DE2829610A1 - POLYPEPTIDES, METHOD FOR THE PRODUCTION THEREOF AND MEDICINAL PRODUCTS - Google Patents

Description

PATENT LAWYERS

patent attorney

PROF. DR. DR. J. REITSTÖTTER 2829610

DR.-ING. WOLFRAM BUNTE DR. WERNER KINZEBACH j

BAUERSTRASSE 22. D-BOOO MUNICH ΔΟ ■ FERNRUF (O89. 37 63 83 ■ TELEX 921S2O8 ISAfI D POSTAL ADDRESS: POST BOX 78O. D-BOOO MUNICH

Munich, July 5, 1978 [

M / 19 167!

AMERICAN KOME PRODUCTS CORPORATION 685, Third Avenue New York, N.Y. 10017 UNITED STATES,

Polypeptides, processes for their production and pharmaceuticals

809883/0 9 94

The present invention relates to a group of polypeptides of the general formula I:

HN-Tyr-D-Ala-Gly-N-Phe-D-Pro-R ² (I)

I I

RR ¹

wherein:

R stands for hydrogen , methyl, allyl or cyclopropylmethyl

stands,
R is hydrogen or methyl,

and
R is -OH, -NH- or -NHC H _{2n + 1} , wherein

η has the meanings 1, 2, 3 or 4, as well as their pharmaceutically acceptable salts.

All chiral amino acid residues in formula I and in the rest of the description are in the natural or L configuration unless otherwise noted.

Preferred compounds are the compounds of the general formula II:

R-NH-Tyr-D-Ala-Gly-N-Phe-D-Pro-R (II)

R ¹

R is hydrogen, methyl, allyl or cyclopropylmethyl, R represents hydrogen or methyl:. ,

and
R has the meanings -NH "or -NHC ₂ H ₅ ,

and their pharmaceutically acceptable acid addition salts.

809B83 / 0994

^{M / l9167} - ⁸ - 28296Ί0

Pharmaceutically acceptable salts of the invention Polypeptides are acid addition salts of the free base with organic or inorganic acids, in particular Hydrochloric acid, phosphoric acid, maleic acid, acetic acid, citric acid, succinic acid, malic acid and like that. The term "pharmaceutically acceptable salts" also includes salts of free peptide acid, especially those Sodium, potassium, ammonium and lower alkyl amine salts. These salts are prepared and isolated in a conventional manner.

The analgesic polypeptides of the invention are manufactured using typical solid-phase processes, either using a resin based on benzhydrylamine-polystyrene for the production of the C-terminal amide, or a chloromethylated or hydroxymethylated with Divinylbenzene crosslinked polystyrene resin used to produce the C-terminal carboxylic acids or lower alkyl amides. The polypeptide is recovered from the resin carrier with the aid of hydrogen fluoride in a conventional manner and through Gel filtration purified. In a similar manner, the invention Compounds can be obtained by conventional solution methods, using the individual amino acid reagents can be coupled via activated derivatives in any order, with reactive functional groups that tend to compete are temporarily protected.

The in the preparation of the compounds according to the invention N-substituted tyrosine and phenylalanine reagents used are easily converted by the reaction of methyl chloride, Allyl chloride or cyclopropylmethyl chloride with a Boc-tyrosyl ester or Boc-phenylalanine ester in the presence made of silver oxide. The N-alkylated product will then saponified and hydrolyzed to give the desired compound.

809883/0994

M / 19

The analgesic activity of the polypeptides of the invention is made using the method of D'Amour and Smith, J. Pharmacol. Exp. Ther.f 7_2_, 74 (1941). The representative Polypeptide according to the invention Tyr-D-Ala-Gly-Phe-D-Pro-NH " showed the following in the standard "rat tail flick" test Results:

dose

Intravenous mg / kg

5.0 2.5 1.0 0.5 0.25

subcutaneous mg / kg

5.0 20.0 ⁺

oral mg / kg

50
100
200
00

reaction 60 Number of d α animals with analgesia / Anz- d “tested
animals 3/6 15th Minutes after administration 1/6 5/5 30th 1/6 6/6 5/5 0/6 4/6 5/6 administration 2/6 3/6 90 120 0/6 2/6 0/6 0/6 0/6 5/6 3/6 30th Minutes after administration 3/6 60 90 120 6/6 0/6 0/6 0/6 6/6 0/6 0/6 30th Minutes after 0/6 0/6 0/6 60 2/6 2/6 0/6 1/6 0/6 0/6 3/6 0/6 2/6

In a group of 6 rats, one died prior to testing; two of the remaining 5 rats showed respiratory depression and one died when she was put back in the cage-

809883/0994

The entire group of 6 rats showed moderate respiratory depression upon administration of 20 mg / kg subcutaneously.

The test results show that the compounds according to the invention cause analgesia when administered intravenously at a single dose of ~ 0.5 mg / kg or higher. For practical use, based on the foregoing test results, it is recommended that an intravenous dose of about 0.5 to about 20 mg / kg in single or multiple doses be used as the appropriate dosage to achieve the degree of analgesia desired for various uses. For oral administration, a dose results in about 400 mg / kg or above the desired effect _t The exact dose to be administered will naturally vary somewhat, depending on the particular compound used, the patient and the desired degree of analgesia. The determination of an exact dose to achieve a desired effect can easily be determined empirically by a physician.

The following examples are intended to facilitate the preparation of the inventive Explain polypeptides in more detail.

809883/0994

Example 1 L-Tyrosyl-D-Alanylglycyl-L-Phenylalanyl-D-Prolinamide

9.2 g of benzhydrylamine resin are placed in a 200 ml reaction vessel (3.7 mmol free amine content). Then treat the resin as follows:

1 "Three times with methylene chloride,

2. A pre-wash is carried out for 5 minutes with 30% strength trifluoroacetic acid in methylene chloride (vol / vol) which contains 0.5 % dithioaerythritol;

3. then treated for 25 minutes with the above Trifluoroacetic acid, ■ ■

4. treated three times with methylene chloride ,

5. Treated with triethylamine-dimethylformamide for 10 minutes

(Vol / Vol),

6. three times with dimethylformamide and

7. three times with methylene chloride.

Unless otherwise stated, the contact time for each wash is 2 minutes.

The resin is then carefully stirred with tert-Boc-D-proline and 1-hydroxybenzotriazole (HOBT) in 50% methylene chloride-dimethylformamide (3.17 g, 14.7 mmol of t-Boc.D-Pro and 3, 4 g, 22 mmol HOBT). After adding the above reactants, the mixture is treated with 2.6 ml of diisopropylcarbodiimide (the DIC is added in two equal portions _{over 30 minutes) o} After stirring overnight, the peptide-resin is washed successively with dimethylformamide (twice) , 12% triethylamine-dimethylformamide (once) and three times with methylene chloride. In order to check the reaction for completeness, the peptide-resin is subjected to a ninhydrin

809883/0994

Staining test according to the method of E. Kaiser et al., Analytical Chemistry, 3-4,595, (1970).

The deprotection of the bound amino acid is as above carried out in steps (2) to (7) described.

The following amino acid residues are then introduced one after the other: t-Boc-L-phenylalanine (3.90 g, 14.7 mmol, 3.4 g, 22 mmol HOBT and 2.6 ml, 16.2 mmol DIC), t- Boc-glycine (2.58 g, 14.7 mmol, 3.4 g HOBT and 2.6 ml 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 peptide-resin is dried, the yield is 16.9 g.

The pentapeptide resin described above (16.9 g) is im Vacuum with. anhydrous liquid hydrogen fluoride (100 ml) and anisole (10 ml) for 1 hour at 0 °. Hydrogen fluoride and anisole are removed under reduced pressure, the residue is suspended in 2N acetic acid, filtered ' and the filtrate is lyophilized, 7.0 g of the title product being obtained.

example

Purification and characterization of

L-Tyrosy1-D-Alanylglycyl-L-Phenylalanyl-D-Prolinamide

The crude product obtained according to Example 1 is purified as follows: 2.3 g of the material are dissolved in a minimal amount Amount of 2N acetic acid and apply it to a Sephadex G-10 column (2.5 χ 200 cm) in 2N acetic acid. The column is eluted with 2N acetic acid and fractions of 8.2 ml are collected. The column effluent is monitored at 254 nm.

809883/0994

ORIGINAL INSPECTED

Fractions 50-59 are combined and lyophilized, 0.780 g of the desired compound being obtained in the form of the acetate. The product (0.780 g) is further purified by placing the material in a small volume of the upper phase of B: A: W = 4: 1: 5 (n-butanol: acetic acid % water) on a column (2.5 χ 100 cm) Sephadex G-25 medium, which has previously been equilibrated with the lower phase and then with the upper phase of the above system. The column is eluted with the upper phase B: A: W and fractions of 4 ml * are collected. The outflow is monitored as above. By thin layer chromatography systems 4: 1: 5; Rf = 0.40 and 7: 7: 6 (isoamyl alcohol: pyridine: water); Rf = 0.72 on silica gel shows that the contents of tubes 35 to 47 are homogeneous. The thin-layer chromatograms are visualized using ninhydrin and chloropeptide reagent.

Amino acid analysis results after hydrolysis with methanesulfonic acid the following ratios:

Per 1.00; GIy 0.94; Tyr 1.01; Phe 1.00; AIa 0.98. '

example

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

Chloromethylated polystyrene resin is prepared by the method of 'Gisin, HeIv. Chim. Acta., 56 , 1976 {1973) esterified with Boc-D-Pro-OH j, the ester polymer is then after the! Method of Example 1 for the introduction of Boc-Phe-OH, Boc-Gly-OH, Boc-D-Ala-OH and Boc-Tyr (BzI) -OH. The resulting peptide-resin is then treated by the procedure of Example 2, the desired pentapeptide acid being recovered.

809883/0994

^{M / 19 167} " ¹⁴ " 2823610

Example 4 L-Tyrosyl-D-Ala-Glycyl-L-Phenylalanyl-D-Pro-Ethylamide

If the peptide resin is treated according to Example 3 in a sealed flask with ethylamine for 10 hours then excess ethylamine, extracted with DMF, filtered and the filtrate is evaporated to give the desired one Ethylamide.

Example 5

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

The procedure of Example 1 is repeated with the exception that the last one is in the solid-phase reactor introduced amino acid around the Boc-N-methyl-L-tyrosyl (BzI) -OH acts. The peptide resin is cleaved and processed according to the method of Example 2, wherein the title compound receives.

example

N-methyl-L-tyrosyl-D-alanylglycyl-L-phenylalanyl-D-proline-ethylamide

The procedure of Example 3 is repeated with the exception that the amino acid coupled last is Boc-N-methyl-Tyr (BzI) is -OH. Treat the peptide-resin with excess ethylamine in a glass pressure flask overnight. The crude product mixture is filtered and the excess ethylamine is removed in vacuo. The product will be out Dioxane freeze-dried. The protecting groups are using

80988 3/0994

M / 19 167

of hydrogen fluoride and anisole are removed, and excess hydrogen fluoride and anisole are removed under reduced pressure. The residue is dissolved in 2N acetic acid and lyophilized. The crude product is then purified by the process of Example 2. In thin-layer chromatography with isoamyl alcohol: pyridine: water 4: 1: 5, Rf = 0.53; 7: 7: 6, RF = 0.70 on silica gel shows that the contents of tubes 27 to 37 are homogeneous.

The amino acid analysis gave the following ratio values: Per 1.00; Phe 1.00; GIy 1.02; AIa 1.00; N-MeTyr not determined.

The product according to Example 6 is made according to the method of D'Amour and Smith, J. Pharmacol, Exp. Ther »7_2 '74 (1941) investigated, with the following results:

Dose mode of administration mg / kg

5.0 i.v.

1.0 IV

0.5 i.v.

1.0 see Co

5.0 s.c.

0.1 i.v.

5.0 s.c.

1.0 s.c.

Number of animals showing analgesia / number of animals tested (time minutes)

15th 30 60 5/6 90 120 j 576 (2/6 dead) 1/6 i
l 5/6 (1/6 dead) l
i
1
; 5/6 5/6 ^: 3/6 0/6 5/6 0/6 5/6 5/6 2/6 0/6

809883/0994

example

N-methyl-tyrosyl-D-alanyl-glycyl-N-methyl-phenylalanyl-! D-proline ethylamide

The procedure of Example 3 is used with the
The exception is that N-methyl-phenyl-jalanine is introduced into the peptide resin as the second amino acid residue. The product is then worked up and purified according to the procedure of Example 2, the title compound then being obtained.

Thin layer chromatography on silica gel butanol: acetic acid: '. Water (4: 1: 5) Rf = 0.34.

The amino acid analysis resulted in the following ratios:

Pro 0.92; GIy 1.00; AIa 0.99; N-Me-Phe not determined;
N-MeTyr not determined.

The rat tail flick test with the product according to Example 7
showed the following results:

dose
mg / kg Mode of administration number the Animals with Analgesia / I.
i
!
Minutes) 5
1 iv
iv number the tested animals (time 60_ j
4/4;
0/6 y I ⁵ .
5/5
1/6 30th
5/5
1/6

809883/0994

Claims (8)

Claims 1. / Polypeptides of the general formula HN-Tyr-D-Ala-Gly-N-Phe-D-Pro-R ² I. RR ¹ wherein R is hydrogen, methyl, allyl or cyclopropylmethyl, R stands for hydrogen or methyl / and
R ² for -OH, -NH "or -NHC H _{3 +} . is where η denotes 1, 2, 3 or 4, and their pharmaceutically acceptable salts. 2. Tyr-D-Ala-Gly-Phe-D-Pro-NH ₂ and its pharmaceutically acceptable salts. 3. Tyr-D-Ala-Gly-Phe-D-Pro-OH and its pharmaceuticals tolerable. Salts .. 4. Tyr-D-Ala-Gly-Phe-D-Pro-NHCpH, - and its pharmaceutical compatible salts 809883/0994 ORIGINAL INSPECTED 5. N-Methyl-Tyr-D-Ala-Gly-Phe-D-Pro-NH- and its pharmaceutical compatible salts. 6. N-methyl-Tyr-D-Ala-Gly-Phe-D-Pro-NHC ^ EL and its pharmaceutically acceptable salts _u 7. N-Methyl-Tyr-D-Ala-Gly-N-methyl-Phe-D-Pro-NHC ₂ H ₅ and its pharmaceutically acceptable salts » 8. Process for the preparation of compounds of the general formula: HN-Tyr-D-Ala-Gly-N-Phe-D-Pro-R ² I I RR ¹ wherein R stands for hydrogen, methyl, allyl or cyclopropylmethyl stands; R is hydrogen or methyl, and
R ^{2 is} -OH, -NH ₂ or -NHC _n H _{2n + 1} , in which η is 1, 2, 3, or 4; and their pharmaceutically acceptable salts, characterized in that that one has the required amino acids or their activated derivatives with the formation of -CO-NH- bonds in each desired order condensed, whereby you get all reactive functional groups, which to competing reactions temporarily protects. 809883/0994 m / 19 167 - ^ -3 28296-1Q ο Pharmaceutical agents containing at least one compound according to claims 1 to 7 in a pharmaceutically acceptable carrier, optionally together with customary Additives. 809883/099Λ M / 19 167 yf Enkephalin, a natural opiate receptor agonist in the brain, was found as a mixture of two pentapeptides: H-Tyr-Gly-Gly-Phe-Met-OH (methionine-enkephalin) and H-Tyr-Gly-Gly-Phe-Leu-OH (Leucine-enkephalin) identified [cf. Huges et al., Nature , 256 , 577 (1975)]. Both peptides mimick the ability of morphine to block electrically induced contractions of the vas deferens in mice and ileum in guinea pigs, and both inhibit stereospecific receptor binding of the opiate antagonist 3H-naloxone in brain homogenates. It has been assumed that enkephalin receptors are the stems on which morphine-like drugs exert their analgesic effects, and that enkephalin could be the modulator or transmitter for pain suppression or analgesia in the brain system. It has been reported that methionine-enkephalin and leucine-enkephalin, when administered by injection into the cerebral ventricles of rats, produce deep analgesia which is completely reversible by naloxone. [See. Belluzi et al., Nature , 260 , 625 (1976) 1. However, the enkephalins are ineffective when administered peripherally and are believed to: that the enkephalins are rapidly destroyed by the enzymes in the blood and / or are poorly transported across the blood-brain cupboards will. The amino acid sequence of methionine-enkephalin is identical to the n-terminal part of the C-fragment (ß-endorphin or a-LPH [61-69]]) of the peptide ß-lipotropin, which in high 809883/0994 Ι Μ / 19 167 Concentration in the pituitary gland and in much smaller con- ■ concentrations in the brain are found. There are others too naturally occurring ß-lipotropin fragments known, \ in particular α-endorphin (β-LPH [61-76]) and γ-endorphin (β-LPH [61-77]). Both, ß-lipotropin and the endorphins ^; show morphine-like properties in various experiments, and it has been assumed that methionine-enkephalin is a degradation product of the large opiate-like peptides. Enkephalin, its relationship to β-lipotropin and the endophins, and their pharmacological properties are summarized in an article by Iversen et al., In Nature , 262 , 738 (1976). Recent developments are also detailed in Proceedings of the International Narcotics Research Club Meeting, Aberdeen, UK, July 19-22, 1976, published in Opiates and Endogenous Opioid Peptides , North Holland Publishing Company _s Amsterdam, 1976 * Various structural variations of methionine-enkephalin and leucine-enkephalin are 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, is described by Chang et al., In Life Sciences, 18 , 1473 (1976) . A long-acting, synthetic pentapeptide, Tyr-D-Ala-Gly-Phe-Met-amide, is also described by Pert et al., Science, 194 , 330 (1976) which: like the natural enkephalins, is ineffective when administered peripherally . Baxter et al., British Journal of Pharmacology, March 2, 1977, pages 455P-156P and 523P indicate that the compound Tyr-D-Ala-Gly-Phe-D-Leu is effective when administered intracerebroventricularly. 809883/099 U