DD287264A5 - PROCESS FOR PREPARING NUTRITIONAL PHOSPHIVES OF PEPTIDE DERIVATIVES - Google Patents

Abstract

The invention relates to a process for the preparation of nutritionally effective peptide derivatives (preferably L-tyrosyl-L-proline L-phenylalanine pyrrolidide) which significantly increase insulin release both in vitro and in the whole animal (rat), promote amino acid uptake through the intestinal wall, food utilization improve by delayed intestinal passage of the porridge and are characterized by good tolerability. The invention is of importance for animal nutrition {method; Peptide derivatives; L-tyrosyl-L-proline-L-phenyl-alaninpyrrolidid; Insulin release; Animal nutrition}

Description

Field of application of the invention

The invention relates to a method for producing nutritionally active peptide derivatives having a digestive stimulant in the mammalian organism by controlling gastrointestinal functions, and the like. a. the increase in insulin release, improve food utilization (absorption) and lead to a potential increase in body weight, especially in the development and growth stage. The invention is of importance for use in agriculture / animal nutrition for the reproduction of livestock.

Characteristic of the known state of the art

The great importance of the broad range of gsatrointestinal peptide hormones for the regulation of the digestive process in the mammalian organism has long been known and studied intensively.

Since 1978 it has been known that various dietary proteins, i.a. Gluten and life, in the gastrointestinal tract, opiate-like, short-chewed poptide fragments (oligopeptides) are released, which can trigger opiate-like and non-opiate effects both on the intestine and after their absorption on other organs. So z. B. from the ß-casein of milk by digestive enzymes or microwave degradation peptide fragments of bovine beta-casein A2 of the partial sequence 60 to 67, the so-called .beta.-casomorphine formed (see V.Brand and H.Teschemacher, Naunyn Schmledeberg's Arch. Pharmacol., 306, 301 to 304 [1979], U. Hamel, G. Kielwein and H. Teschemacher, J. Diary Res., 52, 139 to 148 (19851) Opiates, as is known, act on intestinal peristalsis in the sense of promoting digestion (see W Kramer, W. Preulaff and F. Walnoff Life Sci 26, 1857-1865 [1980]).

It is also known that β-casomorphine peptido release insulin both on isolated rat pancreas islets of Langerhans and in vivo on a pancreas of dogs pre-stimulated with glucose and amino acids, the insulin release not correlating with the opiate activity of the peptides (cf. Schusdziarra, A. Holland, A. Schiok, A. de Fuente, M.Klier, V.Maier.V.Brantl and EFPfeiffer, Diabetology 24,113 to 116 [1983], V.Schusdziarre.R.Schiek, Ade la Fuante, A. Holland, V.Brantl and EF Pfeiffer, Endocrinology 112, 1948 to 1951 [1983]).

In addition, it has been shown that β-casomorphine peptides, in particular β-casomorphine-4, after repeated feeding in rats (dose 100 mg / kg body weight) lead to an increase in body weight in the first days of life compared to control animals (cf. Reetanl, A. Volterra, N. Brunello, V. Brantl, G. Racognl, G. L ₁ GaIIi, Advances in Bloscienes

These results indicate a nutritional physiological function of the caromorphins, the action being triggered by digestion of the precursor proteins or by indirect application of the active peptides. Results of non-native derivatives of casomorphins as well as studies on structure-activity relationships are not yet known.

Object of the invention

The aim of the invention is to provide peptide derivatives ri , which control gastrointestinal functions as simple, novel, stable, well tolerated active ingredients with long-term effect under favorable nutritional parameters, lead to an improvement in food absorption and a potential increase in body weight and as such also optionally combined with known feed additives in animal nutrition are of importance.

Explanation of the essence of the invention

task

The invention has for its object to develop processes for the preparation of nutritionally effective peptide derivatives, which act as regulators of gastrointestinal functions in the acid organism, improve food intake and lead to a potential increase in body weight.

Features of the invention

The object is achieved in that linear peptides of Casomo '^ type which are C-terminal modified, preferably by a heterocyclic C-terminal Amldstruktur the general formula I.

Tyr-Pro-A-T (I)

and their salts are synthesized as active components, wherein A and T are defined as follows:

A = phenylalanine, homophenylalanine, N-alkyl- (preferably N-methyl) -phenylalanine, tyrosine, N-alkyl- (preferably N-methyl) tyrosine, the corresponding ring-substituted compounds, preferably with halogen, amino, nitro, lower

linear or branched alkyl or alkoxy radicals, tyronine, tyroxine, valine, leucine, isoleucine in the L configuration T = NHR ¹ , NR ² R ³ , R ⁴ , where R ¹ , R ² , R ³ and R ⁴ have the following meaning

R ¹ = lower alkyl, aralkyl or alicyclic radical,

R ² and R ¹ = identical or different alkyl or aralkyl radicals

R ⁴ - N-heterocyclic radicals, preferably pyrrolidyl

 The nutritionally effective peptide derivatives prepared according to the invention have u. a. the following advantages:

1. Simple molecular structure

2. Good compatibility (qur.si endogenous, metabolizable substances)

3. Modulation of the effective parameters by targeted structural modification

4. Influence of lipophilicity and other physicochemical parameters in terms of improving the transport and penetration properties through targeted structural modification

A preferred compound is L-tyrosyl-L-prolyl-L-phenylalanine pyrrolidide:

H - Tyr - Pro - Phe -

The structure of the peptidic peptide according to the invention is carried out both by stepwise extension of the peptide chain from the C-terminal end and by segment condonation. The stop-by-step condensation is characterized in that the C-terminal amino acid HAT with X-Pro-Y and X-Ty (S) -Y according to the coupling methods customary in peptide chemistry (compare E. Wünsch: Synthesis von Peptiden In Houben-Weyi, Volume 15/11, Methods of Organic Chemistry, Ed. E. Muller, Georg-Thieme-Vorlag Stuttgart, 1974) to the protected peptide of formula (II) is extended.

X-Tyr (S) -Pro-A-T,

wherein Λ and T are as previously defined and

X is tert -butyloxycarbonyl, benzyloxycarbonyl, p-methoxycarbonyl, biphenylisopropyloxycarbonyl, a, a'-dimethyl-3,5-

dimethoxybenzyloxycarbonyl, o -nitrophenylsulfenyl, 9-fluorenylmethyloxycarbonyl Y for OH, pentafluorophenyl, pentachlorophenyl, trifluorophenyl, 4-nitrophenyl, hydroxysuccinimide, N-hydroxy-5-norbornene

2,3-dicarboximide ester and hydrazide S for H, tort. Butyl, benzyl

stand.

Preferably, the stepwise construction of the peptides of the formula II is carried out by the mixed anhydride technique, the DCCI / Additiva method, the activated ester method and using 2- (1H-benzotriazol-1-yl) -1,1,3, 3, -tetramethyluronium salts as coupling reagents. As a-amino protecting group is preferably the tert. Butyloxycarbonyl radical used.

The correspondingly protected peptides of the formula II are purified on a silica gel by chloroform / methanol or benzene / acetone / acetic acid elution or Sephadex LH 20 (eluent preferably methanol) or by recrystallization, preferably from ethyl acetate / petroleum ether, ethyl acetate / diisopropyl ether or isopropanol and then the protective groups with the customary in Peptldchemle Debiockierungsverfahren (see E. Wünsch, see above), in particular by means of hydrochloric acid / dioxane, hydrochloric acid / glacial acetic acid, hydrochloric acid / ethyl acetate, trifluoroacetic acid, HBr / glacial acetic acid or by catalytic hydrogenation, transfer hydrogenolysis or Piperidine / dimethylformamide, cleaved. The resulting peptides of foimel I are preferably isolated and characterized as hydrochlorides.

The second strategic variant for the preparation of peptides of the formula I, the segment condensation, is characterized in that protected peptides of the formula III

X-Tyr (S) -Pro-Y (III)

with the amino acid derivatives H-A-T,

wherein A, S, T, X, Y are as defined above, according to the coupling methods customary in peptide chemistry (see E. Wünsch, see above) linked to peptides of the general formula II. Preferably, the segment condensation is carried out via the mixed anhydride method, the DCCI / Additiva method and the activation by means of 2- (1 H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium salts. After purification of the protected peptides according to formula II and cleavage of the corresponding a-amino protecting group are d ^; e desired peptides corresponding to formula I.

The nutritionally-active peptide derivatives obtained according to the invention can be used as such or as an option or supplemented by known feed additives, in animal nutrition, preferably in customary feed combinations.

The invention will be explained in more detail by means of embodiments, without limiting it.

Ausführungsbelsplele

List of abbreviations:

Amino acid symbols according to IUPAC-IUB Commission on Biochemical Nomenclature, J. Biol. Chem. 247,977 (1972) AcOH acetic acid

Boc tert. butyloxycarbonyl

CAIBE isobutyl chloroformate

TLC thin-layer chromatography, chromatographic

EE ethyl acetate

Mp melting point

h hours

HOBt N-hydroxybenzotriazole

vacuo. in a vacuum

LM solvent

MeOH methanol

Min. Minutes

NEM N-ethylmorpholine

OTcp trichlorophenyl ester

PE petroleum ether

RT room temperature

TEA triethylamine

THF tetrahydrofuran

Dec. decomposition

Under. Usual workup * means:

After completion of the coupling reaction, the respective crude product is taken up in ethyl acetate and the solution washed successively twice with water (saturated NaCI), three times with 5% KHSO ^ solution, twice with water, three times with saturated sodium bicarbonate solution and three times with water. The organic phase is dried over Na ₂ SO ₄ and the crude product isolated by evaporation of the solvent in vacuo.

The following solvent systems (by volume) for thin layer chromatography on silica gel precast panels (Silufol UV254, CSSR) are used:

BAE benzene-acetone-acetic acid 50 + 20 + 1

BAW 2-butanol-formic acid-water 75 + 13.5 + 11.5

BEWE I-butanol-oleic acid-water-ethyl acetate 20 + 20 + 20 + 20

BPEW 1-butanol-pyridine-citric acid-water 30 + 20 + 6 + 24

CM chloroform-methanol 90 + 10

EPEW Esslgester-pyridine-acetic acid-water 90 + 15 + 4,5 + 8,3

example 1

Synthesis of H-Tyr-Pro-Phe-N · HCl

Variant I: gradual construction

1.1. Boo-Fhe-N ^ |

5.31 g Boc-Phe-OH are dissolved in 50 ml of THF and treated at -15 ⁰ C with 2.6 ml of NEM and 2,54ml CAIBE. After 6min. Add 1.42 g of pyrolidine to the reaction mixture. The mixture is stirred for 1 h at 15 ⁰ C and 12 h at RT and worked up as usual.

The product is crystallized from diisopropyl ether. Yield: 4.3 g (67% of theory)

Mp: 93 ⁰ CWsSe ⁰ C

(a) 'g:' ^e +47.1 (c = 1, AcOH)

DC: uniform in CM, BAE and EPEW

1.2. H-Phe-N ^ | · HCl 3.4 g Boo-Phe-H ^ |

are mixed with 32ml 1.1 N HCI / AcOH. After 30min. at RT, the LM is distilled off. The product crystallizes upon addition of EE.

Yield: 2.5 g (92% of theory)

Mp: 203 ^o C to 205 ° C

Ia] ² S: +87.3 "(C = 1, AcOH)

DC: uniform in BAW, BEWE and BPF.W

1.3. Boo-Pro-Phe-H ^ |

2.15 g Boc-Pro-OH in 2OmI THF are added at -15 ⁰ C with 1,27ml NEM and 1.3 mL CAIBE. After 6min. be 2.55g

, HCl

Phe-1 ^ I

and 1.27ml NEM in 10ml THF. The reaction mixture is worked up after a reaction time of ¹ h at -15 ° C. and 12 h at RT in the customary manner. The product is recrystallised from EE / PE.

Yield: 3.0 g (72% of theory)

Mp: 98 ⁰ CbIs 100 ° C

[a] ² 8: -48.3 ⁰ Ic = 1, AcOH)

DC: uniform in CM and BAE

1.4. H-Pro-Phe-N ^ | , HCl

2.5 g Boo-Pro-Phe-K ^ |

be 30min. treated at RT with 19 ml of 1.1 N HCl / AcOH. The product crystallizes out after the addition of ether.

Yield: 2.0 g (95% of theory)

Mp: 184 ^C C-187 ° C

(αΐ'δ: +3.6 ° (c = 1, AcOH)

DC: uniform in BAW, BEWE and BPEW

1.5. Boo-Tyr-Pro-Phe-ϊζ

Ί, 84 g Boc-Tyr-OTcp are dissolved in 15 ml of THF and at ^{0 0} C with

1.41 β H-Pro-Phe-N ^ | , HCl,

0.54g HOBt and 0.56ml TEA added. The mixture is stirred at ⁰ ° C. for 1 h and at RT for 12 h. The batch is worked up as usual and the product obtained is recrystallized from EE / PE.

Yield: 1.48 g (64% of theory)

Mp: 114 ⁰ Cb! S118 ° C

[Q) ⁷ I: -24.0 ° (c = 1, AcOH)

DC: uniform in CM and BAE

1.6. H-Tyr-Pro-Phe-IC

HCl

200 mg Boo-IPyr-Pro-Phe-N ^ |

are treated at RT for 30 min. with 3 ml of 3.4 N HCl / dioxane. The product is filled with ether, filtered off and washed with ether and reprecipitated from MeOH / ether.

Yield: 150 mg (84% of theory)

Mp: 165 ⁰ C to 163 ⁰ C.

[α] ² g: -21.1 "(c = 1, AcOH)

DC: uniform in BAW, BEWE and BPEW Variant II: Segment condensation

II. 1. Boo-Tyr-Pro-Phe-N ^ H

946rng Boc-Tyr-Pro-OH in 15 ml of THF are added at -15 ⁰ C with 317μΙ NEM and 325μΙ CAIBE. After 6min. been

637 mg H-Phe flk ~ n. HCl

(obtained as described under I.2.) and 317 μM NEM in 5 ml of THF. The reaction mixture is worked up after a reaction time of 1 h at -15 ⁰ C and 12 h at RT in the usual manner. The product is recrystallized from EE / PE. Yield: 800mg (55% d.Th.) Mp: 118 ° C ⁰ Cbls121

Ia] ⁷ I: -27.0 "(C = 1, AcOH) DC: uniform in CM and BAE

II. 2. H-Tyr-Pro-Phe-N; ^]. HOl 680 mg Boc-Tyr-Pro-Phe-H ^ ~

become 30min at RT. treated with 10 ml of 3.4 N HCI / Dloxan. The product is precipitated with ether, filtered off and washed with ether. Dropping from MeOH / ether, the DC provides uniform product.

Yield: 526 mg (87% of theory) Mp: 163 ⁰ CbIsISS ⁰ C

[a] ² 8: -22.0 «(c = 1, AcOH)

DC: uniform in BAW, BEWE and BPEW

Example 2 Effect of

H-Tyr-Pro-Phe ~ N ^ [

on insulin release

The experiments are carried out on isolated Langerhans islands of neonatal rats. The investigated peptide derivative becomes

in a concentration of 10 ~ ⁴ mol / l, 10 ~ 'mol / l and 10 ~ * mol / l used. Accompanying glucose concentrations are determined to be 1.5 x 10 -6 mol / l and 6 x 10 -5 mol / l in the experimental batch. The amount of insulin released is determined radioimmunologically. The evaluation of the test results (see Table 1) is expressed as a percentage change compared to the corresponding controls. The results obtained are checked for significant differences by pair comparison.

Results see Table 1.

Example 3 Effect of

H- Ty r »Pr ó-

on the leucine uptake on isolated rat colon in the influx chamber. The small intestine is isolated, sliced longitudinally, and clamped in an influx chamber so that the intestinal mucosa is lapped by Krebs-Henseleit-Ringer's solution. Liquid scintillation is used to measure the uptake of L-'H-leucine into the tissue at 37 ° C. The Mettzeit is 1 minute.

H-type r

leads to a significant increase in leucine intake (see Table 2).

-6- 287 204

Table 1 Influencing insulin secretion (in%)

H-Ty r-Pro-Phe-N ^ |

in the presence of glucose, based on the corresponding control values of insulin secretion in the presence of glucose without peptide (n = number of experiments)

O η 1.5-10- ³ mol / l glucose concentration η 6 10- ³ mol / l significance 15 32 14 Insulin secretion (increase in%) 15 32 14 Insulin secretion (% increase) 0.05 0.01 0.001 H-Tyr-Pro-Phe-N Conc.:mol/I + 120 ± 48 • +162 ± 89 + 94 ± 24 significance +78 ± 29 +75 ± 29 +73 ± 30 10 "· 10-» 10- · 0.01 0.01 0.001

Table 2 Effect of

H-Tyr-Pro-Phe-H ^ |

on leucine uptake on isolated rat small intestine η = number of experiments, incubation tent 1 minute, L- / 4.5- ³ H / -leucine concentration: 0.6 to 1.7 · IQ-mg / l

Konz

 (minor)

Leucine uptake (% increase)

significance

H-Tyr-Pro-Phe-N

12/12

35 ± 18

0.02

Claims (5)

1. A process for the preparation of nutritionally active peptide derivatives of the general formula I. Tyr-Pro-A-T (I) and their salts, wherein A and T are defined as follows: A - phenylalanine, homophenylalanine, N-alkyl- (preferably N-methyl) phenylalanine, tyrosine, N-alkyl (preferably N-methyl) tyrosine, the corresponding ring-substituted compounds, preferably with halogen, amino, nitro, lower linear or branched alkyl or Alkoxy radicals, tyronine, tyroxine, valine, leucine, isoleucine in the L configuration T = NHR ¹ , NR ² R ³ , R ⁴ , in which R ¹ , R ² , R ³ and R ^{4 have the} following meaning: R ¹ = lower alkyl, aralkyl or alicyclic radicals, R ² and R ³ = identical or different alkyl or aralkyl radicals, R ⁴ = heterocyclic radicals, preferably pyrrolidyl, characterized in that linear casomorphine-type peptides which are C-terminally modified are prepared by stepwise construction or by a (2 + 1) segment condensation from the C-terminal end according to the methods customary in peptide chemistry. 2. The method according to claim 1, characterized in that linear peptides are used, which are preferably modified by a heterocyclic C-terminal amide structure. 3. Use of nutritionally active peptide derivatives of the general formula I, characterized in that they are used as Nutritive broad spectrum of action in the animal feed. 4. Use of the peptide derivatives according to claim 3, characterized in that they are used as such or optionally or earmarked supplemented by known feed additives in animal nutrition, preferably in customary feed combinations. 5. Use of the peptide derivatives according to claim 3 and 4, characterized in that as Nutritiva in particular L-tyrosyl-L-proline-L-phenylalanlnpyrrolidid (Tyr-Pro-Phe-N I) used