IE59489B1 - New polypeptide compounds, their preparation, their use as medicaments and compositions containing them - Google Patents

Abstract

Polypeptidic compounds saving the calcium of the organism, preparation thereof, application thereof as medicaments and compositions containing them. Analogues of natural hypocalcemic dotriacontapeptides characterized by the presence of asparagine respectively: a) either in position 2 with a cysteine in position 1,7; b) or in position 1 with an aminosuberic residue in position 1,7. Said polypeptides may be used as active principle for new medicaments which are particularly indicated for osteoporoses of various etiologies.

Description

European Patent Application EP 0146842 describes chicken calcitonin. This calcitonin has an activity similar to that of salmon calcitonin, and inferior to the products of the present application.

The subject of the present invention is new polypeptide compounds, wherein they are chosen from the group formed by the products with the formula (1^): Cys-Asn-Ag-Leu-Ser-Thr-Cys-Ag-Leu-Gly-A^-A^-AjgGln-Ai5-Aig-Ai7-Lys-Aig-A20-ThrA22-Pro-A24-Thr-A26 (IA) -A27-Gly-A2g-Gly-Ag1-Pro NH2 where Ag = Ser, Gly or Ala Ag = Val or Leu Aji = Lys or Thr Aj2 = Leu or Tyr ^13 = $er or Ajg = Glu or Asp Ajg = Leu or Phe Ap = His or Asn Ajg = Leu or Phe A20 = Gin or His A22 = Tyr or Phe A24 ~ Ar9 or A2g = Asp, Asn or Ala A27 = Val, Thr or lie - 2 A2g = Ala, Ser or Val A31 = Thr, Val or Ala and the products of formula (Ιθ) which contain ^-amino-suberic acid (Asu) of which theώ-carboxyl is fixed to the amino group of Asn: —-(CH2) 5CO-Asn-A'2-Leu-Ser-Thr-NHCHCO-A’?-Leu-Gly-A'1θA'11"A'i2GlnA'14"A'15"A'16LySA'18’ A' ig^hr-A'^-Pro-A'^-Thr-A'^-A'^-GlyA' 28"θ^"Α' 30"Pro NHg where: A'2 = Ser, Gly, or Ala A'y = Val or Leu Α'ιθ = Lys or Thr Α'ϋ = Leu or Tyr A'i2 = Ser or Thr A'i4 = Glu or Asp A'15 = Leu or Phe Α'ιθ = His or Asn Α'ιθ = Leu or Phe A'ig = Gin or His A'2i = Tyr or Phe A23 = Ar9 or GlnA*25 = Asp' Asn or A^aA 26 ~ Va^' Thr orA28 = A^a' $er or ValA30 = Thr’ Val or Ala The products with the formula (Ιθ) are structural analogues of natural peptides, such as K. Jost and J. Rudinger realized in the case of neurohypophyseal hormones (Collect. Czech. Chem. Comm. 1967, 32, 1229).

The products with the formulae (1^) and (Ιθ) can be - 3 regrouped into the following formula: D-Α1y-Leu-Gly-A'jq-A1jj-A1^-Gln-A1^-A1^g-A1jg-Lys-A' -Ϊ9 -Thr-$i -proi3 -Thri5 -ii -^-ii -G1*-$0 Pro NH. in which A'? Λ I Λ I Δ 18' 19’ the previous a ' δ a· A1 A1 -A1 A 10, A n, A 12, ft 14, rt 15, rt 16, 21’ A23’ A25’ A 26' A<28 and A 30 have significance and D represents: - either the residue Cys-Asn-Ag-Leu-Ser-Thr-Cys- in which Ag has the previous significance -or the residue Γ-CH2) 5-1 cO-Asn-A12-Leu-Ser-Thr-NHCHC0in which A'2 has the previous significance.

The subject of the invention is in particular compounds corresponding to the general formulae (IA) and (Ιθ), as defined previously, in which Αθ and A'? represent a valine.

The subject of the invention is more particularly compounds answering to the following general formulae (IA) and (Ιθ): -Cyn-Asn-Ser-Leu-Serg-Thr-Cys-Val-Leu-Gly^g-Lys-Leu-Ser-Gln-Glujg -Leu-His-Lys-Leu-Gln2g-Thr-Tyr-Pro-Arg-Thr2g-Asp-Val-Gly-Ala-GlygQ -Thr-Proamide; -CO-Asn-Ser-Leu-Ser-Thrg-NH-CH-CO-Val-Leu-Gly-LySjg-LeuSer-Gln-Glu-Leujg-His-Lys-Leu-Gln-Thr2g-Tyr-Pro-ArgThr-Asp2g-Val-Gly-Ala-Gly-ThrgQ-Proamide. - 4 The polypeptides according to the invention lower calcemia and phosphatemia; they inhibit bone destruction, accelerate formation of new bones, increase intestinal absorption of calcium and decrease calciurea. They fix calcium inside the cells. They possess an important anti-inflammatory and antalgic effect.

These properties justify the use of polypeptides with the formulae (1^) and (Ιθ) as defined previously, as medicaments.

The subject of the invention is therefore also polypeptides with the formulae (IA) and (Ιθ) as defined previously as medicaments.

The subject of the invention is more particularly, as medicaments, the preferred polypeptides mentioned above.

The main indications of the medicaments according to the invention are represented by Paget's disease, osteoporoses of various etiologies (common, poro-malacic, cortisonic, post-traumatic, of immobilization and idiopathic), renal osteodystrophy, fractures, hypercalcemias, osteroarticular pains, in particular those resulting from bone metastases, spasmophilia and normocalcemic tetany.

The subject of the invention is also pharmaceutical compositions containing as active principle at least one of the medicaments as defined previously.

These pharmaceutical compositions can be, for example, solid or liquid and are presented in the pharmaceutical forms currently used in human medicine, like, for example, simple or sugar-coated tablets, capsules, granules, liposomes, aerosols, suppositories, injectable preparations; they are prepared according to the usual methods. The active principle or principles can be incorporated in the excipients usually employed in these pharmaceutical compositions, such as talc, gum arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous or non-aqueous vehicles, fatty substances of animal or vegetable origin, paraffin derivatives, glycols, various moistening, dispersing or emulsifying agents, and preservatives. - 5 The subject of the invention is in particular pharmaceutical compositions as defined previously, wherein they are intended for parenteral administration, oral administration in the form of liposomes, or nasal administration in pulverised form.

The subject of the invention is also a preparation process for compounds as defined previously, wherein amino acids, peptides or their combinations, are submitted to condensation reactions in the order of the sequence of amino acids with the formulae (1^) and (Ιθ), and when the products with the formula (Ιθ) are prepared, the solid phase technique for obtaining the 10-31 sequence and the classic liquid phase technique for obtaining the 1-9 sequence are combined.

As previously indicated, the synthesis of peptides with the formula (1^) and that of the 10-31 sequence of products with the formula (Ig) are carried out by using the solid phase technique.

This brings into play benzylhydrylamino resin (P.G. Pietta and G.R. Marshall, Chem. Commun., 1970, 650), of which the preparation and use have been described (P. Rivaille, A. Robinson, M. Kamen and G. Milhaud, Helv., 1971, 54, 2772).

In a preferred way of preparation of products with the formula (IA), the group N-X-butyloxycarbonyl (t-Boc) protects the 1) of benzyl ester for the carboxylic chain of aspartic and glutamic acids, 2) of benzyl ether for the hydroxyl of serine, threonine and of 2,4-dichlorobenzyl ether for tyrosine, 3) of methoxybenzyl ether for the sulphhydryl group of cysteine, 4) of dinitro-2,4-phenyl for the imidazol of histidine, ) of the benzoylcarbonyl group for the E-amino function of lysine. - 6 The different amino acids are incorporated in the peptide chain by dicyclohexylcarbodiimide (DCCI) and hydroxybenzotriazol (HOBT) by making protected aminoacids in excess in relation to the proline first fixed on the resin react successively. The reaction is allowed to continue for two hours. The t-Boc groups are eliminated by trifluoroacetic acid in solution in methylene chloride; after washing with methylene chloride, neutralisation is carried out by triethylamine or diisopropylethylamine (DIEA) in the same solvent.

Asparagine and glutamine are introduced in the form of their para-nitrophenyl esters, in solution in dimethylformamide (DMF). If the control test for coupling to ninhydrin according to the method of Kaiser and Colescott is positive, the mixture is treated by pivalic acid (1%); if the blue colouration still persists in this test, the DMF/1% AcOH mixture is saturated with urea. However, even if this test is negative (no blue colouration), each coupling can be repeated twice systematically. If the coupling is not total at the end of 48 hours of reaction, as is the case for glutamine in position 20, the threonine which has not reacted is acetylated by acetyl-imidazol in methylene chloride or by acetic acid and DCCI.

Finally, the protector group of the imidazol of histidine is eliminated, while the peptide is still linked to the resin, at pH 8, with mercapto-ethanol in solution in DMF. The peptide is separated form the resin by treatment with liquid hydrofluoric acid in the presence of anisole and methionine. All the protector groups are eliminated during this operation.

The peptides are purified by filtration on gel. The fractions corresponding to the main peaks are assembled, lyophilized, taken up again in a buffer at pH 8 and oxidized for 24 hours by an air current in order to form the disulphide bridge between the two cysteine residues.

The fraction corresponding to the peak possessing the biological activity sought is purified again on ion exchange resin CMC 32 by an acid elution gradient. The peptide obtained is homogeneous to electrophoresis on paper and to chromatography on cellulose. - 7 The 10-31 sequence of products with the formula (Ig) is realized in the same conditions as those indicated previously for the preparation of products with the formula (IA). What was indicated for glutamine in position 20 for products with the formula (IA) is of course valid for position 19 for products with the formula In a preferred way of carrying out the preparation process for products with the formula (Ig), the synthesis of the 1-9 sequence is carried out in a liquid medium. The fragment where A'2 has the above significance and R = ester, after cyclization, is condensed with tripeptide A'y-Leu-Gly. Use is made of the protector groups generally used for the synthesis of peptides.

Peptide with the formula (Ig) is then obtained as follows: The above protected nonapeptide is converted into N-hydroxy-succinimide ester, then coupled with the 10-31 fragment fixed on the resin in a DMF medium at 30°C. The peptide is separated from the resin by treatment with liquid hydrofluoric acid in the presence of dimethyl-sulphide and p-cresol (J.P. Tam,· W.H. Heathand and R.B. Merrifield, J.A.C.S., 1983, 105. 6442). All the protector groups are eliminated during this operation.

The peptides are purified by filtration on gel.

The examples which follow illustrate the invention without however limiting it.

Example 1 Obtaining peptide with the following formula (IA): Cys-Asn-Ser-Leu-Serg-Thr-Cys-Val-Leu- 8 GlyjQ-Lys-Leu-Ser-Gln-GlUjg-Leu-HisLys-Leu-Gln2g-Thr-Tyr-Pro-Arg-Thr2g- (IA) Asp-Val-Gly-Ala-GlygQ-Thr-Proamide The t-Boc aminoacids carrying the lateral functions come from BACHEM (Dubendorf, Switzerland) or are prepared in the laboratory by Schnabel's technique (E. Schnabel, Liebigs Ann. Chem., 702, 188, 1967) in an autotitration apparatus. Their purity is determined by polarimetry, melting point and chromatography on thin layer silica.

The hydrolysis of the purified peptide is carried out in a sealed tube under vacuum in a 6N HCl medium (110°C, 24 hours).

I - SYNTHESIS OF THE SEQUENCE a) Preparation of the resin (P. Rivaille, A. Robinson, M. Kamen and G. Milhaud, Helv., 1971, 54, 2772).

Benzyl-ketoresin In a 2 1 three-necked flask, fitted with an agitator, 50 g of polystyrene with 1% of divinylbenzene Bio-rad SXj (200-400) is left to swell for 2 hours, in 350 ml of nitrobenzene. In another flask, g of AlClg is dissolved in 300 ml of nitrobenzene to which 80 ml of benzoyl chloride is added. The mixture is poured into the first flask under a good agitation which is maintained for 2 hours at ambient temperature. If the resin swells too much, nitrobenzene is added (about 100 ml). The mixture is filtered, washed with dioxan (3 x), with acetic acid (3 x), with methanol (3 x) and finally, alternately, with CH2C12 and CHgOH in order to make the resin swell and contract each time leaving a sufficient contact time. The mixture is dried in a dessicator under vacuum all night (yield 75 g).

Benzylhvdrvlamine resin In a 1 1 flask fitted with Dean Stark and an agitator, 500 g of ammonium formiate is heated to 120-130°C, for 1 to 2 hours. The resin (50 g) is added in one go and the temperature is allowed to - 9 rise to 160-165°C, over half an hour, under efficient agitation which is maintained for 6 hours. The mixture is washed with water, then treated as above for the benzylketoresin. The resin dried under vacuum is heated to reflux and agitated for 8 hours in 6N HCl. It is then filtered and washed as previously. The fixation capacity varies, according to the nature of the amino acid, between 0.3 and 0.5 mMole/g. b) Preparation of the peptide The apparatus used enables mechanical agitation of a 25 ml pyrex tube the central compartment of which contains the resin (2 g). At the ends of the tube, fritted glass filters are intended for the introduction and then the elimination of the reagents (about 15 ml).

The coupling of the amino acids involves the following times: CYCLE 32 Coupling: g of resin (fixation capacity 0.5 mMoles/g) is agitated (10 min) in a (CHg^NCHO (DMF) medium (1:1, v/v) in the presence of Boc-L-proline (2mM, 0.43 g). DCCI (4 mMoles) and 1-hydroxybenzo- triazol (HOBT) (4 mMoles) are added and agitation is resumed for 2 hours. The resin is washed successively with CH2Cl2 (1 x, 2 min), CHgOH abs (3 x, 2 min), and (3 x, 2 min). The reaction to ninhydrin is carried out on a sample of resin: it is negative.

Liberation of amino group: The resin is treated by a CFgCOOH-^C^ mixture (1:1, v/v) for 1 min; the mixture is filtered and the operation is repeated leaving in contact for 15 min. The resin is washed with CHgC^ (3 x, 1 min). CFgCOOH is eliminated by treating the resin twice with an EtgN/^Clg mixture (12.5 : 87.5 v/v) (contact time 1 min then 5 min). It is washed with CHgClg (8 x, 1 min). The - 10 L-proline resin is titrated: it contains 0.5 mMole/g of proline.

CYCLE 31 Coupling: The L-prolyl resin (1 mMole) is agitated (10 min) with 4 mMoles of Boc-O-Benzyl-L-threonine (1.23 g) in CH2C12-DMF (1:1 v/v) (10 ml). DCCI (4 mMoles) and HOBT (4 mMoles) are added and the agitation is resumed (2 hrs). The resin is washed successively: CHgC^ (1 x, 2 min), CHgOH abs (3 x, 2 min), CH2C12 (3 x, 2 min). The reaction to ninhydrin is negative.

Liberation of the amino group - See cycle 32.

CYCLES 30 TO 25 The above coupling and liberation processes are used in making react for: - Cycle 30 4 mMoles - Cycle 29 4 mMoles - Cycle 28 4 mMoles - Cycle 27 4 mMoles - Cycle 26 4 mMoles - Cycle 25 4 mMoles Boc-glycine (0.71 g) Boc-L-ala (0.76 g) Boc-glycine (0.71 g) Boc-L-valine (0.87 g) B-benzyl ester of Boc-L-aspartic acid (1.29 Boc-O-benzyl-L-threonine (1.24 g). g) CYCLE 24 Coupling: The resin-peptide of cycle 25, previously washed with DMF (2 x), is agitated (10 min) with 4 mMoles of Boc-N-y-tosyl-L-arginine (1.41g) in a solution of DMF and then the process is as described for cycle 31. - 11 CYCLE 23 Coupling: The peptide resin of cycle 24 is agitated (10 min) with 4 mMoles of Boc-L-proline (0.88 g in a CHgClg medium) (10 ml). DCCI/HOBT is added as described for cycle 32. At the end of the reaction period, the reaction to ninhydrin is negative.

Liberation - See cycle 32.

CYCLES 22 TO 21 The coupling and liberation processes used in cycle 23 are used to make react: - Cycle 22 4 mMoles Boc-O-Benzyl-L-tyrosine (1.49 g) - Cycle 21 4 mMoles Boc-O-Benzyl-L-threonine (1.24 g).

CYCLE 20 Coupling: The resin-peptide of cycle 21, previously washed with DMF (2 x) is agitated (10 min) with 4 mMoles of Boc-L-glutamine p-nitrophenylester (1.5 g) and 10 ml DMF with the addition of 1% acetic acid. DCCI (4 mMoles) and HOBT (4 mMoles) are added and the mixture is agitated for 48 hours. The resin is washed with DMF, CH2C12, CHgOH, CH2C12 (2 x).

Liberation - See cycle 32.

CYCLES 19 TO 15 The process for cycle 31 is used to make react: - Cycle 19 4 mMoles Boc-L-leucine (1.0 g) - Cycle 18 4 mMoles Boc-£-carbobenzyloxy-L-lysine (1.52 g) - 12 - Cycle 17 4 mMoles Boc-dinitro-2,4-phenyl(im)L-histidine (1.23 g) - Cycle 16 4 mMoles Boc-L-leucine (1.0 g) - Cycle 15 4 mMoles ^-benzyl ester of Boc-L-glutamic acid (?) (1.3 9)· CYCLE 14 The process is as for cycle 20.

CYCLE 13 The process is as for cycle 31, by using 4 mMoles of Boc-o-benzyl-L-serine (1.0 g).

CYCLES 12 TO 9 The process is as for cycle 31 making react: - Cycle 12: the derivative used in cycle 19.

- Cycle 11: the derivative used in cycle 18.

- Cycle 10: the derivative used in cycle 30.

- Cycle 9: the derivative used in cycle 19.

- Cycle 8: the derivative used in cycle 27.

CYCLE 7 The process for cycle 31 is used making react 4 mMoles of S-p-methoxybenzyl ether of Boc-L-cysteine (1.13 g).

CYCLE 6 TO 3 - Cycle 6: - Cycle 5: - Cycle 4: - Cycle 3: identical identical identical identical to cycle 31. to cycle 13. to cycle 19. to cycle 13. - 13 CYCLE 2 Coupling: The peptide resin from cycle 3 previously washed with DMF (2 x) is agitated (48 hrs), with 4 mMoles of p-nitrophenyl ester of Boc-L-asparagine (1.45 g) in a DMF medium, in the presence of HOBT (4 mMoles). The resin is washed successively with DMF, CHgClg» CHgOH, CHgClg. The reaction to ninhydrin is negative.

Liberation - The process is as described for cycle 32.

CYCLE 1 mMoles of S-p-methoxybenzyl ether of Boc-L-cysteine (1.13 g) is made to react in the conditions of cycle 31.

II - ELIMINATION OF THE DINITROPHENYL GROUP OF HISTIDINE ml of mercaptoethanol is added to 15 ml of dimethylformamide (DMF), and the pH is adjusted to 8 with triethylamine. This reagent is introduced into the apparatus and the mixture is agitated for 14 hours. The resin is filtered and washed alternately with methylene chloride (3 x), and with methanol (3 x). After drying under vacuum, the weight of the resin is 2.85 g.

III - LIBERATION OF THE PEPTIDE OF THE RESIN AND ELIMINATION OF THE PROTECTOR GROUPS ml of hydrofluoric acid is distilled in a mixture of 1.5 g of resin-peptide and 1.5 ml of anisol. The mixture is agitated for 1 hr at 0°C and for 30 min at ambient temperature. The acid is expelled under vacuum and the residue is taken up again in 3 times 5 ml of acetic acid and precipitated by ethyl ether free from peroxide. The precipitate is centrifuged cold, washed several times with ether, and dried under vacuum. The precipitate is taken up again by water and the insoluble residue is eliminated by centrifuging. The lyophilized solution provides 800 mg of product. - 14 IV - PURIFICATION OF THE PEPTIDE Two hundred milligrams of crude peptide are placed at the top of a column (diameter 2.5 cm, length 90 cm) of Biogel P6, (50-100 mesh), and are eluted with 0.1 M acetic acid and fractions of 12 ml are collected by following elution with a 280 nm recording device. The purified peptide is collected in the 23-28 fractions.

V - CYCLIZATION The fractions containing the purified peptide are lyophilized and the residue is taken up again in 500 ml of 1 M ammonium hydrogenocarbonate buffer, pH 8, and submitted to a bubbling of air through a porous plate for 24 hrs. The solution is lyophilized and the residue purified on a CMC 52 Whatman column by an ammonium acetate gradient of 0.6 mho to 7 mho, pH 4, with an LKB Ultrograd 11300 apparatus. After acid hydrolysis, the composition in amino acids is the following, referred to proline = 2 (theoretical figures in brackets): Ala (1) 1.05; Arg (1) 0.83; Asp (2) 2.10; Cys (2) 1.80; Glu (3) 3.10; Gly (3) 3.30; His (1) 0.80; Leu (5) 5.2; Lys (2) 1.90; Pro (2) 2.0; Ser (3) 3.10; Thr (4) 4.15; Tyr (1) 0.85; Val (2) 2.1.

Example 2 Obtaining the peptide with the following formula (Ιβ): (ch2)5 'O-Asn-Ser-Leu-Ser-Thrg-NH-CH-CO-Val-Leu-Gly-LySjg-LeuSer-Gln-Glu-Leujg-His-Lys-Leu-Gln-Thr2g-Tyr-Pro-Arg- (Ιθ) Thr-Asp2g-Val-Gly-Ala-Gly-ThrgQ-Proamide.

The t-Boc aminoacids carrying the lateral functions are obtained as indicated in example 1. - 15 In the same way, the hydrolysis of the purified peptide is carried out in a sealed tube under vacuum in a 6N HCl medium (110°C, 24 hours). 1. SYNTHESIS OF THE 10-31 SEQUENCE OF THE PEPTIDE la) Preparation of the resin (P. Rivaille, A. Robinson, M. Kamen and G. Milhaud, Helv., 1971, 54, 2772).

Benzyl-ketoresin In a 2 1 three-necked flask, fitted with an agitator, 50 g of polystyrene with 1% of divinylbenzene Bio-rad SXj (200-400 mesh) is left to swell in 350 ml of nitrobenzene. In another flask, 66 g of AlClg is dissolved in 300 ml of nitrobenzene to which 80 ml of benzoyl chloride is added. The mixture is poured into the first flask under a good agitation which is maintained for two hours at ambient temperature. If the resin swells too much, nitrobenzene is added (about 100 ml). The mixture is filtered, washed with dioxan (3 x), with acetic acid (3 x), with methanol (3 x) and finally, alternately, with and CHgOH in order to make the resin swell and contract each time leaving a sufficient contact time. The mixture is dried in a dessicator under vacuum all night (yield 75 g).

Benzvlhydrvlamine resin In a 1 1 flask fitted with Dean Stark and an agitator, 500 g of ammonium formiate is heated to 120-130°C, for 1 to 2 hours. The resin (50 g) is added in one go and the temperature is allowed to rise to 160-165°C, over half an hour, under efficient agitation which is maintained for 6 hours. The mixture is filtered, washed with water, then treated as above for the benzylketoresin. The resin dried under vacuum is heated to reflux and agitated for 8 hours in 6N HCl. It is then filtered and washed as previously. The fixation capacity varies, according to the nature of amino acid, between 0.3 and 0.5 mMole/g. lb) Preparation of the 10-31 sequence of the peptide.

The apparatus used allows a 25 ml pyrex tube, the central compartment of which contains the resin (2 g), to be agitated mechanically. At the ends of the tube, 2 fritted glass filters are intended for the introduction and then the elimination of the 5 > reagents (about 15 ml). CYCLE 31 10 Coupling: 15 2 g of resin (fixation capacity 0.5 mMoles/g) is agitated (10 min) in a medium of CHgClg, (CHg)gNCHO (DMF) (1:1, v/v) (10 ml) in the presence of Boc-L-proline (2 mM, 0.43 g). DCCI (4 mMoles) and 1-hydroxybenzotriazol (HOBT) (4ntf1oles) are added and agitation is resumed for 2 hours. The resin is washed successively with CHgCLg (1 x, 2 min), CHgOH abs (3 x, 2 min), CH Cl 2 (3 x, 2 min). The reaction with ninhydrin is carried out on a sample of resin: it is negative. 20 Liberation of the amino group: 25 The resin is treated by a CFgCOOH-CHgClg mixture (1:1, v/v) for 1 min; the mixture is filtered and the operation is repeated leaving in contact for 15 min. The resin is washed with CHgClg (3 x, 1 min). CFgCOOH is eliminated by treating the resin twice with an ETgN/CHgClg mixture (12.5:87.5 v/v) (contact time 1 min then 5 min). The mixture is washed with CHgClg (8 x, 1 min). The resin-L-proline is titrated: it contains 0.5 mMole/g of proline. 30 CYCLE 30 35 Coupling: The resin-L-prolyl (1 mMoles) is agitated (10 min) with 4 mMoles of Boc-O-Benzyl-L-threonine (1.23 g) in CHgClg-DMF (1:1 v/v) (10 ml). DCCI (4 mMoles) and HOBT (4 mMoles) are added and agitation is resumed (2 hrs). The resin is washed successively: CHgClg (1 x, 2 min), CHgOH abs (3 x, 2 min), CHgClg (3 x, 2 min), CHgOH - 17 abs (3x, 2 min), CHgClg (3 x, 2 min). The reaction with ninhydrin is negative.

Liberation of the amino group - See cycle 31.

CYCLE 29 TO 24 (.

The above coupling and liberation processes are used in making react for: - Cycle 29 4 mMoles - Cycle 28 4 mMoles - Cycle 27 4 mMoles - Cycle 26 4 mMoles - Cycle 25 4 mMoles - Cycle 24 4 mMoles Boc-glycine (0.71 g) Boc-L-ala (0.76 g) Boc-glycine (0.71 g) Boc-L-valine (0.87 g) B-benzyl ester of Boc-L-aspartic acid Boc-0-benzyl-L-threonine (1.24 g). (1.29 g) CYCLE 23 Coupling: The resin-peptide of cycle 24, previously washed with DMF (2 x) is agitated (10 min) with 4 mMoles of Boc-N- -tosyl-L-arginine (1.41 g) in a solution of DMF and the following process is as described for cycle 30.

CYCLE 21 TO 20 Coupling: The resin-peptide of cycle 23 is agitated (10 min) with 4 mMoles of Boc-L-proline (0.88 g) in a medium of CHgClg (10 ml).

DCCI/HOBT is added as described for cycle 31. At the end of the reaction period, the reaction with ninhydrin was negative.

Liberation - See cycle 31. - 18 CYCLES 21 TO 20 The coupling and liberation processes used in cycle 22 are used in making react: - Cycle 21 4 mMoles Boc-O-Benzyl-L-tyrosine (1.49 g) - Cycle 20 4 mMoles Boc-O-Benzyl-L-threonine (1.24 g).

CYCLE 19 Coupling: The resin-peptide of cycle 20, previously washed with DMF (2 x) is agitated (10 min) with 4 mMoles of Boc-L-glutamine p-nitrophenyl-ester (1.5 g) and 10 ml of DMF with the addition of 1% acetic acid. DCCI (4 mMoles) and HOBT (4 mMoles) are added and the mixture is agitated for 48 hrs. The resin is washed with DMF, CH2C12, CHgOH, CH2C12 (2 x).

Liberation - See cycle 31.

CYCLES 18 TO 14 The process for cycle 30 is used in making react: - Cycle 18 - Cycle 17 - Cycle 16 - Cycle 15 - Cycle 14 CYCLE 13 mMoles Boc-L-leucine (1.0 g) mMoles Boc-B-carbobenzyloxy-L-lysine (1.52 g) mMoles Boc-dinitro-2,4 phenyl(im)L-histidine (1.23 g) 4 mMoles Boc-L-leucine (1.0 g) mMoles ^-benzyl ester of Boc-L-glutamic acid (1.3 g).

The process is as for cycle 19.

CYCLE 12 The process is as for cycle 30 using 4 mMoles of - 19 Boc-O-benzyl-L-serine (1.0 g).

CYCLES 11 TO 10 The process is as for cycle 30 in making react: - Cycle 11: the derivative used in cycle 18.

- Cycle 10: the derivative used in cycle 17. 2. SYNTHESIS OF THE 1-9 SEQUENCE OF THE PEPTIDE 2a) Partial sequence Boc-L-Thr (Bzl)-Asu OMe (A).

Z-Asu OMe (11.2 g) is introduced into MeOH-HgO (2:1, v/v) and the mixture is hydrogenated in the presence of palladiated carbon (14 hrs). The catalyst having been eliminated, the mixture is concentrated under vacuum. Dioxan (40 ml) is added, then triethylamine (4.2 ml) is added, while cooling, then Boc-L-Thr (Bzl) OSu (16 g) is added. After agitation (72 hrs), N,N-dimethylamino1,3-propane diamine is introduced, and the mixture is agitated (4 hrs) then concentrated to one third. It is extracted with ethyl acetate (AcOEt), the AcOEt is washed with HCl (IN), then with HgO; then distilled under vacuum. The oily residue is taken up in ether, extracted with NaHCOg (5%), re-extracted with AcOEt and washed successively with HgO, HCl (IN), HgO. The extracts are dried on anhydrous sodium sulphate and the AcOEt is expelled. A is obtained in the form of an oily product (10 g). 2b) Partial sequence Boc-L-Ser (Bzl)-L-Thr (Bzl) Asu OMe.CHA (B).

Product A (5 g) is dissolved with cooling in TFA (15 ml). At ambient temperature, TFA is expelled under vacuum and the residue is dried under vacuum, then dissolved in DMF (10 ml) and the pH is brought to 6 by triethylamine (4 ml) while cooling. Then HOBT (5 g) and Boc-L-Ser (Bzl) OSu are added with agitation (3 days) at ambient temperature, and at pH 6. N,N-dimethylamino-l,3-propanediamine is added, with agitation, (1 hr), HgO is added, followed by extraction by AcOEt as described above. Cyclohexylamine (CHA) is added to the - 20 dried AcOEt, followed by distillation under reduced pressure. The oily product B is obtained (4 g). 2c) Partial sequence Boc-L-Asn-L-Ser(Bzl)-L-Leu-NH-NH2 (C). g of Boc-L-Asn-L-Ser-(Bzl)-L-Leu OEt is dissolved in 20 ml of CHgOH, 10 ml of 80% NH2-NH2-H20 is added, and the mixture is left for 14 hours at ambient temperature. It is then precipitated with ethyl ether, the precipitate is washed with ethyl ether and recrystallized from a mixture of CHgOH-AcOEt-ethyl ether. 2.5 g of expected product is obtained (C). 2d) Partial sequence Boc-L-Asn-L-Ser (Bzl)-L-Leu-L-Ser (Bzl)-L-Thr (Bzl)-Asu OMe (D).

Product B (2 g) is introduced into AcOEt in the presence of HCl (IN); the mixture is dried on anhydrous sodium sulphate and concentrated under vacuum. TFA (6 ml) is added cold, with agitation at ambient temperature (0.5 hr), then the TFA is eliminated under vacuum and the residue is dried under vacuum. The residue is dissolved in DMF (2 ml), and after neutralization with triethylamine is slowly introduced at -40°C into a solution of C Boc-L-Asn-L-Ser(Bzl)-L-Leu-NH-NH2 (1.7 g) in DMF (6 ml) to which dioxan (2.8 ml) has previously been added, in the presence of HCl (IN) and isoamyl nitrate (0.6 ml). The pH is adjusted to 7 with triethylamine, and reaction takes place at 5°C (72 hrs).

The reactional mixture is slowly introduced at -5°C into 0.5N HCl (60 ml). The precipitate is washed with H20. Extraction is done by CHClg (100 ml), the extracts being washed successively with aqueous HCl (IN) NaCl. The CHClg is eliminated. Product 0 is precipitated by a mixture of CHClg-n-hexane (2 g). 2e) Cyclization -L-Asn-L-Ser (Bzl)-L-Leu-L-Ser (Bzl)-L-Thr (Bzl) Asu1,7-NH-NH2 (E).

Product D (1.6 g) is dissolved in anhydrous pyridine (15 ml). TFA-ONP (2.5 g) is added, with agitation at 45°C (3 hrs), followed by concentration under vacuum, precipitation by ethyl ether, treatment with TFA as above, then cyclization in anhydrous pyridine at 50°C for 5 hours. Extraction is carried out by CHClg and the extracts are washed as described above, then concentrated under vacuum, and precipitated by n-hexane.

The precipitate (2 g) is dissolved in DMF (5 ml) and CHgOH (25 ml). 15 ml of hydrazine (80%) is added, the mixture is agitated at ambient temperature (14 hrs) and HgO is added. The precipitate is filtered and washed with HgO. CHgOH (50 ml) is added and the mixture is heated to reflux; the product E is precipitated (0.8 g). 2f) Preparation of the 1-9 sequence: -L-Asn-L-Ser (Bzl)-L-Leu-L-Ser (Bzl)-L-Thr (Bzl)-Asu1,7-L-Val-L-Leu-Gly OH (F).

Product E (1 g) is put into suspension in DMF (4 ml), dioxan and 4N HCl (1.7 ml) are added at -5°C. Isoamyl nitrate (0.3 ml) is added at -10°C under agitation. L-H-Val-L-Leu-Gly (0.9 g) is added at -5°, the pH being brought to 7 by triethylamine. The mixture is agitated in an ice bath (48 hrs), then product F (1 g) is precipitated by 0.5N HCl, (150 ml). 3. PREPARATION OF PEPTIDE 3a) HOBT (111 mg) and DCCI (150 mg) are added to product F (500 mg) dissolved in DMF (2 ml). Agitation is carried out for 72 hours in the presence of peptidyl resin 10-31 (300 mg) after elimination of t-Boc of LySjg. 3b) Cleavage of the peptide according to Tam et al. JACS (1983), 105. 6442.

The quantities are referred to 1 g of peptidyl resin. 1) HF (2.5 ml) is distilled in a mixture of resin-peptide (1 g), dimethylsuphide (6.5 ml) and p-cresol (250 mg), with agitation (1 hr) at 0°C. The acid and the dimethylsulphide are expelled under vacuum and the residue is taken up 3 times by 5 ml of AcOEt. - 22 2) The dried peptidyl resin suspended in dimethylsulphide (1 ml), is treated by liquid HF (10 ml) at 0°C(lh). After elimination of the HF and the dimethylsulphide under vacuum, the resin is washed with ether (3 times, 10 ml); the peptide is taken up with acetic acid, precipitated by ethyl ether, centrifuged cold, washed several times with ether, and dried under vacuum. The precipitate is taken up again by water and the insoluble residue is eliminated by centrifuging. The lyophilized solution produces 500 mg of product. 3c) Purification of the peptide. 200 mg of crude peptide is placed at the top of a column (diameter 2.5 cm, length 90 cm) of Biogel P6, 50-100 mesh. It is eluted by 0.1M acetic acid and fractions of 12 ml are collected by following the elution with a 280 nm recording device. The purified peptide is collected in the fractions 23-28. These fractions are lyophilized and the residue is purified on a Whatman CMC 32 column by ammonium acetate gradient of 0.6 mho to 7 mho, pH 4, with an LKB Ultrograd 11300 apparatus.

After acid hydrolysis, the composition in amino acids is the following, referred to proline = 2 (theoretical figures in brackets): Ala (1) 1.15; Arg (1) 0.85; Asp (2) 2.15; Glu(3) 3.10; Gly (3) 2.9; His (1) 0.83; Leu (5) 5.3; Lys (2) 1.95; Pro (2).0; Ser (3) 3.20; Thr (4) 4.10; Tyr (1) 0.80; Val (2) 2.1; Asu (1) 0.93.

Abbreviations AcOEt = ethyl acetate Asu = φζ-aminosuberic acid Bzl = Benzyl CHA = Cyclohexylamine DCHA = Dicyclohexylamine ONP = p-nitrophenyl ester OSu = N-hydroxysuccinimide ester Z = Benzyloxycarbonyl - 23 BIOLOGICAL ACTIVITY OF THE PRODUCTS OF THE EXAMPLES This is determined using male rats weighing from 100 to 120 g and having been without food for 16 hours. The activity is expressed in MRC units starting with the decrease in calcemia, measured one hour after intravenous injection of the peptide under test suitably diluted in a buffer of 0.1N sodium acetate pH 6 and 0.1% albumin, in comparison with the activity of the Research Standard B suitably diluted. The biological activity of the peptide of example 1 is higher than 4000 MRC units per mg and that of the peptide of example 2 is higher than 4500 MRC units per mg.

In the light of this activity, the usual dose, which is variable depending on the product used, the subject treated and the cause of infection, could be for example from 1 to 100 MRC units per day intramuscularly or subcutaneously.

Claims (11)

1. New polypeptides, wherein they are chosen from the group formed 5 by the products of formula (1^): Cys-Asn-Ag-Leu-Ser-Thr-Cys-Ag-Leu-Gly A ll A 12 -A 13“ Gln_A 15 -A 16” A 17“ Lys · Α 19 Α 20 ΤΙίγ “ Α 22 Ργο Α 24 Τ ^ γ “ Α 26 Α 27“ 10 Gly-A^g-Gly-Agj-Pro NH 2 where: Ag = Ser, Gly or Ala 15 Αθ = Val or Leu Ajj = Lys or Thr Aj 2 = Leu or Tyr A 13 = $ er 0Γ T * ir Ajg = Glu or Asp 20 Α|θ = Leu or Phe Ap = His or Asn Ajg = Leu or Phe Α 2θ = Gin or His A 22 = Ty r or Phe 25 A 24 = Arg or Gin Α 2 θ = Asp, Asn or Ala A 2 y = Val, Thr or Ile A 2 g = Ala, Ser or Val A 31 = Th r » Val or Ala and the products of formula (I R ): (CH 9 ) 2. '5' 35 CO-Asn-A' 2 -Leu-Ser-Thr-NH0HC0-A' ? -Leu-Gly-A' 1θ A '11 A '12 G1n A '14 A '15 A '16“ LyS A '18 A'“ig-Thr-A 1 21-Pro-A' 2 g-Thr-A 1 2θ-Α 1 2g“G1y A '28 G1 y _A, 30 Pro NH 2 (I n ) - 25 where: A' A' A 1 A' A' I I 16 18 19 21 '23 26 28 30 A' A' A' Ser, Gly, or Ala Val or Leu Lys or Thr Leu or Tyr Ser or Thr Glu or Asp Leu or Phe His or Asn Leu or Phe Gin or His Tyr or Phe Arg or Gin Asp, Asn or Ala Val, Thr or Ile Ala, Ser or Val Thr, Val or Ala

2. New polypeptides corresponding to the formula (I A ) as defined in claim 1 in which the symbol Αθ represents a valine.

3. New polypeptides corresponding to the formula (Ιθ) as defined in claim 1 in which the symbol A 1 ? represents a valine.

4. The following product with the formula (I A ) according to claim 1 Cys-Asn-Ser-Leu-Serg-Thr-Cys-Val-LeuGlyiQ-Lys-Leu-Ser-Gln-Glu^-Leu-HisLys-Leu-GlngQ-Thr-Tyr-Pro-Arg-ThrggAsp-Val-Gly-Ala-Gly^Q-Thr-Proamide.

5. The following product with the formula (Ιθ) according to Claim 1 (ch 9 ) 2'5 CO-Asn-Ser-Leu-Ser-Thrg-NH-CH-CO-Val-Leu-Gly-LySjQ-LeuSer-Gln-Glu-LeUjg-His-Lys-Leu-Gln-ThrgQ-Tyr-Pro-Arg(Io) - 26 Thr-Asp25-Val-Gly-Ala-Gly-ThrgQ-Proamide

6. Process for the preparation of the new polypeptides specified in claims 1 to 5, wherein amino acids, peptides or their combinations are submitted to condensation reactions in the order of sequence of the amino acids with the formulae (I A ) and (Ι β ) and during the preparation of the products of the formula (Ιθ) the solid phase technique for obtaining the 10-31 sequence and the standard liquid phase technique for obtaining the 1-9 sequence are combined.

7. As medicaments, the polypeptides mentioned in claims 1 to 5.

8. Pharmaceutical compositions containing one medicament according to claim 7 as active principle.

9. The pharmaceutical preparations containing as active principle one or more polypeptides mentioned in claims 1 to 5, as well as appropriate vehicles intended for parenteral administration, oral administration in the form of liposome and nasal administration in the form of a spray.

10. A process for the preparation of polypeptides as claimed in claims 1 to 5, substantially as hereinbefore described by way of Example.

11. Polypeptides according to Claim 1 whenever prepared by a process' as claimed in Claim 6 or Claim 10.