. Yields 11.0 g (100 ^ of theory) "

0098A1 / 1980 originally inspected

:;: ■■ '. . 2ΟΗ8ΌΟ

Example 15 .

Benzyloxycarbonyl-L-leucyl-L-glutaminyl- ^ nitro-D-arginyl-L-leucyl-L-leucyl-L-glutaminylglycyl-L-leucyl-L-valine amide 11.1 g of the protected octapeptide are powdered and added to 50 ml of acetic acid with stirring. 50 ml of 4 η hydrobromic acid in acetic acid are slowly added to the suspension. Stirring is continued until all of the substance has dissolved, and then another 30 minutes. A total of 3 hours are required. The amine hydroteromide is precipitated with 1 liter of ether, filtered, washed with ether and briefly under reduced pressure | dried over sodium hydroxide. The hydrobromide is dissolved in 100 ml of dimethylformamide and 10.4 ml of triethylamine and then 5 _{t of} 0 g of benzyloxycarbonyl-L-leucine-p-nitrophenylesteinzUo are added to the cooled solution. After standing for about 15 hours at room temperature, the mixture is diluted with 2 liters Ethyl acetate. The precipitate that forms is filtered off and washed successively with 200 ml portions of ethyl acetate, chloroform, hot chloroform and hot ethyl acetate. The product is dried at 50 g under reduced pressure. Yield: “12.1 g ( $ 99 of theory) ο

Example 16 K - »Benzyloxycarbonyl-nitro-D-arginyl-L-leucyl-L-glutaminyl-

nitro-D-arginyl - L-leuc.yl ~ L - leucyl-L - glutaminylglycyl-L ^ -leuc.vl-

L-valine amide

24.2 g of the protected nonapeptide amide are suspended in 100 ml Acetic acid and slowly add about 100 ml to the suspension 4 η hydrobromic acid in acetic acid added. After about 1 hour a homogeneous solution is obtained, which can be used for an additional hour

009841/198 0

" ¹⁶ " 20H800

Maintains room temperature. Then add 2 liters of ether to the solution. The precipitated hydrobromide. is filtered off, with ether .Washed and briefly under reduced pressure over sodium hydroxide Then it is dissolved in 250 ml of dimethylformamide. The solution is cooled while it is washed with 14 ml of triethylamine is made alkaline. 25.8 g of benzyloxycarbonyl-nitro-D-arginine-2,4-dinitrophenyl ester are then added and then add another 5 ml of triethylamine. After 3 hours at room temperature, the solution no longer shows any ninhydrin reaction. After about After standing for 24 hours, dilute with 2 liters of ethyl acetate and filter the precipitate and washes it successively with 500 ml of ethyl acetate, 500 ml of chloroform, 1.5 liters of hot chloroform and 500 ml of hot ethyl acetate. The product is dried at 50 ° C. under reduced pressure. Yield: 25.1 g (88 # der Theory).

Example 17 Benzyloxycarbonyl-L-alanyl-nitro-D-arginyl-L-leucyl-L-glutaminyl-

nitro-D-arginyl-L-leucyl-L-leucyl-L-glutaminylglycyl-L-leucyl-L-

valine amide. _r

w . ^ _n

A solution of 120 ml of about 4 η is added to a solution of 25.5 g of the protected decapeptide amide in 120 ml of acetic acid Add hydrobromic acid in acetic acid. After 1.5 hours at The hydrobromide precipitates at room temperature by adding about 2 liters of ether. It is washed with ether and then briefly dried under reduced pressure over sodium hydroxide. Then it is dissolved in 180 ml of dimethylformamide. One adds to the solution first 18 ml of triethylamine and then 9.3 g of benzyloxycarbonyl-L-alanine-p-nitrophenyl ester added. You leave the reaction to it

00 98 A1 / 1980

2Ό14800

about 15 hours by itself, then filter the mixture, wash the triethylammonium bromide with 50 ml of dimethylformamide, adds another 3.1 g of the active ester and 1 ml of triethylamine and narrow the solution. After 2 hours, dilute with 2 liters Chloroform »The precipitated crystals are washed with chloroform and dried in air. Yield: 25.6 g (95 $ der Theory ^

Example 18 .

N-tert-butyloxycarbonyl-O-benzyl-L-seryl-L-alanyl-nitro-D- | arginyl-L-leucyl-L-glutaminyl-nitro-D-arginyl-L-leucyl-L-leucyl-L-glutaminyl-glycyl-L-leucyl-L-valine amide To a suspension of 24.0 g of the protected Hendeeapeptids in 160 ml of acetic acid are slowly added 160 ml of a 4 η hydrobromic acid in acetic acid. After about 2 hours at room temperature, the solution is diluted with 2 liters of ether, the precipitate is filtered off, washed with ether and dried under reduced pressure over sodium hydroxide . The hydrobromide is dissolved in 235 ml of methanol, the solution is cooled in an ice water bath and neutralized with 25 ml of triethylamine. It forms a thick Kristallbreio The suspension is diluted with 500 ml of chloroform, the crystals filtered off, washed with 1 liter of chloroform and dried in air and then at 40 ⁰ C under reduced pressure. Yield: 18.4 g ($ 80 of theory). of monohydrobromide.

Treating 18.0 g of the Monohydrobromids in 800 ml of dimethylformamide in the presence of 1.5 ml of triethylamine with N-tert "-Butyloxyearbonyl-0-benzyl-L-serine-p-nitrophenyl ester. This active ester is made from 9.4 g of the corresponding acid,

0098A 1/1980

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5.5 g of p-nitrophenol and 6.6 g of dicyclohexylcarbodiimide in 40 ml of ethyl acetate. The Ν, Ν'-dicyclohexylurea is filtered off and the solvent is removed from the filtrate under reduced pressure. The oily residue, which does not crystallize, is used further without purification. After one day at room temperature, the mixture is concentrated to about 50 ml under reduced pressure. 1.5 liters of ethyl acetate are then added, the precipitate is filtered off and washed successively with it. 500 ml of ethyl acetate, 200 ml of chloroform and again with 500 ml of ethyl acetate. During treatment with chloroform, the substance turns into a crystalline mass. Then drying the protected dodecapeptide under reduced pressure at 40 ⁰ C. Yield; 19.1 g ( $ 93> of theory).

Example 19

tert-Butyloxycarbonyl-ß-benzyl-L-aspartyl-O-benzyl-L-seryl-L-alanyl-nitro-D-arginyl-L-leucyl-L-glutaminyl-nitro-D-arginyl- 'L-leucyl- L-leucyl-L-glutaminylglycyl-L-leucyl-L-valine amide. 18.8 g of the protected dodecapeptide amide are dissolved in 120 ml of trifluoroacetic acid. After about 15 minutes, most of the trifluoroacetic acid is evaporated under reduced pressure and the syrupy residue is diluted with 500 ml of ether. The precipitate is filtered off, washed with ether and dried under reduced pressure over sodium hydroxide. The free amine trifluoroacetate (19.0 g) does not have a pronounced melting point. 12.35 g of dieBer substance are dissolved in 500 ml of dimethylformamide, 0.70 ml of triethylamine and 3.35 g of tert-butyloxycarbonyl-β-benzyl-L-aspartic acid p-nitrophenyl ester are added to the solution, and a second portion of p-nitrophenyl ester is added 2 hours later 3.35 g of the active ester and

009841/1980

0.35 ml of triethylamine are added. The mixture is left to stand at room temperature for about 15 hours. After the addition of a further 3 "35 g of the active ester and 0.35 ml of triethylamine to the mixture concentrated under reduced pressure to about 100 ml _β Finally, there is the final portion of 3.35 g of active ester and 0.14 ml of triethylamine is added and the the mixture stand for about 15 hours at room temperature. Most of the solvent is then removed under reduced pressure and the residue is diluted with 160 ml of ethyl acetate. The protected tridecapeptide is filtered off, washed with ethyl acetate and dried in air and then at 40 ° C. under reduced pressure. Yield: 13.05 g (94 # of theory).

Example 20

N ^tf - »tert-butyloxycarbonyl-nitro-D-argin.vl-ß-benzyl-L-aBpartyl-O-benzyl-L-beryl-Ir-alan.yl-nitro-D-arginyl-L-leucyl-L -glutamin.ylnitro-D-arginyl-L-leucyl-L-leucyl-L-glutaminylgl-ycyl-L-leucyl-L-valine-amide '

Mine dissolves 13.0 g of the protected tridecapeptide in 100 ml of trifluoroacetic acid. After about 15 minutes at room temperature, most of the trifluoroacetic acid is removed under reduced pressure Pressure and rub the residue with 900 ml of ether. The free amine trifluoroacetate is filtered off, washed with ether and _ dry it under reduced pressure over sodium hydroxide. Yield: 13.3 g. To a solution of the trifluoroacetate in 145 ml 6.0 g of terto-butyloxycarbonyl-nitro-D-arginine-2,4-dinitrophenyl ester are added to dimethylformamide and then add 1 ^ 0 ml of triethylamine. In the following 7 hours you add more Add triethylamine (1.1 ml in total) in several proportions,

00 9 84 1/198 0.

20U800

to maintain a weak alkalinity of the mixture. After about 24 hours at room temperature, a further 2 g are added. of the active ester and 0.7 ml of triethylamine are added and, after a further day, most of the solvent is evaporated under reduced pressure. The residue is triturated with about 1 liter of ethyl acetate, the solid substance is filtered off and washed successively with ethyl acetate and ether. After drying in air and under reduced pressure at 40 ^° C., the crude protected tetradecapeptide has a weight of 13.75 g.

^{The N 0} ^ -tert-butyloxycarbonylnitro-D-arginyl-ß-benzyl-L-aspartyl-O-benzyl-L-seryl-L-alanylnitro-L-arginyl-L-leucyl-L-glutaminyl is obtained in an analogous manner -nitro-L-arginyl-L-leucyl-L-leucyl-L-glutarainylglycyl-L-leucyl-L-valine amide.

Example 21

D-Arginyl-L-aspartyl-L-seryl-L-alanyl-D-arginyl-L-leucyl-L-glutaminyl-D-arginyl-L-leucyl-L-leucyl-L-glutaminylglycyl-L-leucyl-L- vali n-amide

13.7 g of the protected tetradecapeptide are dissolved in 105 ml of trifluoroacetic acid. After standing for 15 minutes at room temperature, the solution is concentrated under reduced pressure and the residue triturated with 1 liter of ether. The free amine trifluoroacetate is filtered off, washed with ether and under dried over sodium hydroxide under reduced pressure. Yield" 13.9 g.

13.9 g of the free amine trifluoroacetate are dissolved in 150 ml of 80 jars Acetic acid. 1.5 g of 10 # palladium-on-carbon are added to the solution added and hydrogenated for 48 hours. The catalyst

00984 1/1980

is filtered off and the filtrate freeze-dried yield:

12.2 go ■■ ■ ·

Example 22 ·. '

D-Histidyl-L-seryl-L-aspartylglycyl-1-threony1-L-phenylalany1-L ~ threonyl-L-seryl-L-glutamyl-L-leucyl-L-seryl-L-arginyl - L-leucyl-L -arginyl-L-aspartyl-L-seryl-L-alanyl-L-arginyl-L-leucyl-L-glutaminyl-L-arginyl-L-leucyl-L-leucyl-L-glutaminylglycyl-L- · leucyl-L- valine amide "

Dissolve 500 rag of the protected heptacosapeptide, benzyloxy-Jj carbonyl-D-histidyl-O-benzyl-L-seryl-ß-benzyl-L-aspartylglycyl-L-threonyl-L-phenylalanyl-L-tiireonyl-O-benzyl-I-seryl -./'- benzyl-Ii- glutamyl-L-leucyl-O-benzyl-L-seryl-nitro-L-arginyl-L-leucylnitro-L-arginyl-ß-benzyl-L-aspartyl-O-benzyl-L-seryl-L-alanylnitro-L- arginyl-L-leucyl-L-glutaminyl-nitro-L-arginyl-L-leucyl-Ir-leucyl-L-glutaminylglycyl-L-leucyl-L-valine amide. in 50 ml of 80% acetic acid. 400 mg of 5% acetic acid are added to the solution Palladium-on-barium sulfate is added and the device 48 is hydrogenated Hours. The catalyst is removed by centrifugation at 1500 rpm and the supernatant solution is freeze-dried » Yield: 444

Example 23

tert-Butyloxycarbonyl-O-benzyl-D-seryl-L-aspartylglycine benzyloxy-carbonyl hydrazide

10.8 g of tert-butyloxycarbonyl-β-tert-nutyl-L-aspartylglyein-benzyloxycarbonylhydrazine are dissolved in 50 ml of ice-cold trifluoroacetic acid and the solution is kept at room temperature for 1 hour. The trifluoroacetic acid is then removed under reduced pressure Pressure at room temperature and triturate the residue with

0 Ο 9 0 K 1/1 9 8 D '

Ether. The pesticide is filtered off, washed with ether and dried under reduced pressure over KOH. Yield; 7.1 g (16 mmol). This trifluoroacetate is dissolved in ice-cold dimethylformamide, neutralized with 4.62 ml (33 mmol) of triethylamine and the mixture is allowed to react with tert-butyloxycarbonyl-O-benzyl-D-serine-p-nitrophenyl ester, which is obtained from 7.1 g (24 mmol) tert-butyloxycarbonyl-O-benzyl-D-serine has been prepared. The reaction mixture is kept for 2 hours at room temperature, it is diluted with 400 ml of ethyl acetate and extracted once with ™ 20 56% citric acid and three times with water. The organic layer is dried over magnesium sulfate and the solvent is removed under reduced pressure. The residue crystallized from Ithylacetato Yield: 7.7 g (80 i "of theory).

Example 24

tert-Butyloxycarbonyl-L-histidyl-O-benzyl-D-seryl-L-aspartylglycine-benzyloxycarbonylhydrazide

840 mg (12 mmol) of sodium nitrite are added to a solution of 5.22 g (12 mmol) of tert-butyloxycarbonyl-D-histidine hydrazide in a mixture of 48 ml of ethyl acetate and 36 ml of 1 η hydrochloric acid with stirring in an ice / salt bath. After 3 minutes you add 9.6 ml of a 50 $ aqueous solution of potassium carbonate are added, interrupted the stirring after a further 3 minutes and allowed the mixture to separate into 2 phases. The aqueous phase is with extracted a second portion of 12 ml of ethyl acetate and discarded. The combined organic phases are over at 0 C. Dried magnesium sulfate.

This ethyl acetate solution of tert-butyloxycarbonyl-L-hintLd Ln-USiLdH is added to an ice-cold solution of O-benzyl-1) -

0098 A1 / 19 8 0

seryl-L-aspartylglycine-benzyloxycarbonylhydrazide-trifluoroacetate, that of 5.0 g of terto-butyloxycarbonyl-protected tripeptide with Removal of the tert-butyloxycarbonyl group using 8 mmol of trifluoroacetic acid has been prepared in a mixture of 24 ml of dimethylformamide and 2.24 ml of triethylamine. The reaction mixture is kept at 5 ° C. After 24 hours you add another Add part of the tert-butyloxycarbonyl-D-histidine azide, which has been prepared from 1.07 g of the corresponding hydrazide. The reaction mixture is concentrated 24 hours after this addition under reduced pressure and the residue crystallizes twice from 50 # aqueous ethanol. Yield: 4.4 g (70 # of the theory). .

00 98A1 / 1980

Claims (9)

- ²⁴ - 20U800 Claims IJ Peptide of the general formula R-His-R-Ser-Asp-Gly-Thr-R-Phe-Thr-Ser-Glu-Leu-Ser-R-Arg-Leu-R-Arg-Asp-Ser-Ala-R -Arg-Leu-Glu (NH ₂ ) -R-Arg-Leu-Leu-GIu (NH ₂ ) -Gly-Leu-VaI-NH ₂ and their pharmaceutically acceptable salts, where R has the meaning D or L and at least one RD is ₀ 2. Peptide of the general formula ρ (protected) -R-His-O- (protected) -R-Ser-ß- (protected) -L-Asp-Gly-I-Thr-R-Phe-L-Thr-O- (protected) -L -Ser - / "- (protected) -L-Glu-L-Leu-O-tprotectedJ-L-Ser - ^ - tprotectedJ-R-Arg-L- Leu- (J - (protected) -R-Arg-ß - (protected) -L-Asp-O- (protected) -L-Ser-L-Ala-6J- (protected) -R-Arg-L-Leu-L-Glu (NH ₂ ) -u? - (protected ) -R-Arg-L-Leu-L-Leu-L-Glu (NH ₂ ) -Gly-L-Leu-L-VaI-NH ₂ with the meanings given for R in claim 1. 3. Peptide of the general formula U) - (protected) -R-Arg-ß- (protected) -L-Asp-O- (protected) -L- P Ser-L-Ala-id- (protected) -R-Arg-L-LeU-L-GIu (NH ₂ ) -α) - (protected) -R-Arg-L-Leu-L-Leu-L- Glu (NH ₂ ) -Gly-L-Leu-L-VaI-NH ₂ with the meanings given for R in claim 1. 4. Peptide of the general formula R-Arg-L-Asp-L-Ser-L-AIa-R-Arg-L-Leu-L-Glu (NH ₂ ) -R-Arg-L-Leu-L-LeU-L-GIu (NH ₂ ) -Gly-L-Leu-L-VaI-NH ₂ and their pharmaceutically acceptable salts with the meanings given for R in claim 1. Q09841 / 1980 5, peptide of the general formula ^ - (protected) -L-Glu-L-Leu-O- (protected) -L-Ser-4? - (protected) -R-Arg-L-Leu-6tJ '- (protected) - R-Arg-ß- (protected) -L-Asp-O- (protected) -L-Ser-L-Ala-iJ - (protected) -R-Arg-L-Leu- L-GIu (NH ₂ ) - u) - (protected) -R-Arg-L-Leu-L-Leu-L-Glu (NH ₂ ) -Gly-. 1-LeU-L-VaI-NH ₂ with the meanings given for R in claim 1. 6. Peptide of the general formula L-Glu-L-Leu-L-Ser-R-Arg-L-Leu-R-Arg-L-Äsp-L-Ser-L-Ala-R-Argr-L-Leu-L-Glu (NH ₂ ) -R-Arg-L-Leu-L-Leu-L-Glu (NH ₂ ) -GIy-L-LeU-L-VaI-NH ₂ and its pharmaceutically acceptable salts with the meanings given for R in claim 1, 7 · Peptide of the general formula L-Thr-R-Phe-L-Thr-O- (protected) -L-Ser- / '- (protected) -L- GIu-L-LeU-O- (protected) -L-Ser- cü - ( protected) -R-Arg-L-Leu-iJ (protected) -R-Arg-ß- (protected) -L-Asp-O- (protected) -L-Ser-L-AIa-i «i- (protected ) -R-Arg-L-Le U-Lr-GIu (NH ₂ ) -iJ- (protected JR-Arg-L-Leu-rL-Leu-L-Glu (NHg) -GIy-L-LeU-L- VaI-NH ₂ with the meanings given for R in claim 1, 8. Peptide of the general formula I-Thr-R-Phe-L-Thr-L-Ser-L-Glu-L-Leu-L-Ser-R-Arg-L-Leu-R-Arg-L-Asp-L-Ser-L- Ala-R-Arg-L-LeU-L-GIu (NH ₂ ) -R-Arg-L-Leu-L-Leu- L-Glu ( NH ₂ ) -Gly-L-Leu-L-VaI-NH ₂ and their pharmaceutically acceptable salts with those for R in Claim 1 given meanings. , 009841/138 9. Process for the preparation of the D-analogs of secretin according to claims 1 to 8, characterized in that the D-forms of histidine, serine · «· phenylalanine or arginine with amino acids to form the desired D-analogs of secretin of the general formula R-His-R-Ser-Asp-Gly-Thr-R-Phe-Thr-Ser-Glu-Leu-Ser-R-Arg-Leu-1 2 3 4 5 6 7 8 9 IO 11 12 13 R-Arg-Asp-Ser-Ala-R-Arg-Leu-Glu (NH ₂ ) -R-Arg-Leu-Leu-Glu (NH ₂ ) 14 15 16 17 18 19 20 21 22 23 24 GIy-LeU-VaI-NH ₂ 25 26 27 in which R has the meaning of D or L and at least one R is D, can react. 1Oo method according to claim 9i, characterized in that Glu-Ieu-Ser-R-Arg-Leu with R-Arg-Asp-Ser-Ala-R-Arg-Leu-Glu (NH ₂ ) -R-Arg-Leu Leu-Glu (NH ₂ ) -GIy-LeU-VaI-NH ₂ converts, the resulting product can react with Thr-R-Phe-Thr-Ser and finally with His-R-Ser-Asp-Gly, with at least one R the reactant has the meaning of D. 0 0 9 8 '1! 080 ORIGINAL INSPECTED