PT95779A - Process for the preparation of peptide derivatives antagonistic to irreversible reduced bombesin - Google Patents

Abstract

The present invention relates to a process for the preparation of a peptide of the general formula R---A---B---C---Trp---Ala---Val---X---Y---T---W I which involves replacing one or more peptide bonds (CONH) with reduced peptide bonds (CH2NH), and of the salts thereof that are acceptable in pharmacy, which are antagonists of bombesin.

Description

FARMITALIA CARLO ERBA S.R.L. The present invention relates to a process for the preparation of peptidic derivatives. The present invention relates to a process for the preparation of peptidic derivatives. The present invention relates to a process for the preparation of peptidic derivatives.

In this specification the symbols and abbreviations used are those commonly used in the chemistry of peptides (see Eriol, Bioohem, 138, (1984) 9-37) .Three amino acid symbols are therefore referred to to the L configuration of the chiralic amino acids. Amino acids of configuration D, are represented by lower case letters: for example, ala * D-Ala. Other symbols and abbreviations are used: AA, amino acid; Ac, acetyl; AcOEt, ethyl acetate; BBS, bombesin; Boc, t-butoxycarbonyl; BuOH, n-butanol; BOP, benzotriazolioxy-tris / dimethylamino / -phosphonium hexafluorophosphate; N, N'-bis (2-chloroethyl) amino] benzoyl; dec., decomposition; DCC, δ, δ'-dicyclohexylcarbodiimide; DGH &dicyclohexylamine; DCU, -dicyclohexylurea; DMP, 4-dimethylaminopyridine; DMP, freshly distilled dimethylformamide; DMSO, dimethylsulfoxide; Dnp, 2,4-dinitrophenyl; EGP, epidermal growth factor; EtOH, ethanol; FAB-MS (or DG), fast atom bombardment mass spectrometry (or field desorption); ECC ethyl chlorocarbonate; EM-IE, electronic impact mass spectrometry; Et2 O, ethyl ether; Glp, L-pyroglutamic acid; h-GRP (or p-GRP), peptide that releases human gastrin (or porcine); HCl / AcOH, anhydrous HCl in glacial acetic acid; HOBt, 1-hydroxybenzotriazole; D.I, internal diameter; HOSu, N-hydroxysuccinimide; Mel, bis (2-chloroethylamino) -L-phenylalanine; MeOH, methanol; m.p., melting point; mod. modification; n.d., not determined; Rle, L-norleucine; 1MM, 1-methylmorpholine; HM, nuclear magnetic resonance; OSu, N-hydroxysuccinimide; Pd / C, palladium on charcoal; EP, petroleum ether, fraction 40-70 ° C; RP-HPLC, high resolution reverse phase chromatography; SCLC, small cell carcinoma of the lung; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TLC, thin layer chromatography; Tos, R-toluenesulfonyl, p-toluenesulfonic acid. The uppercase letter psi between two amino acids indicates a substitution of an amide bridge for the function indicated in parentheses. The present invention provides a process for the preparation of a peptides of general formula (I)

 € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒR-A-B-C-Trp-Ala-Val-X-Y-T-W in which R represents a group of the formula 4- (ClCH2CH2) 2E-G6H4-GH2CH (EHR1) CO- (py); 3- (C 1 H 2 CK 2) 2 U-C 6 H 4 -CH 2 CH (H 2 O) CO- (mMel); 4- (GlCH 2 CH 2) 2 T-C 6 H 4 -CO- (Cab); 3- (C H 2 CH 2) 2 L-C 6 H 4 -CO;

ClGHâ,,CHâ,,NHCOâ,ƒ; C1CH = CH-C0-, BrCH * CH-CO-, CH2 = CC1C0-CH2 = GBrCO- (cis or trans isomers) -CH-CH2-CO-; CH = C-CO-; C H ₂CH₂CH₂CH₂OH;

O

ClCH 2 CO-GH (R 2)] 3HCO (CH 2) 2 CO-; A is a valence bond or a residue of Gly, Leu-Gly, Arg-Leu-gly, or Gln-Arg-Leu-Gly, B is a valence bond or a residue of Asn, Thr or of phe; C is a residue of Gin or His; X is ala or Gly; 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 M2, MH (GH2) 2, GH3, MH (CH2) 2 C H2 H3, or a group of the formula OR2, Met-R3, Leu-R4, ILe-R4 or Rle- ; R4 is hydrogen, a Boc group or an acyl group having 1 to 11 carbon atoms; R6 is hydrogen, a linear or branched aliphatic chain having 1 to 11 carbon atoms, a benzyl group or a phenyl group; the preferred aliphatic chains which Rg may represent include methyl, ethyl, n-propyl, isopropyl, n-butyl and isobutyl groups. R4 is H, Cl, Dnp or Bzl; and R4 is BHg, RH-Mg or a group of formula OR2; in addition, one or more peptide bridges (COM) are substituted by reduced peptide bridges (CH 2 M).

Preferred acyl groups which R4 may represent are aliphatic acyl groups, such as the acetyl, formyl, propionyl and butyryl groups or aromatic groups, such as benzoyl optionally substituted by nitro, methoxy, amino groups or by halogen atoms.

Preferably, in the general formula I, R 1 represents pMel or R 2, R 2 represents a hydrogen atom,

Boc is an acetyl group, A represents a valence bond, B represents a valence bond or a phe residue, C is a residue of Gin, X is a His (Dnp), His or Gly residue, preferably a Gly residue, Y is a valence bond, T is a Leu residue, W is a group of the formula Leu-IHg or Nle-Mg © is a reduced peptidic value (CHMM ) lies between T and W.

Salts of these peptides of pharmaceutically acceptable acids are within the scope of the present invention. These acid addition salts may be derived from various inorganic and organic acids, such as sulfuric, phosphoric, hydrochloric, hydrobromic, hydroiodic, nitric, sulfamic, citric, lactic, pyruvic, oxalic, maleic, succinic, tartaric, cinnamic, acetic, trifluoroacetic, benzoic, saccharic, glonic, ascorbic and related acids. The process for the preparation of the peptides of the present invention is carried out according to standard methods. Synthesis consists essentially of 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.

Amino acids and peptides, which can be condensed according to the methods known in the peptide chemistry, contain amino and carboxy groups, which do not intervene in the formation of the peptide bridge, blocked by suitable protecting groups capable of being removed by alkaline treatment or acid or by hydrogenolysis.

For protection of the amino group, for example, the following groups may be used: benzyloxycarbonyl, t-butoxycarbonyl, trityl, foroyl, trifluoroacetyl, o-nitrophenylsulfonyl, 4-methoxybenzyloxycarbonyl, 9-fluorenylmethoxycarbonyl, 3- , 5-dimethoxy- < / RTI > dimethylbenzyloxycarbonyl or methylsulfonylethoxycarbonyl.

For the protection of carbonyl groups, the following protecting groups may for example be used; methyl, ethyl, t-butyl, benzyl, β-nitrobenzyl or fluorenylmethyl groups, amide, hydrazide, t-butoxycarbonyl hydrazide or benzyloxycarbonyl hydrazide.

The hydroxy, hydroxy amino acid and imino functions of histidine may be protected with suitable protecting groups (throughout the synthesis or only during some of the steps) or they may be unprotected. For the protection of the hydroxy function the following protecting groups may for example be used; t-butyl, benzyl, acetyl groups. For the protection of the imino function of the imidazole the following groups may be used, for example the following groups: 2,4-dinitrophenyl, tosyl, benzyl. The protective reactions are carried out according to methods known in the art of peptide chemistry.

The condensation between an amino group of one molecule and a carboxyl group of another molecule may be effected to form the peptide bond through an activated acyl derivative such as a mixed anhydride, an azide or an activated ester, or by direct condensation between a free amino group and a free carboxyl group, in the presence of a condensing agent, such as dicyclohexylcarbodiimide, alone or together with a racemization-preventing agent, such as an N-hydroxysuccinimide or 1-hydroxy group benzotriazole, or together with an activating agent, such as 4-dimethylaminopyridine. Condensation can be carried out in a solvent, such as dimethylformamide, dimethylacetamide, pyridine, acetonitrile, tetrahydrofuran or 1-methyl-2-pyrrolidone.

A reduced peptide bridge is formed by condensation of an E-pro-protected amino acid aldehyde with a C-protected amino acid or peptide in the presence of a reductant such as KaBH3 CE. , the aldehyde, generally by condensation of an E-protected amino acid with E, N-dimethylhydroxylamine and reducing the resulting amide with a suitable reductant, such as LiAlH 4. The reaction temperature is from -30 ° C to room temperature. The reaction time is generally 1 to 120 hours. The synthesis scheme, the protecting groups and the condensing agents are chosen so as to avoid the risk of racemization.

Biological Activity · Μ Η Η Η Η ΜΜΙΒ ΜΜΙΒ ΜΜΙΒ ΜΜΙΒ ΜΜΙΒ ΜΜΙΒ ΜΜΙΒ ΜΜΙΒ ΜΜΙΒ ΜΜΙΒ ΜΜΙΒ ΜΜΙΒ ΜΜΙΒ ΜΜΙΒ ΜΜΙΒ

The peptides of the present invention are endowed with potent antagonism against in vitro and in vivo induced effects by bombesin, such as the concentration of smooth musculant, modifications of central origin behavior and mitogenesis. Bombesin is a tetradecapeptide of the formula Glp-Gln-Arg-Leu-Grly-Asn-Gin-Trp-Ala-Val-Gly-His-Leu-Met-HHg, initially isolated from the skin of a frog. The biological activity resides in the C-terminal portion of the molecule: the nonapeptide BBS (6-14) which is as active as the progenitor, human equivalent compound of bombesin is a 27 amino acid peptide, known as the release peptide of gastrin-8- (h-GRP). Bombesin and bombesin-like peptides exhibit various biological activities (J.H. Walsh, " Brain Peptides ", D.T. Krieger, M.J. Brownstein and J.B.

Martin (eds.), Wiley Interscience Publ., 1983, pp. 941-960), including autocratic effects " autocrine " (SCLC) (P, Cuttitta et al., Canoer furvey, 4, (1985) 707-727), autocratic and / or paracractic stimulation " para-crine " of the proliferation of human prostate cancer cells (M. Bologna et al., Cancer, in press), and EGF receptor modulation (I. Zachary and E. Rozengurt Cancer Surveys (1985) 729-765).

A bombesin antagonist, competing with the natural ligand for the receptor (s), would inhibit the disruption of cascading phenomena leading to abnormal cell proliferation. several approaches have been taken in this direction by different research groups. A series of n-peptides and C-terminal bombesin decapeptides made by elimination, inversion or amino acid substitution were the object of our prior application (European Patent Application 12 89102283.2) . however these peptides, like other BBS antagonists, usually exhibit moderate affinity for the BBS receptor,

The compounds of the present invention, due to radical alkylation, exhibit a higher affinity for the receptor than the protected peptides and behave as receptor antagonists, even when administered in combination with bombesin or when administered 24 hours prior to stimulation with sill . In addition, due to the presence of reduced peptide bonds, the solubility of water is increased and, in many cases, the antagonistic properties are also increased.

Results of Biological Trials

The binding affinity of the compounds of the present invention to the bombesin receptors (I, Zachary and E. Rozengurt Proc, Ratl, Acad, Sci, USA, 82 (1985), 7616- 7620) (Table 1).

The effect on mitogenesis in immotile and confluent Swiss 3® cells that are preserved in serum-free medium (A. R, Corps et al., Biochem J, 231 (1985), 781-785) was determined.

In the first set of assays, analogs were administered alone or in combination with bombesin. In the second set of assays, the cells were pretreated with the alkylation peptides, washed, left for 24 hours at 37 ° C, and then stimulated with bombesin. In both cases, the synthesis of ADR was calculated as the incorporation of (HH) -thymidine (Chart, è).

In addition, exposure of these peptides in the range of 0.1-50 Âμg was associated with a significant reduction of growth of SCLC cell lines (such as RCI-H345, RCI-R592, RCI-H96, RCI-H128 ), as well as prostate carcioma lines (such as DU145 and PC3)

(Qudro 3) * & parenteral administration of these peptides at doses ranging from 1 ng / kg to -100 mg / kg to nude mice was associated with a significant reduction of growth of the human prostate and SCLC transplanted cell lines referred to above. TABLE 1 AFFINITY CONNECTION OF BOMBESIN ALKYLATION ANALOGS IN SWISS 3T3 RAT FIBROBLASTS

COMPOUND IC 50 (nM) *

I II III IV V 839 +! 78 28 + 1 2340 + 291 2.3 + 1.0 0.9 + 0.5 Reference peptides: BBS Spastide for proparesparan ZLeu133 / (CH2-NH) Leu: average value + SEM 12.6 + 0.65 11100 14000 214 + 30 12 TABLE 2 INCORPORATION OF Î "H / TIMIDINE IN SWISS 3T3 RIB EIBROBLASTS

COMPOUND NUMBER OF TIMES OF INCREASE% INHIBITION IN PRE-IN RELATION TO THE BASIC VALUE OF

25 nM BBS 5nM 50nM 0.5μl 5æm M 0.5p! 5 JM B 0.5 JM 5 JM I ndnd 1.8 1.7 54 + 5 75 + 4 69 + 2 84 + 3 II ndnd 0,8 0,8 86 + 3 90 + 3 55 + 5 86 + 6 III ndnd 0, 8 0.7 34 + 21 86 + 1 46 + 14 61 + 16 IV ndnd X 1.3 79 + 7 85 + 8 0 85 + 7 V ndnd 1.1 0.9 83 + 8 85 + 7 0 39 + 7 Reference peptides: BBS 3.0 + 1 > (GK2-m) LeviUj338 1 1 29 + 10 56 + 4 0 0 A = the analogs were administered in combination with BBS. B = cells were pretreated with analogs, washed and left at 37 ° C for 24 hrs and then stimulated with BBS. - 13 TABLE 3

ACTIVITY " IN VITRO " OF THE ALLOYING AMLOGO ON THE SCLC COMPOUND SCL OILS NCI-N592 ^ q (ejM) HCI-H69 II 110 940 IV 700 783 Reference peptide: / Leu13 ^ (CH2-M) Leu147BBS 520 1,660 -

For this reason, the peptides of Formula I are applied in the therapy of human neoplasms which are modulated by their growth and progression by peptides of the GRP family either directly or in conjunction with other growth factors.

In addition, these alkylation analogs can be used in organizing clinical symptoms associated with these diseases and due to hypersecretion of GRP-like peptides,

The compounds of the present invention may be administered by the usual routes, such as, for example, parenterally, for example by intravenous injection or infusion, or by intramuscular, subcutaneous, intracavitary and intranasal administration. The dosage depends on age, weight and patient status and route of administration.

Based on data obtained from rats, "in vitro and in vivo", it can be calculated that therapeutic doses in humans range from 1 ng / kg to 100 mg / kg, from one to six times per day.

In addition, the toxicity of the peptides of the present invention is rather insignificant.

The pharmaceutical compositions of the present invention are usually prepared according to conventional methods and administered in a pharmaceutically acceptable form.

Intravenous injection or infusion solutions may contain as carrier, for example, sterile water or preferably may be in the form of sterile isotonic sterile aqueous solutions.

Suspensions or solutions for intramuscular injection may contain, in combination with the active ingredient, a pharmaceutically acceptable carrier, such as, for example, sterile water, olive oil, ethyl oleate, glycols (for example propylene glycol) and, if desired, an effective amount of lidocaine hydrochloride.

Chemistry Methods: a) TLC was performed on plates precoated with silica gel 60 (Merck), layer thickness 0.25 mm, length 20 cm, with the following eluents:

System A: n-butanol / acetic acid / water = 600/150/150 by volume

System B: chloroform / methanol = 99/1 by volume

System C: chloroform / methanol = 90/10 by volume

System D: toluene / ethyl acetate / acetic acid / water = 100/10/20/10 by volume. b) Analytical RP-HPLC was run on a Hewlett Packard Mod. 1084 apparatus in a 250 x 4mm D, Li, HCl RP-18 (Merck) LiChrosorb batch, I. particle diameter 5æ. The following eluents were used: 20 mM KHgPO4, pH 3.5, acetonitrile 9/1 by volume; 20 ml, pH 3.5.5 / acetonitrile 3/7 by volume. - 1* -

The elution was graded with a linear gradient of 60-90% B over a period of 20 minutes (System A) or 30-70% B over a period of 15 minutes (System B) and , then isoeratically over 15 minutes at a flow rate of 1 ml / minute.

The peptides were characterized according to their retention time. c) Preparatory RP-HEBC was performed using a Delta Prep 3000 (laters) apparatus on a Deltapack (Waters) column, 300 x 19mm ID, 10 μm particle diameter. The following eluents were used: 05% ΪΡΑ in water; B = 0.05% TFA in acetonitrile / water 7/3 by volume.

Flow rate = 24 ml / detection wavelength = 220 nm.

Elution methods are referred to in the examples.

In each case, the fractions were examined by analytical RP-HPLC and those having a purity greater than 98% were collected. After removal of the acetonitrile, the solutions were lyophilized. d) Amino acid analysis was performed on hydrolysates (at 110 ° C for 22 hours in 6% HCl + 0.1% phenol or at 100 ° C for 16 hours in mercaptoethanol sulphonic acid JK9 both under a nitrogen atmosphere ). Only the residues of the natural amino acids were determined. Due to partial decomposition under normal conditions of hydrolysis, Trp was determined only on the hydrolysates - 17 which were carried out with mercaptoethane sulfonic acid.

Example 1 Preparation of

Boc-pMel-Gln-Trp-Ala-Val-Gly-His (Dnp) -Leu (CH3) 2 H-Met-NH2 (I). Step 1

Boc-Val-Grly-QBzl (Ia)

43.4 g (200 mmol) of Boc-Val-OH was dissolved in 500 ml of anhydrous,, cooled to -25 ° C and treated with 22.48 ml (200 mmol) of After stirring for 2 minutes at -12 ° C, a pre-cooled solution of 67.47 g (200 mmol) was added, of H-Gly-OBzl. TsOH and 22.48 ml (200 ml) of HMM in 500 ml of anhydrous DMF. The reaction mixture was stirred for 2 hours at -12 ° C, then the salts were filtered off and the solution under reduced pressure. The oily residue was dissolved in 1200 ml EtOAc and the solution was successively washed with 10% citric acid (5 x 100 ml), brine, NaHCO 3, 5% (5 x 100 ml) and concentrated sodium chloride solution to neutrality. After drying over Na2 SO4, the solvent was evaporated and the residue was purified by flash chromatography on silica gel, eluting with 95: 5 AcOEt / MeOH. 56.68 g (78% yield) of the title compound were obtained. Compound Ia, after recrystallization from petroleum ether: mp 76-78 ° C; β'ΰ'ψ -28.0 ° (= = 1, MeOH);  € ƒâ € ƒâ € ƒMSC-D: m / z 365 (100, - 1.8 - [MH +]); Rf-g 0.70; RT 11.8.

Step 2: H-Yal-Gly-OBzl-. HCl (1¾)

56.40 g (154.75 mmol) of Boe-Val-Gly-OBzl (Ia) was reacted for 30 minutes at room temperature with 570 ml of HCl / AcOH 1.33H.

The solvent was removed under reduced pressure and the residual DMP oil was evaporated twice and Et 2 O washed. There was obtained 44.2 g (95% yield) of compound Ib as an oil; ES-MS: m / z 265 (100, MH +) as a free base; Rf = 0.54; RT-g 6.7.

Step 3

Boc-Ala-Val-Gly-QBzl (Ic) α. was prepared from 27.81 g (147 mmol) of Boc-Ala-OH and 44.2 g (147 mmol) of H-Yal-Gly-OBzl.HCl (Ib) and acting as described for the preparation of 1a, but replacing AcOEt by CH 2 Cl 2 in the washings gave 54.82 g (68% yield) of Compound Ic after recrystallization from methylene chloride / petroleum ether: m.p. f. 142 - 146 ° C; FDMS: m / z 436 (100%) +; Rfg 0.26; R? PA 9.4 ·

Step 4: Preparation of Boc-Ala-Yal-Gly-OBzl (Ic) from 27 g (62 mmol) and proceeding as described for N- , preparation of Ib yielded 22.65 g (98% yield) of compound Id after recrystallization from MeOH / AcOEt / EP; mp 178-181 ° C; ES-MS: m / z 336 (100, MH +) as a free base; RfA 0.53; RT-g

Step 5

Boc-Trp-Ala-Val-Gly-QBz (Ie)

The condensation of the Boc-Trp-OH 18.41 g (60.5 mmol) and H-Ala-Val-Gly-OH (22.50 g, 60.5 mmol) GBzl (Id). The crude product was then dissolved in dimethylformamide and precipitated by adding the solution dropwise to an aqueous 10% citric acid solution. The precipitate was collected and washed with water to yield and then dried at 40% over PgCl2. 35 * 70 g (95% yield) of the compound Ies were obtained, mp 154 ° -177 ° C (dec.); FDMS: m / z 621 (100, M + +); RfB 0.10; R1A 13.2.

Step 6: H-Trp-Ala-Yal-Gly-QBzl. HCl (If)

33.75 g (54.28 mmoles) of Boc-Trp-Ala-Val-Gly-OBzl (Ie) were reacted with 340 ml of HCl, 1.33 R / AcOH, 34 ml of anisole and 17 ml of 2- -mercaptoethanol for 30 minutes at room temperature. The solvents were removed under reduced pressure and the oily residue was evaporated twice in dimethylformamide. The product was precipitated in a methanol / petroleum ether mixture and washed several times with petroleum ether and then with ethyl ether. - - 2: or

26.75 g (88% yield) of compound If: mp 118-122 ° C; MS-DC: m / z 521 (100, M + +) as the free base; RfA 0.66; R 3 = A 5.8.

Step 7

Boc-Gln-Tro-Ala-V al-Gly-OBzl (Ig) From 11.73 g (47.66 mmol) Boc-Glu-H and 26.6 g (47.66 mmol) H-Trp-Ala-Val-Gly-OBzl.HCl (If) and proceeding in the same way as for the course of 1e, there was obtained 29.86 g (83% yield) of compound VII in MeOH / CH2 Cl2 / Et2O / EP: pyf. 208-211 ° C (dec.); ES-MS: m / z 749 (100, M + +); Rf 0.51; RTA 7.6.

Step

Boc-Gln-Ir-Ala-Val-Gly-OH (Ih)

53 ml of a solution of 12 ml (318 mmol) of 99% formic acid and 33 ml (300 mmol) of 1-methylmorpholine in 1 liter of methanol were added, with stirring, to a suspension of 3 g ( 4 mmol) of Boc-Gln-Trp-Ala-Val-Gly-OBzl (Ig) and 1.86 g of 10% Pd / C in 80 ml of dimethylformamide.

The reaction mixture was stirred for 1 hour at ambient temperature, the catalyst was filtered off and the solvent was evaporated in vacuo. The residue was sedimented with EtOAc to provide 2.2 g (84% yield) of compound Ih: El-DC: m / z 682 (100, M + a), 659 (40, +);

RfA 0.52; RTb 8.0 - 21

Step 9

Boc-Lea-g (CH3) OCH3 (II)

24.93 g (100 mmol) of Boc-Leu-OH-H20 were dehydrated by evaporation from 200 ml of DMI and redissolved in 350 ml of CH3 Cl2. 9.95 g (102 mmol) of HCl · HCl (CH3) 3 CH3 and 3.05 g (2 mmol) of DMAP were added with stirring at 40Â ° C, followed by a few drops of DMP to give almost clear solution. Then, a solution of 20.65 g (100 mmol) of DCG in 130 ml of 11,111 ml solution of 11.24 ml (100 mmol) of D-methylmorpholine in 130 ml ml CH 2 Cl 2, over 30 minutes, maintaining the reaction temperature at 0 ° C. After a further 1 hour at room temperature, the reaction mixture was separated by filtration of the salts and DCU and evaporated. The residue was dissolved in AcOEt, quenched with DCU and washed successively with 10% citric acid (5 x 100 ml), 5% NaHCO 3 (15 x 100 ml ) and with saturated sodium chloride solution until neutrality is achieved. After purification of the solvent, the oily residue was purified by flash chromatography on silica gel eluting first with 85/15 EtOAc (to remove faster-moving impurities) and then with ΕΡ / Ε ( , Ο to 1/1. 17.38 g (57% yield) of pure compound 1I was recovered as an oil: MS EI: m / z 201 (4, M-OtBu), 173 (2, M-Boe); Rfg 0.44; RTa 10.4? RTB 19.1.

Step 10

Boc-Leu-H (1.1)

8.4 g (30.58 mmol) and Boc-Leu-N-CH3 OCH3 (l) were dissolved in Et2 O (350 ml) and reacted at 0 ° C with 3.48 g 91.74 mmol) of LiAlH 4, which was added portionwise over 15 minutes. The reaction mixture was stirred for 15 minutes at 0Â ° C and then 175 ml EtOAc was added, followed by 700 ml of 10% citric acid, maintaining the reaction mixture at 0 ° C. After 30 minutes of stirring the reaction mixture was extracted with AcOEt (5 x 300 ml), the combined organic layers were washed with citric acid 10%, and then with a saturated sodium chloride solution until neutral, and dried over Na2 SO4. Evaporation of the solvent gave 6.26 g (95% yield) of crude lithium compound: ESI-MS: m / z 186 (7, M-CHO); Rf-g 0.38; RIT 7.7; RT-g 15.

Step 11

To a solution of 6.14 g (28.52 mmol) of Boc-Leu-H (Ij) in 100 ml of 1% AcOH in anhydrous methanol was added HCl.H-Met-HHg was added 4.24 g (28.52 mmol), followed by 4.21 g (57 mmol) of NaBH4 CH3, which was added portionwise over 30 minutes at room temperature. After a further 40 minutes of stirring, the solution was evaporated, the residue taken up with 300 ml of 5% NaHCO3 and

The product was washed with EtOAc (5 x 100 ml), the organic phase was washed with concentrated sodium chloride solution, and, to neutrality, dried over Na2 SO2 and concentrated. There was obtained 5.30 g (53% yield) of pure compound Ik: mp 124-126 ° C; ES-MS: m / z 347 (100, M + e); Rfg 0.16; RTA 6.2; RTb 12.4.

Step 12: H-heptane (CHCl3). 2 HCl (II)

A solution of Boc-Leu (1.04 g, 3 mmol) in CH2 Cl2 (Ik) in 10 ml of 1.33 N, HCl / AcOH containing 1 ml of anisole and 0.5 ml of 2-mercaptoethanol. The solvents were removed under reduced pressure and the oily residue was evaporated three times from DM 3 - and once from MeOH, and then triturated with AcTOEt and Et 2 O. There was obtained 1.44 g (98.3% yield) of compound IX in two portions; ES-MS: m / z 247 (1, M +), 203 (6, M-COOH) in the form of a free base; RfA 0.58; RTg 3.6. - 24 -

Step 13

Boc-His (Dnp) -Leu (CH 2 HH) Met-M 2 (Im)

Boc-His (Dnp) -OH.iPrOH (1.29 g, 2.68 mmol) was evaporated three times from MI to remove the crystallizing isopropyl alcohol, then dissolved in 15 ml of DMI, cooled to -25 ° C and reacted with 0.30 ml (2.68 mmol) of N, N,. followed by 0.27 ml (2.68 mmol) of ICO. After stirring for 3 minutes at -12 ° C a cold solution of 0.858 g (2.68 mmol) of H-Leu-CH 2 MOMet-2 · 2 · 01 (II) and 0.60 ml (5.36 g) mmoles) of MM in 15 ml of DMI. The reaction mixture was kept for 60 minutes at -12 ° C and then for 30 minutes at 0 ° C. The solvent was removed in vacuo and the residue was dissolved in EtOAc, washed with 5% NaHCO 3, and then concentrated with sodium chloride solution. After drying over RaSO4 the solvent was evaporated and the oily residue was purified by flash chromatography on silica gel eluting with ethyl acetate containing increasing amounts of methanol (from 0.5% to 10% ). The product was recovered by evaporation of the solvents and trituration with ethyl ether yielded 1.23 g (70.7% yield) of compound Im: mp 70øC (mod.) - 90øC (dec.); FDMS: m / z 651 (100, MH +); Rf 0.57s RTA 12.0.

The title compound was prepared from 1.16 g (1.78 mmol) of Boc-HisCDnpJ-LeuCHgMjMet-HHg (Im) and H-His (Dnp) -Leu (GH2 NH) by following the procedure of Step 12, 1.09 g (98% yield) of compound In was obtained by recrystallization from ethyl acetate: 110Â ° C (mod.) - 200Â ° C (dec.); MS-DC: m / z 551 (100, 1H +) as the free base; Rf = 0.41; RIA 4.2; RT-g 7.1,

Step 15

Boc-Ala-Val-Gly-His (Dnp) -Leu (CH 2 CH)

156 mg (1.16 mmol) HOBt, 239 kg (1.16 mmol) DCC, 660 mg (1.06 mmol) H-His (Dnp) -Leuy (CH2m) Met- To a solution of Boc-Gln-Ir-Ala-Val-Gly-OH (700 mg, 1.06 mmol) in dichloromethane 8 ml of DMF. The reaction mixture was stirred at 0Â ° C for 1 hour and at room temperature overnight and then filtered and evaporated in vacuo. The oily residue was dissolved in BMP and poured into ethyl acetate , with stirring, in a 5% aqueous RaHCO 3 solution. The suspension was pelleted and the product was washed with water until neutral. The crude product was purified by flash chromatography using an eluent system consisting of 8/2 EtOAc / MeOH. 820 mg of compound Io (65% yield) in MeOH / AcQEt / Et 2 O: 128øC (mod.) - 145øC (dec.); FAB-MS: m / z 1192 (23, MH +); Rfc 0.10; R $ A 10.6. - 26 -

Step 16 H-Gln-Epi-Ala-Val-Gly-His (Dnp) -Leu / (CH 3) H Met-H 2 O. 2 HCl (ip)

800 mg (0.67 mmol) of Boc-Gln-Trp-Ala-Val-Gly-His (Dnp) -Leu (CH 2 M) Met-NH 2 (Io) was dissolved in 8 ml of 1: 0.8 ml of anisole and 0.4 ml of 2-mercaptoethanol and reacted for 90 minutes at room temperature. The solvent was removed in vacuo and the residue was sedimented with Et2 O to give 0.765 mg (98% yield) of compound Ip; m.p. 165 ° C (mod.) - 220 ° C (dee +); FAB-MS: m / z 1092 (6, 1H) + in the free base form; Rf = 0.20; RT = 4.5; RTB 12.1.

Step 17

Boc-pMel-Gln-Irp-Ala-Val-Gly-His (Dnp) -Luvre (0H2IH) Met-IH2 (I)

321 mg (0.64 mmol)) of Boc-pMel-OSu / prepared extemporaneously from 259 mg (0.64 mmol) of Boc-pIel-OH was added dropwise (see our patent application of U.S. Patent No. 8906000.9), 77 mg (0.67 mmol) of HOSu and 132 mg (0.64 mmol) of DCC in 5 ml of DMF was added dropwise to a cooled (0øC) solution of 500 mg , 43 mmol) of H-Gln-Trp-Ala-V al-Gly-His (Dnp) -LeuVACyynOMet-lffig. 2HCl (Ip) and 0.096 ml (0.87 mmol) of MM in 10 ml of DMF. The reaction mixture was stirred overnight at ambient temperature and then poured into 5% aqueous NaHCO 3 solution dropwise. The suspension was stirred for 10 minutes at ambient temperature and then filtered and washed with water until neutral. The crude product-277 (520 mg, 78% yield) was purified by preparative RP-HPLC which developed in a gradient of 80% to 100% of the eluent B in the eluent A for 20 minutes at a flow rate of 30 ml / minute, 286 mg (45% yield) of compound 1 mp, 140 ° C (mod.) = 170 ° C (dec.); (1) (I), Trp, His (Dnp) and Leu (CH2Iff) Met-M2 nd, ); FAB-MS: m / z 1478 (10, MH +); RfA 0.50; R 27.0.

Example 2 Preparation of

Boc-pMel-Gln-Trp-Ala-Val-Gly-His-Leu-CGHgNiOMet-IirHo (II)

Boc-pMel-Gln-Trp-Ala-Vl-Gly-His (Dnp) -Leu (CH2 NH) Met-EH2 (I) (180 mg) was suspended in 7.2 ml of KH 2 PO 4 (0.8M) (which was adjusted to pH 8 with EaOH 1E), followed by addition of 7.2 ml of 2-mercaptoethanol. The resulting solution was stirred for 20 minutes at ambient temperature and then concentrated in vacuo and poured into Et 2 O dropwise. The crude product was filtered and purified first by flash chromatography on silica gel using the solvent system AcOEt / MeOH 7/3, v / v; then by preparative RP-HPLC in a gradient of 30% to 90% of the eluent B in the eluent A, over a period of 20 minutes at a flow rate of 24 ml / minute. 82 mg (52% yield) of compound II: m.p. 75øC (mod.) - 120ø (dec.); amino acid ratios: Glu 1, Gly 0.97 (1), Ala 0.99 (1), Val 1.02-28

(1), His 0.94 (1) (pMel, Trp and Leu (CH 2 TH) Met-M 2 n.d.); FAB-MS: m / z 1312 (7H, +); RfA 0.14: Râ, A A 18.1.

Example 3 Preparation of

Ac-pMel-Gln-iPrp-Ala-Val-Gly-His (Dnp) -Leu-CH3-Methyl-

Ac-pMel-OH (IIIa)

A solution of acetylimidazole (0.991 mg, 9 mmol) in dimethylformamide (10 ml) was added dropwise with stirring to a solution of 500 mg (1.5 mmol) H-pMel-OH (SIGMA ) in 10 ml of dimethylformamide. The reaction mixture was stirred for 5 hours at room temperature, then the solvent was evaporated in vacuo. The crude product was purified in the form of its dihexylhexylamine salt. 312 mg (60% yield) of compound IIIa were obtained from AcOEt: Et2 O: m.p. 52-54 ° C; EI-MS m / z 346 (2, m + e); RfD 0.33; R% 12.8.

Step 2

Ac-pMel-Gln-Irp-Ala-Val-Gly-His (Dnp) -Leu (NH2) Met-3β2 (IC)

70 mg (0.2 mmol) of Ac-ple1-OH in 5 ml of dimethylformamide was dissolved, then 233 mg (0.2 mmol) of H-Gln-Trp-Ala-Val-Gly-His (Dnp) -Leu (CH3) 2 -Met-2 HCl (Ip), followed at 5 ° C, 0.066 ml - 29

(0.6 mmol) of E-methylmorpholine and 88.5 mg (0.2 mmol) of BOP. The reaction mixture was stirred at room temperature for 4.5 hours and then poured into drop in AcOEt. The crude product was washed, washed with AcOEt and purified by preparative RP-EPLC with a gradient from 60% to 90% of eluent B in eluent A for 40 minutes at a flow rate of 24 ml / min . There was obtained 128 mg of compound III (45% yield); mp. 124 ° -150 ° C (de.); amino acid relationships; Glu 1, Gly 0.89 (1), Ala 0.98 (1), Val 0.94 (1), (Trp, His (Dnp) and Leu (CH2SH) Met-IH2 n.d.); FAB-MS: m / z 1420 (16, MH +); RfA 0.57% RTA 18.15.

Example 4 Preparation of

(IV) From 0.20 g (0.172 mmol) H-Gln-Trp-Ala-V al-Gly-His (Dnp) -6ψψ (ΗΗ ^) Val-Gly-His (Dnp) -Leu vp (CH 2) H 2 -H 2 NH 2 (Ip), 0.068 g (0.258 mmole) [N -bis- (2-chloroethyl) amino] benzoic acid (Cab-OH ), 0.115 g (0.258 mmol) of BOP and 0.057 ml (0.516 mmol) of NMM and proceeding as described for the preparation of compound III gave a crude product which was purified by preparative RP-HPLC with a gradient from 30% to 90% of the eluent B in the eluent A for 20 minutes at a flow rate of 24 ml / minute. There was obtained 0. 138 g (60% yield) of compound III: m.p. 128Â ° -150Â ° C (dec.); amino acid ratios: Glu 1.02 (1), Gly 1, Ala 1.00 (1), Val 0.95 (1) (Trp, His (Dnp) and Leu 30 (CH 2 NH) Met-WH 2 n.d.); EM-BAR; m / z 1336 (13, MH +); RfA 0.47; RTa 19.9.

Example 5

Preparation of

Cab-Gln-Trp-Ala-V al-Gly-His-Leu VK CH2IH) Het-IEU (V)

Cab-Gln-Trp-Ala-Val-Gly-His (Dnp) -Leu (CH2IH) Met-M2 (IV) was suspended in 10.5 g ml of 0.1M KH2 PO4 (which was adjusted to pH 8.1 with KOH), and then 10.5 ml of 2-mercaptoethanol was added. The resulting solution was stirred for 30 minutes at ambient temperature and then concentrated in vacuo. The product was extracted with butanol and the organic layer was washed twice with water and evaporated. The residue was dissolved in methanol and precipitated with ethyl ether. The crude product was purified by preparative RP-HPLC which developed with a gradient from 50% to 90% of the eluent B in the eluent A for 30 minutes at a flow rate of 24 ml / min; 96 mg (55% yield) of compound V: m.p. 128 ° -150 ° C (dec.); amino acid relationships; Glu 1.08 (1), Gly 1, Ala 0.90 (1), Val 0.91 (1), Trp 1.10 (1), His 1.09 (1) (Leu (GHglH) let HHg nd ); FAB-MS m / z 1170 (23, MH +); Rf 0.39; RIA 14.1. 31

Proceeding in the same manner as in the previous Examples, the following peptides were also prepared: H-pMel-Gln-Trp-Ala-V al-Gly-Leu ψ (CH₂POLeu-Hg HH-pMel-Gln-Trp-Ala-Val-Gly -cyclohexyl-HHg

Ac-pMel-Gln-Trp-Ala-Val-Gly-Leuy (GH2lH) Ile-lH2

Brom-pMel-phe-Gln-Trp-Ala-Y-allyl-Gly-LeuM (GH 2 HH) ## STR1 ## (CH2) 3 -H-pMel-Phe-Gln-Trp-Ala-Y-allyl-Gly-Leu (CH 2 NH) -Glyn-Trp-Ala-Ala-Gly-Leu (CH g NH) L-I'TH 2 C ab -Gln-T (-CH 2 -H-Gl-Trp-Ala-V al-Gly-Leu CH 2 M i) Met-H 2

(-CH 2 CH 2) -CH 2 -CH 2 -CH 2 -NH -CH 2 -CH 2 -CH 2 -NH -CH 2 -CH 2 -CH 2 -NH

Claims (1)

Process for the preparation of a peptide of formula RABC-Trp-Ala-Val-XYTW (I) in which R represents a group of the formula: 4- (C 1 -C 2 CH 2) 2 N-Cg H 4 -CH 2 CH (NHR 1) (NHCH 2 CH 2) 2 N-C 8 H 4 -CH 2 CE (NHR; L) CO-; 4- (ClCH 2 CH 2) 2 N-C 6 H 4 -CO-? 3- (ClCE2CH2) 2N-CgH4-CO-; C H ₂C H ₂-; 3rCII = CH-CO-; CH2 = CClCO-; CH2 = CBrCO- (both cis and trans isomers); CH 2 -CH-CH 2 -CO-; CH = C-CO-; C 1 -C 2 CH 2 CH 2 N (NO) CO-; < / C1CH2C0-CH (R2) NHC0 (CH2) 2CO-; A represents a valence bond or a Gly moiety, Leu-Gly, Arg-Leu-Gly, or Gin-Arg-Leu-Gly; B represents a valence bond or an Asn, phe or Thr residue; C represents a radical Gin or His; X represents a Gly or ala residue; Y represents a valence bond or a His (R3), his (R3), Phe, phe, Ser, ser, Ala or ala residue; T represents a valence bond or a Leu, leu, Phe or phe moiety; W represents a group of the formula O R 2, NH 2, NH (CH 2) 4 CH 3, NH (CH 2) 2 Cg H 5, Met-R 4, Leu-R 4, Ile-R 4 or Nle-R 4; R 2 represents a hydrogen atom, a Boc group or a C 1 -C 4 acyl group; R 2 represents, a hydrogen atom, a straight or branched chain aliphatic chain having 1 to 1 carbon atoms. 11 carbon atoms, a benzyl group or a phenyl group; R3 represents a hydrogen atom or a group of Cl, Dnp or Bzl; R4 represents a group NH2, NH-NH2 or OR2, one or more peptidic bonds (CONH) are substituted by reduced peptide bonds (CE2 NH), and pharmaceutically acceptable salts thereof, characterized in that the amino acid and / or amino acid derivatives in the desired sequence and / or peptide fragments containing amino acids or derivatives thereof in the desired sequence to provide the desired peptide, the carboxy terminal carboxylic acid group being activated for peptide bonding and the remaining groups being protected and the resulting compound being deprotected and / or of converting the resulting peptide into a pharmaceutically acceptable salt thereof. Lisbon, November 2, 19.90 The Official Agent of Industrial Property