CS199674B2 - Method of producing polypeptides - Google Patents

Description

(LH) from the pituitary. It has now been found that the substitution introduction of azaglycine at position 10 associated with substitution introduction of various Da-anrino acids at the 6-luliberin position or the substitution introduction of azaglycine or azalanine at the 6-position of luliberin associated with luliberin terminal glycinamide replacement luliberin in terms of their ability to release luteinizing hormone.

Accordingly, the invention provides a process for the production of polypeptides of Formula I

Tyr-A-8-Ars- ^Pr ° ^-E - ^F (1) and their pharmaceutically or veterinarily usable acid addition salts.

In the above general formula I and throughout the text, amino acid residues are referred to by their standard abbreviations (see Pure and Applied Ohemietry, 1974, 40, 317-331). The α-aza-amino acid residue is one in which the α-CH-amino acid group is replaced by nitrogen. The abbreviation α-aza-aminophacid is derived from the abbreviation of the corresponding amino acid by including the prefix "Az". Thus, Azgly is azaglycine and Azala is azalanine. In cases where it is not the configuration of the respective amino acid has the following amino acid (except for a-aza-amino acids which adjacent carboxyl groups do not contain any center of asymmetry] natural α-configuration. Also, the abbreviations Me and Bu ¹ are common in the art and standard used and represent a methyl or tert-butyl group, respectively.

Particularly suitable groups of compounds produced by the process of the invention are:

a compound of formula (I) above wherein:

A is D-Tyr, D-Tyr (Me), D-Ser, D-Ser, (Bu ¹ ), D-Phe, D-Ala, or D-Trp,

B represents Leu or MeLeu,

E is Azgly and F is amino;

a compound of formula (I) above wherein:

And it means Azgly or Azala,

B represents Leu,

E represents a direct bond and

F is ethylamino;

a compound of formula (I) above wherein:

A means D-Tyr (Me), D-Ser, D-Ser (Bu ¹ ),

D-Phe, D-Ala, or D-Trp,

B represents Leu or MeLeu,

E is Azgly and

F represents an annincte group.

A preferred group of compounds produced by the process of the invention are the compounds of formula (I) in which:

A is D-Tyr (Me), D-Ser (Bu ¹ ) or D-Phe,

B represents Leu or MeLeu,

E is Azgly and

F is amino.

The three preferred compounds produced by the process of the invention have the following structures:

^ i ^ HiS-Trp-Ser- ^ - D-SertBu-Leu-Ars-Pro-Az ^ -NH ^

ΓΊ _ (~ I _{U-His-Trp-Ser-Tyr-Tyr-O} FME) -Leu-N £ -Pro-Azgly-NH _of

Suitable pharmaceutically or veterinarily useful acid addition salts of the compounds of the invention are, for example, hydrochlorides, phosphates, citrates or acetates.

The method of the invention consists in cleavage of one or more conventional peptide protecting groups from the respective protected polypeptide to form a compound of formula I.

In the process of the invention, the starting material may contain as many protecting groups as there are residues needing protection, such as some or all of the residues occurring in the product as free hydroxyl groups or basic imino groups.

The protecting groups used in the process of the invention may be those described in standard peptide chemistry textbooks such as M. Bodansky and M. A. Ondetti, "Peptide Symthesis", Interscience, New York, 1966, Chapter IV; F. M. Finn and K. Hofmann, "The Proteins", Vol. II, editors H. Neurath and R. L. Hill, Academic Press Inc,. New York, 1976, p.106, "Amino-acids, Peptides and Proteins" (Specialist Periodical Reports), The Chemical Society, London, Vol. Various methods of cleavage of protecting groups are also described in the above publications.

A particularly suitable imino protecting group is benzyloxycarbonyl and a particularly suitable hydroxyl protecting group is benzyl. Both of these protecting groups are readily cleaved by hydrogenolysis, for example in the presence of palladium on carbon catalyst.

Another particularly suitable amino protecting group is a tert-butoxycarbonyl group, and another particularly suitable hydroxyl protecting group is a tert-butyl group. Both of these groups are readily cleaved by treatment with an acid such as hydrogen chloride or trifluoroacetic acid.

Another particularly suitable protecting group

Imino groups are benzyloxycarbonyl or tert-butoxycarbonyl and a particularly suitable hydroxyl protecting group is tert-butyl. These protectors

The groups are readily cleaved by treatment with hydrogen bromide in acetic acid.

Starting materials for the process of the invention may be prepared from known compounds by standard peptide chemistry coupling reactions, standard deprotection reactions known from peptide chemistry, and standard cleavage reactions known from peptide chemistry. These reactions are described, for example, in Examples 1 and 2 below.

As mentioned above, the compounds which can be produced by the method of the invention exhibit luliberin agonist properties, i.e. mimic the action of luliberin. Luliberin is a natural hormone secreted by the hypothalamus that acts on the pituitary gland by releasing the luteinizing hormone (LHj and follicle-stimulating hormone (FSHj). These two pituitary hormones control the reproductive processes, the latter, the follicle-stimulating hormone, acting on the ovaries to help The compounds of formula I are unexpectedly more effective than luliberin in their ability to release luteinizing hormone and are therefore useful for controlling and / or improving reproduction in animals. for the multiplication of large domestic animals during the anesthesia and all types of artificial insemination to more accurately control the ovulation time The compounds of the invention can also be used to improve infertility in men and women.

The agonistic properties of the disclosed compounds to luliberin can be demonstrated, for example, by their ability to induce ovulation in androgen-sterilized constant ester rats, or by their ability to release luteinizing hormone and follicle-stimulating hormone, as measured by double radioimmunoassay for blood plasma antibodies of immature male rats or anesthetic blood plasma. or diestral sheep.

The above test on androgen-sterilized rats is performed as follows:

Androgeneta sterilized female rats, prepared by administering 100 µg of testosterone propionate to rats on the third, fourth and fifth day of age, have sustained estrus as evidenced by vaginal swabs and the presence of numerous pre-ovulatory follicles in the ovaries. Administration of luliberin and its active analogues results in the release of an ovulatory dose of luteinizing hormone and follicle-stimulating hormone, as evidenced by the presence of eggs in the fallopian tubes and fresh yellow bodies in the ovaries.

All of the compounds mentioned herein are more potent than luliberin in their ability to induce ovulation in constant ester rats and, moreover, show no signs of toxicity when administered at a dose at least four times their minimum effective dose. Particularly preferred compounds of the invention, which are the compounds of Examples 6, 8 and 9, are approximately 100 times more potent than luliberin and have no toxic effects when administered at a dose 100 times their minimum effective dose. The compounds produced by the process of the invention may be used in the form of pharmaceutical or veterinary compositions containing as active ingredient a compound produced by the process of the invention, in combination with a pharmaceutically or veterinarily acceptable diluent or carrier.

The composition may, for example, be in a form suitable for oral or perlingual administration, for example in the form of a tablet, capsule, solution or suspension, for nasal administration, for example in the form of snuff, nasal spray or nasal drops, vaginal or rectal administration, for example in the form of suppositories, or for parenteral administration, for example in the form of a sterile injectable solution or suspension.

In general, the above compositions may be prepared in conventional manner using conventional excipients, but in the case of oral compositions, it may be advantageous to coat the composition with a polypeptide protecting the active component from the action of enzymes in the stomach.

The disclosed compositions may contain, in addition to the polypeptide produced by the process of the invention, one or more known drugs selected from the group consisting of prostaglandin derivatives, for example prostaglandin Fα, clopirostenol or fluprostenol, and other drugs such as clomiphene or tamoxifen.

A preferred composition is a preparation for oral administration in unit dosage form, for example a tablet, capsule, drops or bolus, containing in each unit dose an otd of 2.5 to 500 mg, preferably of 10 to 100 mg, of a polypeptide, or a preparation suitable for parenteral administration. containing from 5 µg to 1 mg of polypeptide per ml solution, preferably from 10 µg to 100 µg of polypeptide per ml solution.

The composition for parenteral administration is preferably a solution in an isotonic saline or isctonic dextrose solution, optionally buffered to a pH of 5 to 9. The composition for parenteral administration may alternatively be in a form from which the active ingredient is slowly released, in which case the amount of polypeptide is a unit dose generally higher than that of conventional injection preparations. A preferred composition for parenteral slow release administration comprises in a unit dose from 100 µg to 1.00 mg of the polypeptide.

The present compositions are normally administered so that, when administered orally, the daily dose is from 50 µg / kg to 20 mg / kg, and the daily dose when administered parenterally, for example by intravenous, sututaneous or intramuscular injection or infusion, is from 0.2 µg. / kg to 100 µg / kg. In humans, this dosage corresponds to a total daily dose of 3.5 mg to 1.4 g for oral administration and a total daily dose of 14 µg to 7 mg for parenteral administration. When administered via mucous membranes, the doses range between the above-mentioned doses for oral and parenteral administration.

The invention is illustrated by the following non-limiting examples.

In these examples, the values of R _(refer to ascending thin layer chromatography on silica gel. The solvent system used in the chromatography are also systems

1-butanol - acetic acid - water (4: 1: 5 v / v) (R _f Aj,

1-butanol - acetic acid - water - pyridine (15: 3: 12: 10 v / v) (R _f B),

2-butanol-3% (w / v) aqueous ammonium hydroxide solution (3: 1 v / v) (R _f C), acetonitrile-water (3: 1 v / v) (R (D), acetone-chloroform ( 1: 1 v / v) (RfE), chloroform - ethanol (1: 4 v / v) (RfF), cyclohexane - ethyl acetate [1: 1 v / v) (RfG), cyclohexane - ethyl acetate - methanol (1: 1 : 1 v / v) (R _£ H), chloroform - methanol - water (11: 8: 2 v / v) (R [I <), chloroform - methanol (19: 1 v / v) (RFP), and chloroform-methanol (9: 1 v / v) (RfQ).

In all cases, the chromatographic plates are examined in ultraviolet light and are detected with fluorescamine, ninhydrin and chlorine with a iodine reagent. Unless otherwise indicated, the inclusion of the symbol R _f indicates that only one spot was detected in these procedures.

Acid hydrolysates of all products in the examples are obtained by heating the peptide or protected peptide with 6 N hydrochloric acid containing 1% w / v phenol in a sealed evacuated vial to 100 ° C for 16 hours. The amino acid composition of each hydrolyzate is determined using an amino acid analyzer (LoCarte) and is in all cases consistent with the expected composition.

The term "worked up" as used in the examples means that after the reaction all solids were filtered off, the filtrate was evaporated to dryness below 40 ° C, the residue was dissolved in ethyl acetate, washed with a 20% citric acid solution, water, saturated sodium bicarbonate solution and again water, dried over anhydrous sodium sulfate, and ethyl acetate was evaporated in vacuo to give the title compound.

Examples 1 and 2

Synthesis of L-pyroglutamyl-L-histidyl-L-tryptolyl-L-seryl-L-tyrosyl-A-B-L-arginyl-L-prolyl-azaglycinamide

General Procedure (Draw Schemes 1 and 2) mg of protected decapeptide derivative [containing SerfBu ¹ ) at position 6 or TyrfBu ¹ ) at position 5] is dissolved in 5 ml of 90% (v / v) aqueous trifluoroacetic acid, 3 drops added ( The solution was allowed to stand at room temperature for 45 minutes, the solvent was evaporated in vacuo and the residue was lyophilized once with water and twice with tert-butanol (yield 90-100%).

The compounds of the invention prepared in this manner are shown in Examples 1 and 2, summarized in the following table:

199874

Table

Example A number (B) E F Method Výě- žek (%) Paper electrophoresis R _f AR _f (based on luliberin) pH 2.1 pH 6.5 1 D-Ser Leu jargly NH2 0 95 0.94 0.96 0.25 2 D-Phe MeLeu jargly NH2 0 46 0.84 0.87 0.35

The starting materials for use in the above Bzl = benzylic procedure can be prepared according to the following Z = benzyloxycarbonyl Schemes 1 and 2 [process (o)]. Boc = t-butoxycarbonyl

In these schemes the following DMF = dimethylformamide abbreviations are used:

The numbers in the circles indicate the respective re OC _P = 2,4,5-trichlorophenyl ester of the action step described below.

199B74

© OJ O \ OJ O + ffi o ^c 1 and with C j 1 of 5 C tc fe £ ΪΤ Μ J ffi O and © © © © © 1 LO t> and £ H jy H -> i ~ d Bzl / i — 1 N m · \ Bzl t 0 [> q t> q t- Cs J Π m 1 * 1 4 © 3 4 3 d

-Ser (Bu cr co

Dř

CD C \ J OJ CD C \ J 3 3 x 3 3 3 3 3 3 13

© CD O 3 + 3 \ 4 · 3 \ + \ <n a s Ž O c and C > fe > c tt D 2! 1 3 CD > 5 2 O 3 S O © © © © © ISJ t 0 q * 3 5 ca c -1 P 3 G 3 PQ \ P G ca \ P G ca \ P G ca \ P G ca \ P · G 3 l 1. 3 t 0 t and t> q ] f CD j s -t m 24 © 4 ·? 5

Stupea

The N-tert-butoxycarbonyl derivative was dissolved in ethyl acetate and treated with 3 N hydrogen chloride in ethyl acetate (4 equivalents) at room temperature for 1 hour.

E R _F = 0.49, Rf F 0.66, _Rf H = U 69, R _f Q 0.70.

It works the same as the procedure

Stupe a

5.4 mmol of L-pyroglutamyl-L-histidyl-L-tryptophyll-L-serine methyl ester are dissolved in 70 ml of dimethylformamide and treated with 100 mmol of hydrazine hydrate for 4 hours. The dimethylformamide was evaporated in vacuo, the residue was triturated with ethanol, the product collected, washed with ethanol and ether, and dried. 184 DEG-189 DEG. The yield was 88.2 percent;

R _f = 0.18 A, RFB = 0.55, R _f = C '0.39, _Rf D 0.27, _Rf K 0.58.

Grade® D-Ti / ROME>]

A solution of 3.17 g (9.64 mmol) of ZD-Tyr (Me) -OH, 1.92 g (10.6 mmol) of H-Leu-OHe.HCl, 2.6 g (19.2 mmol) of 1- hydroxybenzotriazole and 1.6 ml (11 mmol) of triethylamine in 30 ml of dimethylformamide are cooled to 0 DEG C. and 2.29 g (11.1 N, N'-dicyclohexylcarbodiimide) are added and the reaction mixture is stirred at 4 overnight. ^Q C, then worked up the usual manner. After recrystallization from hot cyclohexane gave the protected dipeptide derivative in a yield of 1.41 g (95.2%);

D R _f - 0.83, Rf E O 69, R _f P 0.72, R _f Q 0.76.

3.54 g (5.0 mmol) of the hydrazide from step

The reaction mixture was dissolved in 10 ml of dimethylformamide, cooled to -20 degrees Celsius with stirring, and then 3.38 ml (20 mmol) of 5.9 M hydrogen chloride in dioxane was added, followed by 0.6 ml (5.25 mmol). ) tert-butylnitrite. After 2 minutes, a pre-cooled solution of 2.04 g (5 mmol) of H-Arg (NO 2) -Pro-Azgly-NH 2 HCl and 3.55 ml of triethylamine (25 mmol) in 10 ml of dimethylformamide was added and stirring continued through overnight at 4 ° C. The reaction direction was worked up in the usual manner and the crude product was purified by silica gel column chromatography using chloroform, 5% (v / v) methanol in chloroform and 10% (v / v) methanol in chloroform as eluents. Yield: 3.72 g (70.9 percent);

R _f = 0.64, R _f B1 = _f , 72, R _f C = 0.55, R _f D = 0.66, R _f F = 0.40, R _f H = 0.52.

The degree ^ £) £ Á ^D-Ser (Su jj

The work is the same as in step

The product was crystallized from aqueous methanol. The yield was 90.4%; melting point 107 to 108 degrees Celsius,

R _f D = 0.80, R _f Ei = 0.68, R _f F = 0.73, R _f H = 0.72, R _f P = 0.72, R _f Q = 0.74.

To a solution of 4.59 g (6.4 mmol) of Z-Tyr (Bzl) -D-Tyr (Me) -Leu-OMe in 25 mL of dimethylformamide and 50 mL of methanol was added 12.9 mmol of hydrazine hydrate and left overnight stand at room temperature. The methanol was evaporated in vacuo, the product precipitated with water, collected, washed with water and dried. Mp 212-213 ° C,

Sets © | ^ A = D-Ser

Five hour catalytic reduction in the presence of 5% (w / w) palladium on carbon, in a mixture of dimethylformamide and water (8: 2).

Stage® [/ UD-Ser

A solution of 19.17 g (32.7 mmol) of Z-Tyr (Bzl) -OCp and 32.7 mmol of Η-Ser (BiúJ-Leu-OMe in 100 ml of dimethylformamide was allowed to stand at room temperature for 72 hours. The solid was collected, washed with ether and dried to give 17.6 g (79%), mp 135-137 ° C;

Rf D 0.80, _Rf H = 0.77, R _f Q 0.81.

Degree ® ^ = D-Ser

Work is the same as step (59)

The product was recrystallized from aqueous methanol. Yield: 56.2%; mp 134 to 136 ^Q C,

Rf D 0.66, _Rf H = 0.64, R _t Q -0.64.

Degrees | a * DSer (fiiZ1J

Work is the same as in degrees

The product was purified by silica gel column chromatography using chloroform and 5% (v / v) methanol in chloroform as eluents. The yield of the product is

38.5%; mp 142-145 ^{O C,}

RfA - 0.64, R _f B = 0.71, R _f O 0.55, _Rf D 0.65, _Rf = 0.46, R _f Hl = 0.43, R _f Q = 0.16.

(5ř)

Degree

To a solution of 5.99 g (20 mmol) of Z-Phe-OH a

2.2 ml (20 mmol) of N-methylmorpholine in 60 ml of dimethylformamide, cooled to -15 ° C, was added 1.8 ml (18 mmol) of ethyl chloroformate. After 2 minutes, a solution of 4.8 g (20 mmol) of H-MeLeu-OMe. HBr was added

2.8 ml (20 mmol) of triethylamine in 20 ml of dimethylformamide, previously cooled to -15 DEG C., is stirred for 30 minutes at 0 DEG C. and then overnight at room temperature, then worked up in the usual manner. The yield of the oily product was 7.54 g (90%).

in (75)

Step Three-hour catalytic reduction in methanol containing hydrogen chloride equivalent in the presence of 5% (w / w) palladium on carbon.

Stuperx

16.02 g (43.2 mmol) of Z-Tyr (Bu ¹ ) -OH and 13.15 g (40.0 mmol) of HD-Phe-MeLeu-OMe.HCl were condensed in the same manner as in the step. The product was purified by silica gel column chromatography using chloroform and 5% (v / v) methanol in chloroform as eluents. The yield of the oily product was 60%;

G R _f = 0.48;

R _f 'P = O, 71, RTQ = 0.73.

_t in (77)

Degree.

A solution of 6.52 g (0.76 mmol) of Z-TyrfBu-D-Pihe-MeLeu-OMe and 97.6 mmol of hydrazine hydrate in 50 mL of methanol was allowed to stand overnight at room temperature, then the methanol was evaporated in vacuo. The hydrazide was crystallized from methanol / ether, washed with methanol / water (1: 1 v / v) and ether, and dried. Yield 5.2 g (80%); melting point 135 ° C,

R _f D = 0.75,

R _f E '—0.69,

R _f = 0.66,

_Rf H = 0.79,

R _f Q = 0.73.

Work is the same as in degrees

During work-up, the product is precipitated with ethyl acetate, filtered off, washed with ethyl acetate and ether, and dried. Yield 58.5%; mp 145-148 ° C,

RfD = 0.72,

R _f = 0.40,

R, H = ^! 0,53,

Q R _f = 0.18.

Examples 3 to 12

Using the general procedure described in Examples 1 and 2, the following compounds can be prepared:

_LGlu-Hts -Trp-Ser-A-B-Ar & -Pro- £ -F

Example number AND (B) E F Paper electrophoresis R _{ (based on luliberin) R _t A pH 2 PH 6.5 3 Azgly Leu - NHC2H5 0.98 1.03 0.28 4 Azala Leu - NHC2H5 0.97 1.0 0.30 5 D-Ala Leu Azgly NH2 0.97 1.0 0.30 6 D-Phe Leu Azgly NH2 1.0 0.71 0.27 7 D-Trp Leu Azgly NH2 0.53 0.37 0.37 8 D-Tyr Leu Azgly NH2 0.92 0.87 0.25 9 D-Ser (Bu ¹⁾ Leu Azgly NH2 0.94 0.96 0.25 10 D-Tyr MeLeu Azgly NH2 0.87 0.90 0.34

PURPOSE OF THE INVENTION

Claims (5)

1. A process for the production of polypeptides of the formula I -Giu- / Vis-'I '- ^p- ® ^{r' r} Y ^r where A means D-Tyr, D-Tyr (Me), D-Ser, D-Ser (Bu ¹ ), D-Phe, D-Ala, or D-Trp, B represents Leu or MeLeu, E is Azgly and F is amino, or And it means Azgly or Azala, B represents Leu, E is a direct bond and F is ethylamino, and their pharmaceutically and veterinarily usable acid addition salts, characterized in that one or more protecting groups common in peptide chemistry are cleaved from the respective protected polypeptides to form a compound of formula (I) above, and optionally the resulting product in an unprotonated form by reaction with an acid providing a pharmaceutically or veterinarily usable anion is converted to its salt. 2. A method according to claim 1, wherein the starting material is a protected polypeptide in which the imino protecting group is a benzyloxycarbonyl group-AB-Ar &apos; -Pro- [beta] -F (I) pin and / or a benzyl hydroxyl protecting group. group. 3. The method of claim 1, wherein the protected substance is a protected polypeptide in which the imino protecting group is a tert-butoxycarbonyl group and / or a hydroxyl protecting group is a tert-butyl group. 4. The method of claim 1, wherein the protected substance is a protected polypeptide in which the imino protecting group is a benzyloxycarbonyl or t-butoxycarbonyl group and / or a hydroxyl function protecting group is a tert-butyl group. 5. The method of claim 1, 2 or 3, wherein the starting polypeptide is A is D-Ser (Bu ¹ ), B represents Leu, E is Azgly and F is amino, the starting material also containing one or more protecting groups cleavable by hydrogenolysis or by treatment with hydrogen chloride or trifluoroacetic acid.