DD298108A5 - IRREVERSIBLE BOMBESINANTAGONISTS - Google Patents

Abstract

The invention describes a peptide of the formula I: RABCTrpAlaValXYTWIworin mean: R is a group of the formula 4 (ClCH2CH2) 2NC6H4CO, 3 (ClCH2CH2) 2NC6H4CO, ClCH2CH2NHCO, {irreversible bombesin antagonists; Peptide; cis or trans isomers; linear or branched aliphatic chain; benzyl; phenyl; pharmaceutically acceptable salts; pharmaceutical composition}

Description

4- (CICH ₂ CH ₂ J ₂ NC ₆ H ₄ -CO-, 3- (CICH ₂ CH _{2) 2} NC ₆ H 4 CO-, CICH ₂ CH ₂ NHCO-, CICH-CH-CO-, BrCH = CH -CO-, CH ₂ -CCICO-, CH ₂ = CBrCO-

(either cis or trans isomers),

CH ₂ -CH-CH ₂ -CO-, HC = C-CO-, ClCH ₂ CH ₂ CH ₂ N (NO) CO-,

\ / O ClCH ₂ CO-CH (R ₁ ) NHCOiCH ₂ ) ₂ C0-;

A is a valence bond or a Gly, Leu-Gly, Arg-Leu-Gly, Gln-Arg-Leu-Gly residue; B is a valence bond or an Asn or Thr radical;

C is a GIn or His residue;

X is a glycine ala residue;

Y is a valence bond or a His (R ₂ ), his (R ₂ ), Phe, phe, Ser, ser, Ala or ala radical; T is a valence bond or a Leu, leu, Phe or phe residue; W is a group of the formula OH, NH ₂ , NH (CH ₂ ) ₄ CH ₃ , NH (CH ₂ ) ₂ C ₆ H ₅ , Met-R ₃ , Leu-R ₃ , Me-R ₃ , or

Nle-R _3; R ₁ hydrogen atom, a linear or branched aliphatic chain with 1 to

11 carbon atoms, a benzyl or phenyl group; R _{2 is} hydrogen or Tos, Dnp or Bzl group and R _{3 is} an amino, hydroxy, methoxy or Hydrazingruppe

and pharmaceutically acceptable salts thereof.

A pharmaceutical composition comprising a peptide of claim 1 or a pharmaceutically acceptable salt of such a peptide in admixture with a pharmaceutically acceptable diluent or carrier.

3. A process for the preparation of a peptide according to claim 1, characterized in that amino acids and / or amino acid derivatives in the desired sequence and / or peptide fragments containing these amino acids or their derivatives in the desired sequence are condensed to give the desired peptide in that the carboxylic acid end groups are activated for the peptide bond, that the remaining groups are protected and that the protective groups are eliminated in the resulting compound and / or that the resulting peptide is converted into a pharmaceutically acceptable salt thereof.

4. Use of the peptides according to claim 1 for the preparation of a suitable pharmaceutical preparation for the treatment of human neoplasms.

5. Use of the peptides according to claim 1 for the preparation of a pharmaceutical preparation which is useful as an antagonist against effects caused by bombesin.

Field of application of the invention

The present invention relates to peptide derivatives, to pharmaceutical compositions containing them, to processes for their preparation and to their use as therapeutic agents.

In the present specification, the symbols and abbreviations are the same as those commonly used in peptide chemistry (see Eur. J. Biochem., 11984, 138, 9-37). Thus, the three letter amino acid symbols represent the L configuration of the chiral amino acids. D amino acids are indicated by small letters · ζ. B. ala = D-Ala. Other symbols and abbreviations used are: AA, amino acid; AcOEt, ethyl acetate; BBS, Bombesin; Boc, t-Butoxycarbonyi; BuOH, n-butyl alcohol; BOP, benzotriazolyloxy-tris (dimethylamino) phosphonium hexafluorophosphate; DCC, N, N'-dicyclohexylcarbodiimide; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; Dnp, 2,4-dinitrophenyl; EGF, epidermal growth factor; EtOH, ethyl alcohol; FAB (or FD) -MS, mass spectroscopy by means of collision processes by excited

Atoms (or Feide desorption); ECC, ethyl chlorocarbonate; Et ₂ O, diethyl ether; GIp,! .- pyroglutamic acid; h-GRP (or p-GRP), gastrin releasing peptide from human (or porcine); HOBt, 1-hydroxybenzotriazole; ID, inner diameter; MeOH, methyl alcohol; mp. Melting point; nd, not found; Never, L-norleucine; NMM, N-methylmorpholine; NMR, nuclear magnetic resonance; OSu, N-hydroxysuccinimidyl; PE, petroleum ether 40-70 ° C; RP-PLC, reverse phase high pressure liquid chromatography; SCLC, small cell lung carcinoma; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TLC, thin layer chromatography; Tos, p-toluenesulfonyl.

Explanation of the essence of the invention

This invention provides a peptide of formula I:

R-A-B-C-Trp-Ala-Val-X-Y-T-W I

wherein R is a group of the formula

4- (CICH ₂ CH ₂ I ₂ NC ₆ Hc-CO-, 3- (CICH ₂ CH ₂ I ₂ NC ₆ H ₄ -CO-, CICH ₂ CH ₂ NHCO-, CICH = CH-CO-, BrCH = CH -CO-, CH ₂ = CCICO-,

(either cis or trans isomers),

CH ₂ -CH-CH ₂ -CO-, CH = C-CO-, ClCH ₂ CH ₂ N (NO) CO-,

O ClCH ₂ CO-CH (R _3. ) NHCO (CH ₂ J ₂ CO-;

A is a valence bond or a Gly, Leu-Gly.Arg-Leu-Gly.Gln-Arg-Leu-Gly residue; B is a valence bond or an Asn or Thr radical;

C is a GIn or His residue;

X is a glycodereinala residue;

Y is a valence bond or a HiS (R ₂ ), his (R ₂ ), Phe, phe, Ser, ser, Ala or ala residue; T is a valence bond or a Leu, leu, Phe or phe residue;

W is a group of the formula OH, NH ₂ , NH (CH ₂ I ₄ CH ₃ , NH (CH ₂ I ₂ C ₆ H ₅ , Met-R ₃ , Leu-R ₃ , He-R ₃ , or NIe-R ₃ R _{1 is} hydrogen atom, a linear or branched aliphatic chain of 1 to 11 carbon atoms, a benzyl or phenyl group;

R _{2 is} hydrogen atom or Tos, Dnp or Bzl group and R _{3 is} an amino, hydroxy, methoxy or hydrazine group.

Salts of these peptides with pharmaceutically acceptable acids are included in this invention. Such acid addition salts can be derived from a variety of inorganic and organic acids, for example, sulfuric acid, phosphoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid. Nitric, sulfamic, citric, lactic, pyruvic, oxalic, maleic, succinic, tartaric, cinnamic, acetic, trifluoroacetic, benzoic, salicylic, gluconic, ascorbic and related acids. Preferred aliphatic chains, which can be represented by R 1, include methyl, ethyl, n-propyl, iso-propyl, η-butyl and iso-butyl groups.

The preparation of the peptides according to the invention can be completed by classical solution methods. The preparation consists essentially of suitable successive condensations of protected amino acids or peptides. The condensations are carried out so that the resulting peptides have the desired sequence of amino acid residues.

The amino acids and the peptides that can be condensed by the methods known in peptide chemistry are blocked at the amino and carboxyl groups that are not involved in peptide bond formation by suitable protecting groups that can be obtained by acid or alkali treatment or by Hydrolysis can be removed. For example, the following protecting groups may be used to protect the amino group: benzyloxycarbonyl, t-butoxycarbonyl, trityl, formyl, trifluoroacetyl, o -nitrophenylsulphenyl, 4-methyloxybenzyloxycarbonyl, S-fluorenylmethoxycarbonyljS.B-dimethoxy-alpha-alpha'-dimethylbenzyloxycarbonyl or methylsulphonylethoxycarbonyl. For protecting the carbonyl group, the following protecting groups may be used, for example: methyl, ethyl, t-butyl, benzyl, p-nitrobenzyl or fluorenylmethyl, amide, hydrazide, t-butoxycarbonylhydrazide or benzyloxycarbonylhydrazide. The hydroxy functions of the hydroxyamino acids and the imino function of histidine may be protected by appropriate protecting groups (throughout the synthesis, or only during some manufacturing steps) or may remain unprotected. For protecting the hydroxy function, the following protecting groups may be used, for example: t-butyl, benzyl, acetyl. For example, to protect the imidazolimino functionality, the following groups can be used: 2,4-dinitrophenyl, tosyl, benzyl. Deprotection reactions are carried out by methods known as such in peptide chemistry.

The condensation between an amino group of one molecule and a carboxyl group of another molecule to form the peptide bond can be carried out by means of an activated acyl derivative, for example a mixed anhydride, an azide or an activated ester, or also by direct condensation between a free amino group and a free carboxyl group, in the presence of a condensing agent, for example dicyclohexylcarbodiimide atleine or together with an agent which prevents the racemization, for example N-hydroxysuccinimide or 1-hydroxybenzotriazole, or

together with an activating agent, for example 4-dimethylaminopyridine. The condensation can be carried out in a solvent, for example dimethylformamide, dimethylacetamide, pyridine, acetonitrile, tetrahydrofuran or N-methyl-2-pyrrolidone.

The reaction temperature may be in a range from -30 ⁰ C to room temperature. The reaction time is generally from 1 to 120 hours.

The production scheme, protecting groups and condensing agents are selected to avoid the risk of racemization.

Biological activity

The peptides of the present invention exhibit strong antagonism against "in vitro" and "in vivo" effects caused by bombesin, for example, smooth muscle contraction, modification of central origin behavior, and mitogenesis.

Bombesin (BBS) is a tetradecapeptide of the formula Glp-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-VaM3ly-His-Leu-Met-NH2 which was originally isolated from the skin of a frog. The biological activity is in the C-terminal region of the molecule: BBS (6-14) nonapeptide is as effective as the parent compound. The human counterpart of bombesin is a 27 amino acid peptide known as gastrin releasing peptide (h-GRP). Bombesin and bomby-like peptides display a number of biological activities (JH Walsch [1983] in Brain Peptides, DT Warrior, MJ Brownstein and JB Martin [ed.], Wiley Interscience Publ., Pp. 941-960), including autocrine growth promoting effects in human small cell lung carcinoma (SCLC) (F.Cutfrtta et al. [1985] Cancer Survey, 4,707-727), autocrine and / or paracrine stimulation of prostate cancer cell proliferation in humans (M.Bologna et al. in press) and the modulation of the EGF receptor (I.Zachary and E. Rozengurt [1985] Cancer Surveys, 4,729-765).

A bombesin antagonist could, by competition with the natural ligand for the receptor (s), inhibit the triggering of the cascade of events leading to abnormal cell proliferation.

Various approaches in this direction have been attempted by various research groups. A series of C-terminal bombesin nona and decapeptides characterized by amino acid deletion, inversion or substitution has been the subject of a prior patent application by the present applicants (European Patent Application No. 89102283.2). However, these peptides, like other BBS antagonists, usually show moderate affinity for the BBS receptor.

The compounds of the present invention having an alkylation moiety behave as receptor antagonists either when administered in combination with bombesin or when administered 24 hours prior to exposure to bombesin.

Biological test results

The binding affinity of the compounds according to the invention for bombesin receptors was determined on mouse Swiss 3T3 fibroblasts (I.Zachary and E.Rozengurt 11985] Pro Natl Acad., USA, 82 7616-7620). (Table 1).

The effect of mitogenesis was determined on resting and confluent Swiss 3T3 cells maintained in a serum-free medium (AN Corps et al., (1985) Biochem., J. 231, 781-785). In a first set of experiments, analogues were administered alone or in combination with bombesin. In a second set of experiments, the cells were previously pretreated with the alkylating peptides, washed, left at 37 ° C for 24 hours and then exposed to bombesin. In both cases, DNA synthesis was determined to be (H ³ ) thymidine incorporation (Table 2).

The mitogen activity of bombesin and its analogs has also been determined to be the activation of protein tyrosine kinase that phosphorylates a 115KD protein (p115) associated with the bombesin receptor complex (D.Cirillo et al. [1986] Mol. Cell. Biol. 6, 4641-4649) (Table 3). In addition, exposure to these peptides was in the 0.1 to 50 pM range with a significant reduction in the growth of SCLC cell lines (e.g., NCI-H345, NCI-N 592, NCI-H128) as well as the prostatic ones Carcinoma cell lines (for example, DU 145 and PC3).

Parenteral administration of these peptides at doses ranging from 1 ng / kg to 100 mg / kg of nude mice was associated with significant growth reduction of the above transplanted human SCLC and prostatic carcinoma cell lines.

Table 1

Binding affinity of bombesia

Alkylation analogues on mouse Swiss 3T3 fibroblasts

connection ID ₅₀ (nM) 12.6 + 3.8 I 1363 ± 266 11100 Il 438 ± 111 14000 III 1815 + 330 214 ± 30 IV 0.7 ± 0.2 V 1336 + 199 Vl 648 + 290 reference peptides BBS spantide (per ² ) Spantid [Leu ¹³ Ψ (CH ₂ -NH) LeU ¹⁴ IBBS

Table 2

(H ³ ) thymidine incorporation into mouse 3T3 fibroblasts

connection

Wrinkle increase above basal value

5 nM

5OnM

0.5 μΜ

5μΜ

% Inhibition in the presence of 25 nM BBS

A B

0.5 μΜ 5μΜ 0.5 μΜ 5μΜ

I N.D. N.D. 1 0.6 1 0.6 9 ± 6 56 ± 4 12 ± 3 0 0 53 + 8 It N.D. N.D. 1.0 1.0 0 50 ± 10 0 20 ± 12 III N.D. N.D. 2.3 3.4 0 0 51 ± 11 0 IV N.D. N.D. 3.2 2.2 13 ± 8 32 ± 9 0 29 ± 10 V N.D. 1.8 1.8 2.5 0 0 0 Vl N.D. 1.0 1.0 1.0 0 0 39 ± 14 reference peptides BBS 3, C ) ± 1 [Leu ¹³ Ψ (CH ₂ -NH) LeU ¹⁴ JBBS 29 ± 10 0

A = analogues are given in combination with BBS

B = cells pretreated with analogs, washed, left at 37 ° C for 24 hours and then exposed to BBS.

Table 3

Phosphorylation of the p115 protein linked to the bombesin receptor

connection Minimum active dose (nM) 3 I > 4000 > 10000 Il > 1000 > 10000 III 500 > 200 IV 500 V > 2000 Vl > 1000 Reference peptides: BBS spantide (per ² ) Spantid [LeU ¹³ UMCHR NH) LeU ¹⁴ IBBS

The peptides of formula I are therefore useful in the therapy of human neoplasms, which modulate in their growth and progression by peptides of the GRP family either directly or together with other growth factors

become.

Additionally, these alkylating analogs can be used in the treatment of the clinical symptoms associated with these diseases that occur due to the hypersecretion of the GRP-like peptides.

The compounds of the present invention may be administered in a conventional manner, for example, parenterally, for example by intravenous injection or infusion, or by intramuscular, subcutaneous, intracavitary and intranasal administration.

The dose depends on the age, weight and condition of the patient or on the mode of administration.

Based on "in vitro" and "in vivo" data in mice, it can be estimated that the therapeutic doses in humans range from 1 ng / kg to 100 mg / kg, once to 6 times a day.

The invention also provides pharmaceutical compositions containing a compound according to formula (I) as active substance in association with one or more pharmaceutically acceptable carriers.

The pharmaceutical compositions according to the invention are usually prepared by conventional methods and administered in a pharmaceutically suitable form. For example, solutions for intravenous injection or infusion as carriers may contain, for example, sterile water, or they may preferably be in the form of sterile aqueous

Isotonic saline solutions are present.

Suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, for example sterile water, olive oil, ethyl oleate, glycols (eg, propylene glycol) and, if necessary, an appropriate amount of lidocaine hydrochloride.

Further, according to the present invention, there is proposed a method of treating neuroendocrine neoplasms (e.g., small cell lung carcinomas and protastatic carcinomas) or treating the clinical symptoms associated with these diseases in patients suffering therefrom, the method being to treat such patients a

Composition of the invention is administered.

Chemistry procedure:

a) TLC was applied to precoated silica gel 60 F ₂₅₄ (Merck) plates, layer thickness 0.25 mm. Length 20cm, with the following

Eluents performed:

System A: n-butanol / acetic acid / water = 600/150/150 (volume) System B: chloroform / methanol / NH 3 OH 30% = 500/364/154 (volume) System C: chloroform / methanol = 90/10 (Volume) System D: dichloromethane / diethyl ester = 90/10 (volume) System E: acetonitrile / water / formic acid = 80/20/10 (v / v)

b) Analytical RP-HPLC was performed using a Hewlett Packard Mod. 1084 instrument on a LiChrosorbHibarRP-18 column (Merck) 250 χ 4 mm I.D.,

Particle diameter 5 μιη carried out. The following solvents were used:

A = KH ₂ PO ₄ 2OmM, pH 3.5 / acetonitrile 9/1 (volume)

B = KH ₂ PO ₄ 2OmM, pH 3.5 / acetonitrile 3/7 (by volume)

The elution would be adjusted to a linear gradient of 60% to 90% B over a period of 20 minutes (System

A) or from 30 to 70% B over a period of 15 minutes (System B) and then the elution was carried out isocratically for a period of 15 minutes, at a flow rate of 1 ml / min.

The peptides are characterized by their retention time (RT)

c) Preparative RP-HPLC was performed using a Delta Prep 3000 instrument (Waters) with a Deltapak column (Waters), 300 χ 19 mm I.D.,

Particle diameter IOpm, was used. The following solvents were used:

A = 0.05% TFA in water

B = 0.05% TFA in acetonitrile / water 7/3 (by volume)

Flow rate = 24ml / min; Wavelength of detection = 220nm.

The elution procedures are given in the individual examples.

In each case fractions were assayed by analytical RP-HPLC and those fractions having greater than 98% purity were collected. After removal of acetonitrile, the solution was lyophilized.

d) An amino acid analysis was carried out with acid hydrolysates (either for 22 hours at 110 ⁰ C in 6 N HCl + 0.1% phenol or 16 hours at 100 ° C in 3 N mercaptoethanesulfonic acid (in both cases, under nitrogen atmosphere). Only natural Amino acid residues were determined Due to the partial decomposition under normal hydrolysis conditions, Trp was determined only in hydrolysates with mercaptoethanesulfonic acid.

example 1

Preparation of 4- {CICH ₂ CH ₂ ) ₂ N (yH ₄ CCM "hr-Gln-Trp-Ala-VaW3ly- eu-4Vlet-NH ₂ (l).

25.2 mg (0.096 mmol) of (p-bis (2-chloroethyl) amine) benzoic acid were dissolved with 5 ml of distilled DMF and 60 mg (0.064 mmol) of H-Thr-Gln-Trp-Ala-Val-Gly-Leu-Met -NH2 · HCl (EP Patent Application No. 89102283.2) were added sequentially; The solution was cooled to 5 ° C and 0.0176 ml NMM (0.16 mmol) and 0.0425 g BOP (0.096 mmol) were added. The reaction mixture was stirred overnight at room temperature, then it was added dropwise at a temperature of 5 ° C in 100 ml of a 10% citric acid solution. The mixture was stirred at a temperature below 10 ° C for one hour, then filtered and washed with water until neutral: 66.1 mg of the crude product (90% yield) was obtained. The product was purified by preparative RP-HPLC using a gradient of 60% to 90% of solvent B in eluent A over a period of 30 minutes (flow rate 35 ml / min): 33 mg of product I (50% yield) obtained: Rf _A 0.61; RT _A = 8.66; FAB-MS: m / z1147 (MH ⁺ ); AAV ratios: Thr 0.98 (1), Glu 1, Ala 0.97 (1), VaI 0.95 (1), Gly 1.04 (1), Leu 1.02 (1), Met 0.90 ( 1) (Trp nd).

Example 2

Production of

4- (CIC ₂ CHj) JNC ₆ H ₄ CO-ThT-GIn-TrP-Ala-VaI-Gly-LeU-NIe-NH ₂ (II).

0.2 g (0.216 mmol) of H-Thr-Gln-Trp-Ala-Val-Gly-Leu-Nle-NHz.HCl (prepared by the process described in European Patent Application No. 89102283.2 for H-Thr-Gln. Trp-Ala-Val-Gly-Leu-Met-N-HCl) were mixed with 0.085 g (0.324 mmol) of (p-bis (2-chloroethyl) amine) benzoic acid, 0.143 g (0.324 mmol) of POB and O, 059 ml of NMM (0.537 mmol) in 17 ml of distilled DMF reacted as described in Example 1. The crude product was purified by preparative RP-HPLC, this with a gradient of 45% to 90% of solvent B in solvent A on a duration of 20 minutes was carried out: 0.122 g (50% yield) of product II were obtained: RF _A 0.75; RT _A 10.39; FAB-MS: m / z 1129 (MH ⁺ ); AA Ratios: Thr 0.97 (1), Glu 1, Ala 034 (1), Val 0.96 (1), Gly (D, Le 0.92 (1), Never 0.87 (1) (Trp nd).

Example 3

Production of

₂₂₂₆₄ ργ (ρμ ₂ ()

Following the same procedure as described for Example 1, 63 mg (0.24 mmol) of (p-bis (2-chloroethyl amino benzoic acid and 200 mg (0.16 mmol)) were added.

HCl · H-Thr-Gln-Trp-Ala-Val-Gly-His (Dnp) -Leu-Met-NH ₂ (prepared from Boc-Thr-Gln-Trp-Ala-Val-Gly-OH) (own European Patent Application No.89102283.2) and HCI. H-His (DnpM-eu-Met-NH ₂ (F.Angelucci and R. de Castiglione (19751 Experientia, 507-508) was reacted using the method described in the same patent application for analogous compounds, whereby 161 mg (70% yield) of crude Compound III The product was purified by preparative RP-HPLC run with a gradient of 60% to 90% of solvent B in eluent A over a period of 20 minutes: 60 mg (m.p. 26% yield) of product III were obtained: Rf _A 0.70; RT _A 14.45; RT _B 24.82; FAB-MS: m / z 1451 (MH ⁺ ); AA ratios: Thr 1.05 (1 ), Glu1, Gly 1.03 (1), AlaO, 99 (1), VaI 0.95 (1), Met 0.90 (1), Leu 0.97 (1), (Trp and His nd).

Example 4 Preparation of

65 mg (0.05 mmol) of Compound (III) were suspended in 3 ml of a 0.02M phosphate buffer of pH 8 and treated with 2-mercaptoethanol. The resulting clear solution was allowed to react for 20 minutes, then poured into 300 mL of Et ₂ O with stirring. The precipitate was filtered, washed thoroughly with Et ₂ O, then between Bu-OH and water

distributed. The organic phase was concentrated to a small volume and the peptide was precipitated by dilution with AcOEt / Et ₂ O: 50 mg of crude compound (IV) (71% yield) was obtained. The product was purified by preparative RP-HPLC run at a gradient of 40% to 90% of solvent B in eluent A for 30 minutes: 25 mg (36% yield) of IV were obtained: Rf _A 0, 39; RT _A 9.26; RT "19.41; FAB MS: m / z 1284 (MH ⁺ ); AA ratios: Thr 1.05 (1); GIn (1), GIy 1.03 (1), Ala 0.99 (1), VaI 0.95 (1), Met 0.88 (1), His 0.91 (1) (Trpn.d.) ,

Example 5

Preparation of CICH ₂ CHjN (NO) CO-THr-GIn-TrP-Ala-VaI-Gly-LeU-NIe-NH ₂ (V).

Step 1

CICH ₂ CH ₂ NHCO-OSu (Va)

5,75g (0,05 mol) N-hydroxysuccinimide were dissolved in 125 ml of AcOEt and at a temperature of 0 ⁰ C and 12.92 ml (0,075mmol) N-ethyl-diisopropy (amine and 5,12ml (0,060mol) 2 The reaction mixture was stirred vigorously for 48 hours, then the solvent was evaporated, and the residue was partitioned between AcOEt and water, and the organic phase was dried over Na ₂ SO ₄ and evaporated . FAB-MS melting point 104-107 ⁰ C; Rf 0.43 _c:: m / evaporated a small volume, the product crystallized on standing in the cold: 7,68g (70% yield) of compound (Va) were obtained z 220 (M ⁺ ); ¹ H-NMR (200MHz, DMSO-d ₆ ) delta (ppm): 8.57 (t, 1H, CH ₂ NHCO), 3.64 (t, 2H, CICH ₂ CH ₂ ), 3.39 (m, 2H, CH ₂ CH ₂ NH), 2.75 (s, 4H, succinimide), elemental analysis (C ₇ H ₉ NjO ₄ Cl): C, 38.10 (38.11), H 4,10 (4,11), N 12,67 (12,70), Cl 16,00 (16,07) CICH _z CH ₂ N (NO) CO-OSu (Vb)

1,100g (0,005mol) of the compound (Va) were dissolved in 12ml CH ₂ CI ₂ and at a temperature of O ° C with 0,800ml (0,010mol) of pyridine and 14.0 ml (0.020 mol) of 1.4 N NOCl in CH ₂ CI ₂ reacted. The reaction mixture was stirred vigorously for 5 hours, then washed with cold water, dried over Na ₂ SO ₄ and concentrated to a small volume. The crystallization started at room temperature and was completed in the cold. 0.80 g (64% yield) of compound (Vb) were obtained: mp 101-103 ⁰ C; Rf _c 0.87; FAB MS: m / z 249 (M ⁺ ); ¹ H-NMR (200MHz, DMSO-d ₆ ) delta (ppm): 4.13 (t, 2H, CICH ₂ CH ₂ ), 3.69 (t, 2H, CH ₂ CH ₂ NNO), 2.89 ( s, 4H, succinimide); Elemental analysis (C ₇ H ₈ N ₃ O ₆ CI): C 33.70 (33.68); H3,25 (3,23), N 16,82 (16,83), Cl 14,80 (14,20)

CICH ₂ CH ₂ N (NO) CO-Thr-Gln-Trp-nAla-Val-Gly-Leu-Nle-NH ₂ (V) 0.185g (0.2mmol)

HCI-H-Thr-Gln-Trp-Ala-VaI-Gly-LeU-NIe-NH ₂ (see Example 2) were suspended in a mixture of 0.5 ml of hexamethylphosphorylamide and 0.5 ml of N-methylpyrrolidone and incubated at room temperature with 0.028 ml (0.2 mmol) of triethylamine, then 0.100 g (0.4 mmol) of compound (Vb) was reacted. After stirring for 2 hours, the reaction mixture was diluted with water: 0.150 g (73% yield) of the crude compound (V) was obtained. A 50 ml sample was purified by preparative RP-HPLC running at 10% to 90% gradient! A was run in eluent B for a period of 30 minutes: Rf ₈ 0.59; RT _B 16.10; FAB-MS: m / z 1020 (MH ⁺ ); ¹ H-NMR (200 MHz, DMSO d ₆ ) delta (ppm) La: 3.60 (t, 2H, CICH ₂ CH ₂ ); AA ratios: Thr 0.91 (1), GIu 1.07 (1), Ala 1.03 (1), VaI 1, GIy 1.02 (1), Leu 1.01 (1), Never 0.90 (1) (Trpn.d.).

Example 6

Production of

CH ₃ (CH ₂ ) ₃ CH (COCH ₂ Cl) NHCO (CH ₂ ) ₂ CO-Thr-Gln-Trp-Ala-Val-Gly-Leu-Met-NH ₂ (VI) Step 1

BoC-NIE-CH = N ₂ (VIa)

4.60 g (0.02 mol) of Boc-Nle-OH in 40 mL of anhydrous THF were added at -12 ° C with 2.20 mL (0.02 mol) NMM and 2.62 mL (0.02 mol) isobutyl chlorocarbonate Reaction brought. After one minute, the reaction was quenched by addition of 50 mL of Et ₂ O and by cooling the solution to -35 ° C. The salts were filtered off and the filtrate was cooled to -70 ⁰ C.

In a separate reaction flask was added 3,96g (0,06 mol) of KOH which dissolved in 20 ml of 50% EtOH was dissolved within 10 minutes at 0 ⁰ C was added dropwise to a solution of 6.43 g (0.03 mole) of N Methyl-N-nitroso-p-toluenesulfonamide in 100ml Et ₂ O and 10ml ethylene glycol monomethyl ether added. The thus formed diazomethane was directly distilled into the reaction flask containing the previously formed and cooled mixed anhydride. After a 20-hour stirring the reaction mixture at a temperature of 0 ⁰ C, the solvent was evaporated under reduced pressure, and the residue was partitioned between AcOEt and 0.5 M aqueous KHCO. ₃ The organic layer was washed to neutrality with brine, dried over Na ₂ SO ₄ and concentrated in vacuo to give an oil which crystallized on standing in the cold: 2.9 g (57% yield) of compound (VIa) obtained: mp 83-85X; Rf ₀ 0.51; ¹ H-NMR (200MHz, DMSO-d ₆ ) delta (ppm) generally: 6.00 (s, 1 H, CH = N ₂ ); FD-MS: m / z 255 (M ⁺ ) Elemental Analysis (C ₁₂ H ₂₁ N ₃ O ₃ ): C 56.50 (56.45), H 8.30 (8.29), N, 16.47 ( 16,46).

step 2

HCl · H-Ni-CH ₂ Cl (VIb)

2.4 g (0.01 mol) of BoC-NIe-CH = N ₂ (VIa) were dissolved in 20 ml of 4.4 N HCl in dioxane. After 5 minutes at room temperature the product was precipitated with Et ₂ O: 1.56 g (78% yield) of compound (VIb) were obtained:

Melting point 153-154 "C; Rf _E 0.63; ¹ H-NMR (200MHz, DMSO-d ₆₎ delta (ppm) ia: 4,84,4,74 (2d, 2H, CH ₂ CI); FD MS: m / z 199 (m ⁺⁾ Elemental analysis (C ₇ H _1S NOCI _2); C42,02 (42.01), H 7.54 (7.55), N 6.96 (6.99) Cl 35.42 (35.43).

HOOC-CHzCH ₂ -CO-NiE-CH ₂ Cl (VIc)

0.60 g (3 mmol) of HCl H-Ni-CH ₂ Cl (VIb) and 0.33 ml (3 mmol) of NMM were dissolved in 10 ml of DMF and reacted at 0.degree. C. with 0.30 g <3 mmol) succinic anhydride in 5 ml of DMF , which was added dropwise within 10 minutes. After 30 minutes, the reaction mixture was brought to room temperature and stirred for an additional 4 hours at this temperature.

The solvent was removed under reduced pressure, the residue was taken up in AcOEt and, after filtration

of the insoluble material was washed with 0.02 M HCl, brine and finally dried over Na ₂ SO ₄ . Evaporation of the solvent and trituration with Et ₂ O / PE gave 0.35 g (44% yield) of Compound (VIc): mp 114-116 ° C, R f _B 0.33; ¹ H-NMR (200MHz, DMSO-d ₆ ) delta (ppm) generally: 4.57 (s, 2H, CH ₂ -CI); Elemental analysis (Ci ₁ H ₁₈ NO ₄ Cl): C 49.74 (50.10), H 5.34 (5.31), Cl 13.16 (13.44).

CHJ (CH ₂ ) JCH (COCH ₂ Cl) NHCO (CH ₂ ) ₂ CO-Thr-Gln-Trp-HAIa-VaM3ly-Lβu-Mβt-NH _I (VI) 100mg (0.106mmol) HCl.

H-Thr-Gln-Trp-Ala-Val-Gly-Leu-Met-N ^ (European Patent Application Nr.89102283.2) and 0,006ml (0,106mmol) NMM in 2 ml DMF were added at 0 ⁰ C einerTemperaturvon with 31 mg (0.120 mmol) HOOC- (CHj) ₂ CO-NIe-CH ₂ Cl (VIC), 19 mg (0.143 mmol) HOBT and 27 mg (0.133 mmol) DCC. The reaction mixture was then stirred for 4 days at room temperature. The solvent was removed under reduced pressure, the residue was triturated with warm isopropyl alcohol and the insoluble material was isolated by filtration: 50 mg (40% yield) of compound (VI) were obtained: Rf _A 0.42; RT _B 15.10; FAB MS: m / z 1149 (MH ⁺ ); AA ratios: ThrO, 95 (1), Glu 1, Ala 1.08 (1), Val 0.97 (1), Gly 1.01 (D.Leu 1.00 (1), Met 0.93 OMTrpn.d. ).

Claims (1)

-1- 298 Claims: 1. A peptide of the formula I: R-A-B-C-Trp-Ala-Val-X-Y-T-W I in which mean: R is a group of the formula