CH634549A5 - POLYPEPTIDES AND THEIR PREPARATION. - Google Patents

Description

The present invention relates to Polypeptides and to their preparation.

Encephalin, which is a natural agonist of opiate receptors in the brain, has been identified [see Hugues et al., "Nature" 256.577 (1975)] as a mixture of two pentapeptides: H — Tyr— Gly — Gly — Phe — Met — OH (methionine / encephalin) and H — Tyr — Gly — Gly — Phe — Leu — OH (leucine / encephalin). These two peptides mimic or reproduce the ability of morphine to block the electrically induced contractions of the mouse vas deferens and the guinea pig ileum, and they both inhibit binding to the stereospecific receptors of the antagonist d opiate consisting of 3H-naloxone in brain homogenates.

It has been suggested that encephalin receptors may be sites where morphine-type drugs exert their analgesic activities, and that encephalin may be the modulator or transmitter of analgesia or pain-relieving effects in the brain systems . Methionine / encephalin and leucine / encephalin, when given by injection into the ventricle of the rat brain, have been reported to produce deep analgesia which is completely reversible with naloxone [see Belluzzi et al., "Nature 260.625 (1976)]. Encephalins, however, are inactive during peripheral administration and are believed to be rapidly destroyed by enzymes in the blood and / or poorly transported across the blood-brain barrier.

The amino acid sequence of methionine / encephalin is identical to that of the N terminal portion of fragment C (ß-endorphin or a-LPH [61-91]) of the ß-lipotropin peptide, which is found in concentrations important in the pituitary fluid and in much lower concentrations in the brain. Other natural fragments of ß-lipotropin are known, for example: a-endorphin (ß-LPH [61-76]) and y-endorphin (ß-LPH [61-77]). Ss-lipotropin and endorphins all show properties resembling those of morphine in various test systems and it has been suggested that methionine / encephalin is a dissociative of important opiate peptides. Encephalin, its relationships with ß-lipotropin and with endorphins, and its pharmacological properties have been highlighted in an article by Iversen et al., "Nature" 262,738 (1976). Recent developments have also been described in detail in "Proceedings of the International Narcotics Research Club Meeting", Aberdeen, Great Britain, July 19-22,1976 ", published in" Opiates and Endogenous Opioid Peptides ", North Holland Publishing Company, Amsterdam, 1976.

Various structural variations of methionine / encephalin and leucine / encephalin have been described in the literature. For example, the pentapeptide H — Tyr — Gly — Gly — Phe — Thr — OH, in which the fifth amino acid residue (methionine or leucine) is replaced by threonine, has been described by Chang et al., “ Life Sciences ”18.1473 (1976). Likewise, a long-acting synthetic pentapeptide, Tyr-D-Ala-Gly-Phe-Met-amide has been described by Pert et al., "Science" 194, 330 (1976); this compound, like the natural encephalins, is inactive during peripheral administration. Baxter et al., “British Journal of Pharmaco-logy”, March 2 (1977), pp. 455-456 and 523, reported that the compound Tyr-D-Ala-Gly-Phe-D-Leu shows activity upon intracerebroventricular administration.

According to the present invention, a group of polypeptides, active in medicine, of the formula:

H — N — Tyr — D — Ala — Gly — N — Phe — D — Pro — R2

I I (D

R R1

in which:

R represents hydrogen or a methyl, allyl or cyclo-propylmethyl radical;

R1 represents hydrogen or the methyl radical, and

R2 represents —OH, - NH2, or —NHCnH2n + 1, where n is 1,2,3 or 4.

The invention also encompasses the pharmaceutically acceptable salts of these compounds.

All chiral amino acid residues, in the preceding formula I and in the following description, are in the natural configuration or L, unless otherwise indicated.

The preferred compounds are those which correspond to the following formula II:

R — NH — Tyr — D — Ala — Gly — N —Phe — D - Pro —R2 (H)

R1

5

10

15

20

25

30

35

40

45

50

55

60

65

3

634549

in which:

R represents hydrogen or a methyl, allyl or cyclo-propylmethyl radical;

R represents hydrogen or the methyl radical, and

R2 represents —NH2 or —NHC2H5 5

or the pharmaceutically acceptable acid addition salts of these preferred compounds.

The pharmaceutically acceptable salts of the polypeptides according to the invention are acid addition salts of the free base, in which the acid can be organic or inorganic, for example hydrochloric acid, phosphoric acid, maleic acid, acetic acid, citric acid, succinic acid, malic acid and the like. Likewise, salts of the free peptide acid are encompassed by the term pharmaceutically acceptable salts and these include, in particular, the sodium, potassium, ammonium and lower alkylamino salts. The salts are prepared and isolated using traditional methods.

The analgesic polypeptides of the present invention can be prepared by conventional solid phase methods, using either a benzhydrylamine / polystyrene resin for the production of C-terminal amides, or a cross-linked polystyrene resin. divinylbenzene, chloromethylated or hydroxy-methylated for the production of the carboxylic acid or lower C-terminal alkylamides. The polypeptide is traditionally separated from the resin support by hydrofluoric acid and is purified by gel filtration. Likewise, the compounds of the present invention are easily prepared by traditional methods in solution, by which the individual amino acid reagents are combined, via activated derivatives, in the appropriate order with temporary protection of the functional groups. competitively reactive nels in order to obtain the desired product.

The N-substituted tyrosine and phenylalanine reagents used in the production of the compounds described herein are readily prepared by the reaction of methyl chloride, allyl chloride or cyclopropylmethyl chloride with a Boc-tyrosyl ester or a Boc-phenylalanine ester in the presence of silver oxide. The N-alkylated product is then saponified and hydrolyzed to obtain the desired reagent.

The analgesic activity of the polypeptides of the present invention has been demonstrated by the method of D'Amour and Smith, "J. Pharmacol. 40 Exp. Ther. " 72.74 (1941). The representative polypeptide of the invention, namely Tyr-D-Ala-Gly-Phe-D-Pro-NH2, gave the following results in the conventional rat tail touch test.

45

Reply

Number of rats showing analgesia / Number of rats treated

200,400

0/6 3/6

0/6 2/6

0/6 2/6

0/6 2/6

Intravenous dose

Minutes after administration

(mg / kg)

15

30

60

5.0 *

5/5

5/5

2.5

6/6

5/6

3/6

1.0

4/6

3/6

1/6

0.5

2/6

2/6

1/6

0.25

0/6

0/6

0/6

Subcutaneous dose

Minutes after administration

(mg / kg)

30

60

90.

120

5.0

3/6

0/6

0/6

0/6

20.0+

6/6

6/6

5/6

3/6

Oral route Minutes after administration

(mg / kg) 30 60 90 120

50 0/6 1/6 0/6 0/6

100 0/6 0/6 0/6 0/6

* In a group of 6 rats, one died before the test; 2 of the 5 remaining rats showed respiratory depression and one died after being caged.

+ The full group of 6 rats showed moderate respiratory depression at 20 mg / kg subcutaneously.

The test results demonstrate that the compounds of the present invention cause analgesia when administered with a single intravenous injection of about 0.5 mg / kg or more. For practical reasons, it has been considered, on the basis of the previous test results, that an intravenous dose of about 0.5 to about 20 mg / kg in a single dose or in multiple doses is the appropriate dose to achieve the degree of analgesia desired in various applications. When administered orally, a dose of approximately 400 mg / kg or more produces the desired effect. The exact dose to be used will obviously depend to some extent on the specific compound used, the patient and the degree of analgesia desired. The determination of a precise dose for obtaining a desired effect is easily done empirically by the doctor.

The following examples further illustrate the preparation of the polypeptides according to the invention.

Example 1:

L- Tyrosyl — D — A lanylglycyl - L-Phenylalanyl — D — Pro — Amide

9.2 g of benzhydrylamine resin (free amine content of 3.7 mmol) are placed in a 200 ml reaction vessel. The resin is then treated in the following manner;

1. methylene chloride (three times);

2. 5 min prewash with methylene chloride / trifluoroacetic acid (30%) (volume / volume), containing 0.5% dithio-erythritol;

3. treatment for 25 min with the above trifluoroacetic acid;

4. methylene chloride (three times);

5. treatment for 10 min with triethylamine / dimethyl-formamide (volume / volume);

6. • dimethylformamide (three times);

7. methylene chloride (three times).

A contact time of 2 min is allowed for each wash unless otherwise indicated.

The resin is moderately stirred with t-Boc-D-Proline and 1-hydroxybenzotriazole (HOBT) in dimethylformamide / methylene chloride (50%) (3.17 g, 14.7 mmol det — Boc — D — Pro and 3.4 g, 22 mmol HOBT). After the addition of the preceding reagents, the mixture is treated with 2.6 ml of diisopropylcarbodiimide (DIC) (the latter is added in two equal portions over a period of 30 min). After stirring overnight, the resin-peptide is washed successively with dimethylformamide (twice), dimethylformamide / triethylamine (12%) (once) and methylene chloride (three times). To verify the completeness of the reaction, the resin-peptide is subjected to the ninhydrin staining test following the method of E. Kaiser et al., "Analytical Chemistry" 34, 595 (1970).

The removal of the protection of the attached amino acid is carried out as described in phases 2 to 7 above.

The following amino acid residues are then introduced in succession: t - Boc-L-phenylalanine (3.90 g, 14.7 mmol, 3.4 g, 22 mmol of HOBT and 2.6 ml, 16.2 mmol of DIC) , t — Boc — glycine (2.58 g, 14.7 mmol, 3.4 g of HOBT and 2.6 ml of DIC), t — Boc — D — alanine (2.78 g, 14.7 mmol, 3.4 g HOBT and 2.6 ml DIC), t — Boc — O — benzyl — L — tyrosine (5.46 g, 14.7 mmol, 3.4 g HOBT and 2.6 ml DIC ). The resin-peptide is dried (production of 16.9 g).

The resin-pentapeptide described above (16.9 g) is treated under vacuum with anhydrous liquid hydrofluoric acid (100 ml) and

634549

4

anisole (10 ml) at 0 ° C for 1 h. The hydrofluoric acid and the anisole are separated under reduced pressure and the rest is suspended in 2N acetic acid, then filtered, and the filtrate is lyophilized to give the product mentioned in heading above, in an amount 7.0 g.

Example 2:

Purification and characterization of L-Tyrosyl — D — Alanylglycyl— L-Phenylalanyl —D — Pro — Amide

The crude product from Example 1 is purified as follows: 2.3 g of material are dissolved in a minimum amount of 2N acetic acid and the solution is applied to a column (2.5 x 200 cm) of Sephadex G-10 in 2N acetic acid. The column is eluted with 2N acetic acid and 8.2 ml fractions are collected. The column effluent is checked at 254 nm. The fractions 50-59 are combined and lyophilized to obtain 0.780 g of product mentioned in heading in the acetate form. The product (0.780 g) is further purified by applying the material in a small volume of an upper BAE phase, 4/1/5 (n-butanol / acetic acid / water) on a column (2.5 x 100 cm ) of a Sephadex G-25 medium previously balanced with a lower phase of the previous system and then balanced upwards with a lower phase of the previous system and then an upper phase. The column is eluted with an upper phase B-A-E and 4 ml fractions are collected. The effluent is controlled as before. Tubes 35-47 were shown to be homogeneous in 4 / 1/5 thin layer chromatography systems; Rf = 0.40, and 7/7/6 (isoamyl alcohol / pyridine / water); Rf = 0.72 on silica gel. Thin layer chromatograms were visualized with ninhydrin and a peptide / chlorine reagent.

Analysis of the amino acids, after hydrolysis with methanesulfonic acid, gave the following proportions: Pro 1.00; Gly 0.94; Tyr 1.01; Phe 1.00; Ala 0.98.

Example 3:

L-Tyrosyl —D — Ala — Glycyl -L-Phenylalanyl — D — Pro — OH

A chloromethylated polystyrene resin is esterified with Boc-D-Pro-OH according to the method of Gisin, "Helv. Chim. Acta. " 56.1976 (1973), and the polymeric ester is treated according to the method of Example 1 for the incorporation of Boc — Phe — OH, Boc — Gly — OH, Boc — D — Ala — OH, and from Boc — Tyr (Bzl) —OH. The resulting resin-peptide is treated according to the method of Example 2 to give the pentapeptide acid mentioned in the heading.

Example 4:

Lr Tyrosyl — D — Ala — Glycyl -L-Phenylalanyl — D — Pro— Ethylamide

Treatment of the resin-peptide of Example 3 with ethyl amine in a stoppered flask for 10 h, then separation of the excess ethylamine, extraction with dimethylformamide, filtration and evaporation of the filtrate , gives the ethylamide cited in the heading.

Example 6:

N-Methyl — L-Tyrosyl — D — Alanylglycyl — L-Phenylalanyl— D_ — Pro — Ethylamide

The process of Example 3 was repeated except that the last combined amino acid is Boc-N-Methyl-Tyr— (Bzl) —OH. The resin-peptide is treated with excess ethylamine overnight in a pressure glass bottle. The crude mixture is filtered and the excess ethylamine is separated under vacuum. The product is lyophilized at the start of dioxane. The removal of the protection is carried out with HF and anisole and the excess of this HF and this anisole is separated under reduced pressure. The remainder is dissolved in 2N acetic acid and lyophilized. The crude product is purified by the method of Example 2. Tubes 27-37 contain the homogeneous product, which is demonstrated by thin layer chromatography with isoamyl alcohol systems. pyridine / water, 4/1/5, Rf = 0.53; 7/7/6, RF = 0.70 on silica gel.

Amino acid analysis gives the following proportions: Pro 1.00; Phe 1.00; Gly 1.02; Ala 1.00; N — MeTyr: not determined.

The product of Example 6 was tested according to the method of D'Amour and Smith, "J. Pharmacol. Exp. Ther. " 72.74 (1941) with the following results:

Number of rats with analgesia /

25

Number of rats tested

Dose

Minutes after administration

(mg / kg)

Administration

15 30

60

90 120

5.0

intravenous

4/6 (2 deaths)

1.0

intravenous

5/6 (1 death)

30 0.5

intravenous

5/6

1.0

subcutaneous

3/6

5.0

subcutaneous

5/6

0.1

intravenous

0/6

5.0

subcutaneous

5/6

5/6

5/6 5/6

35 1.0

subcutaneous

2/6

1/6

0/6 0/6

50

L-Phenylalanyl — D—

Example 5:

N-Methyl— L-Tyrosyl — D — Ala — Glycyl -Pro — Amide

The process of Example 1 was repeated except that the last amino acid introduced into the solid phase reactor was Boc-N-methyl-L-tyrosyl (Bzl) -OH. The resin-peptide is split and treated according to the method of Example 2 to give the compound mentioned in the heading.

Example 7:

N-Methyl - Tyrosyl — D — Alanyl - Glycyl — N-Methyl— Phenyl-alanyl — D — Pro — Ethylamide

The process of Example 3 was repeated except that N-methyl-phenylalanine was introduced into the resin-peptide as the second amino acid residue. The product is treated and purified by following the process of Example 2 to obtain the compound mentioned in the heading. Thin layer chromatography is carried out on silica gel with the butanol / acetic acid / water system (4/1/5), Rf = 0.34.

Amino acid analysis gives the following proportions: Pro: 0.92; Gly: 1.00; Ala: 0.99; N — Me — Phe: not determined; N — MeTyr: not determined.

During a test by the conventional method of touching the tail of the rat, the product of Example 7 gives the following results:

Number of rats with analgesia / Number of rats tested

Dose Minutes after administration

(mg / kg)

Administration

15

30

60

5

intravenous

5/5

5/5

4/4

1

intravenous

1/6

1/6

0/6

Claims (8)

634,549 1. Therapeutically active polypeptides corresponding to the formula: H — N — Tyr — D — Ala — Gly — N — Phe — D — Pro —R2 I I R R1 in which: R represents hydrogen or a methyl, allyl or cyclo-propylmethyl radical; R1 represents hydrogen or a methyl group, and R2 represents —OH, —NH2 or - NHCnH2n + i, where n is 1, 2,3 or 4; as well as their pharmaceutically acceptable salts. 2. Compounds according to claim 1, characterized in that it is Tyr-D-Ala-Gly-Phe-D-Pro-NH2 or its pharmaceutically acceptable salts. 2 3. Compounds according to claim 1, characterized in that it is Tyr-D-Ala-Gly-Phe-D-Pro-OH or its pharmaceutically acceptable salts. 4. Compounds according to claim 1, characterized in that they are Tyr-D-Ala-Gly-Phe-D-Pro-NHC2 H 5 or its pharmaceutically acceptable salts. 5. Compounds according to claim 1, characterized in that it is N-méthy I — Tyr — D — Ala — Gly — Phe — D — Pro — NH2 or its pharmaceutically acceptable salts. 6. Compounds according to claim 1, characterized in that it is N-methyl -Tyr - D - Ala - Gly - Phe - D - Pro - NHC2H5 or its pharmaceutically acceptable salts. 7. Compounds according to claim 1, characterized in that it is the N-methyl — Tyr — D — Ala — Gly — N-methyl - Phe — D— Pro — NHC2H5 or its pharmaceutically acceptable salts. 8. Process for producing a compound corresponding to the formula: H —N — Tyr — D — Ala — Gly — N — Phe — D — Pro — R2 I I R R1 in which: R represents hydrogen or a methyl, allyl or cyclo-propylmethyl radical; R1 represents hydrogen or methyl, and R2 represents —OH, - NH2 or - NHCnH2n + i, where n is equal to 1, 2,3 or 4, or of a pharmaceutically acceptable salt of such a compound, characterized in that it comprises the condensation of the corresponding amino acids or of their activated derivatives with formation of —CO — NH bonds — in the desired order of succession, with temporary protection reactive functional groups.