DD150199A1 - METHOD FOR THE PRODUCTION OF NEW PEPTIDE MATERIALS - Google Patents

Abstract

The invention relates to a process for the preparation of novel peptide active ingredients which contain both cyclic and linear structures in the molecule. The object of the invention is the production of biologically active substances having improved properties, in particular an increase in activity, an increase in stability, an improvement in the transport properties and an influence on the pharmacological and physiological properties. The task of developing such active substances has been solved by Dasz peptide active ingredients or biologically active analogs, including truncated or modified partial sequences, by acid amide bonds - either by free alpha or omega-amino bzw.Carboxylgruppen or by Brueckenreagenzien - covalently bound to cyclic peptides be bound. The invention is of importance to the pharmaceutical industry, clinical pharmacology and diagnostics.

Description

T o o u r th e rs 11 u nc ;. yo_n_ji eii e η Pep ti dwi rk st ο f _u fe η ^

Field of application of Erf.in..du _T ng

The invention relates to a process for the preparation of new peptide drugs, which contained both cyclic and linear structures in the molecule of the inventive binding linear peptide active ingredients or "analogues biologically active agent, including truncated or modified partial sequences (hereinafter also referred to as active ingredient) to cyclic peptide structures are drug Cyclopcptid conjugates having potential effect amplification, including a prologierten or differentiated biological activity is obtained, which can throw modified by substitution of certain functional groups over a wide spectrum, the invention is of importance for the pharmaceutical industry, the clinical Pharmacology and diagnostics.

There are a large number of methods for the formation of peptide bonds known. Also, methods are described that lead to cyclization of linear peptides (see E. Wünsch, synthesis of peptides -. Houben -. Weyl Vol 15/1 and II, methods Oer organic chemistry, Ed "E * Müller, Georg Thiems Verlag, Stuttgart, 1974 ).

Although an anchoring of active ingredients, including pharmacological and agrochemical type, to polymeric carriers in particular

with immunological task and depot effect, has been known for some time (GE Allan et al., Nature, 234-349 (1971); HG Gatz et al., Angew. Chem. 84, 1189 (1972); K. Hofmann Acad., U.S.A. 73, 3516 (1976), a covalent fixation of peptide drugs to cyclic peptide structures with the goal of enhancing activity has not previously been described in the literature.

The object of the invention consists in the production of peptide active agents or biologically active active substance analogues with improved properties, in particular an increase in activity, increase in stability, improvement in transport properties and an influence on the pharmacological and physiological properties.

Loan of the essence of the invention

 mn »Μ ιι -ιιιιιιιιΛ ι ι ι ιΐ mi * V.i.ninn ι ιι.ιι" .μ »μ .im t.mmimimi ... ιι ι in μπιιμ ι ι mi ιι n 11 ιιι ι Πιι'ι

The invention is based on the object bzvj Peptidwirkstoffe. to develop biologically active drug analogues which have the following advantages:

a) Potential effect amplification of the respective test compound, including prolonged or differentiated biological effects due to change of the molecular parameters, especially dor hydrophobicity of the molecular structure

b) Potential improvement of the transport properties of the active substance concerned

c) influencing the pharmacological and physiological properties of the active substance in question by appropriate substitution on suitable functional groups

the peptide chains that alter the lipophilicity of the molecular structure and other physico-chemical parameters

d) Increasing the stability of the active substance concerned against enzymatic degradation »

The object has been achieved by peptide active substances or

biologically active analogues, including truncated or modified subsequences, are covalently bound to cyclic peptides by acid amide linkages-either via free c £ or έ -amino or carboxyl groups, or via bridging reagents

 The compounds represented by the process according to the invention are represented by the general formulas I to IV

L _x . (X) _n - _Y J

- -w

Z - (X) _n TT] III

Z - (X) _n - Y - W ^; , IV

in which

W - for the amino acid sequence of an active ingredient X - for any amino acid residue

Y - for Asp, Glu (compound type I to III), Lys, Orn, Dab (compound type I, III, IV) (abbreviations for amino acids and peptide derivatives, see IUPAC)

IUB Commission on Biochemical Nomenclature - 0. Biol. Chem. 247, 977 (1972)

Z- in the case of Y «= Glu, Asp, Lys, Om, Dab for Lys, Om and Dab (compound type III) and in the case of Y · = Orn, Lys, Dab for Glu and Asp (compound type IV)

stand and

η - has the meaning O, 1, 2, 3, 4, 8, 10.

The linear precursor of the desired cyclic peptide structure having selectively protected side chain function (connection type I and III) or with selectively protected C-terminal carboxyl group of Y (compound of type II and IV) is established by the known methods of peptide chemistry (see. E * Yi / ünsch , so), then into one for the intended cyclization strategy

DCCI process with additional components (A) mixed anhydride method (B) - azide process (C)

Active ester aminolysis (D)

required structure of the general formulas V to VIII RRS ι * ι

H ₂ N - X - (X) _n - Y - T (V)

RRT • · I

H ₂ N - X - (X) _n -YS (VI)

H ₀ NO p. I: l

SZ- (X) _n -Y-T (VII)

- 5 - d ZX TR NH ₀

1: 1

SZ- (X) _n -Y-S (VIII) ₁

in which X ₁ Y, Z and η are as defined above "and R, S _t have the following meanings:

R - depending on the synthetic strategy possibly required side chain protection in trifunctional amino acids

S - according to the definition of Y and Z is an ovoid or to amino or 6 ° - or Co -Carboxylschutzgruppe

T - OH (cyclization variant A or B) NH-NH "(cyclization variant C), conversion into the azide-activated ester (cyclization variant D)

transferred and subjected to one of the above strategies of the ring-closing reaction. After purification on ion exchangers and Sephadex gel filtration as intermediate, the cyclic peptides of the general formulas IX to XII are obtained

(IX)

• 11 ι

RRS

U ~ (X) _n - Y - S (X)

I f

1 η

R R

SZ- (X) _n - YY (XI)

j I

RS

SZ- (X) _n -Y-S (XII),

R.

wherein X, Y, Z, R ₁ S and η have the abovementioned meaning.

Upon selective release of the side chain function at Y, the intended linear peptide drug is covalently attached to the cyclic peptide. It is also possible to use shortened or modified partial sequences of the linear peptide active substance. Use is made of sequences which have partially protected structures suitable for covalent bonding and binds via a free N-terminal amino or C-terrainal carboxyl group or a free ζύ- amino or carboxyl function in the side chain or via so-called bridging reagents, such as Dicarboxylic acids or alkyldiamines of different chain length, according to the usual methods of peptide chemistry *

Subsequently, the remaining protecting groups under the usual in peptide chemistry conditions by acidolysis, hydrogenolysis, alkaline hydrolysis or hydrazinolysis according to the target position are completely or partially removed. After purification, preferably by column chromatography on silica gel or Sephadex, the desired active compound-cyclopeptide conjugate of the general formulas I to IV is obtained. The compounds produced according to the invention have increased or prolonged or differentiated biological activity.

The invention will be explained below with reference to exemplary embodiments.

The following abbreviations are used:

- 7 -

Amino Acid Symbols According to IUPAC-IUB Commission on Biochemical Nomenclature, 0. Biol. Chem. 247, 977 (1972)

AcOH acetic acid

Boc tert-butyloxycarbonyl

TLC thin-layer chromatogram, chromatogram

graphic

DCCI N, N'-dicyclohexylcarbodiimide _f

DHF dimethylformamide

HE ethyl acetate

Mp melting point

i. Vak. in a vacuum

fiin * minutes

OBut terto butyl ester

OMe methyl ester

OPfp pentafluorophenyl ester

RT room temperature

TFA 'trifluoroacetic acid

THF tetrahydrofuran

Z is benzyloxycarbonyl

The following solvent systems (by volume) for thin layer chromatography on silica gel precast panels (Silufol UV 254, CSSR) were used:

BAW 2-butanol-formic acid-water 80 + 20 + 20

BEW I-butanol-acetic acid-water 75 + 13.5 + 11.5

CM chloroform-methanol 90 + 10

EPEW ethyl acetate-pyridine-acetic acid-water 10 + 10 + 10 +

In the amino acid analyzes, the amino acid marked with *) represents the reference fourth.

Example I

Covalent binding of substance P (7-11) ~ pentapeptide to cyclo (Glu-Pro)

cyclo (Glu - Pro)

^x I-phe-Phe-Gly-Leu-Met-NH ₂

I »1. cyclo (GIu - Pro)

OH

4.5 g (10 mmol) of Z-Glu (OBut) -Pro ~ OMe in methanol is treated with Pd / carbon (10%), the benzyloxycarbonyl protecting group at 30 ⁰ C removed by hydrogenolysis. Thereafter, the catalyst is filtered off and the filtrate i · Vak. concentrated to about 30 ml. After adding a few drops of 32% ammonia solution, the mixture is allowed to stand for 24 h at about 45 C, then i. Concentrated, concentrated with ether, filtered off and the residue obtained to remove the tert. Butylestergruppe treated with 20 ml of trifluoroacetic acid at RT. After 45 minutes, the cleavage reagent i. Vak. stripped off and the oily residue was digested with ether, filtered off with suction, washed with ether and dried over,

Yield: 0.85 g (38% of theory, over 3 steps.). Mp: 173-175 ⁰ C.

DC: uniform in BEW, CM, EPEW

₄ (226.33) Ber · C 53.09 H 6.24 N 12.38 'Gef. C 52.89 H 6.58 N 12.70

I. 2 "H-Phe - Phe - Gly-Leu-Met-NH _£ . HCl

The construction of the pentapeptide was carried out starting from 3.0 g (10 EiMoI) of H-Lou-Mot-NHg.HCl - (H.-D. Dakubke, Ch. Kleßen, K. Neubert, 3. prakt. Chem., 319, , 640 (1977) supported by gradual attachment of the corresponding. "butyloxycarbonylamino ~ acid pentafluorophenyl ester (13 mmol) in DMF (K. Neubert, B. Hartrodt, S. Koch, M. Bienert, E. Klauschenz, H.-D. Dakubke, The Pharmacy, in press.) For the deblocking of N ^ -Aminoschutzgruppen each served 1.1 η HCl / AcOH - 30 min. At RT.

Yield: 5.2 g (80 % of theory ) mp: decomp. from 140 ⁰ C

: 77.6 ^w (c-1,

DC: uniform in BEW, EPEW

I. 3. cyclo (Glu - Pro)

- Phe - GIy »Leu - Met -

a) Koyalente bond after the mixed anhydride Tec hnik.

0.4 g (1.77 mmol) of cyclo (Glu-Pro) are dissolved in 20 ml of THF / DMF (4: 1) and after cooling to -15 ° C. with 0.22 ml (1.77 mmol) of N- Ethylmorpholine is added to the solution. 0.23 ml (1.77 mmol) of isobutyl chloroformate are added to the solution. After 8 minutes, 0.13 ml (1.42 mmol) of N-ethylmorpholine are added and immediately afterwards 0.92 g (1, 42 mmol) of H-Phe-Phe-Gly-Leu-Met-NHg.HCl in 10 ml

THF added. The mixture is stirred for 2 h at -15 ° C and 12 h at RT. Thereafter, the solvent is i. Vak. The residue was stripped with water, filtered off with suction and washed successively with 5% strength KHSO 4 solution, water, saturated NaHCO 3 solution and water on a frit, and i. Vak «over PoOc dried» Subsequently

- 10 -

the crude product is extracted twice each with 25 ml of methanol and ether and reprecipitated from acetic acid / ether.

Yield: 0.44 g (38 % of theory ) mp: 172.5-173.5 ⁰ C Md ^2; ~ 43.2 ° (c-1, AcOH) DC: uniform in CM, EPEW

Amino Acids Analysis: GIu Pro Phe GIy Leu Met

Ber · 112 11 1 Gef. 0.89 0.92 2.04 1.00 1.00 0.87

Bi o-lοqische ^ Testu ^ a: contraction on isolated Meerscbwein

chen-ileum

Relative activity: 27 % (substance P £ 100 %) Comparative value for substance P (7~11) -pentapeptide: 1.4%

b) j ^ ya ^ njtLg__j ^

To a cooled to -5 C solution of 0.3 g (1.33 mmol) cyclo (Glu-Pro), 0.84 g (1.3 mmol) of H-Phe-Phe-Gly-Leu-Met-NI- ^ .HCl, 0.17 ml (1.3 μmol) of N-ethylmorpholine and.0.2 g (1.46 mmol) of N-hydroxybenzotriazole in 8 ml of DMF are added O, 3 g (1.46 mmol) DCCI in 2 ml of DMF and the mixture is stirred for 0.5 h at -5 ⁰ C, 2 h at ^{0 0} C and 12 h at RT. After addition of a few drops of AcOH, the reaction mixture is cooled to ⁰ ° C., the dicyclohexylurea formed is separated, i. Vak. concentrated and precipitated the crude product by addition of 5% KHSO.-solution. The solid is worked up as described under a).

Yield: 0.35 g (33% .d.Th.) Mp: 172.- 174 ⁰ C.

ψ ι - .43.0 ° (CI, AcOH) uniformly in CMj EPEW _ΛΛ

- αϊ - *

Seismic II

Covalent binding of substance P (6-ll) ~ hexapeptide to cyclo (Glu-Pro)

cyclo (Glu-Pro)

- Phe - Phe - Gly - Leu - Met - NH ₂

0.23 g (1 mmol) of cyclo (Glu-Pro) are dissolved with stirring in 12 ml of THF / DMF (3: 1) and at -15 ⁰ C with 0.13 ml (1 mmol) of N-ethylmorpholine and 0, 13 ml (1 mmol) Chlorobohlensäureisobutylester added. After 8 minutes, 0.1 ml (0.8 mmol) of N-ethylmorpholine is added immediately followed by 0.62 g (0.8 mmol) of H-Gln-Phe-Phe-Gly-Leu-Met, -ΝΗ ₂ × Η0ί (obtained after 1.2. By reaction of H ~ Phe-Phe-Gly-Leu ~ Met-NH ₂ with Boc-Gln-QPfp and subsequent deblocking with 1.1 η HCl / AcOH ~ 10 min. At 10 ⁰ G, U-crystallized from methanol / EA, mp: 228 - 233 ⁰ C,

p: - 43.0 ° (c ~ 0.5, DMF) in 10 ml of DMF). The mixture is stirred for 2 h at -15 ° C and 12 h at RT and worked up as described under 1.3.a).

Yield: 0.32 g (42% of theory.). Mp: 209 to 210.5 ⁰ C

² : ~ 56.3 ° (c-0.5; AcOH) DC: uniform in CM, EPEW

Amino Acids Analysis: GIu Pro Phe GIy Leu Met

Bar .2 1 2 11 1 Gef _s 2.00 0.91 2.07 1.00 1.00 0.85

S ^ SiSEa ^ EilS-Xf-alidilS-i contraction on isolated guinea pig

ileum

Relative Activity: 227 % (substance P £> 100 %) Comparative value for substance P (6-> ll) ~ hexapeptide: 50 %

Claims (3)

Invention claim 1, Process for the preparation of novel peptide active ingredients which contain both cyclic and linear structures in the molecule, characterized in that active compound-cyclopeptide conjugates of the general formulas I to IV L _x _ _(X ) _n _yJ L _x _ _{(x) n} " _Y II III IV wherein Vi - for the atainic acid frequency of an active ingredient X - for any amino acid residue Y - for Asp, GIu (compound type I to III), Lys, Orn, Dab (compound type I, III, IV) Z - in the case of Y << GIu, Asp, Lys, Orn, Dab for Lys, Orn and Dab (compound type III) and in case of Y »Orn, Lys, Dab for GIu and Asp (compound type IV) stand and η - has the meaning O, 1, 2, 3, 4, 8, 10, of cyclic peptides of the general formulas IX to XII, which arise by ring closure of linear structures of the general formulas V to VIII RRRS RRT X- (X) _n -YT H ₂ MX (X) _n -ys VI ~ 2 NH ^ R S - Z - (X) _n - YT VII R NH ₀ :1 SZ- (X) _n -Y-S VIII - γ - S S_ y _ XI "., Γ! Rs SZ- (X) _n -Y-S XII, wherein X, Y, Z and η are as defined above and depending on the synthetic strategy, any necessary side chain protection for trifunctional amino acids S - according to the definition of Y and Z is a dL - or 0 amino or o £ - or fo -Carboxylschutzgruppe T - OH, NH - NH ₀ , activated ester mean, and linear peptide agents or biologically active analogs, including truncated or modified partial sequences, by covalent linkage * 2 * Method according to item 1, characterized in that the cyclic peptides with partially protected depending on äer amino acid sequence and taking into account essential structural elements Peptidwirks.toffen or biologically active analogs, including shortened or modifizier - 3 -IB- partial subsequences, linked via a .free N-terminal amino or * C-terminal carboxyl group or a free CO amino or carboxyl function in the side chain or via bridging reagents , such as dicarboxylic acids or alkyldiamines, via acid amide bonds, then the remaining protective groups be completely or partially removed and after purification, the drug-cyclopeptide conjugates are obtained. 3. The method according to item 1, characterized in that the cyclic peptides of the general formulas IX to XII are formed by ring closure of the linear structures V to VIII, being selected as the cyclization strategy, the DCCI process with additional components or the mixed anhydride method, if T OH is the azide method chosen when T is NH - NH ₂ and active ester aminolysis is chosen when T is activated ester.