DE3518086A1 - BIOLOGICALLY ACTIVE TRIDECAPEPTIDES, METHOD FOR THE PRODUCTION THEREOF AND MEANS THAT CONTAIN THEM - Google Patents

Description

FARMITALIA CARLO ERBA S.p.Α., MILAN / ITALY

Biologically active tridecapeptides / processes for their production and compositions containing them

The invention relates to biologically active peptides, a process for their production and pharmaceutical
Means containing them.

In the present description, the symbols and abbreviations correspond to those usually used in
peptide chemistry (see Eur. J. Biochem. (1984) 138 , 9-37). Thus, the three letter amino acid symbols represent the L configuration of chiral amino acids. Other symbols and abbreviations used are: AcOEt, ethyl acetate; AcOH, acetic acid; DCC, dicyclohexylcarbodiimide; DCEU, dicyclohexy! Urea; DMF, dimethylformamide; ECC, ethyl chloroformate;

ORDINAL INSPECTED

Et-O, diethyl ether; FC, flash chromatography on Silica gel (Merck) 0.040-0.063 mm; HCl / THF, dry HCl in anhydrous tetrahydrofuran; HOBT, 1-hydroxybenzotriazole; HOTcp, 2,4,5-trichlorophenol; iBCC, isobutyl chloroformate; iPr-O, diisopropyl ether; iPrOH, isopropyl alcohol; MeOH, methyl alcohol; Msc, methyl sulf onylethoxycarbonyl; NMM, N-methylmorpholine; PE, Petroleum ether; THF, tetrahydrofuran.

The invention relates to peptides of the formula

AB B

I I f

Glp-X-Lys-Pro-Tyr-Trp-Pro-Pro-Pro-Y-Tyr-Pro-W-C

wherein

X is a neutral or acidic L-oO-amino acid residue represents;

A is a hydrogen atom or an acyl, alkoxycarbonyl, Aralkoxycarbonyl, alkyl or aralkyl amino protecting group means;

B is a hydrogen atom or an acyl, alkyl, aralkyl, Denotes alkoxycarbonyl or aralkoxycarbonyl protecting group for a phenolic hydroxy group;

Y and W independently represent a neutral L-oO-amino acid residue;
30th

C is a hydroxyl group, an amino group or a group of the formula

- ίο -

OR, NHR, NR ₂ or NH-NH-R ', WOrIn ₇ R is a linear, branched or cyclic (including fused or bridging ring) alkyl group with up to 11 carbon atoms, a phenyl group or an aralkyl group with 7 to 14 carbon atoms and R ^{1 represents} a hydrogen atom, a group as represented by R, a linear, branched or cyclic aliphatic acyl group having 1 to 11 carbon atoms, a linear, branched or cyclic alkoxycarbonyl group having 3 to 11 carbon atoms, or an aralkoxycarbonyl group;

and pharmaceutically acceptable salts thereof. 15th

Preferred L-CO amino acid residues that X can represent include GIu and gin.

Preferred terminal amino protecting groups which A may represent include (of the acyl type) formyl, Acetyl, trifluoroacetyl, propionyl and benzoyl groups; (of the aralkoxycarbonyl type) benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl-, 4-methoxybenzyloxycarbonyl-, 2 ^ -Dichlorbenzyloxycarbonyl-, 2-bromobenzyloxycarbonyl-, 9-fluorenylmethoxycarbonyl and 3,5-dimethoxy-oi. , oC '~ dimethylbenzyloxycarbonyl groups; (of the alkoxycarbonyl type) t-butyloxycarbonyl-, 1-methyl-cyclobutyloxycarbonyl-, Adamantyloxycarbonyl, isobornyloxycarbonyl, and methylsulfonylethoxycarbonyl groups; and from Alkyl and aralkyl type) trityl, benzyl, methyl and Isopropy! Groups.

ORIGINAL INSPECTED

Preferred phenolic hydroxy protecting groups which B may represent include: (of the acyl type) acetyl and Benzoyl groups; (alkyl type) a t-butyl group; (of the aralkyl type) benzyl, 2,6-dichlorobenzyl and 2-nitrobenzy groups; (alkoxycarbonyl type) t-butyloxycarbonyl group; and (of the aralkoxycarbonyl type) benzyloxycarbonyl and 2-bromobenzyloxycarbonyl groups.

Preferred L-oO amino acid residues that represent Y. may include He, Leu, Ahx, and Phe.

Preferred L-oC -amino acid residues which W can represent, include Met, MetO, and Ahx.

Preferred meanings as represented by R and R ¹ include methyl, ethyl, n-propyl, isopropyl, η-butyl, s-butyl, isobutyl, t-butyl, 2,2,2- Trifluoroethyl, cyclohexyl, adamantyl, phenyl, benzyl, phenethyl and fluorenylmethyl groups.

Examples of acyl groups as R ¹ can represent are formyl, acetyl, trifluoroacetyl, propionyl, butyryl, adamantyl-carbonyl, benzoyl, phenylacetyl and cinnamyl. The preferred alkoxycarbonyl and aralkoxycarbonyl groups which R ¹ can represent are the groups of the alkoxycarbonyl and aralkoxycarbonyl type mentioned above as preferred o ^ -amino protective groups A.

Salts of peptides according to the invention with pharmaceutical acceptable acids or bases are also encompassed by the invention. Such acid addition salts

can be derived from a variety of inorganic and organic acids, such as sulfuric acid, phosphoric acid, Hydrochloric acid, hydrobromic acid, hydriodic acid, nitric acid, sulfamic acid, citric acid, Lactic acid, pyruvic acid, oxalic acid, maleic acid, succinic acid, tartaric acid, cinnamic acid, acetic acid, Trifluoroacetic acid, benzoic acid, salicylic acid, gluconic acid, ascorbic acid and related acids. Base addition salts can be derived from a variety of inorganic or organic bases, such as sodium hydroxide, Potassium hydroxide, diethylamine, triethylamine, Dicyclohexylamine.

The present invention also encompasses a process for the preparation of a peptide according to the invention or a pharmaceutically acceptable salt thereof which is characterized by the following steps: Condensing suitable protected amino acids or peptides in the order of the amino acids of the desired one Peptide, removal of protective groups, if necessary, and, if necessary, conversion of the obtained Peptide into a pharmaceutically acceptable salt thereof.

The synthesis essentially consists of suitable successive condensations of protected amino acids or peptides. The condensation is carried out so that the peptides obtained have the desired sequence of Have 13 amino acid residues. The amino acids and peptides produced by known methods of polypeptide chemistry can be condensed, have a through

ORIGINAL! HSFECTED

■ 3513086

suitable protecting groups blocked amino and carboxyl groups that are not involved in the formation of the peptide bond.

It is possible to remove the protecting groups by acid or alkali treatment or by hydrogenolysis. The following protective groups, for example, can be used to protect an amino group: benzyloxycarbonyl, t-butyloxycarbonyl, trityl, formyl, trifluoroacetyl, o-nitrophenylsulfenyl, 4-methoxybenzyloxycarbonyl, 9-fluorenylmethoxycarbonyl, 3,5-dimethoxy-oo-oC ^{1-carbonylethylbenzyloxyl} . The following protective groups, for example, can be used to protect a carbony group: methyl, ethyl, t-butyl, benzyl, p-nitrobenzyl or fluorenylmethyl,

Reactions to remove the protective groups can be carried out according to methods of polypeptide chemistry known per se will.

The condensation between an amino group on one molecule and a carboxyl group on another molecule with the formation of a peptide bond, an activated acyl derivative, such as a mixed anhydride, an azide or an activated ester, or by direct condensation between a free amino group and a free carboxyl group in the presence of a condensing agent such as dicyclohexylcarbodiimide alone or together with an agent to prevent "racemization, such as N-hydroxysuccinimide or 1-hydroxy

benzotriazole are carried out. The condensation can be in a solvent such as dimethylformamide, dimethylacetamide, pyridine, acetonitrile, tetrahydrofuran or N-methyl-2-pyrrolidone. 5

The reaction temperature may be from -30 ⁰ C to room temperature. The reaction time is generally between 1 and 120 hours. The synthesis scheme, the protective groups and the condensation methods are selected so that the risk of racemization is avoided.

Peptides in which C represents the group OR are produced, for example, starting from a C-terminal amino acid which is esterified with a suitable alcohol. Peptides in which C represents the group OH can be produced, for example, by hydrolysis of peptides in which C represents the group OR. Peptides in which W represents the group NH_, NHR or NR ₂ can be produced by ammonolysis of the corresponding esters or starting from C-terminal amino acids amidated with a suitable amine.

Hydrazido and substituted hydrazido derivatives according to the invention are N-protected by condensation Peptides or amino acids with a suitable substituted hydrazine, such as benzyl carbazate, t-butyl carbazate, Adamantyl carbazate, phenylhydrazine or Adamanty! Hydrazine, or by implementing the N-protected peptide or amino acid hydrazide with a suitable alkylating agent such as alkyl chloride,

ORKSiNAL

35T3086

or with a suitable acylating agent, e.g. Benzyl chloroformate, t-butyl chloroformate, di-t-butyl dicarbonate or adamantyl fluoroformate.

The peptides according to the invention and their pharmaceutical acceptable salts are useful for therapeutic treatment of the human or animal Body. When administered intracerebroventricular, the compounds according to the invention stimulate - see above was noted - the secretion of pituitary prolactin, they cause sedative and deconditioning effects in behavioral tests on emotionality of laboratory animals, inhibit the intestinal transit of a meal, especially when tested with a charcoal meal, lower the body temperature and have an antipyrethic effect.

The increase in prolactin release was determined by a method described by GD Niswender, CL Chen., AR Midgley Jr., J. Meites and S. Ellis in Proc. Soc. Exp. Biol. Med., 130 , 793-797 (1969).

The behavioral effects were determined with the aid of experiments carried out on rats according to the method described by J. Archer in Anim. Behav. 2J_, 205-235 (1973) have been carried out.

The inhibition of intestinal mobility was also confirmed with the help of rats, the "Tran- ■ sit charcoal meal test "according to B. B. Brown and

■ "351-8Ό86

H. W. Werner, J. Pharmacol. Exp. Ther. ίΓ7, 157 (1949) was applied. The invention further encompasses a pharmaceutical preparation comprising a compound according to the invention or a pharmaceutically acceptable salt thereof in admixture with a pharmaceutical

an acceptable diluent or carrier; in addition, these preparations be formulated so that they allow a directed or delayed release of the active ingredient. 10

Preferred peptides according to the invention are those listed below:

Glp-Glu-Lys-Pro-Tyr-Trp-Pro-Pro-Pro-Ile-Tyr-Pro-Met-OH _50Me

Olli ———------.-_---——---— · -._- ^ j-j

V-jJLIl - —- - <- - - »---— - - - -. - - -.-_ -_ «-

Leu OH

--—-— _._.—- ΠΜλ

Leu NH ₂

ΛΤ-ϊλτ -—_._.- »__._.__ - ___—. ΓΜΤ

-TlXX-T-. —- ■■ —- V ^ JfI

Phe OH

MetO-OMe

Ahx-OMe

351BO86

The invention is to be explained in more detail with the aid of the following example, without it being restricted thereby. The R ^ values were determined on precoated plates of silica gel 60 F ₅ C ₄ (Merck), the layer thickness being 0.25 mm, the length 20 ciii and the following development systems being used:

System A: benzene / gasoline (60-80) / ethyl acetate = 70/10/40 by volume;

System B: benzene / ethyl acetate / acetic acid / water =

100/100/20/10 by volume (upper phase);

System C: benzene / ethyl acetate / acetic acid / water =

100/100/40/15 by volume (upper phase);

System D: n-butanol / acetic acid / water = 4/1/1 by volume;

System E: n-butanol / acetic acid / water / pyridine = 4/1/1/1 by volume.

"Merck" is a trade name.

TLC (thin layer chromatography) analyzes were carried out at a temperature in the range from 18 to 25 ^° C.: The R _£ values can therefore vary by ± 5%. The melting points were determined in open capillaries with a Tottoli device and are not corrected. Most of the derivatives soften and decompose

'- ■ -'3S-1S-086

before melting. Solvents for crystallization, Precipitation or grinding are given in brackets. The high voltage paper electrophoresis was used with a Pherograph Original Frankfurt Type 64 device on Schleicher and Schüll paper No. 2317 at pH 1.2 (Formic acid! Acetic acid: water = 123: 100: 777) at 1,600 V (40 V / cm) and a pH of 5.8 (pyridine: acetic acid: water = 450: 50: 4500) at 1,400 V (32.5 V / cm). The products are due to their mobility in relation to GIu at a pH

of 1.2 (E _{1 o} ) and a pH of 5.8 (E _co ) characters i, c. ο , ο

ized.

Optical rotation was photoelectric with a Perkin-Elmer 141 polarimeter at one concentration of c = 1 in methanol, unless otherwise stated.

EXAMPLE

Making Glp-Glu-Lys-Pro-Tyr-Typ-Pro-Pro-Pro-Ile-Tyr-Pro-Met-OH (XXIV)
25th

Level_l £

Boc-Tyr-Pro-OH (I)

BzI

14.06 g (30 mmol) of Boc-Tyr-Pro-OH (R. de Castiglione et al, Int. J. Peptide Protein Res. (1981), 17, 263-272),

dissolved in 60 ml of methanol, were hydrogenated at room temperature and atmospheric pressure in the presence of 3.51 g of 10% palladium on charcoal. The catalyst was removed by filtration and the solution concentrated in vacuo. The residue was redissolved in ethyl acetate and concentrated in vacuo until the product was precipitated. Precipitation was completed by diluting with a mixture of diethyl ether and petroleum ether. 11.35 g (quantitative yield) of the compound (I) were obtained: mp 124-132 ⁰ C (foam);

- - OR

Z <* / d -28.0 °; Rf _B 0.45; E ₅ ^ ₃ 0.55. 15th

Boc-Tyr-Pro-Met-OMe (II)
20th

To a solution of 11.35 g (30 mmol) of Boc-Tyr-Pro-OH (I) in 60 ml of anhydrous THF were successively 3.37 ml (30 mmol) of NMM and 2.97 ml (30 mmol) of ECC at a Temperature of -12 ⁰ C added.

After stirring for 2 minutes at this temperature, a cold solution of 5.99 g (30 mmol) of HCl-H-Met-OMe (H. Yajima, Chem. Pharm. Bull (1968), 1 £, 1342) and 3, 37 ml (30 mmol) of NMM in 40 ml of DMF were added. The mixture was stirred for 45 minutes at -12 ° C and 90 minutes at 0 to 15 ⁰ C, then filtered from salts

■■ '■ "■'" ■■ · 351ΒΌ86

and evaporated in vacuo. The residue was dissolved in ethyl acetate and several times successively washed with saturated sodium chloride solutions of 1 M citric acid, 1 M sodium bicarbonate, and water. The organic layer was dried over anhydrous sodium sulfate and the solvent in vacuo removed.

14.92 g (95% yield) of the compound (II) were obtained as a solid foam from AcOEt:

-44.90. _R f _A o.37; Rf _B 0.67.

Level_3i

HCl'H-Tyr-Pro-Met-OMe (III)

14.70 g (28.07 mmol) of Boc-Tyr-Pro-Met-OMe (II) were dissolved in 150 ml of a 3.5 M HCl / THF solution at room temperature. After 30 minutes the removal of the Boc residue was complete and the solution was _{diluted with a large excess of Et 3} O, causing the product to precipitate. After recrystallization from iPrOH / Et ₂ O, 11.75 g of the compound (III) (91% yield) were obtained: melting point 78 ^° C. (foam);

Z ⁵ I / ^ ⁸ "32.7 °; Rf ₀ 0.52; E ^ ₂ 0.78.

ORIGINAL INSPECTED

Level_4 £
Boc-Ile-Tyr-Pro-Met-OMe (IV)

To a precooled (0 ⁰ C) solution of 17.42 g (75.33 mmol) of Boc-Ile-OH in 50 ml anhydrous THF was added 7.77 g (37.66 mmol) of DCC. After 30 minutes the formed DCEU was removed by filtration and a solution of 11.55 g (25.11 mmol) of HCl · H-Tyr-Pro-Met-OMe (III) and 2 ₇ 82 ml (25.11 mmol) of NMM in 50 ml of DMF are added and the mixture is left to stand for 90 minutes at room temperature for reaction. The solvents were removed in vacuo and the product was partially purified by washing steps as described in step 2. The compound (IV) was obtained in pure form after purification by FC while eluting with AcOEt: n-hexane = 9: 1. 10.39 g (65% yield) of the compound (IV) were isolated from Et ^ O / iPr-O: melting point 55 ⁰ C (foam);

/ «I / ß ⁶ " 62.4 °; Rf _A 0.30? Rf _ß 0.69.

Level 5:
25th

HCl-H-Ile-Tyr-Pro-Met-OMe (V)

10.20 g (16.018 mmol) of Boc-Ile-Tyr-Pro-Met-OMe (IV) were dissolved in 100 ml of a saturated solution of hydrogen chloride dissolved in acetic acid at room temperature.

ORIGINAL UiSfECTED

- 22 -

After 30 minutes, the removal of the Boc residue was complete and the solvent was removed in vacuo. The residue was dissolved in methanol and the solution concentrated to dryness. 7/34 g (80% yield) of the compound (V) were obtained from iPrOH / iPr ₂ O: mp 115-129 ⁰ C;

"34.7 °; Rf ₀ 0.68; E ^ ₂ = 0.68

Level_6_ £

Boc-Trp-Pro-OH (VI)

3.45 g. (30 mmol) H-Pro-OH was dissolved in 15 ml of 2 M sodium hydroxide at room temperature. The solution was ^{cooled to 0 °} C., diluted with 60 ml of DMF and the solvents were stripped off ^{at 35 ° C. in vacuo.} The residue was suspended in 120 ml of DMF and 12.09 g (25 mmol) of Boc-Trp-OTcp (W. Broadbent et al, J. Chem. Soc. (C) (1967) 2632-2636) were added. The reaction mixture was stirred at room temperature for 2 hours, then evaporated in vacuo.

The residue was dissolved in water and washed several times with ethyl acetate. The aqueous layer was ^{cooled to 0 °} C., acidified to pH 2 with 5 N aqueous hydrochloric acid solution and then extracted with ethyl acetate. The organic layer was washed up with saturated sodium chloride (aqueous) solution

INSPECTED

35ΊΒΌ86

washed to neutrality and dried over anhydrous sodium sulfate. The solvent was removed in vacuo and the compound (VI) was obtained from Et ₂ O / PE (9.53 g, 95% yield): melting point 100 ^° C.;

Z ° ^ / d - ²⁴ ' ² °' · ^Rf B ° ^{'55; E} 5.8 ° ^'39

Level_7j.

Boc-Trp-Pro-Pro-Pro-OBzl (VII)

Starting from 9.35 g (23.29 mmol) Boc-Trp-Pro-OH (VI) and 7.89 g (23.29 mmol) HCl-H-Pro-Pro-OBzl (CM Deber et al, J. Am Chem. Soc. (1970)! 9_2, 6191-6198) were obtained by the method described in step 2, but using iBCC as the condensing agent, 12.14 g (76% yield) of the compound (VII) from iPrOH / iPr ₂ O obtained: melting point 137 ⁰ C?

? ² -154.9 °; Rf _B 0.50.
25th

Boc-Trp-Pro-Pro-Pro-OH (VIII)
30th

Starting from 11.95 g (17.42 mmol) of Boc-Trp-Pro-Pro-Pro-OBzl (VII), the procedure described in step 1 was followed

■■ '- ■' · ■ - "3Sieö86

- 24 -

Process 7.78 g (75% yield) of compound (VIII) are obtained from iPrOH / iPr ₂ O: melting point 139-147 ° C;

-147.9 °; Rf _c 0.43; E _{5 Q} 0.32.

Boc-Trp-Pro-Pro-Pro-Ile-Tyr-Pro-Met-OMe (IX)

To a pre-cooled ( ⁰ ° C.) solution of 7.39 g (12.40 mmol) of Boc-Trp-Pro-Pro-Pro-OH (VIII) and 1.78 g (12.40 mmol) of HOBT in 100 ml of anhydrous THF was added 2.56 g (12.40 mmol) of DCC. After 5 minutes the activated ester solution became a cold solution of HCl-H-Ile-Tyr-Pro-Met-OMe (V) (7.11 g, 12.40 mmol) and 1.39 ml (12.40 mmol) NMM in 100 ml DMF was added.

For reaction, the mixture was left to ^{stand for 30 minutes at 0 °} C., then at room temperature until the reaction had ended. DCEU was removed by filtration, the solvents were evaporated in vacuo. The product was then purified by washing steps as described in step 2. The compound (IX) was finally _{isolated from iPrOH / iPr 2} O: (9.67 g, 70% yield); Melting point 125-135 ⁰ C;

-155.7 °; Rf _c 0.52.

ORKSINAL WSfECTED

351-86

Level_1_0.L
Boc-Trp-Pro-Pro-Pro-Ile-Tyr-Pro-Met-OH (X)

9.30 g (8.35 mmol) of Boc-Trp-Pro-Pro-Pro-Ile-Tyr-Pro-Met-OMe (IX) were saponified with 80 ml of 1N sodium hydroxide for 1 hour at room temperature. The solution was diluted with water, cooled to 0 ° C., acidified to pH 2 with 5 M aqueous hydrochloric acid solution, then extracted several times with chloroform.

The collected organic layers were washed with saturated (aqueous) sodium chloride solution until neutral and dried over anhydrous sodium sulfate, and then the solvents were removed in vacuo. 7.16 g (78% yield) of the compound (X) were obtained from iPrOH / iPr ₂ O: melting point 147-163 ° C;

/ ctli ² -148.9 °; Rf 0.26; E. _Q 0.17.

HCOOH-H-Trp-Pro-Pro-Pro-Ile-Tyr-Pro-Met-OH (XI)

6.95 g (6.32 mmoles) of Boc-Trp-Pro-Pro-Pro-Ile-Tyr-Pro-Met-OH (X) were dissolved in 70 ml of HCOOH at room temperature. After the removal of the Boc residue is complete the product was precipitated by the solution with a

■ - ■■ · '351δϋ86

large excess of Et ₃ O was diluted.

5.95 g (90% yield) of the compound (XI) was obtained after recrystallization from MeOH / Et ₂ O: melting point 163-171 · ⁰ C;

, -, .- 22 ₁₂ _ 7.3 °. Rf ₀ 0.44; E _{1 2} = .45 Eq.

HCl-H-Tyr-NH-NH-Z (XII)

12.88 g (30 mmol) of Boc-Tyr-NH-NH-Z (Yajima et al (1972), J. Am. Chem. Soc. 9J_, 6170-6178) were added at room temperature to 130 ml of a 3.5 M HCl / THF solution dissolved. After 30 minutes, the removal of the Boc residue was complete and the solvent was removed in vacuo. 9.88 g (90% yield) of the compound (XII) were obtained from PE: mp 87-92 ⁰ C (foam);

-26.6 °; Rf ₀ 0.74; E ^ ₂ 0.82

Boc-Pro-Tyr-NH-NH-Z (XIII)
30th

■ Starting from 5.65 g (26.24 mmol) Boc-Pro-OH and 9.60 g (26.24 mmol) HCl-H-Tyr-NH-NH-Z (XII) were according to the

ORKaINAL ^ .S ^ E

Process as described in step 2, 11.05 g of the crude product (XIII) are obtained. The compound (XII) was further purified by FC while eluting with AcOEt: n-hexane = 8: 2, 8.43 g (61% yield) being obtained from MeOH / iPr ₂ O: melting point 105-117 ⁰ C;

^ "55.9 °; Rf _A 0.34; Rf _ß 0.68.

Level_1_4 .:

HCl.H-Pro-Tyr-NH-NH-Z (XIV)

Starting from 8.30 g (15.76 mmol) of Boc-Pro-Tyr-NH-NH-Z (XIII). were prepared by the method as described in Step 3, 7.30 g (90% yield) of the compound (XIV) from iPrOH / iPr ₂ O: mp 128-133 ⁰ C (foam);

57 ^ ² -28.1 °; Rf ₀ 0.49; E ^ ₂ 0.76 GIu.

§_1i

BOC-LyS (MsC) -OTCp (XV)

To a cold (0 ⁰ C) solution of 7.30 g (20 mmol) of Boc-Lys (Msc) -OH (Eberle A. et al (1975) Helv. Chim.

Acta 58, 2106-2129) and 3.95 g (20 mmol) HOTcp in 200 ml

- 28 -

AcOEt was added to 4.127 g (20 mmol) of DCC. After 30 minutes at ⁰ ° C., the mixture was left to react overnight. DCEU was removed by filtration and the solvent evaporated in vacuo. 7.83 g (68% yield of the compound (XV) were obtained from AcOEt / iPr ₂ O: mp 112-117 ⁰ C;

& Jv ² -24.7 °; Rf ₃ 0.67.
10

Level_1_6_i

Boc-Lys (Msc) -Pro-Tyr-NH-NH-Z (XVI)

To a solution of 6.15 g (13.28 mmol) of HCl'H-PrO-Tyr-NH-NH-Z (XIV) and 1.49 ml (13.28 mmol) NMM in 70 ml DMF were 7.65 g (13.28 mmol) Boc-Lys (Msc) -OTcp (XV) was added and the mixture was left to react overnight at room temperature.

The solvent was removed in vacuo and the residue by FC, eluting with AcOEt, which increasing Containing amounts of MeOH (from 2 to 5%), purified. 7.48 g (70% yield) of the compound (XVI) were obtained from iPrOH / iPr_O: melting point 74-82 ° C (foam);

-51.0 °; Rf _c 0.42.

ORIGINAL If ^ EC

351SÜ86

- 29 -

HCl-H-Lys (Msc) -Pro-Tyr-NH-NH-Z (XVII)

7.35 g (9.13 mmol) of Boc-Lys (Msc) -Pro-Tyr-NH-NH-Z (XVI) were dissolved in 75 ml of a 3.5 M HCl / THF solution. After 40 minutes, the removal of the Boc residue was complete and the solvent was stripped off in vacuo. The residue was dissolved in methanol and treated with activated charcoal. 5.75 g (85% yield) of the compound (XVII) were obtained from MeOH / AcOEt / PE: m.p. 105-117 ⁰ C (foam);

-26.8 °; Rf ₀ 0.37; E ^ ₂ 0.62.

HCl · · H-GlU (OBzI) -ONb (XVIII)

Starting from 7.09 g (15 mmol) of Boc-Glu (OBzI) -ONb (R. Schwyzer and P. Schieber (1959) HeIv. Chim. Acta 42 , 972-977), the same procedure as in step is g (XVIII) / obtain 5.83 (95% yield) of the compound from MeOH / iPrOH iPr ₂ O 12 described: mp 112-119 ⁰ C;

+ 6.7 °; Rf ₀ 0.70; E ^ ₂ 0.96.

Level_1_9 £
Z-GIp-GIu (OBzI) -ONb (XIX)

Starting from 3.64 g (13.82 mmol) of Z-GIp-OH and 5.65 g (13.82 mmol) of HCl-H-GIu (OBzI) -ONb (XVIII), the same procedure as described in Step 2 is described, 7.25 g (85% yield) of the compound (XIX) obtained from MeOH / AcOEt / iPr, 0: melting point 147-151 ⁰ C; 10

"p ² -12.6 °; (c = 1, DMF); Rf _A 0.43; Rf _β 0.64.

5 level_20i

Z-Glp-Glu (OBzI) -OH (XX)

3.6 g were added to a solution of 7.10 g (11.49 mmol) of Z-GIp-GIu (OBzI) -ONb (XIX) in 300 ml of a 90% strength acetic acid which had been ^{cooled to 0 ° C.} Zinc dust added in portions.

After 2 hours the excess zinc dust was removed by filtration and the solvent removed in vacuo. The oily residue was taken up with AcOEt, saturated with water, and the precipitate was filtered off. The organic layer was extracted several times with 1 M sodium bicarbonate solution. The aqueous solution was ^{cooled to 0 °} C., acidified to pH 2 with 5 N aqueous hydrochloric acid solution, then several times

ORIGINAL INFE

extracted with AcOEt. The collected organic layers were washed with saturated (aqueous) sodium chloride solution washed until neutral and dried over anhydrous sodium sulfate.

4.71 g (85% yield) of the compound (XX) were obtained from MeOH / HO: Rf _ 0.30; Rf_ 0.65; E _{c Q} 0.54.

0 level e_2H £

Z-Glp-Glu (OBzI) -Lys (Msc) -Pro-Tyr-NH-NH-Z (XXI)

Starting from 3.64 g (7.55 mmol) Z-Glp-Glu (OBzI) -OH (XX) and 5.60 g (7.55 mmol) HCl · H-Lys (Msc) -Pro-Tyr-NH -NH-Z (XVII) 7.07 g of the partially purified product were obtained by the same procedure as described in step 2, but using methylene chloride as solvent during the washing steps. Further purification was achieved with FC, eluting with CH ₂ Cl ₂ : MeOH = 95: 5. 5.74 g (65% yield) of the compound (XXI) were obtained from iPrOH / iPr ₂ O: Rf 0.17; Rf ₀ 0.67.

Glp-Glu-Lys (Msc) -Pro-Tyr-NH-NH ₂ (XXII) 30

5.60 g (4.79 mmoles) of Z-Glp-Glu (OBzI) -Lys (Msc) -Pro-Tyr-NH-NH-Z (XXI) in 70 ml of DMF were in the presence of 2.8 g

10% palladium on carbon hydrogenated at room temperature. The catalyst was removed by filtration and the solvent removed in vacuo. 3.30 g (85% yield) of the compound (XXII) were obtained from iPrOH / iPr ₂ O: Rf ₀ 0.24; E _{1 2} 0.52.

Level_23_ £
10

Glp-Glu-Lys (Msc) -Pro-Tyr-Trp-Pro-Pro-Pro-Ile-Tyr-Pro-Met-OH (XXIII)

To a solution of 4.04 g (3.87 mmol) of HCOOH-H-TrP-PrO-Pro-Pro-Ile-Tyr-Pro-Met-OH (XI) in 40 ml of DMF, cooled to 0 ^° C., were added 1.16 ml of a 4 M solution of hydrogen chloride with anhydrous THF were added. The solution was concentrated in vacuo, the residue was taken up in 40 ml of DMF and concentrated again in vacuo.

To a solution of 3.10 g (3.87 mmol) of Glp-Glu-Lys (Msc) -Pro-Tyr-NH-NH ₂ (XXII) in 50 ml of anhydrous DMF was added 2.42 ml (9.67 mmol) a 4 M solution of hydrogen chloride in anhydrous THF and 0.50 ml (4.25 mmol) of n-butyl nitrite were added ^{successively at a temperature of -3O 0 C.} After stirring for 30 minutes at -15 ° C., 1.52 ml (13.53 mmol) of NMM and then a cold (-3O ⁰ C) solution of 4.01 g (3.87 mmol) of HCl «H-Trp- Pro-Pro-Pro-Ile-Tyr-Pro-Met-OH (prepared as described above in this step) and 0.87 ml (7.73 mmol) NMM in 40 ml DMF were added. The reaction mixture was left to react at -9 ⁰ C for 3 days

OFUGINAL INSPECTED

351ÖÖ86

left, then the salts were filtered off; the solvents were removed in vacuo and the product was partially purified by gel chromatography on Sephadex LH-20, eluting with DMF. 3.99 g (58% yield) of the compound (XXIII) were obtained from iPrOH / AcOEt / iPr _o 0: Rf ₇ . 0.43; Rf_ 0.58; E- _Q 0.36.

Level_24_ £

Glp-Glu-Lys-Pro-Tyr-Trp-Pro-Pro-Pro-Ile-Tyr-Pro-Met-OH-HCl (XXIV)

3.7 g (2.08 mmol) of Glp-Glu-Lys (Msc) -Pro-Tyr-Trp-Pro-Pro-Pro-Ile-Tyr-Pro-Met-OH (XXIII) were dissolved in 37 ml of MeOH at room temperature dissolved. 184 ml of a solution consisting of dioxane / methanol / 8 M sodium hydroxide = 7.5: 2.25: 0.25 were added. The mixture was left to stand for 5 minutes at room temperature for reaction, then - after cooling to ⁰ ° C. - 46 ml of 1N HCl were added. The solvents were removed in vacuo and the crude product was isolated _{from MeOH / Et 2 O.}

The compound (XXIV) was obtained by countercurrent partitioning using the n-butanol / solvent system. Acetic acid / ethyl alcohol / water = 5: 1: 1: 8, 1.25 g (60% yield) being obtained from MeOH / iPrOH / iPr ₂ O: Rf _ 0.23; Rf_ 0.40; E _{1 o} 0.38; E _{c Q} 0.13.

Claims (21)

HOFFMANN · EITLE & PARTNER PATENT- AND LAWYERS PATENTANWÄLTE DIPL.-ΙΝβ. W. EITLE. DR. RER. NAT. K. HOFFMANN. DIPL.-ΙΝΘ. W. LEHN DIPL.-ING. K. FCICHSLE DR. RER. NAT. B. HANSEN. DR. RER. NAT. HA. BRAUNS DIPL.-ΙΝβ. K. GORQ D1PL.-ING, K. KOHLMANN. LAWYER A. NETTE 41 861 m / wa FARMITALIA CARLO ERBA S.p.A., MILAN / ITALY Biologically active tridecapeptides, processes for their production and means containing them. PATENT CLAIMS 1. Peptide of the formula AB B Glp-X-Lys-Pro-Tyr-Trp-Pro-Pro-Pro-Y-Tyr-Pro-W-C 5 wherein X denotes a neutral or acidic L-oü-amino radical;
10 A is a hydrogen atom or an acyl, alkoxycarbonyl, Aralkoxycarbonyl, alkyl or aralkyl ARABELLASTRASSE A ■ D-SOOO MÖNCHEN 81. TELEPHONE COSO} Ο11Ο87. TELEX 5-29619 CPATHE} ■ TELECOPIER SI83CB «n Means amino protecting group; B is a hydrogen atom or an acyl, alkyl, aralkyl, alkoxycarbonyl or aralkoxycarbonyl protecting group represents a phenolic hydroxy group; Υ and W independently represent a neutral L-oO-amino acid residue; C represents a hydroxyl group, an amino group or a group of the formula OR, NHR, NR ₂ or NH-NH-R ¹ , where R is a linear, branched or cyclic alkyl group with up to 11 carbon atoms, a phenyl group or an aralkyl group with 7 to 14 Denotes carbon atoms and R ¹ denotes a hydrogen atom, one of the groups as specified for R, a linear, branched or cyclic aliphatic acyl group having 1 to 11 carbon atoms, a linear branched or cyclic alkoxycarbonyl group having 3 to 11 carbon atoms or an aralkoxycarbonyl group; and the pharmaceutically acceptable salts thereof. 2. A compound according to claim 1, characterized in that g e k e η η draws that X represents GIu or GIn. 3. A compound according to claim 1 or 2, characterized in that A is a formyl, Acetyl, trifluoroacetyl, propionyl, benzoyl, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl-, 2,4-dichlorobenzyloxycarbonyl-, 2-bromobenzyloxycarbonyl-, 9-fluorenylmethoxycarbonyl-, 3,5-dimethoxy- oO, oO'-dimethylbenzyloxycarbonyl-, t-butyloxycarbonyl, 1-methylcyclobutyloxycarbonyl, Adamantyloxycarbonyl, isobornyloxycarbonyl, methylsulfonylethoxycarbonyl, Represents trityl, benzyl, methyl or isopropyl group. 4. Connection according to one or more of the preceding Claims, characterized in that B is an acetyl, benzoyl, t-butyl, Benzyl, 2,6-dichlorobenzyl, 2-nitrobenzyl, t-butyloxycarbonyl, Represents benzyloxycarbonyl or 2-bromobenzyloxycarbonyl group. 5. Compound according to one or more of the preceding claims, characterized in that Y He, Leu, Ahx or Phe represents. 6. A compound according to one or more of the preceding claims, characterized in that W represents Met, MetO or Ahx. 7. A compound according to one or more of the preceding claims, characterized in that R or R ^{1 is} methyl, ethyl, n-propyl, isopropyl, η-butyl, sec-butyl, isobutyl, tert-butyl, 2,2,2-trifluoroethyl, cyclohexyl, adamantyl, phenyl, benzyl, phenethyl or fluorenymethyl represent.
5 8. Compound according to one or more of the preceding claims, characterized in that R ^{1 is} formyl, acetyl, trifluoroacetyl, propionyl, butyryl, adamantylcarbonyl, benzoyl, phenylacetyl, cinnamyl, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-methoxybenzyloxycarbony1, 2,4-dichlorobenzyloxycarbonyl , 2-bromobenzyloxycarbonyl, 9-fluorenylmethoxycarbony1, 3,5-dimethoxy- Oo , cL'-dimethylbenzyloxycarbonyl, t-butyloxycarbonyl, 1-methylcyclobutyloxycarbonyl, adamantyloxycarbonyl, isobornyloxycarbonyl or methylsulfonylethoxycarbonyl. 9. A compound according to claim 1, characterized in that this Glp-Glu-Lys-Pro-Tyr-Trp-Pro-Pro-Pro-Ile-Tyr-Pro-Met-OH represents. 10. Pharmaceutical agent or pharmaceutically acceptable salt thereof, characterized in that it is a peptide according to Claims 1 to 9 in a pharmaceutically sufficient amount together with a pharmaceutically acceptable amount Includes carrier or extender. 11. Pharmaceutical agent or salt thereof according to Claim 10 for stimulating the secretion of prolactin from the pituitary gland. 12. Pharmaceutical agent or salt thereof according to Claim 10 as a sedative or deconditioning agent. 13. Pharmaceutical agent or salt thereof according to Claim 10 for slowing down the passage of a meal through the intestinal tract. 14. Pharmaceutical agent or salt thereof according to Claim 10 for lowering body temperature or as an antipyretic. 15. A process for the preparation of a peptide according to claim 1 or a pharmaceutically acceptable one Its salt, marked by Condensing appropriately protected amino acids or peptides in the order of the amino acids of the desired peptide, removing the protecting groups as necessary and optionally converting the resulting peptide into a pharmaceutical one acceptable salt thereof. 16. The method according to claim 15, characterized in that - indicates that the starting C-terminal amino acid W is esterified to form a peptide in which C represents the group OR. 17. The method according to claim 16, characterized in that on this Thus obtained peptide is hydrolyzed to obtain a peptide in which C is a hydroxy group represents. 18. The method according to claim 16, characterized in that the on this The peptide obtained in this manner is subjected to ammonolysis or aminolysis in order to obtain a peptide in which C represents the group NH ₂ , NHR or NR ~.
15th 19. The method according to claim 15, characterized in that the C-terminal starting amino acid W is amidated in order to obtain a peptide in which C represents the group NH ₂ / NHR or NR ₂ . 20. The method according to claim 15, characterized in that the C-terminal starting amino acid W or the N-protected peptide obtained is condensed with a substituted hydrazine in order to obtain a peptide in which C represents the group NH-NH-R 'or that a condensation with hydrazine and then with an alkylating agent takes place in order to obtain a peptide in which C represents the group NH-NH-R ¹ , in which R ^{1 represents} an alkyl group as defined in the claim. 21. Process for the preparation of a peptide according to Claim 1, characterized in that the method is essentially as in that above example is carried out.