CH615904A5 - Process for the preparation of L-leucine-13-motilin - Google Patents

Description

The invention relates to an improvement and further development of the main patent, from which a process for the production of L-norleucine-13-motiline is known, which differs from the naturally occurring motiline in that it is the 13th of a total of 22 amino acids that build up the polypeptide chain instead of an L-methionine residue of the formula

-nh-ch-co-

»

ch_

1 2

ch0

I 1

s an L-norleucine residue of the formula

-nh-ch-co-Ì

ch0 I 2

ch "

I 2

ch_

I 2 ch3

contains. The synthetic motilin derivative has practically the same biological activity as the natural motilin (cf. JC Brown in Demling et al. “Die Oberbaucheinheit - The Upper Gastro-Intestinal Tract, a Functional Entity”, Schattauer Verlag, Stuttgart — New York, 1974, pages 11-13).

It has now been found that correspondingly advantageous results can be achieved if, in the 13-position of the motiline, an L-leucine residue of the formula instead of the L-norleucine residue

-NH-GH-CO-

I.

CH «

I *

CH-CH,

I 3

is installed. Compared to the well-known norleucine motilin, leucine motilin has the advantage that it can be produced at a lower cost with the same good biological activity, because L-leucine is cheaper than L-norleucine, and that it is particularly easily broken down in the body because L-leucine is a naturally occurring amino acid that plays a key role in protein building.

As is known, motilin is a polypeptide which stimulates motor activity in both the antral and fundus gland areas of the stomach and has been isolated from the duodenal mucosa of the small intestine of pigs (cf., for example, «Can. J. Physiol. Pharmacol. ", Vol. 49, 1971, pages 399-405," Gastroenterology ", Vol. 62, 1972, pages 401 to 404 and" Can. J. Biochem. ", Vol. 51, 1973, pages 533 to 537) , which stimulates pepsin release without changing the ^ secretion, and is chemically and physiologically different from the gastrointestinal hormones that have now been elucidated. The Motilin is therefore of particular importance both as a therapeutic and as a diagnostic, since with its help z. B. a controlled release of pepsin, for example with the aim of determining or isolating pepsin or regulating the level of pepsin, and targeted antibody formation can be effected.

The original structure of motilin revealed the amino acid sequence -Met-Glu-Glu- in the 13-14-15 position, which was considered to be correct on the priority date on which the main patent is based. In the meantime, a correction of this amino acid sequence has been reported, according to which glutamine should be present in the 14-position instead of glutamic acid, so that the above partial sequence should be called Met-Gln-Gluhe (see “Can. J. Biochem.” Vol. 52, 1974, Pages 7-8). Accordingly, the motiline derivative known from the main patent with 13-L-norleucine-14-desamido-motilin and the now claimed motilin derivative with 13-L-leucine-14-desamido-motilin would be designated. However, as long as it is not certain that further corrections to the amino acid sequence are superfluous, the previous names L-norleucine-13-motiline and L-leucine-13-motiline for the motiline derivatives having a glutamic acid residue in the 14-position will still be used

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where it has been shown that the amide group in the side chain of 14-glutamic acid has no influence on the biological activity (see, for example, the right column on page 8 of the above-mentioned publication "Can. J. Biochem.", Vol. 52, 1974).

According to the main patent, the problem with all natural products that isolation from animal organs, for example, is incredibly time-consuming and costly and can only be achieved in minimal yields, was achieved by carrying out a total synthesis of motiline, replacing the methionine residue in the 13-position with a norleucine residue, based on the consideration that, according to the current state of peptide synthesis, the medium-sized arginyl-methionyl bond (amino acids 12 and 13 of motiline) is practically hardly synthesizable, but on the other hand the norleucine-13 docosapeptide corresponding to motilin is easier to synthesize is accessible, a comparatively quick structure determination is guaranteed and is able to provide important information about the biological effects specificity of methionine.

After purification, L-nor-leucine-13-motiline, which is fully synthetically produced in high yields, has practically the same activity as naturally occurring motilin, which is an indication that the sequence position of methionine can also be taken by norleucine. This finding was surprising in that the replacement of methionine with norleucine in naturally occurring human gastrin I leads to a synthesis product which has only about 10% gastrin activity.

According to the present invention, a further improvement is achieved in that an equally advantageous motilin derivative is made available as in the main patent, but which is less expensive to produce and more readily biodegradable.

The invention relates to a process for the preparation of L-leucine-13-motiline according to main patent no. 602 598, characterized in that a peptide defined in the main patent is produced, in which a leucine is used in the 13-position instead of norleucine. The L-leucine-13-motiline has the short formula

H-Phe-Val-Pro-Ile-Phe-Thr — Tyr-Gly-Glu-Leu-GIn-Arg-

(1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12)

Leu-Glu-Glu-Lys-Glu-Arg — Asn-Lys-Gly-Gln-OH (13) (14) (15) (16) (17) (18) (19) (20) (21) (22 )

in which the numbers given in brackets indicate the position of the amino acid residues numbered from 1 to 22 (apart from glycine, all amino acids in the L configuration) in the peptide chain and in which the name of the amino acid residues is abbreviated in the usual known manner.

The process is characterized in that the partial sequences

Fragment I, consisting of arginyl (hydrobromide) asparaginyl-Ne-tert-butyloxy-carbonyl-lysyl-glycyl-glutamine-tert-butyl ester (amino acids 18-22),

Fragment II, consisting of Na-benzyloxycarbonyl-glutamyl- (y-tert-butyl ester) -glutamyl (y-tert-butyl ester) -Ne-tert-butyloxycarbonyl-lysyl-glutamic acid-y-tert-butyl ester ( Amino acids 14-17),

Fragment III, consisting of Na-benzyloxycarbonyl-Nó, Nw -di-benzyloxycarbonyl-arginyl-leucine (amino acids 12-13),

Fragment IV, consisting of Nß-benzyloxycarbonyl-glutamyl- (y-tert-butyl ester) -leucyl-glutamine (amino acids 9-11),

Fragment V, consisting of O-tert-butyl-threonyl-O-tert-butyltyrosyl-glycine (amino acids 6-8) and

Fragment VI, consisting of Na-tert-butyloxycarbonyl-phenylalanyl-valyl-propyl-isoleucyl-phenylalanine (amino acids 1-5),

in which all side chain functions of the polyfunctional amino acids carry easily cleavable groups and the complex functions of argin are masked either by Nó, o »-diacylation or by protonation caused by salt formation with hydrogen bromide, condenses, cleaves the protecting groups and the crude L-leucine -13-Motilin isolated.

The L-leucine-13-motiline obtained is advantageously column-chromatographically analogously to the process of the main patent on a strongly basic anion exchange resin and then on a strongly acidic cation exchange resin.

This agent is usually used in such doses that about 0.05-0.5 y, corresponding to 50-500 ng of motilin derivative, is omitted per kg of body weight. Mainly physiological saline is used as a carrier. The application is intravenous, intramuscular or subcutaneous. So z. B. lyophilized Motilinderivat depending on the intended application in amounts of 1-100 ylml physiological saline, the z. B. can be stabilized by mannitol. For intravenous administration to a person weighing approximately 80 kg, 2 ml of solution for injection containing 8 y motilin derivative gives a dosage of 0.1 yl kg body weight. For infusion into a human of the specified weight, 8 y motilin derivative can be dissolved in 30 ml of physiological saline and 0.5 ml of this solution applied per minute, which is about 0.1 y motilin derivative / kg body weight / hour. corresponds. Comparable dosages have been found to be suitable in animal experiments. B. Naturally occurring Motilin in amounts of 0.05 ylkg body weight administered intravenously to dogs, which caused a strong increase in motor activity (see, for example, the specified publication "Can. J. Biochem.", 51 [1973], 533 , left column, paragraph 1).

For the production of leucine-13-motiline, the synthesis plan designed for norleucine-13-motiline could be used. The synthesis of fragments I, II, IV, V and VI was therefore carried out according to the method described in the main patent, as was the condensation of fragments I and II to the partial sequence (14-22b) and fragments V and VI to the partial sequence (1-8 ).

The fragment III (amino acids 12-13) with C-terminal leucine was synthesized and converted with the condensed peptide fraction I-II (14-22b) to the condensed peptide fraction I-II-III (12-22a), from which hydrogenol- The corresponding undecapeptide (12-22b) was isolated in the form of the hydrobromide after the benzyloxycarbonyl protective group had been split off. Further reaction with fragment IV (9-11) yielded the condensed peptide fraction I-II-III-IV (9-22a), which was deacylated by catalytic hydrogenolysis in the presence of hydrobromic acid to the hydrobromide of tetra-decapeptide (9-22b) and finally condensation was carried out with the condensed peptide fraction V-VI (1-8) to the completely protected process product I-II-III-IV-V-VI (1-22a), from which the protective groups were removed by means of trifluoroacetic acid and subsequent ion exchange treatment and lyophilization desired leucine-13-motilin (1-22b) was obtained as a crude product.

The invention is explained in more detail by the accompanying drawing, in which:

1 shows the synthesis scheme for the production of fragment I,

2 shows the synthesis scheme for the production of fragment II,

3 shows the synthesis scheme for the production of fragment VI,

Fig. 4 shows the synthesis scheme for the production of fragment III and for linking fragments I + II + III and

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Fig. 5 shows the synthesis scheme for the preparation of fragment IV and for linking fragments IV with (I-III), VI with V and (I-IV) with (V-VI).

The following examples are intended to explain the invention in more detail. The following abbreviations and symbols, which are common in peptide chemistry, are used in the figures and examples:

Amino acid abbreviations made up of 3 letters, which are usually the 3 first letters,

Numbers in brackets which represent the position within the polypeptide chain and which do not receive any further addition if the compound in question does not form another derivative, but which are continuously provided with the addition a, b, c etc., if further derivatives starting from a getting produced,

L as an indication of the configuration,

Z for the benzyloxycarbonyl radical (cf. e.g.

Bergmann et al. in «Reports d. German Chem. Ges. » Vol. 65, 1932, 1192ff.), BOC for the tert-butyloxycarbonyl radical (cf. e.g.

McKay et al. in «J. At the. Chem. Soc. » 79, 1957, p. 4686 and Anderson et al. in «J. At the. Chem. Soc. » 79.1957, p. 6180),

DCCD for the N, N'-dicyclohexylcarbodiimide process

drive (cf. e.g. Sheehan et al. in "J. Am. Chem. Soc." Vol. 77, 1955, p. 1067), DCCD / HOSU for the N, N'-dicyclohexylcarbodiimide-N-Hy- droxysuccinimide method (cf., for example, Wünsch et al. in "Reports of the German Chem. Ges." Vol. 99, 1966, p. 110 and Weygand et al. in "Zeitschr. f. Naturforschg." Vol. 216, 1966, p. 426 and Koenig et al. In “Reports of Dtsch. Chem. Ges.” Vol. 103, 1970, p. 788), DCCD / HOBt for the N, N-dicyclohexylcarbodiimide-N-Hy- droxybenzotriazole process (see, for example, Koenig et al. in "Ber. d. Dtsch. Chem. Ges." "Vol. 103, 1970, p. 788),

PAM for the phosphorazo method (see e.g.

Goldschmidt in «Angew. Chem. » Vol. 62, 1950, p. 538 and Goldschmidt et al. in "Liebigs Ann." 580, 1953, p. 68), SU for the N-hydroxysuccinimide residue,

NP for the p-nitrophenyl radical,

tBu for the tert-butyl radical,

DBSI for dibenzene sulfimide as salt former,

Me for the methyl residue,

TFE for trifluoroacetic acid,

i.V. for «in vacuum»,

Educ. for yield,

i.e. the (according to yield data in%) for «der

Theory",

F for «floating point» and

(Z) (after the pour point) for «under

Decomposition".

The production of the fragments used for the synthesis is first described below.

Preparation of fragment I (partial sequence 18-22)

According to the main patent, H-Gln-OtBu (22b), which had been prepared from Z-Gln-OH (22a) by esterification with tert-butyl acetate with sulfuric acid catalysis and subsequent hydrogenolytic removal of the benzyloxycarbonyl protective group, was also used Z-Gly-OSU (21) linked to the benzyloxycarbonyl dipeptide ester (21-22a); the compound H-Gly-Gln-OtBu (21-22b) obtained after hydrogenolytic removal of the N-protecting group was also used

Z-Asn-Lys (BOC) -OH (19-20) according to the N, N'-carbodiimide-N-hydroxysuccinimide method described by Wünsch and Weygand in the cited references or according to that described by Koenig et al. modifications to the benzyloxycarbonyl-tetrapeptide-tert-butyl ester (19-22a) described in the cited literature reference are linked. The head component (19-20) used was obtained by aminoacylation of H-Lys (BOC) -OH (20) with Z-Asn-ONP (19) under customary known conditions.

The benzyloxycarbonyl protecting group was removed from Z-Asn-Lys (BOC) -Gly-Gln-OtBu (19-22a) by catalytic hydrogenation to give the tetrapeptide ester derivative (19-22b), which was treated with Z-Arg (ó , co-Z2) -ONP (18) to form Z-Arg- (ó, w-Z2) -Asn-Lys (BOC) -Gly-Gln-OtBu (18-22a).

The experimental procedure was as follows:

A. L-glutamine-tert-butyl ester-dibenzene sulfimide salt 103 g of benzyloxycarbonyl-glutamine-tert-butyl ester (obtained by the method described by Schnabel et al. In "Liebigs Ann.", Vol. 686, 1965, p. 229) in 2 l of methanol were hydrogenated catalytically (palladium black) with the dropwise addition of 91 g of dibenzenesulfimide in 500 ml of methanol at pH = 3.5.

The filtrate was i. V. evaporated, finally with azeotropic distillation with benzene; crystallization from the ethyl acetate solution of the oily residue occurred upon addition with diethyl ether. Crystals of F = 135-136 ° resulted from methanol / diethyl ether; [a] D20 = + 10.78 ° (c = 1.0; in methanol). Educ. = 146 g (97% of theory).

B. Benzyloxycarbonyl-glycyl-L-glutamine tert-butyl ester 115 g of L-glutamine tert-butyl ester-dibenzenesulfimide salt suspended in 800 ml of dichloromethane were treated with 32.2 ml of triethylamine and then with 70.5 g of benzyloxycarbonyl-glycine -N-hydroxysuccinimide ester added with stirring. Stirring at room temperature for 24 hours, evaporating down in vacuo, taking up the oil in ethyl acetate, washing this solution as usual with citric acid, potassium hydrogen carbonate solution and water, drying over sodium sulfate and evaporating down in vacuo. resulted in an oil. Educ. = 87 g (93% of theory).

C. Glycyl-L-glutamine-tert.-butyl ester-dibenzenesulfimide salt 80 g of benzyloxycarbonyl-glycyl-L-glutamine-tert.-butyl ester (according to B.) in 900 ml of methanol were deacylated by hydrogenolysis as described under A (60 g Dibenzenesulfimide) and worked up. The result was an oily residue which crystallized on trituration with petroleum ether; F = 92 ° (Z); [a] D20 = -8.5 ° (c = 1.0; in methanol). Educ. = 110 g (98% of theory).

D. Benzyloxycarbonyl-L-asparaginyl-Nf-tert-butyloxycarbonyl-L-lysine 51 g of Ne-tert-butyloxycarbonyl-L-lysine were converted into the benzyltriethylammonium salt in a conventional manner and then in 700 ml of dimethylformamide with 81 g of benzyloxycarbonyl -L-asparagin-4-nitrophenyl ester with the addition of 1 equiv. Pyridine stirred at 20 ° for 48 hours. The vacuum evaporation residue was treated with ethyl acetate and potassium hydrogen sulfate solution at the same time; the thick precipitate formed was filtered off and then recrystallized from ethanol / petroleum ether. F = 174-175 ° (Z); [a] D20 = + 13.8 ° (c = 1.0; in pyridine). Educ. = 70 g (71% of theory).

E. Benzyloxycarbonyl-L-asparaginyl-Ne-tert-butyloxy-

carbonyl-L-lysyl-glycyl-L-glutamine tert-butyl ester 61.5 g glycyl-L-glutamine tert-butyl ester dibenzenesulfimide salt and 54.5 g benzyloxycarbonyl-L-asparaginyl-Ne-tert.-

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Butyloxycarbonyl-L-lysine in 700 ml of dimethylformamide was initially mixed with stirring at 0 ° C. with 15.5 ml of triethylamine and after 15 minutes with 13 g of N-hydroxysuccinimide and 23 g of N, N'-dicyclohexylcarbodiimide. After 3 hours, the mixture was stirred at room temperature for a further 24 hours. The filtrate from the N, N'-di-cyclohexylurea was i.V. evaporated; the residue crystallized when treated with ethyl acetate. The product was recrystallized from methanol / water and isopropanol / ethyl acetate. F = 155-156 °; [a] D20 = -20.8 ° (c = 1.0; in ethanol).

Educ. = 63 g (78% of theory).

F. L-asparaginyl-Ne-tert-butyloxycarbonyl-L-lysyl-glycyl-L-glutamine-tert-butyl ester hydrochloride 47.5 g benzyloxycarbonyl-L-asparaginyl-Ne-tert.-butyl-oxycarbonyl-L- Lysyl-glycyl-L-glutamine tert-butyl ester (according to E) in 800 ml of methanol was hydrogenated as usual while keeping the pH constant (pH = 5.5; 25.2 ml of 2.5 N-chlorine hydrogen solution in methanol). The I. V. evaporated filtrate gave an oil; colorless powder was formed after trituration with diethyl ether. F = 98 °, [a] D20 = + 9.5 ° (c = 1.0 in methanol). Educ. = 40 g (97% of theory).

G. Na, Nó, Ncj-Tris-benzyloxycarbonyl-L-arginyl-L-asparaginyl-Nf-tert-butyloxycarbonyl-L-lysyl-glycyl-

L-glutamine tert-butyl ester 31.9 g L-asparaginyl-Nf -tert. -butyloxycarbonyl-L-lysyl-glycyl-L-glutamine tert.-butyl ester hydrochloride (according to F) and 33.68 g Na, Nó, Nw-tris-benzyloxycarbonyl-L-arginine-N-hy-hydroxysuccinimide ester in 11 Dimethylformamide was mixed with 0 ml of triethylamine at 0 ° C; after stirring for 24 hours at room temperature, evaporating i. V., reprecipitation of the residue from methanol / water and then recrystallization from methanol resulted in a colorless powder; [cc] d20 = -7.6 ° (c = 1.0; in glacial acetic acid). Educ. = 46.5 g (80% of theory).

H. L-arginyl (hydrobromide) -L-asparaginyl-Ne -tert. -butyloxy-carbonyl-L-lysyl-glycyl-L-glutamine-tert.-butyl ester-hydrobromide dihydrate 45.5 g Na, Nó, Nai-Tris-benzyloxycarbonyl-L-arginyl-L-asparaginyl-Ne-tert.- butyloxycarbonyl-L-lysyl-glycyl-L-glutamine-tert.-butyl ester (in accordance with G) in 1.5 l of dimethylformamide / methanol (2: 1) were hydrogenolytically deacylated while maintaining the pH in a customary known manner ( pH = 1.5; 80 ml of 1 N-hydrobromic acid). Evaporation of the filtrate i. V. and reprecipitation of the residue from ethanol / diethyl ether resulted in an amorphous powder: [a] D20 = —4.8 ° (c = 1.0; in 80% acetic acid). Educ. = 37 g (98% of theory).

Yield 55% over all stages, based on H-Gln-OtBu (22b) as starting material;

[«1d20 = -7.6 ± 1 ° or [a] S4620 = -9.4 ° (c = 1; in acetic acid);

chromatographically pure in n-butanol / glacial acetic acid / water 3: 1: 1 and n-heptane / tert-butanol / glacial acetic acid 5: 1: 1;

Analysis:

calc .: C 57.98 H 6.69 N 13.29 O 22.02 found: C 57.70 H 6.56 N 13.29 O 22.45 by subsequent removal of the three benzyloxy-carbonyl masks The desired fragment I, namely H-Arg (HBr) -Asn-Lys (BOC) -Gly-Gln-OtBu-HBr (18-22b), was obtained by catalytic hydrogenolysis with the addition of two equivalents of hydrogen bromide.

Yield: 98%; [a] D20 = —4.8 ± 1 ° or [a] 54620 = -6.05 ° (c = 1; in 80% acetic acid);

chromatographically pure in n-butanol / glacial acetic acid / water 3: 1: 1 and tert-amyl alcohol / pyridine / water 35:35:30; Analysis:

calc .: C 40.21 H 6.85 N 16.12 Br 1672 found: C 40.46 H 6.93 N 15.89 Br 16.51

Preparation of Fragment II (Partial Sequence 14-17) According to the main patent, Z-Lys (BOC) -OSU (16) and H-Glu (OtBu) -OMe (17) were combined without difficulty to give the benzyloxycarbonyl dipeptide ester (16-17a) . The subsequent alkaline ester saponification and subsequent catalytic deacylation led to H-Lys (BOC) -Glu (OtBu) -OH (16-17c) via the dipeptide derivative (16-17b). This compound was reacted twice in succession with Z-Glu (OtBu) -OSU (15 and 14), the glutamic acid derivative being used as the top component in each case. In this way, fragment II, namely Z-Glu (OtBu) -Glu- (OtBu) -Lys (BOC) -Glu (OtBu) -OH (14-17a) was obtained. • The experimental procedure was carried out in an analogous manner to that described for the preparation of fragment I.

Yield: 48% over all stages, based on H-Glu (OtBu) -OMe (17); F = 147-149 °;

Md20 = -9.8 ± 1 ° or [«W20 = 12.3 ° (c = 1; in dimethylformamide);

chromatographically pure in cyclohexane / chloroform / glacial acetic acid 45:45:10;

Analysis:

calc .: C 59.02 H 7.86 N 7.48 found: C 58.70 H 8.04 N 7.72

Preparation of fragment III (partial sequence 12-13) 30 g of Z-Arg (ó, w-Z2) -OSU (12) and. Were used to prepare N «, Nó, Nai-Tris-benzyloxycarbonyl-L-arginyl-L-leucine 11.6 g leucine (13) in dioxane / water (1000: 500 ml)

after adding 89 ml of N-sodium hydroxide solution with stirring for 18 hours. After addition of 89 ml of N-hydrochloric acid, the reaction mixture was taken up in ethyl acetate, the solution obtained was washed with dilute hydrochloric acid and water, dried over sodium sulfate and the solvent i.V. away. The residue was reprecipitated from water / ethanol and ethyl acetate / diethyl ether / petroleum ether.

Yield: 26.6 g (86.6% of theory); F = 126-128 °; [a] D20 = - 5.8 ± 1 ° or [a] s4620 = - 7.1 °. (c = 1; in acetic acid);

chromatographically pure in n-heptane / tert-butanol / acetic acid (5: 1: 1);

Analysis:

calc .: C 62.69 H 6.28 N 10.15 found: C 62.59 H 6.41 N 9.94 amino acid analysis: Arg 1.00 Leu 1.00.

Production of Fragment IV (Partial Sequence 9-11) According to the main patent, this was carried out according to the method described in «Reports d. German Chem. Ges. », Vol. 104, 1971, p. 2430, synthesized the dipeptide H-Leu-Gln-OH synthesized as a sequence section (10-11) and used with Z-Glu (OtBu) -OSU (9) in 74% yield to fragment IV, namely Z-Glu- (OtBu) -Leu-Gln-OH (9-11), implemented.

The experimental procedure was carried out in an analogous manner to that described for the preparation of fragment I.

F = 146-148 °; [a] D20 = -31.6 ± 1 ° or [al54620 = -38.1 ° (c = Ì; in methanol);

chromatographically pure in n-heptane / tert-butanol / glacial acetic acid 5: 1: 1 and n-butanol / glacial acetic acid / water 3: 1: 1;

Analysis:

calc .: C 58.12 H 7.32 N 9.68 found: C 58.05 H 7.24 N 9.48

Production of fragment V (partial sequence 6-8)

(according to main patent)

That after the in «Zeitschr. f. Physiol. Chem. », Vol. 353, 1972, page 1246 described methods synthesized dipeptide H-Tyr (tBu) -Gly-OH was used as a sequence section (7-8). Linking H-Tyr (tBu) -Gly-OH (7-8) with

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Z-Thr (tBu) -OSU (6) gave the benzyloxycarbonyl tripep-tid (6-8a), which, by catalytic hydrogenolysis, gave the desired fragment V, namely H-Thr (tBu) -Tyr (tBu) -Gly-OH ( 6-8b) resulted.

The experimental procedure was carried out in an analogous manner to that described for the preparation of fragment I.

Yield: approx. 81% over both stages, based on the dipeptide used (7-8); F = 126-127 °;

[«1d20 = +7.9 ± 1 ° or [a] s4620 = + 8.9 ° (c = 1; in methanol);

chromatographically pure in tert-amyl alcohol / pyridine / water 35:35:30;

Analysis:

calc .: C 60.88 H 8.30 N 8.88

Found: C 60.69 H 8.31 N 8.91 based on a dipeptide with V4 mol crystal ethyl acetate.

Preparation of fragment VI (partial sequence 1-5)

(according to main patent)

Z-Ile-OH (4) was linked to H-Phe-OtBu (5) by the carbodiimide method given. Subsequent catalytic hydrogenolysis of the intermediate benzyloxycarbonyl dipeptide ester (4-5a) enabled H-IIe-Phe-OtBu (4-5b) to be isolated in over 90% yield.

At the same time, BOC-Val-OH (2a) and H-Pro-OMe (3a) were combined to the tert-butyloxycarbonyl dipeptide ester (2-3a) according to the specified phosphorazo method; Subsequent alkaline ester hydrolysis gave BOC-Val-Pro-OH (2-3b) in over 70% yield. The preparation of (2-3b) was easier and in high yield (83%) by reacting BOC-Val-OSU (2b) with two equivalents of proline (3b).

Both dipeptide derivatives are linked using the carbodiimide method given to form the BOC-Val-Pro-Ile-Phe-OtBu, (2-5a); Trifluoroacetic acid exposure to (2-5a) led to the free tetrapeptide H-Val-Pro-Ue-Phe-OH (2-5b), on which BOC-Phe-OSU (1) in the usual known manner leads to BOC-Phe-Val -Pro-Ile-Phe-OH (1-5a) (fragment VI) was grown.

The experimental procedure was carried out in an analogous manner to that described for the preparation of fragment I.

Yield: 63% over the last three stages based on (4-5b); F = 218 °;

[«1d20 = 64.2 ± 1 ° or [als4620 = -75.82 ° (c = 1; in acetic acid);

chromatographically pure in n-butanol / glacial acetic acid / water 3: 1: 1 and tert-amyl alcohol / pyridine / water 35:35:30; Analysis:

calc .: C 64.88 H 7.64 N 9.70

Found: C 64.72 H 7.70 N 9.61

example 1

Condensation of fragments I to VI (total sequence 1-22)

With the carboxyl-terminated fragment I (18-22b), fragment II (14-17a) was combined by condensation with the aid of the indicated dicyclohexylcarbodiimide-N-hydroxysuccinimide method and the N-benzyloxycarbonyl-nonapeptide tert-butyl ester obtained (14-22a) was obtained by catalytic hydrogenolysis in H-Glu (OtBu) -Glu (OtBu) -Lys (BOC) -Glu (OtBu) -Arg (HBr) -Asn-Lys (BOC) -Gly-Gln-OtBu ( 14-22b).

The combination with fragment III again with the aid of the mentioned method led to the formation of Z-Arg (<3, &> - Z2) -Leu-Glu (OtBu) -Glu (OtBu) -Lys (BOC) -Glu (OtBu) - Arg- (HBr) -Asn-Lys (BOC) -Gly-Gln-OtBu (12-22a).

The three benzyloxycarbonyl protective groups were removed from the unde-capeptide (12-22a) by catalytic hydrogenation and by neutralization of the released guanido

Function with hydrobromic acid during the hydrogenation resulted in H-Arg (HBr) -Leu-Glu (OtBu) -Blu (OtBu) -Lys- (BOC) -Glu (OtBu) -Arg (HBr) -Asn-Lys (BOC) -Gly -Gln-OtBu (12-22b) in the form of the hydrobromide.

The undecapeptide ester obtained was combined with fragment IV (9-11) according to the specified dicyclohexylcarbodiimide-N-hydroxybenzotriazole method by condensation, and the tetradecapeptide derivative (9-22a) obtained after hydrogenolytic cleavage of the benzyloxycarbonyl protective group H- Glu (OtBu) -Leu-Gln-Arg (HBr) -Leu-Glu (OtBu) -Glu (OtBu) -Lys (BOC) -Glu (OtBu) -Arg- (HBr) -Asn-Lys (BOC) -Gly -Gln-OtBu (9-22b) obtained.

During the course of the above reactions, fragments V (6-8a) and VI (1-5a), the latter after being converted into the (N-hydroxysuccinimide) ester (1-5b), were combined. However, BOC-Phe-Val-Pro-Ue-Phe-Thr (tBu) -Tyr (tBu) -Gly-OH (1-8) could not be obtained in pure form, and amino acid analysis showed an impurity of about 10% ( 1-5a) or (1-5b). Attempts to separate this mixture have been unsuccessful.

The obtained "raw" section (1-8) was again with the above section (9-22b) according to the specified dicyclohexylcarbodiimide-N-hydroxysuccinimide method for BOC-Phe-Val-Pro-Ile-Phe-Thr (tBu) - Tyr (tBu) -Gly-Glu (OtBu) -Leu-Gln-Arg (HBr) -Leu-Glu (OtBu) -Glu (OtBu); Lys (BOC) -Glu (OtBu) -Arg (HBr) -Asn-Lys (BOC) -Gly-Gln-OtBu (1-22a).

After cleavage of all protective groups using anhydrous trifluoroacetic acid and subsequent exchange of the trifluoroacetate and bromide ions by ion exchange chromatography on a weakly basic ion exchange resin (acetate form) known as “Dowex 44”, a “crude leucine-13-motiline was obtained »(1—22b), which, due to the use of« impure »section (1-8), had to have a« wrong sequence », which was also confirmed during the cleaning operation.

The experimental procedure for fragment condensation was as follows:

A. Condensation I with II

According to the main patent, 9.2 g of fragment I (according to example 1 H), 9.36 g of fragment II (according to example 2) and 1.4 ml of triethylamine in 200 ml of dimethylformamide were mixed at 2.3 ° C. with 2.3 g N-hydroxysuccinimide and then 3.1 g of N, N'-dicyclo-hexylcarbodiimide; the reaction mixture was stirred at 5 ° C. for 24 hours and at room temperature for 4 days, whereupon the filtrate i. V. was evaporated. The residue was digested several times with water and finally reprecipitated twice from methanol / ethyl acetate.

The result was benzyloxycarbonyl-L-glutamyl (y-tert-butyl ester) -L-glutamyl (y-tert-butyl ester) -Nf -tert. -butyloxycarbo-nyl-L-lysyl-L-glutamyl (y-tert-butyl ester) -L-arginyl (hydro-bromide) -L-asparaginyl-Ne-tert-butyloxycarbonyl-L-lysyl-glycyl-glutamine-tert .-Butyl ester in the form of an amorphous powder.

Md2 ° = -14.6 ° (c = 1; in dimethylformamide).

Educ. 14.8 g (84% of theory).

12.3 g of the compound obtained in 800 ml of methanol were catalytically hydrogenated (pH = 5.5; 7 ml of 1N-bromo-hydrochloric acid) while keeping the pH constant in the usual manner. The filtrate was i. V. evaporated, the residue from ethanol / ethyl acetate reprecipitated twice.

The result was L-glutamyl (y-tert-butyl ester) -L-glutamyl- (y-tert-butyl ester) -N £ -tert.-butyloxycarbonyl-L-lysyl-L-glut-amyl (y-tert.- butyl ester) -L-arginyl (hydrobromide) -L-asparaginyl-Ne-tert-butyloxycarbonyl-L-lysyl-glycyl-glutamine-tert-butyl ester hydrobromide as the desired condensation product (14-22b).

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[a] D20 = -21.5 ° (c = 1.0; in methanol).

Educ. 11.4 g (96% of theory).

B. Condensation (I-II) with III A solution of 1.7 g peptide (14-22b), 1.4 g Z-Arg- (ó, cu-Z2) -Leu-OH (12-13), 0 , 3 g of N-hydroxysuccinimide and 0.14 ml of triethylamine in 100 ml of dimethylformamide were mixed with 0.412 g of N, N'-dicyclohexylcarbodiimide at -10 ° and stirred at 4 ° for 24 hours. After stirring for a further 24 hours at room temperature, the precipitated dicyclo-hexylurea was filtered off, the solvent i.V. removed and the evaporation residue from methanol / ethyl acetate fell over. The product obtained was carefully digested with water, filtered off and dried.

The result was Na, Nó, Nw-tris-benzyloxycarbonyl-L-arginyl-L-leucyl-L-glutamyl (y-tert-butyl ester) -L-glutamyl (y-tert-butyl ester) -Nf-tert-butyloxycarbonyl -L-Iysyl-L-glutamyl (y-tert-butyl ester) -L-arginyl (hydrobromide) -L-asparaginyl-Nc-tert-butyloxycarbonyl-L-lysyl-glycyl-L-glutamine tert-butyl ester (12-22a).

Educ. 1.94 g (85% of theory); chromatographically pure in n-heptane / tert-butanol / acetic acid (3: 2: 1) and n-butanol / acetic acid / water (3: 1: 1).

Amino acid analysis:

Lys 2.02, Arg 2.01, Asp 1.00, Glu 3.98, Gly 1.01, Leu 1.00. 1.8 g of peptide in 500 ml of methanol were deacylated by dropwise addition of 15.70 ml of 0.1 N-hydrobromic acid at pH 4.5 by catalytic hydrogenation. The filtrate freed from the catalyst was i.V. evaporated, and the residue obtained was reprecipitated from methanol / ethyl acetate.

The result was L-arginyl (hydrobromide) -L-leucyl-L-glutamyl (y-tert-butyl ester) -L-glutamyl (y-tert-butyl ester) -Ne-tert-butyloxycarbonyl-L-lysyl- L-glutamyl (y-tert-butyl ester) -L-arginyl (hydrobromide) -L-asparaginyl-Ne-tert-butyloxy-carbonyl-L-lysyl-glycyl-L-glutamine-tert-butyl ester hydro-bromide dihydrate (12-22b).

Educ. 1.61 g (98% of theory).

Analysis:

calc .: C 47.12 H 7.43 N 14.07 found: C 47.20 H 7.50 N 14.00

C. Condensation (I-II-III) with IV 1.56 g peptide (12-22b) and 0.87 g peptide (9-11) in 100 ml dimethylformamide were added after adding 0.105 ml triethylamine with 0.210 g N-hydroxybenzotriazole and 0.320 g of dicyclohexylcarbodiimide condensed as described under (B).

The result was N-benzyloxycarbonyl-L-glutamyl (y-tert-butyl ester) -L-leucyl-L-glutaminyl-L-arginyl (hydrobromide) -L-leucyl-L-glutamyl (y-tert-butyl ester) -L -glutamyl (y-tert-butyl ester) -Nf-tert-butyloxycarbonyl-L-lysyl-L-glutamyl- (y-tert-butyl ester) -L-arginyl (hydrobromide) -L-asparaginyl-Ne-tert. -butyloxycarbonyl-L-lysyl-glycyl-L-glutamine tert-butyl ester dihydrate (9-22a).

Educ. 1.6 g (83% of theory).

Analysis:

calc .: C 51.41 H 7.57 N 13.63 Br 6.22 found: C 51.28 H 7.55 N 13.57 Br 6.15 1.6 g peptide (9-22a) in 600 ml of methanol was deacylated by catalytic hydrogenation while maintaining the pH as described under (B). The filtrate freed from the catalyst was i.V. evaporated, and the residue was reprecipitated from methanol / ethyl acetate.

The result was L-glutamyl (y-tert-butyl ester) -L-leucyl-L-glutaminyl-L-arginyl (hydrobromide) -L-leucyl-L-glutamyl- (y-tert-butyl ester) -L-glutamyl ( y-tert-butyl ester) -Nf-tert-butyloxycarbonyl-L-lysyl-L-glutamyl (y-tert-butyl ester) -L-arginyI (hydrobromide) -L-asparaginyl-N £ -tert.-butyloxy-

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carbonyl-L-lysyl-glycyl-L-glutamine tert-butyl ester hydro-bromide tetrahydrate (9-22b).

Educ. 1.5 g (96% of theory);

[alo20 = -5.3 ± 1 and [a] 54620 = -7.2 ° (c = 0.7; in methanol);

chromatographically pure in n-butanol / acetic acid / water (3: 1: 1).

Amino acid analysis:

Lys 2.04, Arg 2.01, Asp 0.98, Glu 6.05, Gly 1.01, Leu 2.00.

D. Condensation V with VI According to the main patent, 21.6 g of fragment VI and 6.9 g of N-hydroxysuccinimide in 400 ml of dimethylformamide were mixed with 6.3 g of N, N'-dicyclohexylcarbodiimide at -5 ° C. The reaction mixture was stirred at 0 ° C for 2 hours and at room temperature overnight. After filtration, the filtrate was i. V. evaporated; the oily residue crystallized from isopropanol.

The result was tert-butyloxycarbonyl-L-phenylalanyl-L-valyl-L-prolyl-L-isoleucyl-L-phenylalanine-N-hydroxysuccini mid ester.

Educ. 21.3 g (88% of theory);

F = 190-192 °; [a] D20 = + 59.28 ° (c = 1.0; in dioxane). 14.2 g of the above succinimide ester obtained from fragment VI were added to 12.2 g of fragment V and 3.8 ml of triethylamine in 400 ml of dimethylformamide; after stirring for 24 hours at room temperature, the reaction mixture was evaporated down i. V. evaporated, and the oily residue was partitioned between ethyl acetate and citric acid solution. The ethyl acetate phase was washed in a conventional manner, dried and i. V. brought to dryness. Colorless crystals of tert-butyloxycarbonyl-L-phenylalanyl-L-valyl-L-prolyl-L-isoleucyl-L-phenylalanyl-O-tert.-butyl-L-threonyl-O-tert were made from ethyl acetate / petroleum ether. -butyl-L-tyrosyl-glycine (1-8) obtained.

Educ. 18.2 g (87% of theory);

[ct] D20 = -44.0 ° (c = 1.0; in ethanol).

E. condensation (I-II-III-IV) with (V-VI) 1.28 g peptide (9-22b) according to (C), 1.17 g peptide (1-8) according to (D), 0, 07 ml of triethylamine and 0.115 g of N-hydroxysuccinimide (or in further experiments 0.20 g of N-hydroxybenzotriazole) in 100 ml of dimethylformamide were mixed with 0.206 g of N, N'-dicyclohexylcarbodiimide at -10 °. The reaction mixture was stirred at 0 ° C for 2 days and at room temperature for 3 days. After removing the solvent i. V. the residue was carefully digested with hot ethyl acetate, the resinous product from methanol / ethyl acetate was reprecipitated, dried after trituration with ethyl acetate and then treated exhaustively with water for 5 hours. It was reprecipitated from methanol / water.

The result was N-tert-butyloxycarbonyl-L-phenylalanyl-L-valyl-L-prolyl-L-isoleucyl-L-phenylalanyl-O-tert-butyl-L-threonyl-0-tert-butyl-L-tyrosyl -glycyl-L-glutamyl (y-tert, -butyl ester) -L-leucyl-L-glutaminyl-L-arginyl (hydrobromide) -L-leucyl-L-glutamyl (y-tert.-butyl ester) -L-glutamyl ( y-tert-butyl ester) -Nf-tert-butyloxycarbonyl-L-lysyl-L-glutamyl (y-tert-butyl ester) -L-arginyl (hydrobromide) -L-asparagmyl-N £ -tert.-butyloxycarbonyl- L-lysyl-glycyl-L-glutamine tert-butyl ester hexahydrate (1-22a).

Educ. 1.65 g (91% of theory).

Analysis:

calc .: C 54.03 H 7.82 N 12.68 Br 4.38 found: C 54.13 H 7.80 N 12.67 Br 4.6 1 g protected peptide (1-22a) according to (E ) was poured with 40 ml of ice-cold trifluoroacetic acid and left to stand at room temperature for 2 hours. Excess trifluoroacetic acid was then added at the lowest possible temperature7

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removed, the remaining material taken up in dilute acetic acid, the solution obtained was treated twice with 4 g of a weakly basic anion exchanger in the acetate form (for example “Dowex 44”), whereupon the exchanger eluate was lyophilized.

The result was L-phenylalanyl-L-valyl-L-prolyl-L-isoleucyl-L-phenylalanyl-L-threonyl-L-tyrosyl-glycyl-L-glutamyl-L-leucyl-L-glutaminyl-L-arginyl-L- Ieucyl-L-glutamyl-L-glutamyl-L-lysyl-L-glutamyl-L-arginyl-L-asparaginyl-L-lysyl-glycyl-L-glutamine.

Educ. 0.74 g.

Example 2

Pure representation and amino acid analysis According to the main patent, the cleaning was carried out based on the pure representation of natural Motilin by ion exchange chromatography using a strongly basic anion exchange resin, e.g. B. the present in the acetate form, known under the name "QAE-Sephadex A-25" resin based on a modified dextran with diethyl (2-hydroxypropyl) aminoethyl residues as functional groups, and then with the aid of a strongly acidic cation exchange resin, e.g. B. in the ammonium form, known under the name "SP-Sephadex C-25" resin based on a modified dextran with sulfopropyl radicals as functional groups.

An amino acid analysis was performed after acidic hydrolysis (6N-HCl), showing that practically the same values were obtained at 20 and 72 hours hydrolysis time. The results obtained are shown in the following table:

Table found calculated io Lys 2.00 2

Arg 1.93 2

Asp 1.00 1

Thr 0.97 1

Glu 6.18 6

15 per 0.98 1

Gly 1.99 2

Val 0.90 1

Ile 0.90 1

Leu 1.94 2

20 Tyr 0.93 1

Phe 1.82 2

The biological activity of the freeze-dried L-leucine-13-motiline was practically the same as that of the L-norleucine-25-13-motiline and thus hardly differed from that of the natural motiline.

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5 sheets of drawings

Claims (2)

615 904 2 CLAIMS Process for the preparation of L-leucine-13-motilin of the formula H-Phe-Val-Pro-Ile-Phe-Thr-Tyr-Gly-Glu-Leu-Gin-Arg - (1) (2) ( 3) (4) (5) (6) (7) (8) (9) (10) (11) (12) Leu-Glu-Glu-Lys-Glu-Arg-Asn-Lys-Gly-Gln-OH (13) (14) (15) (16) (17) (18) (19) (20) (21) (22), characterized in that the partial sequences fragment I, consisting of arginyl (hydrobromide) aspara -ginyl-Ne-tert-butyloxy-carbonyl-lysyl-glycyl-glutamine-tert.-butyl ester (amino acids 18-22), fragment II, consisting of Na-benzyloxycarbonyl-glut-amyl- (y-tert.-butyl ester) -glutamyl (y-tert-butyl ester) -ne-tert-butyloxycarbonyl-lysyl-glutamic acid-y-tert-butyl ester (amino acids 14-17), fragment III, consisting of Na-benzyloxycarbonyl-N <5, N « > -di-benzyloxycarbonyl-arginyl-leucine (amino acids 12-13), fragment IV, consisting of Na-benzyloxycarbonyl-glut-amyl- (y-tert-butyl ester) -leucyl-glutamine (amino acids (9-11), fragment V, consisting of O-tert-butyl-threonyl-O-tert-butyltyrosyl-glycyl (amino acids 6-8) and fragment VI, consisting of Na-tert-butyloxycarbonyl-phenylalanyl-valyl-propyl-isoleucyl-phenylalanine (amino acids 1-5), in which all side chain functions of the polyfunctional amino acids carry easily cleavable groups and the complex functions of the arginine either by Nó, masked by a protonation caused by salt formation with hydrogen bromide, condensed, the protective groups split off and the crude L-leucine-13-motiline isolated. SUB-CLAIMS 1. The method according to claim, characterized in that by aminoacylation of L-leucine (amino acid 13) with the aid of Na, N <3, Na) -Tris ~ benzyloxycarbonyI-L-arginine-N-hydroxy-succinimide ester ( Amino acid 12) available fragment is used. 2. The method according to claim, characterized in that the L-leucine-13-motiline obtained is purified by column chromatography on a strongly basic anion exchange resin and then on a strongly acidic cation exchange resin.